Picosecond hole transfer and microsecond charge-separated states were observed at the perovskite nanocrystal/tetracene interface, which are leveraged to demonstrate the dissociation of up to 5.6 excitons per nanocrystal by multi-hole transfer. Hole transfer (HT) is often kinetically sluggish compared to electron transfer (ET), which increases recombination losses and thus limits the efficiency of many energy conversion devices such as light-emitting diodes, solar cells and solar-fuel production systems. Recently introduced lead halide perovskites and their nanocrystals (NCs) have emerged as an important class of energy conversion materials. Here we report that tetracene molecules can enable ultrafast and efficient HT from perovskite NCs. Transient absorption measurements reveal that HT occurs in 7.6 ± 0.2 ps, and the charge-separated states are extremely long-lived (5.1 ± 0.3 μs). Such exceptional charge separation properties are leveraged to demonstrate the dissociation of up to 5.6 excitons per NC by multi-hole transfer for the first time. These results not only suggest that tetracenes are an effective hole-extracting material for perovskite devices, but also have important implications for using perovskite NCs as sensitizers and tetracenes as redox mediators to drive single and even multi-electron photochemical reactions. Lead halide perovskite nanocrystals (NCs) have emerged as a promising enabler for many optoelectronic applications.In particular, recently, there has been growing interest in Dexter-type triplet EnT in NC–acene hybrids due to the peculiar singlet fission (SF) and triplet–triplet annihilation (TTA) behaviors of acene molecules.3 NCs overlaps well with the absorption spectrum of tetracenes (∼500 nm). The enormously large extinction coefficients of CsPbBr3 NCs (on the order of 107 cm–1 M–1 at 400 nm)Our initial idea for this work was to observe FRET from perovskite NCs to tetracene molecules as the emission spectrum of CsPbBrxBrx3– perovskite NCs with edge lengths within 8–10 nm , confirming the exceptional light-harvesting capability of perovskite NCs. Based on the absorption spectra and extinction coefficients, ∼137 TCAs are bound to each NC. The exact binding configuration has not been characterized in this work, but it is commonly assumed that similar polycyclic aromatic hydrocarbon (PAH) molecules bind to NC surfaces through the carboxyl groups.CsPbClm Fig. S1 and tetrm Fig. S1. The carm Fig. S1. As the xBrx3– with a Cl to Br ratio of ∼1 : 7 because of the very weak confinement of the NCs. Bulk CsPbClxBrx3– values are calculated as the composition-weighted average value using those reported for bulk CsPbCl3 (3.vs. 2.15%), no sign of EnT can be detected. Note that the QY of TCAs was measured using 453 nm excitation light. Thus, HT completely dominates over EnT in NC–TCA complexes. PL lifetime measurements  of ∼0.07, which are dominated by an exciton bleach (XB) feature due to the state-filling signals of band edge electrons and holes.) of ∼0.07, which are dominated by an exciton bleach (XB) feature due to the state-filling signals of band edge electrons and holes.Details on TA set-ups can be found in the ESI.––TCA+). Multi-exponential fit to the kinetics reveals that the HT process has a single-exponential time constant of 7.6 ± 0.2 ps and the CR process has an amplitude-averaged time constant of 5.1 ± 0.3 μs. Compared with the long XB lifetime in free NCs  coincides exactly with the hole contribution to the XB feature for these NCs. Even if this coincidence is true, the fact that the PL of NCs in the NC–TCA complexes is almost completely quenched  would reNote that HT kinetics can in principle also be analyzed using the ground-state-bleach of TCAs or absorption of TCA cations. However, because the extinction coefficient of TCAs is ∼3 orders-of-magnitude lower than that of NCs and because there is a spectral overlap between them, detection of TCA signals is technically difficult. TCA cations were reported to have broadband absorptions in the 500–900 nm range,3 NCs to phenothiazineA possible reason for the ultrafast HT is the strong electronic coupling between NCs and TCAs. It was reported that Dexter EnT from pentacene triplets to PbSe NCs occurred in sub-psvia bypassing the many intermediate recombination steps involved in sequential photon absorption triggered charge transfer reactions. It has been shown that interfacial electron transfer (ET) from NCs to attached molecules is fast enough to dissociate multiple excitons from NCs,We noticed that the HT time is much faster than the biexciton Auger recombination (AR) time  reported for perovskite NCs,N〉 at each pump fluence (see the ESI at each pump fluence (see the ESIN〉 ∼ 0.6 and 〈 ∼ 0.6 and 〉 ∼ 0.6 and 〈N〉 ∼ 0.2 ( ∼ 0.2 (τXX) of 73.2 ± 3.4 ps. Using a well-established statistical scaling law for AR, the lifetime of an N-exciton state is obtained as follows: τNX = 2τXX/N(N – 1).60 buckyballsWe measured multi-exciton dynamics in NCs using pump fluence-dependent TA ; see Fig , as has been done in previous multi-electron transfer studies.N〉 in the range of 20–50 ps. In the <20 ps range, increasing 〈 in the range of 20–50 ps. In the <20 ps range, increasing 〉 in the range of 20–50 ps. In the <20 ps range, increasing 〈N〉 leads to faster decay due to the superposition of AR with HT; note that the smaller initial signal amplitude observed for very large 〈 leads to faster decay due to the superposition of AR with HT; note that the smaller initial signal amplitude observed for very large 〉 leads to faster decay due to the superposition of AR with HT; note that the smaller initial signal amplitude observed for very large 〈N〉 is a result of fast decay convoluted with slow XB formation at very high fluences possibly due to the hot-phonon-bottleneck reported for perovskites and their NCs. is a result of fast decay convoluted with slow XB formation at very high fluences possibly due to the hot-phonon-bottleneck reported for perovskites and their NCs.N〉 from 0.1 to 5.6, beyond which the AR kinetics becomes progressively prominent. This directly evidences that HT can dissociate up to 5.6 excitons per NC: when 〈 from 0.1 to 5.6, beyond which the AR kinetics becomes progressively prominent. This directly evidences that HT can dissociate up to 5.6 excitons per NC: when 〉 from 0.1 to 5.6, beyond which the AR kinetics becomes progressively prominent. This directly evidences that HT can dissociate up to 5.6 excitons per NC: when 〈N〉 is below the maximum dissociation number, all the excitons are dissociated by HT and no AR feature can be observed; in contrast, when 〈 is below the maximum dissociation number, all the excitons are dissociated by HT and no AR feature can be observed; in contrast, when 〉 is below the maximum dissociation number, all the excitons are dissociated by HT and no AR feature can be observed; in contrast, when 〈N〉 exceeds the maximum dissociation number, the remaining, undissociated excitons decay exceeds the maximum dissociation number, the remaining, undissociated excitons decay via AR whose kinetics features in the 50–200 ps range can be detected.The distinguishing kinetics features of HT and AR on different timescales offer an alternative way to determine the number of dissociated excitons. As shown in 60 and QDs. This difference likely arises from the size effect on coulombic charging energy. The NCs used here have a diameter of ∼10 nm; the charging energy of introducing electrons into these NCs dispersed in a nonpolar solvent such as hexane was estimated to be ∼75 meV according to a previous report.This measured number (5.6) is lower than the estimated one (6.5), most likely because the HT rate decreases when the dissociated exciton number increases, as discussed above. However, a small deviation between them suggests that the charging energy effect for the NCs here is not as strong as those reported for Cvia their reversible one-electron oxidation to radical cations.In summary, we observed 7.6 ps HT from perovskite NCs to surface-attached tetracenes and exceptionally long-lived (5.1 μs) charge-separated states. This ultrafast HT was used to demonstrate dissociation of up to 5.6 excitons from each NC. These results demonstrate that small acene molecules are very effective hole scavenging materials for perovskite NCs. This system may find applications in redox-mediated photo-oxidation for organic synthesis because acenes have been demonstrated to be hole transfer mediators The authors declare no conflict of interest.Supplementary informationClick here for additional data file.


