Exposure of human populations to bovine spongiform encephalopathy through contaminated food has resulted in <250 cases of variant Creutzfeldt–Jakob disease (vCJD). However, more than 99% of vCJD infections could have remained silent suggesting a long-term risk of secondary transmission particularly through blood. Here, we present experimental evidence that transfusion in mice and non-human primates of blood products from symptomatic and non-symptomatic infected donors induces not only vCJD, but also a different class of neurological impairments. These impairments can all be retransmitted to mice with a pathognomonic accumulation of abnormal prion protein, thus expanding the spectrum of known prion diseases. Our findings suggest that the intravenous route promotes propagation of masked prion variants according to different mechanisms involved in peripheral replication. It is hypothesised that exposure to bovine spongiform encephalopathy through contaminated food could have resulted in a large proportion of latent variant Creutzfeldt-Jakob disease cases in humans. Here the authors demonstrate that inoculation with blood from non-symptomatic, vCJD infected humans, results in a unique prion-like disorder in mice and macaques. However, the fatal variant Creutzfeldt–Jakob disease (vCJD) in humans has fortunately remained rare, with 177 cases in the UK to date ; the majority of cases were detected between 1995 and 2005, and only three cases from 2012 to 2016. Besides primary contamination through food, inter-human vCJD transmission has been associated with blood transfusions from donors incubating vCJD in the UK, but this has been limited to 4 cases among the 67 recorded recipients of blood from identified vCJD donors (<6%)3. In addition, one possible silent infection was associated with treatment with Factor VIII extracted from a pool of 10,000 plasma donations that included plasma from a preclinical case of vCJD4. Such contamination occurred before the introduction of systematic deleukocytation procedures in 20035 and improvements in techniques for the purification of plasma derivatives. No transmission of vCJD by transfusion has been reported since the implementation of those measures (blood infectivity is presumed to be shared in a ratio of 50:50 between leucocytes and plasma6).Bovine spongiform encephalopathy (BSE) was widely distributed in Europe and most notably in the UK. Overall, more than 2 million undiagnosed infected cattle would have entered the human food chaind) was detected in 16 appendices among more than 32,000 samples in a recent anonymized survey of prevalence in the UK population. This suggests an unexpectedly high prevalence  of asymptomatic infected subjects that is at least 100-fold higher than that of reported clinical cases7. The development of the disease after an extended period of time, may be several decades as observed with Kuru8 and iatrogenic CJD9, cannot be excluded. However, the most probable scenario is that after exposure to BSE, more than 99% of infected individuals will remain as silent carriers, possibly for decades. Such a long-term silent infection of lymphoid tissues represents a risk of secondary transmission that is difficult to assess. With regard to blood transfusion, basic calculations suggest that the risk may be in the order of 1000 potentially contaminated blood donations every year in the UK. Moreover, projections assume that the prevalence of clinical and subclinical infections in most of the other countries of Western Europe would be significant, since no more than 10- to 100-fold lower10. According to these levels of exposure, which are much higher than those of several blood-borne viruses, there is continuing cause for concern about the management of blood, blood components and derivatives, and surgical instruments that supports further evaluation of risks from primary BSE and secondary vCJD in relevant experimental models.In contrast to the limited prevalence of clinical vCJD, abnormal prion protein  and brain infectivity . Nevertheless, a low percentage of the affected recipient animals exhibit specific features of vCJD, whereas a much higher proportion of them develops neurological impairments devoid of the classical biochemical and/or pathological features that currently constitute the diagnostic criteria for prion diseases. These atypical impairments are identified as prion disorders by secondary transmission.In this study, we aim to explore the risk of prion infection from blood-derived products using both conventional mice susceptible to vCJD17. As negative controls, animals were sham-inoculated (40 mice and 6 macaques) or exposed to blood or brain products from healthy donors (90 mice and 2 macaques), while positive controls included animals exposed to crude homogenates of infected brains through the intracerebral (IC) (38 mice and 10 macaques) or IV (12 mice and 4 macaques) route.In a series of independent experiments, we transfused blood products derived from donors exposed to vCJD into 470 wild-type mice and 19 cynomolgus macaques. In parallel, we injected 70 mice and 11 macaques intravenously (IV) with soluble brain extracts derived