Singular traumatic insults, such as bone fracture, typically initiate an appropriate immune response necessary to restore the host to pre-insult homeostasis with limited damage to self. However, multiple concurrent insults, such as a combination of fracture, blunt force trauma, and burns (polytrauma), are clinically perceived to result in abnormal immune response leading to inadequate healing and resolution. To investigate this phenomenon, we created a model rat model of polytrauma.To investigate relationship between polytrauma and delayed healing, we created a novel model of polytrauma in a rat which encompassed a 3-mm osteotomy, blunt chest trauma, and full-thickness scald burn. Healing outcomes were determined at 5 weeks where the degree of bone formation at the osteotomy site of polytrauma animals was compared to osteotomy only animals (OST).We observed significant differences in the bone volume fraction between polytrauma and OST animals indicating that polytrauma negatively effects wound healing. Polytrauma animals also displayed a significant decrease in their ability to return to pre-injury weight compared to osteotomy animals. Polytrauma animals also exhibited significantly altered gene expression in osteogenic pathways as well as the innate and adaptive immune response. Perturbed inflammation was observed in the polytrauma group compared to the osteotomy group as evidenced by significantly altered white blood cell (WBC) profiles and significantly elevated plasma high-mobility group box 1 protein (HMGB1) at 6 and 24 h post-trauma. Conversely, polytrauma animals exhibited significantly lower concentrations of plasma TNF-alpha (TNF-α) and interleukin 6 (IL-6) at 72 h post-injury compared to OST.Following polytrauma with burn injury, the local and systemic immune response is divergent from the immune response following a less severe singular injury (osteotomy). This altered immune response that follows was associated with a reduced capacity for wound healing.The online version of this article (10.1186/s13018-019-1082-4) contains supplementary material, which is available to authorized users. Fracture healing is a complex process that requires the early involvement of the immune system to generate a local inflammatory process necessary to drive the initiation of regeneration. This initial inflammatory response is followed by a resolution phase and quickly progresses toward the proliferation and remodeling phase . DelayedUpon fracture, local damaged blood vessels rupture, leading to the formation of a hematoma, while the surrounding injured tissue releases cytokines and chemokines to recruit neutrophils, macrophages, and lymphocytes to the site of injury. The recruited immune cells induce the initial inflammatory processes required for repair, and loss of an appropriate, sequential immune response has been demonstrated to contribute to disturbed bone regeneration –12. FollInjury is the most common cause of death and disability of young adults and children and represents a small, care-intensive proportion of trauma patients. A recent review of European trauma registries found that approximately 10–16% of all trauma patients present with polytraumatic injuries. For individuals with significant injuries to more than one region of the body, traffic-related accidents were the main mechanism of injury, accounting for 65–82% of all injuries across age groups . AdditioPrevious studies have demonstrated that additive concurrent trauma can delay fracture healing, but few animal models have included a large total body surface area burn as an additive trauma and the impact of thermal injury on fracture healing has not been well characterized , 20. Whin ~  8/group) of trauma representing an increased ISS as follows:A.Osteotomy (OST)B.Osteotomy, blunt chest trauma, and full-thickness burn (polytrauma).All animal procedures were approved by the Institutional Animal Care and Use Committee at the United States Army Institute of Surgical Research and were conducted in compliance with the Animal Welfare Act and in accordance with the principles of the Guide for the Care and Use of Laboratory Animals. Male outbred Sprague-Dawley rats weighing 385 ± 2 g were grouped housed preoperatively in a pathogen-free vivarium at the United States Army Institute of Surgical Research which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. All animals had food and water provided ad libitum and unrestricted activity prior to and after all procedures. Animals were divided into two cohorts (n = 8) to analyze departure from baseline.In some experiments (leukocyte populations), OST and polytrauma groups were also compared to naïve animals  was given at least 15 min prior to trauma to maintain adequate analgesia for 48–72 h after the procedure . AnestheAnimals did not receive any prophylactic antibiotics. Following surgery, each animal was assessed for signs of pain or distress twice a day for the first 72 h and weekly thereafter, evaluating general appearance, behavior, body weight, clinical signs of wound infection , and gait. No animals displayed signs of clinical infection at any point during this study. If the animals were found to be in distress, additional Buprenorphine-SR was administered and additional assessments were performed until resolved. If animals were found to have more than 10% body weight loss relative to surgery, 3 mL of normal saline was given subcutaneously once a day and additional enrichment was provided until resolved. Due to concerns regarding the use of chronic analgesia and bone fracture healing , analges2 PCR or μCT and histology.A 3-mm non-critical sized osteotomy was performed as described previously described using aseptic techniques, with minor modifications Fig. a 28–30]–3028–30]While still in a surgical plane of anesthesia, blunt chest trauma was generated by the energy transfer from a falling weight to a platform resting on the animal’s chest, which generates standardized bilateral lung contusions Fig. b 34]. B. B34]. B2 PCR or microcomputed tomography (μCT) and histology.Full-thickness burns were performed utilizing a protocol developed at the United States Army Institute for Surgical Research as previously described, with minor modifications Fig. c, d 35]35]. AfteTo confirm full-thickness burn, histology of the burn wound area was performed at 72 h post-trauma. Biopsies of normal . Osteotomy regions were scanned at high resolution with a 10.5-μm voxel size at a voltage of 70 kVp, a current of 114 μA, and an integration time of 300 ms. The images were converted to 8-bit bitmap files using Image J  and reoriented with DataViewer  so that the slices were in the plane of the defect border. Regions of interest  scans  were performed on a set of harvested rat femurs at 5 weeks post-injury  centered within the osteotomy defect, and a polygonal area was drawn around the intact cortex proximal and distal to the defect and interpolated throughout the slices to simulate the area in which the intact femur should lie Fig. a, b. A gn = 6/group). Femurs were immediately harvested following euthanasia and stored in 10% neutral buffered formalin for approximately 7 days for fixation. Femurs were rinsed in type 1 ultra-pure water and stored in formic acid bone decalcifier  for approximately 7 days. Femurs were embedded in paraffin and cut in longitudinal section at a thickness of 8 μm, deparaffinized, and stained with H&E. Histologic analysis of the osteotomy (fracture site), K-wire insertions (pin sites), and bone marrow adjacent to the fracture site on each sample was performed using light microscopy. The pin and fracture sites were evaluated for the severity of inflammation, i.e., infiltration of neutrophils and macrophages, and the number of osteoclasts and osteoblasts on a scale of 0 to 4 . The presence of granulation tissue, determined by increased mitotic rate of fibroblasts, was also analyzed at the pin insertion sites. Bone marrow necrosis at the fracture site was scored on a scale from 0 to 4 . All slides were reviewed and scored by a board-certified veterinary pathologist. Prior to analysis, images were converted to black and white at 8-bit and analyzed on open access software .Histological analysis was conducted on femurs at 24 h and 72 h post-trauma , and stored according to manufacturer’s directions. Disruption and homogenization was performed with 750 μl of QIAzol lysis reagent. Samples were incubated at RT for 5 min. Following incubation, 150 μl of chloroform was added, samples were mixed, incubated for 3 min, and the aqueous phase separated by centrifugation. Aqueous phase was transferred to a sample tube and RNA extraction was performed utilizing silica-coated magnetic-particle pre-filled reagent cartridges  including 10-μl RNase-free DNase I and loaded into the EZ1 Advanced XL  using the pre-programmed EZ1 RNA Universal Tissue protocol. Samples were eluted into 50 μl of RNase-free water. The concentration of RNA was determined at OD260/280 using a spectrophotometer , and ribosomal RNA band integrity was analyzed utilizing a RNA ScreenTape on a 2200 TapeStation . Samples with RNA Integrity Numbers of approximately 7 were selected for analysis. First strand complementary DNA (cDNA) was synthesized from RNA using 172 μg of total RNA and the RT2 First Strand Kit  according to manufacturer’s directions. Pathway-focused gene expression analysis was performed for innate and adaptive immune responses  and osteogenesis  from the local fracture site and performed according to the manufacturer’s instructions. Gene expression was performed on a real-time PCR detection system  and data was analyzed using web-based software . Comparisons were made between OST and polytruama at 24 and 72 h for the innate and adaptive immune response (n = 5 /group) and at 10 days post-trauma for osteogenesis (n = 6/group).Immediately after animals were euthanized, the osteotomy site was excised, placed in RNAn = 8/group) and 72 h (n = 6/group) post-trauma from a deeply anesthetized rat by cardiac puncture, followed by euthanasia. Complete blood cell counts were analyzed on a hematology analyzer , for circulating concentration and composition of lymphocytes, monocytes, and granulocytes . Additional aliquots of blood were collected in EDTA tubes; plasma was isolated and stored at − 80 °C for analysis.Whole blood was collected at terminal time points of 24 h (n = 5/polytrauma and n = 3/OST) and 24 h (n = 5/polytrauma and n = 8/OST) post-trauma was assessed via a high sensitivity in vitro enzyme-linked immunosorbent assay  according to manufacturer’s directions. In addition to HMGB1, 23 plasma cytokines were quantified at 24 h and 72 h using a commercially available multiplex kit . Samples were run in triplicate, according to manufacture directions (n = 5 rats/group/time point).Circulating HMGB1 at 6 h . Genes with differences greater than 2 fold (p < 0.05) compared to control group were considered significant.Statistical analysis was conducted using GraphPad Prism 7.01 . Deficit of bone regeneration within the segmental defect of polytrauma animals compared to OST was assessed by one-tailed Student’s t test. Animals receiving polytrauma injury exhibited a significant decrease in the bone volume fraction throughout the defect area (6.73% ± 1.53) compared to OST (13.48% ± 2.94), as determined by one-tailed Student’s t test.The extent of bone healing was assessed by microcomputed tomography by comparing bone volume fraction between the OST and polytrauma cohorts 5-week post-trauma. Polytrauma was hypothesized to be detrimental to bone healing, and therefore, fracture healing in this injury group was compared to standard osteotomy by one-tailed Student’s p = 0.061)   Fig. .Fig. 4Qup < 0.05)  as compared to naïve animals. Circulating lymphocyte counts were significantly reduced in polytrauma animals both 24- and 72 h post-trauma, while monocytes and granulocytes were significantly increased compared to naïve and OST at these time points. While the total number of circulating leukocytes was reduced at 72 h in the polytrauma group compared to osteotomy (6.23 ± 1.27 vs 3.63 ± 0.22), this decrease was not significantly different (p = 0.071)  in the polytrauma cohort when compared to the osteotomy cohort at both time points   in plasma from osteotomy and polytrauma animals at 6 and 24 h post-trauma. The plasma HMGB1 levels were significantly increased .Systemic concentrations of 23 cytokines were assessed using a multiplex immunoassay . The results of this assay are summarized in Table p < 0.05): C-C motif chemokine receptor 6 (Ccr6), C-X-C motif chemokine ligand 10 (Cxcl10), interferon gamma receptor 1 (Ifngr1), interleukin 1 receptor type 1 (Il1r1), NFKB inhibitor alpha (Nfkbia), and mitogen-activated protein kinase 8 (Mapk8). While the same genes were evaluated in the 72 h post-trauma group, none of the six genes that were identified at 24 h were significantly different between the osteotomy and polytrauma cohort. At the 72-h time point, the following genes were significantly downregulated in the polytrauma group: C-C motif chemokine ligand 3 (Ccl3), interleukin 1 beta (Il1b), interleukin 6 (Il6), Jun pronto-oncogene, AP-1 transcription factor subunit (Jun).mRNA expression of 84 genes related to the innate and adaptive immune response was analyzed for differential transcription between the osteotomy and polytrauma cohort. At the 24-h time point, it was found that six genes were significantly up-regulated in the polytrauma group (Tgfbr3), bone morphogenetic protein 6 (Bmp6), and vascular cell adhesion molecule 1 (Vcam1), were significantly downregulated in the polytrauma group  (p < 0.05) . Despite significant decreases to the average body mass in the polytrauma cohort, daily and weekly behavior assessments of all animals did not indicate a decrease in appetite in this group.At 48 h post-trauma, the polytrauma cohort began to significantly decline in body mass, reported as a percent of initial surgery weight, compared to OST . While the osteotomy group never dropped below 96% of initial surgery weight and had surpassed initial body mass by week 2, the polytrauma group dropped to 89.8% of surgery weight at 72 h and did not reach initial body mass until week 5 , as well as the genes regulating expression of Ilr1, the receptor for IL1β, and Cxcl10, or interferon gamma-induced protein 10. These data are consistent with a study of the bone marrow of immunologically restricted patients, which found higher concentrations of IL-1β, IL-6, and IP-10/CXCL10 in the fracture hematoma of a population that later demonstrated reduced rates of bone healing [Ccl3 , Il6, Il1b, and Jun were observed. The significant decrease in local expression of Ccl3 and Il6 match the drop in circulating cytokine levels of MIP-1a and IL-6, indicating the systemic depressed immune response is matched by a local decrease in expression of genes related to a pro-inflammatory state. In general, the temporal patterns of cytokine expression indicated a depressed immune response that did not favor either a systemic pro- or anti-inflammatory response. The severity of the injuries seen in this model may indicate a state of immune hyporesponsiveness that has been previously described in ICU patients [In addition to circulating cytokines, we characterized the local expression of inflammatory genes within the fracture. Interestingly, the immune response within the wound space of polytrauma animals began with a significant increase in expression levels for the receptor for macrophage inflammatory protein-3,  healing . By 72 hpatients –44.Other groups have reported changes to histological analysis was performed to quantify immune cells within the fracture and at the pin sites connecting the polyacetyl plate to the femur. In our study, macrophages were not observed within the fracture gap at 24 or 72 h post-injury, while neutrophils were significantly reduced in the polytrauma within the fracture gap at 24 h. Due to the removal of the periosteum at the time of surgery, periosteal thickness could not be quantified at the fracture gap or pin sites. Formation of granulation tissue at the pin sites was used to assess the mitotic rate; however, there were no significant differences in the formation of granulation tissue between the groups. Furthermore, we observed reduced macrophage numbers at the pin sites at the 24-h time point. In our study, histological evaluation of macrophages and osteoclasts in the fracture gap and the pin sites tended to be reduced in the polytrauma group, though this finding was only significant for macrophages at the pin site at 24- h, and neutrophils were only reduced at the pin site at 24 h. This delay in macrophage migration into the wound site may contribute to delayed fracture healing, as others have found that macrophage depletion during the early anabolic phase of fracture healing resulted in reduced callus formation, a process that could be completely abolished when macrophages were depleted at the time of fracture . We sougPrevious work by our laboratory reported delayed fracture healing was shown by the additive effects of trauma in a less severe model of injury involving volumetric muscle loss and tibial fracture . StudiesPrevious work involving polytrauma patients have found alterations in the circulating leukocyte numbers for 2 weeks following injury . In our Lastly, previous studies found that blunt chest trauma did not induce significant weight loss , 21; howAltogether, our study indicates that the polytrauma model described herein is one of altered systemic and local immune responses, but not necessarily one of systemic inflammation. The local immune and systemic responses in our polytrauma model differ from that of osteotomy alone in profound ways and may be one of the primary factors for the reduced bone volume fraction seen in the polytrauma group. Further investigation is required to better understand the immune responses seen in our study, while testing of intervention strategies, which are severely lacking for polytrauma patients, may help inform clinical guidelines to improve return to duty in severely injured service members along with civilian trauma. We feel that the model described herein represents a valid surrogate of human polytrauma and multi-trauma that will be useful to future research and development of interventional strategies focusing on methods to improve wound healing through immunomodulation.Additional file 1:Assessment Scores. Description: A) Average assessment scores for general appearance over the 5wk survival period B) Average percent weight loss over the 5wk survival period C) Average assessment scores for behavior over the 5wk survival period D) Assessment score rubric. (PPTX 135 kb)


APPSwe/PSEN1M146V) show altered synaptic transmission, compatible with increased physiological function of amyloidβ, before plaques are detected. Recently, the importance of microglia has become apparent in the human disease. Similarly, TASTPM show a close association of plaque load with upregulated microglial genes.Progression of Alzheimer's disease is thought initially to depend on rising amyloidβ and its synaptic interactions. Transgenic mice (TASTPM; CA1 synaptic transmission and plasticity were investigated using in vitro electrophysiology. Microglial relationship to plaques was examined with immunohistochemistry. Behaviour was assessed with a forced-alternation T-maze, open field, light/dark box and elevated plus maze.The most striking finding is the increase in microglial numbers in TASTPM, which, like synaptic changes, begins before plaques are detected. Further increases and a reactive phenotype occur later, concurrent with development of larger plaques. Long-term potentiation is initially enhanced at pre-plaque stages but decrements with the initial appearance of plaques. Finally, despite altered plasticity, TASTPM have little cognitive deficit, even with a heavy plaque load, although they show altered non-cognitive behaviours.The pre-plaque synaptic changes and microglial proliferation are presumably related to low, non-toxic amyloidβ levels in the general neuropil and not directly associated with plaques. However, as plaques grow, microglia proliferate further, clustering around plaques and becoming phagocytic. Like in humans, even when plaque load is heavy, without development of neurofibrillary tangles and neurodegeneration, these alterations do not result in cognitive deficits. Behaviours are seen that could be consistent with pre-diagnosis changes in the human condition.GlaxoSmithKline; BBSRC; UCL; ARUK; MRC. There is a large body of research examining many aspects of phenotypes associated with mouse models of Alzheimer's disease – a PubMed search for the terms Alzheimer* AND mouse returns in excess of 21,000 articles. However, there are few systematic articles pulling together pathological, functional , and behavioural analyses across the life-span of such models. There is also a number of conflicting outcomes, for example reports of impaired versus enhanced synaptic plasticity; cognitive impairments or not.Trem2 and other genes identified as risk-factors in humans are strongly up regulated in close association to plaque development in the mouse model used in this study. Moreover, this previous study predicted two of the most recently identified genes that were identified in GWAS since the publication of our paper.Recently, the importance of microglia in Alzheimer's disease has come to the fore in human genome-wide association studies (GWAS), with variants of a number of microglial genes identified as risk-factors for developing the disease. Interestingly, we have recently reported that We have previously used this model to identify the earliest synaptic changes and shown changes in release of glutamate, the primary excitatory neurotransmitter in the brain, to occur even before plaques are detectable.By studying this transgenic mouse model of Alzheimer's disease, throughout the development of plaques, from prior to detection through to heavy plaque loads, we have been able to identify a clear time course of key phenotypic changes associated with early disease. In particular, this study identifies the very early changes in microglia and can separate the time course of the microglial phenotype. In addition, we detail the changes in synaptic plasticity over time and importantly identify that, like in humans in the absence of Tau tangles or neurodegeneration, considerable synaptic changes and a heavy plaque load can occur without resulting in substantial cognitive loss.Our data indicate that rising amyloidβ prior to detectable plaque deposition results in changes in synaptic function that likely reflect an enhanced physiological effect of amyloidβ. At this stage, microglia proliferate but do not activate. Once plaques begin to appear, microglia migrate to surround the plaque and become phagocytic, likely targeting dystrophic synapses and neurites caused by the cloud of highly-toxic amyloidβ around the plaque. Similarly to humans, who have plaques but no tangles and have yet to develop substantial neurodegeneration, cognitive deficits are not seen, even with a heavy plaque load; behavioural changes are limited to anxiety-like effects.This investigation of the parallel time-course of events highlights the probability that, if progression of disease can be reversed or slowed early enough, before Tau tangles and substantial neurodegeneration occur, the symptoms of cognitive decline could be very largely avoided. Moreover, it suggests that the substantial increases in microglia number and upregulation of their specific gene expression in association with plaques is not associated with cognitive loss and may indeed be protective.Alt-text: Unlabelled Box1The onset and progression of Alzheimer's disease (AD) is most likely initiated by environmental factors interacting with predisposed genetic risks, many of which have now been identified in genome-wide association studies (see reference APP) or presenilin 1 or 2 (PSEN1 or PSEN2). This is an effective approach for initiating the rise in Aβ and the deposition of plaques, albeit by a different trigger than in sporadic AD. According to the ‘amyloid hypothesis’, in the human disease, a rise in Aβ levels and plaque deposition is suggested to lead to altered neuronal ionic homeostasis and increased oxidative stress. Together, these may result in increased kinase activity on microtubule-associated protein Tau, resulting in Tau hyperphosphorylation and neurofibrillary tangle formation [APPSwe and human PSEN1M146V, plaques are first detected at approximately 4 months of age and a considerable plaque load develops by 8 months [Animal models for AD have generally depended on transgenic expression, or more recently knock-in of the gene variants that cause the familial dominantly inherited forms of the disease, particularly amyloid precursor protein  [We recently reported alterations in synaptic transmission preceding the detection of plaques, manifesting as a loss of spontaneous action potentials in Schaffer collateral axons and a concomitant increase in probability of glutamate release . Importaeac.org) .Here we extend our previous studies on TASTPM mice to understand the relationship between early changes in synaptic transmission, synaptic plasticity, cognitive function and microglia. We study the development of plaques in more detail and dissect out the microglial response to distinguish between the numbers of microglia and their phagocytic status. We find that, like synaptic changes, microglia are more prevalent even before plaques are detectable, whereas their phagocytic phenotype is age-related, coming much later. We then proceed to study hippocampal synaptic plasticity and hippocampus-dependent learning, in the forced-alternation T-maze, enabling identification of Aβ phenotypes, finding little cognitive deficit even with a heavy plaque load but clear behavioural changes, probably related to increased levels of anxiety.While we continue to focus mainly on the previously defined TASTPM mice (double hemizygotes), we have broadened the study to investigate dose dependency of the transgene by including mice homozygous for both genes. The effect of the individual genes is also investigated in mice with hemizygous expression of only one or the other gene. When not otherwise defined, TASTPM mice hence refers to the double hemizygous mouse.22.1APPSwe) [PSEN1M146V) [All experiments were performed in agreement with the UK Animals (Scientific Procedures) Act 1986, with approval from the UCL Animal Welfare and Ethical Review Body and in agreement with the GlaxoSmithKline statement on use of animals. Male TASTPM mice and C57Bl/6j mice were supplied by GlaxoSmithKline and bred either at Charles River Laboratories International, Inc.  or at UCL by crossing male homozygous TASTPM with female C57Bl/6j. Age-matched, non-littermate male C57Bl/6j mice were used as wild type controls. In some experiments, double homozygous TASTPM were bred. Single mutant TAS (APPSwe)  or TPM (N1M146V)  mice werIn this study we avoided single housing by keeping mice in large open cages (20 cm × 35 cm × 45 cm) and enriching their environments. Under these conditions, while the aggressive nature of the TASTPM mouse is not completely avoided, it is less of a problem and allows group housing to be maintained over the lifetime of the mice. Thus, cages containing 2–8 male mice were maintained in a 12-h light/12-h dark cycle with food  and water ad libitum. Environmental enrichment consisted of changes of food location, bedding type  and inanimate objects ) within the cage at least once per week. Mice were used for experimentation at the ages stated (± 0·5 months) and, where unavoidable, were single-housed for no longer than 24 h. Tails or ear punches were used for genotyping by standard PCR protocols to ensure the presence of the expected genes.2.22.2.1Briefly, genomic DNA was extracted using the ‘HotSHOT’ lysis method. Alkaline lysis reagent  was added to tissue samples prior to heating to 95 °C for 30 min. The sample was then cooled to 4 °C before the addition of neutralisation buffer . The PCR reaction was performed through addition of MyTaq DNA Polymerase (Bioline) reaction buffer and primer pairs:APPSwe):TAS (5’ GAATTGACAAGTTCCGAGGG 3′5’ GGGTACTGGCTGCTGTTGTAG 3′PSEN1M146V):TPM (5’ GTTACCTGCACCGTTGTCCT 3′5’ GCTCCTGCCGTTCTCTATTG 3′using the cycling parameters: 94 °C (30 s), 58 °C (30 s), 72 °C (30 s), for 30 cycles and a final extension at 72 °C for 4 min. PCR product sizes 366 bp for TAS and 104 bp for TPM.2.2.2Animals were deeply anaesthetised  and transcardially perfused with 0·1 M phosphate buffer saline (PBS) followed by 10% buffered formal saline . Alternatively, single hemispheres were drop-fixed immediately following brain extraction; the opposite hemisphere used for electrophysiology. The brains were post-fixed in 10% buffered formal saline for 24 h and cryoprotected in 30% sucrose/0·03% sodium azide/PBS at 4 °C for at least 24 h before sectioning or storage. Transverse sections were cut at 30 μm through the full left hippocampus using a frozen sledge microtome  and collected into a 24-well plate containing PBS/sodium azide (0·03%) for storage at 4 °C. Serial sections were placed in separate wells until all wells contained a section and collection then continued serially from Well 1 so that within each well the transverse sections were from the length of the hippocampus at least 720 μm apart.Standard immunohistochemical techniques were employed. For Aβ staining only, antigen retrieval was achieved by submerging sections in 10 mM sodium citrate (pH 6·0) in 0·05% Triton X-100 and heated in a water bath at 80 °C for 30 min. Sections for all immunohistochemistry were then washed in PBS, followed by 0·3% Triton X-100 in PBS and subsequent blocking in 8% horse serum/Triton/PBS for 1 h. Incubation with primary antibody  in block2.2.3Sections were imaged for quantification using an EVOS FL Auto Cell Imaging System (Life technologies). Tiled images were taken of the whole transverse hippocampal section using a 20× objective. To determine cell densities, an area of 400 μm × 240 μm was defined in the CA1, CA3 and the inner blade of the dentate gyrus. Cell counts were performed using Adobe Photoshop CS6. A minimum of three sections were used to create a mean for each animal. Sections for any given condition were obtained from the same collection well within the 24-well plate and were therefore a minimum of 720 μm apart, thus avoiding multiple counts of the same cells. Counts of objects touching the boundaries of the area of interests were only included from the north and east borders and excluded from the south and west borders.2.2.42.2.4.13, 1·4 NaH2PO4, 20 d-glucose, 3 MgCl2, 0⋅5 CaCl2, pH 7·4, ~315 mOsm/l). After approximately 2 min in ice-cold dissection ACSF, the brain was prepared for slicing by removing the cerebellum, hemisection of the forebrain and a segment cut away from the dorsal aspect of each hemisphere at an angle of approximately 110° from the midline surface to optimise slicing transverse to the hippocampus. Each hemisphere was then glued with cyanoacrylate  on this surface onto the stage of a vibrating microtome  containing frozen dissection ACSF and 400 μm transverse slices of hippocampus cut. As each slice of a hemisphere was cut, the hippocampus was dissected out, retaining a portion of entorhinal cortex and the resulting smaller slice was placed into a chamber containing ‘Carbogenated’  dissection ACSF at room temperature (approximately 21 °C). After 5 min, slices were then transferred into a fresh chamber held at 36 °C with the same dissection ACSF. At 5 min intervals, they were then consecutively transferred to physiological Ca2+ and Mg2+ ion concentrations (in mM): i) 1 Mg2+, 0⋅5 Ca2+; ii) 1 Mg2+, 1 Ca2+; iii) 1Mg2+, 2 Ca2+.Mice were decapitated and the brain rapidly removed and placed in ice-cold dissection artificial cerebrospinal fluid : 125 NaCl, 2·4 KCl, 26 NaHCO2.2.4.22+, 2 Ca2+ ACSF, slices were allowed to return to room temperature and after at least a further 40 min recovery time, a single slice was transferred to a submerged chamber and superfused with recording ACSF (containing (in mM): 125 NaCl, 2·4 KCl, 26 NaHCO3, 1·4 NaH2PO4, 20 d-glucose, 1 MgCl2, 2 CaCl2, bubbled with Carbogen). Individual CA1 pyramidal or dentate gyrus granule neurones were visualised using infrared-differential interference contrast microscopy on an upright microscope . Glass microelectrodes for patch-clamp were pulled from borosilicate glass capillaries  on a vertical puller . Electrodes (tip resistance approximately 5 MΩ) were filled with a CsCl-based internal solution (containing (in mM): 140 CsCl, 5 HEPES, 10 EGTA, 2 Mg-ATP, pH 7·4, ~290 mOsm/l). Patch-clamp recordings were performed using an Axopatch 1D , and current signals low-pass filtered at 10 kHz then 2 kHz  during digitization  and acquired using WinWCP  and WinEDR . Stimulation was applied via a patch electrode filled with ACSF, placed extracellularly in the appropriate axon path using a square pulse constant-voltage stimulator  triggered by WinWCP.Once transferred to 1 MgWinEDR synaptic analysis software was used for detection of spontaneous and miniature currents and WinWCP used to analyse identified spontaneous, miniature and evoked currents. Criteria for detection of spontaneous or miniature currents was to remain over a threshold of 3 pA for 2 ms. Currents were inspected by eye and only included if the rise time was < 3 ms and faster than the decay.2.2.4.3Slices were transferred as needed to a heated (30 ± 1 °C) submerged chamber and superfused with ACSF and allowed to recover for 1 h in the recording chamber. A glass stimulating electrode  and an identical recording electrode (connected to an AxoClamp 1B via a 1× gain headstage) were both positioned in stratum radiatum of the CA1 field to obtain a dendritic excitatory postsynaptic field potential (fEPSP). Recordings were controlled and recorded using WinWCP software (as above), filtered at 10 kHz and subsequently at 3 kHz and digitized at 10 kHz via a micro1401 interface . Stimuli  were applied at 0·1 Hz and resultant fEPSPs subsequently averaged over consecutive 1-min intervals. Stimulation intensity was set at approximately 30–50% of the intensity required to evoke a population spike or the maximum fEPSP amplitude obtained and a ≥ 15-min stable baseline recorded. LTP conditioning was applied at test-pulse stimulus intensity and consisted of either 3 trains of tetani, each consisting of 20 pulses at 100 Hz, 1·5 s inter-train interval; or 4 trains of theta-burst stimuli, each train consisting of 4 pulses at 100 Hz repeated 8 times at 20 Hz; inter-train interval 1 min. Following conditioning, fEPSPs were evoked at 0·1 Hz for 1 h.2.2.52.2.5.1Previously reported methods, optimised for mouse, were used to assess hippocampus-dependent learning . Mice weThe T-maze was constructed from three arms, each measuring 50 cm × 8 cm with 10 cm colourless Perspex walls and a grey floor, mounted on a table in the centre of a room with numerous distal visual cues, such as black and white posters on the walls. Black barriers were used to block the start and goal arms. Reward consisted of a drop of Nestlé Carnation™ condensed milk that was placed at the end of each goal arm. Arms were cleaned with 70% ethanol between all runs to reduce odour cues. In addition, in an inaccessible well a drop of reward is always present in both arms.Mice received 4 days of habituation to the maze, during which time they were allowed to explore the maze for 5 min with all arms open. During the first two days of habituation, reward was scattered along the floor and in food wells to encourage exploratory behaviour; then restricted to only the food wells at the ends of the goal arms in the last two days.The behavioural regime lasted for three weeks, with five days of training per week. Each day, animals received six trials; each trial consisted of a sample and choice run. In the sample run, one arm was blocked off. The mouse was placed at the starting point at the base of the T, the barrier was removed and the mouse was allowed to go to the available arm and given 20 s to eat a drop of reward from the food well. For the choice run, the mouse was immediately returned to the starting point and the barrier in the previously blocked arm removed. The starting barrier was then raised and the animal allowed to choose between the two arms but only rewarded if it chose the previously unvisited arm. Thus, a correct choice was scored when the mouse selected the arm not visited in the sample run. After the choice run, the mouse was removed from the maze and placed in its holding box. The location of the sample arm (left or right) was varied pseudorandomly across the session and mice received three left and three right presentations, with no more than two consecutive trials with the same sample location. Animals were allowed a maximum of 5 min to make a choice to enter a goal arm in both runs before a trial was aborted. If an incorrect arm was chosen during the choice run, the mouse was confined in the arm with no reward for 20 s and then removed from the maze.During the first two weeks of training the choice run followed immediately after the sample (test) run (there was a delay of approximately 15 s between runs for cleaning and resetting the maze). Data was analysed in blocks of 2 days and hence blocks 1–5 represent the first 2 weeks of training.. For delays the four runs for each delay are averaged.On the first day of the third week, mice received a repeat of the previous training sessions in order to assess retention of the task (block 6). On the following four days, longer delays (2–10 min) were introduced between the sample and choice runs to extend the time that the previous choice was to be held in memory (blocks 7 and 8 in response times). During these intervals, each animal was placed in a separate holding box. Each mouse received two of each of the delay periods per day, varied pseudorandomly both within and across days. Response times were calculated from the time that the starting block was removed until the mouse made a choice of arms and all four paws had crossed the entry point. Squads of 15–17 mice were run per day. During training, data are presented as blocks averaged across 2 days for each animal and expressed as mean ± SEM2.2.5.2The plus-maze was constructed from two enclosed arms (30 cm × 5 cm × 20 cm) and two open arms (30 cm × 5 cm × 0⋅8 cm) connected by a small central platform (5 × 6 cm); the two closed and two open arms were positioned opposite each other, respectively. The maze was elevated 30 cm above a table.Mice were placed in the centre of the plus maze facing an open arm and allowed to freely explore the maze for one 6-min trial. The time spent on each arm (open versus closed) was recorded, as well as the number of entries into the arms. An entry into an arm was defined as all four paws resting on a given arm.2.2.5.3. Albans, U.K) at a sample rate of 50 Hz. The open field was optically divided into a central circle and a peripheral ring, each with equal area. The path of the mouse was analysed offline using ImageProPlus. Path length and dwell times in the periphery and centre were calculated using custom made routines written in Matlab R2010a (MathWorks).The open field consisted of a plastic cylinder  with a white plastic floor. Mice were placed on the periphery of the open field floor and allowed to explore freely for 30 min. The path of each mouse was recorded using dacQUSB recording system  prior to pooling. Thus sample sizes represent the number of animals. Unless stated otherwise, data are presented as mean ± SEM and differences considered significant at p < 0·05.All data analysis was carried out blind to genotype. All statistics were performed using Graphpad Prism 6 with appropriately designed two-tailed 33.12) and these were very sparse , small plaques were detected at 16 months. While plaque density and size increased with age, plaque loads did not reach that seen in the double mutant TASTPM mice, even at the very advanced age of 27 months. In TPM mice (PSEN1M146V), there were no plaques observed at any age.Mice carrying only the single mutations were also examined. In TAS mice . However, note that Aβ levels are already raised by 2 months [2 threshold) may already be depositing at this time. There was also an age-dependent increase in Iba1 positive microglia in both wild type and TASTPM mice at around 10 months of age.Having previously observed that a module of microglial genes increased in expression in Αβ mice (including TASTPM) in close correlation with plaque load , we went2 months  and it in = 5–9, two-way ANOVA p = 0·07). This coincided with the appearance of large plaques . In contrast, the PSEN1M146V TPM mice, which do not develop plaques at any age, showed no difference from wild type mice in their microglial phenotype at 4 months of age for either total or CD68 positive microglia .Sections from single mutant mice were also stained for both IBA1 and CD68. The 6 months f, had a Very similar changes in total microglial and CD68 positive microglial densities were found in dentate gyrus, CA3 and subiculum (data not shown).3.33.3.1A receptor antagonist , the substantial loss of spontaneous activity we have previously reported up to 4 months of age [When spontaneous excitatory currents are recorded from CA1 pyramidal neurones in the presence of a GABAs of age  is largen = 5–8, two-way ANOVA, main effect of genotype p < 0⋅02), while at 18 months of age, the lower paired-pulse ratio was only evident at 25 ms, resulting in a genotype × interval interaction (p < 0⋅05) and a highly significant Sidak post hoc pairwise comparison between genotype at 25 ms (p < 0⋅005).When excitatory activity was evoked by stimulating the Schaffer collaterals in slices from older animals, the previously reported increase in release probability at 4 months, as assessed by a lower paired-pulse facilitation and confirmed by a reduction in failure to release glutamate in response to minimal stimulation , was mai3.3.2A receptor antagonist was included in the perfusion solution, the frequency of currents was decreased to about 5–10% in wild type slices. Consequently, GABAA receptor-mediated activity contributes about 90% of the frequency of the initial recording of mixed spontaneous activity. Note that, if the recording were made in the presence of a glutamatergic antagonist, any currents that were due to spontaneous glutamatergic activity mediating feed-forward or feed-back inhibition would be lost. Hence, the assessment of the drug-free frequency is a better assessment of the contribution of inhibition with the understanding that this will overestimate frequency by about 10%. Under these conditions we find that there is no significant change in inhibitory synaptic activity in TASTPM mice at any age from 4 to 18 months , the best cellular model we have for the laying down and retrieval of memory , could bA receptor antagonist [Field input-output relationship was not significantly changed at any of the ages tested a. Moreovtagonist , in fielAt 2 months of age, LTP is altered in TASTPM mice but this is dependent on the induction protocol. When LTP was induced by tetanic stimulation in these young mice, the magnitude of LTP was greater in slices from TASTPM than from wild type mice. Paired-pulse ratios remained unaltered when the last ten minutes of the post-induction recordings was compared to the baseline in both genotypes c&d. ThisAt 4 months of age, the effects of the different stimulus protocols had changed. By this age, a clear deficit was seen in LTP induced by tetanic stimulation, which was the reverse change to that seen at 2 months. Moreover, similar effects were seen in two separate cohorts of 4-month-old mice . This confirms that, despite deficits in tetanus-induced LTP, TASTPM are capable of expressing LTP under other induction protocols.The deficit in tetanus-induced LTP was maintained at 12 and 18 months of age e.3.3.4APPSwe TAS mice, the magnitude of LTP was smaller than wild type mice .The induction of LTP was also examined in 24 month old single mutant mice f-h. In aype mice f. In con greater g. There 3.43.4.1The hippocampus-dependent forced-alternation T-maze task was used to assess cognition in the TASTPM mice at three ages : 4 monthp = 0⋅01). However, TASTPM performed significantly worse than wild type mice overall , and there was no significant interaction between training block and genotype. Interestingly in Block 6, after 2 days without training, the two groups came together with almost identical performance at 90% correct choices . When the same mice that were run at 8 months were retested at 12 months of age, wild types had, as expected, returned to the baseline response of approximately 75% correct and again improved from ~75% to ~90% during the training period. Unexpectedly however, TASTPM appeared to have retained their previous training, starting the training at ~90% and showed no further improvement over the training period. This was reflected in a significant interaction between training period and genotype in a two-way ANOVA .Animals were again challenged further by the introduction of a delay between sample and choice runs c. While Another interesting difference between the 12-month mice and the younger groups was that the response time for the choice run for TASTPM mice at 12 months was rapid and no different from the wild type mice c. . Furthermore, TASTPM mice spent more time in the peripheral than central area .In the light of the behavioural differences seen in the T-maze trials, locomotor activity and anxiety were assessed using a battery of tests. First, mice were placed in an open field a. TASTPMp < 0⋅05); at 4 and 12 months of age this difference was not evident .Finally, at 4 and 8 months of age, TASTPM mice spent more time in the closed compartment of a light/dark box (12 months not tested). There was a significant interaction between age and genotype , which are either detected directly by microglia, or via secondary messengers, particularly altered pro- or anti-inflammatory cytokine levels within the environment  mice [As has been previously shown in TASTPM  and othend) mice  leading nd) mice ,33.At 8 months of age the synaptic changes observed are almost identical to those we have previously reported at 4 months, with no change in miniature currents but a complete loss of spontaneous activity and an increase in release probability of glutamate. Particularly at early stages, the plaque load in terms of the percentage tissue coverage is very low and so very few of the synapses recorded would be close to a plaque. Moreover, the observation that the change in paired-pulse ratio is similar despite a rising plaque load also suggests that the plaques themselves are unlikely to be causing this effect. Hence, this is presumably the result of very low levels of soluble Αβ throughout the tissue. Increased release probability has been reported to be the physiological effect of Αβ release in wild type rats . As the Trem2 that we have previously reported at this age [It is notable that the decrease in frequency of miniature EPSCs observed at 12 months coincides with the increase in microglial number and activation but that it does not decrease any further by 18 months. This is consistent with the plaque load and indeed the microglial density largely plateauing by 14 months. The very effective microglial proliferation and subsequent phagocytic phenotype, associated with strong upregulation of microglial genes such as this age , may be The biphasic change in LTP induced by a mild tetanic stimulus at the early stages of pathology in Αβ mice is of interest. At 2 months of age, prior to detection of plaques and when LTP is enhanced in the Αβ mice, Aβ is certainly present, albeit at low levels ,16. By 4In concert with  the direct physiological effects of low levels of Aβ, the early increase in LTP magnitude we report could be an interaction with the mutant presenilin 1. Expression of both normal and mutant presenilin 1 has direct effects on synaptic transmission and plasticity. For example, the A246E mutation leads to increased LTP magnitude at young ages ,41, whilAgain, cytokines released as part of the immune response to raised Aβ levels and plaque deposition could offer an additional route for the altered synaptic transmission and plasticity that we observe. It has long been established that cytokines can modulate synaptic plasticity  and indeInterestingly when theta-burst stimulation was used for induction, during which the readily-releasable pool would have a chance to replenish, there was no significant deficit in LTP at 4 months. However, under these conditions at 2 months, we see a decrease in paired-pulse ratio following induction of LTP that remains throughout the experiment, indicating an increase in release probability. As outlined above, the physiological effect of Aβ is to increase release probability, and so this suggests that the theta rhythm itself may be causing local release of Αβ at the stimulated synapses. Despite this apparent presynaptic increase in transmission, the magnitude of LTP remains unaltered, raising the possibility of a postsynaptic decrease balancing this effect. However, when TEA was applied to slices, resulting in a wide-spread depolarisation and thus release of all neurotransmitter vesicles , LTP was induced similarly to wild types confirming that some forms of long-term plasticity remain intact. In our hands, the locus of expression of LTP, induced by tetanus, tends to have a presynaptic component in both genotypes, indicated by a change in paired-pulse profile following induction.In this study, TASTPM mice showed no measurable cognitive deficits at any age, despite a substantial plaque load. There were, however, clear differences in activity and anxiety related behaviours. The TASTPM mice were slower to perform the T-maze task than the wild type mice or failed to do so altogether. Furthermore, in other tests, decreased activity and increased anxiety were evident. Although not specifically measured, the TASTPM mice are also more aggressive than wild type mice .The improved retention of learning in TASTPM mice, requiring no repeat training when the same mice were tested twice at 8 and 12 months, may appear counterintuitive. However, similar outcomes in hippocampal learning have been reported in TASTPM mice at around 4 months, using a contextual fear conditioning regime. While mice were unimpaired in learning the task in these previous reports, they failed to extinguish their learnt fear behaviour when conditioning was reversed ,14,46. IThe subtlety of the cognitive changes reported here in the TASTPM mouse, even at stages where a heavy plaque load is present, is consistent with previous findings ,46. EvenHere we bring together the progression of pathology, microglia, synaptic transmission and other functional changes in a preclinical mouse model of AD. In a following paper we compare these results in mice with only plaques and no neurofibrillary tangles to a transgenic mouse model of later stage dementia in which neurofibrillary tangles occur without plaques .From the present study we conclude that the earliest effects of Αβ on synaptic transmission are probably due to very low levels of soluble Αβ in the volume of the tissue, rather than being directly related to plaques. At this early stage plaques are extremely small and sparse so that they would impinge directly on very few synapses. However, while wide-spread soluble Αβ may also influence early microglial changes these may well be influenced by the earliest seeding of plaques or the release of cytokines into the environment. Later changes most probably relate to the increasing numbers of larger plaques with the cloud of high-level soluble Αβ surrounding them impinging on a, still limited, but much greater area. The direct effects of this localised high concentration of soluble Αβ include loss of active synapses and activation of microglia, likely involved in clearing damaged synapses near or within plaques via phagocytosis and thus preventing wider damage to the affected axons and dendrites. While seeding of new plaques seems to continue throughout the development of plaque pathology, it is only once large plaques are present that these latter changes occur. Moreover, the lack of cognitive deficits is similar to the human situation prior to the build-up of neurofibrillary tangles and neurodegeneration and thus suggests that TASTPM represent a good model of the prodromal phase of the disease. Thus, these phenotypes are likely to represent the early stages in individuals who will go on to develop Alzheimer's disease.The datasets used and/or analysed during the current study are available from the corresponding authors on reasonable request.GlaxoSmithKline (FAE); BBSRC Case studentship with GlaxoSmithKline to PH (FAE); UCL International Studentship to WL (FAE); ARUK and UCL ARUK Network ; MRC grant MR|J011851|1 (FAE); BBSRC grant BB|R009872|1 for LM (FC).A material transfer agreement between GlaxoSmithKline and UCL for use of the TASTPM mice and agreement for the types of experiment was in place prior to experimental design as well as agreement to include GlaxoSmithKline authors. GlaxoSmithKline were not involved directly with the design of the experiments. GlaxoSmithKline (JCR) have read and approved the submitted form of the manuscript. No further input was received from funding sources.FAE reports non-financial support from GlaxoSmithKline and grants from GSK, Medical Research Council UK, and Alzheimer's Research UK during the conduct of the study.PH reports grants from BBSRC, during the conduct of the study.JCR reports other from GlaxoSmithKline, outside the submitted work.No further conflicts of interests declared.EM performed the histological staining and counting of microglia and provided feedback on draft manuscript. TAB carried out behavioural experiments and analysis and comments on draft manuscript. WL performed the LTP experiments at 12 and 18 months of age and comments on draft manuscript. TH performed LTP and behavioural experiments at 4 months of age. PH carried out the chemical LTP experiments and comments on draft manuscript. OTJ performed patch clamp experiments for IPSCs at 4 months of age. KY performed the LTP experiments at 24 months. LM provided training and supervision of all behavioural experiments. FM performed LTP experiments at 2 and 4 months of age. MANS performed behavioural experiments at 4 and 8 months of age. GB carried out histological staining and quantification of plaques. RW performed the microglial histology and counting for the TPM mice. JCR developed the mouse model and comments on the draft manuscript. FC designed and supervised the behavioural experiments and provided comments on the draft manuscript. DAS supervised the histological experiments and commented on draft manuscript. DMC supervised all experiments, performed electrophysiological recordings, wrote first draft of the manuscript, performed all statistical analyses, prepared figures, edited and finalised manuscript. FAE obtained funding, designed experiments, edited and finalised manuscript. All authors read and approved the final manuscript.


Bacteroides use surface-exposed lipoproteins to bind and metabolize complex polysaccharides. Although vitamins and other nutrients are also essential for commensal fitness, much less is known about how commensal bacteria compete with each other or the host for these critical resources. Unlike in Escherichia coli, transport loci for vitamin B12  and other corrinoids in human gut Bacteroides are replete with conserved genes encoding proteins whose functions are unknown. Here we report that one of these proteins, BtuG, is a surface-exposed lipoprotein that is essential for efficient B12 transport in B. thetaiotaomicron. BtuG binds B12 with femtomolar affinity and can remove B12 from intrinsic factor, a critical B12 transport protein in humans. Our studies suggest that Bacteroides use surface-exposed lipoproteins not only for capturing polysaccharides, but also to acquire key vitamins in the gut.Human gut  Eating is the first step in an hours-long process that extracts the nutrients we need to live. It not only nourishes us, but also a vast community of bacteria in our gut called the microbiota. The gut microbiota acts like an extension of our immune system and helps us stay healthy in many ways. For example, it blocks pathogens from making us sick. But too many gut bacteria in the wrong parts of our intestines can be harmful.Some people are prone to developing a dangerous overgrowth of bacteria in their small intestine where most of our dietary nutrients get absorbed. This overgrowth can lead to many problems including vitamin B12 deficiency even when they eat plenty of it. To understand why, scientists must learn how microbes affect our ability to absorb nutrients from food and how the microbes themselves capture nutrients like vitamin B12 as they pass through our digestive tract.Bacteriodes grabs onto vitamin B12 with extraordinary strength. In fact, these bacterial proteins bind to vitamin B12 so strongly that they can even pry it away from our own vitamin B12 collecting protein.Now, Wexler et al. show that some gut microbes may be able to pirate vitamin B12 from us as it passes through the digestive tract. Wexler et al. showed that a protein called BtuG on the surface of a type of gut bacteria called Bacteriodes with and without BtuG were placed in mice with no gut bacteria of their own, bacteria with BtuG rapidly outcompeted those lacking the protein. The experiments suggest that competition for vitamin B12 among microbes has favored bacteria that are better at capturing the nutrient. More studies are needed to learn whether BtuG contributes to vitamin B12 deficiencies in humans with gut bacteria overgrowth and determine the best ways to combat such deficiencies.When  Our understanding of the factors that shape gut microbial community composition is largely based on the primary economy of this ecosystem: the flow of carbon from the diet to bacterial biomass and fermentation products. However, an accompanying secondary economy of essential vitamins and other cofactors, which are much less abundant, also plays a critical role in determining bacterial growth rates and resulting microbiome dynamics . Small m12  is representative of this challenge: de novo biosynthesis requires the coordinated activity of nearly 30 dedicated enzymes (12-dependent enzymes (e.g. B12-independent methionine synthase MetE and ribonucleotide reductase NrdEF), even species that lack vitamin biosynthetic machinery maintain their B12-dependent enzymes  for use when vitamin B12 is available in the environment , transport and utilization proteins and regulatory elements are typically referred to as B12-dependent, in keeping with their initial characterization.The complex organometallic cofactor vitamin B enzymes . Althougironment . In the pathways . Althoug12 and other corrinoids has been studied extensively in Escherichia coli and has been used as a model for TonB-dependent transport. In E. coli, extracellular B12 is transported into the periplasm through the outer membrane β-barrel protein BtuB in a TonB-dependent manner , but these do not play a role in B12 transport  encode a diverse array of Br genome . Bacterot manner . In the ransport .E. coli, Bacteroides accessory proteins can play a critical role in B12 transport. Using the B. thetaiotaomicron homolog of a highly conserved accessory protein  as a representative, we establish that BtuG2 localizes to the cell surface, interacts with BtuB, and determines the ability of B. thetaiotaomicron to transport B12 and persist in the mammalian gut. Furthermore, BtuG2 directly binds vitamin B12 with femtomolar affinity, thereby enabling it to acquire this vitamin from intrinsic factor, a critical B12 transport protein in humans.Here we report that unlike BtuE in Bacteroides B12 transport loci include homologs of a hypothetical gene that is exclusively found in the Bacteroidetes phylum  adjacent to a homolog of btuB . No homologs were detected outside of the Bacteroidetes using the defined BlastP parameters  that complicate the ability to assign functions to specific genes, we established a simplified genetic background that lacks locus1 and locus3 (hereafter referred to as the B. thetaiotaomicron ‘parent’ strain). We created an in-frame, unmarked deletion of btuG2 in this parent strain and tested its ability to grow in minimal media supplemented with cyanocobalamin  or methionine (B12-independent growth). We chose cyanocobalamin concentrations of 37 nM and 0.37 nM because these concentrations repress (37 nM) or activate (0.37 nM) B12 riboswitches in this species , and was even found in the supernatant fraction, consistent with its localization to the interface of the outer membrane of the cell and the extracellular milieu  with varying concentrations of proteinase K (0–100 µg/mL), ran whole cell lysates on an SDS-PAGE gel, and probed by Western blot for BtuG2 or a periplasmically localized control protein SusA (BT3704) appended with a C-terminal HA-tag . While Sr milieu .btuG2 deletion strain both in terms of protein production and B12-dependent growth  on BtuG2 after co-incubation with equimolar cyanocobalamin. At both wavelengths, we observed nearly identical traces corresponding to the elution volume for BtuG2, indicating that BtuG2 can directly bind cyanocobalamin in vitro  also exhibit this function , we used surface plasmon resonance (SPR) to determine a  binding . Both liociation . Notablyociation . The cooociation . TherefobtuG2 mutant cells upon supplementation with supernatants from btuB2 mutant cultures  strains grown to exponential phase in minimal medium lacking cyanocobalamin and supplemented with methionine. Concentrated supernatants were incubated in the presence or absence of 0.37 µM cyanocobalamin, diluted and concentrated repeatedly to remove unbound ligand and residual methionine, and introduced to recipient cells in medium lacking both cyanocobalamin and methionine.Because BtuG2 modulates intracellular corrinoid levels, is surface-exposed, associates with BtuB2 and binds cyanocobalamin directly, we reasoned that it might act as a critical extracellular step in the process of capturing and transporting corrinoids. To test whether BtuG2 can function extracellularly, we measured growth of btuG2 recipient cells grow robustly when provided with BtuG2-containing culture supernatant that had been incubated with cyanocobalamin .Under these conditions, ∆obalamin . By contobalamin . FurtherbtuG2 strain when the two strains are co-cultured , is released from parietal cells in the stomach and binds cobalamin in the duodenum following the degradation of haptocorrin by host enzymes (3 to ~108 cells/g)  .B. thetaiotaomicron can use BtuG2 to acquire vitamin B12 from IF, we first incubated recombinant human IF with cyanocobalamin, diluted and concentrated repeatedly to remove unbound ligand, provided the IF-cyanocobalamin complexes to B. thetaiotaomicron cells in minimal media without exogenous cyanocobalamin or methionine, and measured culture growth over time. The parent B. thetaiotaomicron strain grew readily when provided IF-cyanocobalamin  could have health consequences ; the most extreme of these describes a KD ~5 × 10−15 M, a kon ~7 × 107 M−1s−1 and a koff ~4 × 10−7 s−1  to compete with IF for free cobalamin , but its uptake receptors on epithelial cells are located exclusively in the distal small intestine (ileum). Therefore, IF must maintain its association with cobalamin while traversing several meters of intestinal tract before the host can absorb its nutrient cargo. By contrast, BtuG2 localizes to the bacterial cell surface and likely acts in cis to capture extracellular corrinoids for subsequent delivery to the outer membrane receptor BtuB on the same cell. This difference in protein function may impose differences in selective pressure for koff rates.The Although BtuG2 and IF thus achieve these strong binding affinities by different kinetics, mixing the proteins results in a transfer of cobalamin from IF to BtuG2 . It is uBacteroides each encode dozens of Sus-like systems, defined by the presence of an outer membrane β-barrel protein (e.g. SusC), a cell surface-exposed lipoprotein , and other components that directly interact to digest and import various polysaccharides into the cell , filled with 70% N2, 20% CO2, and 10% H2 by volume, using minimal media with vitamin B12 omitted . Escherichia coli S17-1 lambda pir or BL21 Rosetta (DE3) strains were grown in LB medium and incubated aerobically at 37°C. Culture media were supplemented with antibiotics as needed at the following concentrations: ampicillin 100 μg/mL, chloramphenicol 30 µg/mL, erythromycin 25 μg/mL, gentamicin 200 μg/mL, tetracycline 2 μg/mL, and 5-fluoro-2′-deoxyuridine (FUdR) 200 μg/mL.Culturing of  omitted  supplemeB. thetaiotaomicron were carried out as previously reported using B. thetaiotaomicron ∆tdk ∆locus1 ∆locus3 as a parent strain  introduced in single copy into B. thetaiotaomicron as previously described  and the N-terminal domain of the B. thetaiotaomicron strains grown in minimal medium supplemented with methionine (no B12) were pelleted, washed three times in minimal medium without methionine or vitamin B12, and used to inoculate wells of a 96-well plate in triplicate containing minimal media with methionine or vitamin B12 . The plate was incubated anaerobically under constant agitation for 72 hr at 37˚C and OD600 measurements were taken at regular intervals using a BioTek Eon microplate spectrophotometer.Overnight cultures of B. thetaiotaomicron strains carrying unique oligonucleotide barcodes were co-cultured in minimal media in triplicate as previously described , which was then used to inoculate, at 1:1000, minimal media supplemented with methionine or vitamin B12 . These inoculations were then incubated anaerobically at 37 ˚C under static conditions or shaking (250 rpm). Cultures were passaged at 1:1000 into fresh media every 24 hr and an aliquot was stored at −20 ˚C for gDNA extraction . Each mouse in the first group was gavaged with 108 CFU each of B. thetaiotaomicron ∆tdk ∆locus1 ∆locus3 att1::pNBU2_tetQ_BC01 (‘parent’ strain) and B. thetaiotaomicron ∆tdk ∆locus1 ∆locus3 ∆btuG2 att1::pNBU2_tetQ_BC14 (‘∆btuG2’ strain). Each mouse in the second group was gavaged with 108 CFU each of the parent strain and B. thetaiotaomicron ∆tdk ∆locus1 ∆locus3 ∆btuB2 att1::pNBU2_tetQ_BC14 (‘∆btuB2’ strain). Each mouse in the third group was gavaged with ~107 CFU each of the parent strain, the ∆btuG2 strain, and B. thetaiotaomicron ∆tdk ∆locus1 ∆locus3 ∆btuG2 att1::pNBU2_tetQ_BC16_us1957_btuG2 (‘∆btuG2 + btuG2’ strain). Fecal samples were collected over time and stored at −80°C before genomic DNA extraction. DNA was extracted as described previously  eviously . The releviously  in a qPCB. thetaiotaomicron strains were grown in triplicate anaerobically at 37 ˚C to mid-log phase (OD600 ~0.3) in minimal media supplemented with methionine and vitamin B12 . RNA was extracted using a cell lysis buffer  and an RNeasy kit . DNA was removed using DNA-free DNA Removal Kit , and RNA was again cleaned using an RNeasy kit . cDNA was then made with SuperScript II Reverse Transcriptase  using the manufacturer’s instructions, and RNA was removed with 1 N NaOH at 65 ˚C for 30 min and neutralized with 1 N HCl. Samples were then cleaned using a PCR purification kit (Qiagen) and cDNA was quantified using a Qubit . Quantitative PCR was performed using SYBR FAST Universal Mastermix  and gene-specific primers .Detection of BtuG2 from B. thetaiotaomicron strain expressing an HA-epitope tagged allele of the periplasmic protein SusA  and supernatant was filtered through 0.2 µm filter and stored temporarily on ice. Pellets were resuspended in breakage buffer , lysed at 4˚C by sonication , and clarified lysates were ultracentrifuged at 100,000 x g for 1 hr at 4˚C to separate membranes (insoluble) from cytoplasm/periplasm (soluble) fractions. Membrane fractions were resuspended in 250 µl of breakage buffer, while the cytoplasm/periplasm fraction was concentrated by centrifugal filtration  to 250 µl. Membrane and cytoplasm/periplasm fractions were temporarily stored on ice. Filtered supernatant was utracentrifuged at ~100,000 x g for 1 hr at 4˚C to remove outer membrane vesicles. The soluble fraction was then concentrated by centrifugal filtration to 250 µl. 20 µl each of membrane, cytoplasm/periplasm, and supernatant fractions were loaded onto SDS-PAGE gels and analyzed by Western blot. PVDF membranes were probed with rabbit anti-BtuG2 and mouse anti-RpoB  as a cytoplasmic control.B. thetaiotaomicron strains were grown to OD600 ~0.6 in minimal media with methionine. Cells were pelleted, supernatant was removed, and cells were lysed with BugBuster reagent. Co-immunoprecipitation was carried out using FLAG HA Tandem Affinity Purification kit  according to the manufacturer’s instructions. Eluates were probed with rabbit anti-BtuG2 by Western blot.B. thetaiotaomicron strains were grown in minimal media with methionine. Supernatant donor strains were subcultured (1:100) into 60 mL fresh minimal media with methionine and allowed to grow to OD600 ~0.6. Supernatant recipient strains were washed 3 times in minimal media without methionine or vitamin B12, subcultured to a final OD600 ~0.001 in 1 mL minimal media without methionine or vitamin B12, and incubated at 37 ˚C anaerobically for 8 – 12 hr. Supernatant donor cultures were pelleted and supernatant filtered (0.2 µm). Filtered supernatants were then ultracentrifuged at 100,000 x g for 1 hr at 4 ˚C to remove outer membrane vesicles and concentrated to <2 mL by centrifugal filtration . Where indicated, concentrated supernatants were supplemented with 0.37 µM vitamin B12 or PBS and incubated for 20 min at room temperature aerobically on a nutator. Supernatants were then washed 4 times in 70 mL minimal media without methionine or vitamin B12 by centrifugal filtration (30K). Washed, concentrated supernatants were then applied to recipient cell cultures and incubated at 37 ˚C anaerobically. CFU measurements were taken at regular intervals. For assays involving IF, recombinant human IF (Xeragenx LLC) was incubated in 0.37 µM vitamin B12 or PBS for 20 min at room temperature on a nutator, and washed 4 times in 70 mL minimal media without methionine or vitamin B12 via centrifugal filtration (30K). IF (± vitamin B12) was provided either alone to recipient cell cultures  carrying a modified pET21 vector. Cells were grown to OD600 ~0.6 before being induced for 3 hr in 0.5 mM IPTG at 37 ˚C under constant agitation. Cell pellets were lysed using BugBuster reagent . Lysates were incubated for 1 hr at 4 ˚C with Ni-NTA agarose beads  and washed with 12 – 18 mL of wash buffer , and eluted with 6 mL elution buffer . BtuG2-6xHis or BtuG2-10xHis fractions were dialyzed overnight in 20 mM Tris pH eight before being spun through a PierceTM strong anion exchange column  according to the manufacturer’s instructions. Eluted proteins were dialyzed twice for 4–8 hr in 4 L of PBS, pH 8. Proteins were quantified by Bradford assay  according to the manufacturer’s instructions. BtuG2-6xHis was incubated at a 1:1 molar ratio of cyanocobalamin to protein for 30 min at 25 ˚C and analyzed by SEC-MALS. BtuG2-10xHis was enriched for protein monomers by size exclusion chromatography and quantified using a NanoDrop spectrophotometer  for use in SPR experiments (described below).Recombinant BtuG2-6xHis or BtuG2-10xHis was expressed and purified from pET21_NESG was used to express and purify C-terminal 6xHis-tagged versions of the BtuG homologs BVU2056, BACUNI04578 and BACCOPRO02032 as described above for BtuG2. Each BtuG homolog, or BSA or PBS as controls, was incubated at a 1:1 molar ratio of cyanocobalamin to protein for 30 min at 25 ˚C before being spun through a centrifugal filter  to elute unbound cyanocobalamin. Proteins were washed 3 times with 400 µl PBS. Retained protein-cyanocobalamin complexes were resuspended in 100 µl PBS and analyzed by spectrophotometry for absorbance at 360 nm, corresponding to cyanocobalamin.E. coli BL21 Rosetta (DE3) as described above and immobilized on a NTA chip. Cyanocobalamin was injected at 0.8, 0.4, 0.2, 0.1, and 0.05 nM and the binding was monitored in single cycle kinetics in PBS . Recombinant BtuG2-10xHis was purified from s in PBS . Binding In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.12 from their Environment and Host via Cell Surface-Exposed Lipoproteins" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Wendy Garrett as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Jan Peter van Pijkeren (Reviewer #3).Thank you for submitting your article "Human Gut Bacteria Capture Vitamin BThe reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:12 uptake in an important gut commensal. The authors center their study on BtuB, an outer membrane lipoprotein from Bacteroides thetaiotaomicron. They perform a series of careful biochemical analyses that reveal that BtuG is essential for B12 transport into the cell. They further show that BtuG is key for the in vivo fitness of B. thetaiotaomicron. A major claim is that BtuG is involved in "vitamin piracy" by competing with human intrinsic factor for vitamin B12 binding.The reviewers agreed that this paper represents a very interesting, well-designed, and well-conducted study of vitamin BB. thetaiotaomicron vitamin B12 uptake is convincing. The reviewers felt that overall, the work provides valuable new insight into nutrient acquisition strategies of intestinal commensal bacteria. However, as detailed below, there were concerns about the basis for the vitamin piracy claim that need to be addressed in a revised manuscript.Overall, the study presents novel insight into how gut commensals obtain essential vitamins in the complex gastrointestinal environment. The study is well-conducted, well-written, and the data on BtuG function in Essential revisions:12 from human intrinsic factor. The experiments supporting this claim were all conducted in vitro. In vivo experiments showing that the wild-type bacteria capture B12 from the host, while the mutant does not, would greatly strengthen this central claim of the paper. However, the reviewers recognized that such an in vivo experiment could pose some difficult technical challenges and might not be doable in a restricted time-frame. In that case, we suggest that the title and Abstract be revised with less emphasis on the idea of B12 capture from the host. In addition, the discussion would need to be improved by discussing alternative possibilities as suggested in point 3 below.1) A major claim of this study is that BtuG is involved in "vitamin piracy" by capturing vitamin B∆btuG2 and ∆btuB2 are outcompeted by the parent strain in a germ-free mouse . Since this is the only in vivo data demonstrating the role of these proteins in intestinal fitness, it is important that – at least for one of these proteins – that its function is complemented to conclusively demonstrate that the reduction in fitness is attributable to deletion of btuG2 or btuB2.2) 12 availability in the colon is an essential underpinning of the vitamin piracy claim. In the discussion amongst the reviewers, there were questions raised about whether vitamin B12 is likely to be freely available in the colon given that it is typically absorbed in the small intestine. This may also be a point of confusion for some readers, and thus we suggest some further discussion and/or clarification of this point. The following points were raised as possible avenues for clarification:3) Vitamin BB. theta  – is it possible that the piracy is therefore occurring primarily among these small intestinal populations rather than those present in the colon?a) A reviewer pointed out that there can be small intestinal populations of 12 to emerge.b) Is it possible that BtuG could be a factor that has evolved to allow successful competition with other microbes, rather than with the host? This would plausibly constitute enough selection pressure for the high affinity of the protein for BB. thetaiotaomicron utilize pseudovitamin B12, and – if so – how does the utilization (i.e. growth) compare to vitamin B12? Pseudovitamin B12 is produced by select gut bacteria , food-associated microbes (i.e. Arthrospira platensis), or used as a food additive. Although pseudovitamin B12 is another source of B12, it cannot be absorbed in the human GI tract (it does bind IF). If B. thetaiotaomicron can utilize pseudovitamin B12 more efficiently than B12, then B. thetaiotaomicron would be beneficial for human health rather than detrimental, as the authors speculate in their Discussion (third paragraph).c) Can  Essential Revisions:12 from human intrinsic factor. The experiments supporting this claim were all conducted in vitro. In vivo experiments showing that the wild-type bacteria capture B12 from the host, while the mutant does not, would greatly strengthen this central claim of the paper. However, the reviewers recognized that such an in vivo experiment could pose some difficult technical challenges and might not be doable in a restricted time-frame. In that case, we suggest that the title and Abstract be revised with less emphasis on the idea of B12 capture from the host. In addition, the discussion would need to be improved by discussing alternative possibilities as suggested in point 3 below.1) A major claim of this study is that BtuG is involved in "vitamin piracy" by capturing vitamin BWe agree with this suggestion and have revised the title, Abstract, and Discussion accordingly.2) ∆btuG2 and ∆btuB2 are outcompeted by the parent strain in a germ-free mouse . Since this is the only in vivo data demonstrating the role of these proteins in intestinal fitness, it is important that – at least for one of these proteins – that its function is complemented to conclusively demonstrate that the reduction in fitness is attributable to deletion of btuG2 or btuB2.btuB2 mutant in vivo can be complemented by expression of the gene in single copy from a heterologous chromosomal locus . We have added this information to the paper.It has been previously shown that the competitive fitness defect of the ∆btuG2, and complemented strains in gnotobiotic mice. These new data have been added to the paper as Figure 1—figure supplement 1.In response to this comment, we conducted new experiments measuring the fitness of wildtype, 12 availability in the colon is an essential underpinning of the vitamin piracy claim. In the discussion amongst the reviewers, there were questions raised about whether vitamin B12 is likely to be freely available in the colon given that it is typically absorbed in the small intestine. This may also be a point of confusion for some readers, and thus we suggest some further discussion and/or clarification of this point. The following points were raised as possible avenues for clarification:3) Vitamin Ba) A reviewer pointed out that there can be small intestinal populations of B. theta  – is it possible that the piracy is therefore occurring primarily among these small intestinal populations rather than those present in the colon?B. thetaiotaomicron can use BtuG2 to acquire cyanocobalamin from human intrinsic factor” and Discussion, paragraph six).We agree with the reviewer and have suggested this possibility (see subsection “12 to emerge.b) Is it possible that BtuG could be a factor that has evolved to allow successful competition with other microbes, rather than with the host? This would plausibly constitute enough selection pressure for the high affinity of the protein for BYes, we think this is the most likely explanation for our observations .12, and – if so – how does the utilization (i.e. growth) compare to vitamin B12? Pseudovitamin B12 is produced by select gut bacteria , food-associated microbes (i.e. Arthrospira platensis), or used as a food additive. Although pseudovitamin B12 is another source of B12, it cannot be absorbed in the human GI tract (it does bind IF). If B. thetaiotaomicron can utilize pseudovitamin B12 more efficiently than B12, then B. thetaiotaomicron would be beneficial for human health rather than detrimental, as the authors speculate in their Discussion (third paragraph).c) Can B. thetaiotaomicron utilize pseudovitamin BB. thetaiotaomicron can utilize [Ade]Cba (pseudovitamin B12); growth rates in minimal medium lacking methionine and supplemented with either [Ade]Cba or cyanocobalamin are equivalent . Others have reported that the binding affinity of IF to [Ade]Cba is an order of magnitude weaker than IF to B12 .We appreciate this useful point. We have previously shown that 12 and cobinamide with similar affinity . Thus, future studies will need to examine binding of diverse corrinoids  with many proteins in the corrinoid transport loci in addition to BtuG.We are reluctant to speculate about the interactions between BtuG, IF and [Ade]Cba because we have not studied the interactions between these proteins and [Ade]Cba. We think that it is unlikely that BtuG2 acts as a “filter” to distinguish corrinoids, because it binds B