from BSE- or vCJD-infected donors; the extracts were obtained according to protocols designed to mimic blood infectivityd (PrPres) was detected in their brains using classical biochemical diagnostic methods (ELISA and western blot), as a confirmation of their positive prion infection status, as expected. None of the negative control animals exhibited neurological signs.All the IC- and IV-inoculated positive control animals (50/50 mice and 14/14 macaques) developed vCJD . We further investigated the neurologically affected animals but PrPres negative (53/94 mice and 9/24 macaques) that corresponded, (depending on the inoculum), to between 9 and 80% of the animals developing neurological impairments  or endogenous preparations (infected blood), a proportion of the animals (94/540 mice and 24/30 macaques) developed fatal neurological impairments with incidences ranging from 10 to 100% depending upon the inoculum. A higher incidence of fatal neurological impairment was obtained in animals exposed to soluble infected brain extracts that were expected to harbour more infectivity than the infected blood. Among the affected animals, classical biochemical diagnostic methods detected PrPres; these animals were thus initially suspected of developing the same prion disease as the positive controls. Seventy-four (80%) of the mice with neurological impairments and similar numbers of mice exposed to infected material and devoid of neurological signs (64 mice considered as apparently negative) and control mice  were sampled for histology and for detection of PrPd by immunohistochemistry  to the 50 positive control animals. This was independent of the inocula they received and their expression of PrPres positive) presented a classical vCJD profile: they exhibited significant neuronal lesions , cerebral and spinal sponjpgorm change  in both brain and spinal cord .Among the 94 neurologically affected mice, 29 mice : they exhibited the same pattern of neuronal lesions and PrPd deposits in the whole central nervous system (CNS) as vCJD mice, but spongiform change was restricted to the brainstem and spinal cord  and spongiform change both of which were strictly limited to their spinal cords  animals constitute the first group of atypical phenotypes with lesions focused on the spinal cord.The first group included the five remaining PrPord Fig. . An accurds Fig. . These ad  and an absence of detectable PrPd Fig. . Fifteenres negative animals with neurological signs could not be sampled for histology and thus cannot be specifically subclassified within the S, C, B or NL phenotypes. They were thus classified as non-vCJD. No significant neuronal lesions or spongiform change were observed either in 41 healthy negative control mice sampled for histology ) or were transfused (8/19 = 42%) with blood derived from donors with detectable accumulation of PrPd or infectivity in their peripheral organs, including donors with the status of healthy carriers (Supplementary Note d).In contrast to the mice, the 9 PrPThe 9 macaques that developed this atypical neurological impairment began their long clinical phases (2–6 months) with impaired visual acuity, dysmetry of the forelimbs and they gradually stopped using their hands Table . Hind-liThe animals exhibited almost no lesions in the cerebral hemispheres: the cortices and basal ganglia were virtually spared; there was moderate rarefaction of cerebellar granule cells but no spongiform change was observed. However, all animals presented severe bulbar and spinal lesions Fig.  and we cd in the CNS  of any of the nine myelopathic macaques , was able to detect PrPues Fig. , 6 excepues Fig. , 5. At tues Fig. –4.Fig. 4d negative and PrPd positive myelopathic macaques were injected IV into 72 and 71 Swiss mice, respectively, while similar blood products derived from vCJD macaques and mice were injected into 222 and 105 Swiss mice, respectively. Altogether these mice correspond to the 470 mice exposed to blood products as described above. In parallel, mice were inoculated by intracerebral injection with brain material derived from myelopathic and vCJD macaques .Blood samples  from PrPd accumulation in their spinal cords  or blood 8/143, 6%) samples derived from the myelopathic macaques: none of the mice developed a complete vCJD profile, but they exhibited the same incomplete vCJD phenotypes as those described above, including the spinal (S) phenotype with PrP% samples/10, 50% χ2-test) was observed in terms of rates of transmission, of PrPd positive or PrPres positive animals, suggesting that infectivity from plasma is similar in vCJD and myelopathic macaque donors. Conversely, a gradient for the same criteria was observed in the groups exposed to BC samples derived from vCJD mice >vCJD macaques >PrPd positive myelopathic animals (only three AS mice) >PrPd negative myelopathic animals, for which no disease was transmitted: buffy coats from myelopathic macaques appeared to harbour limited infectivity, if any.Among the four groups of mice exposed to plasma from the different sources, no statistically significant difference (res positive phenotypes (vCJD and BS), with recipients exhibiting PrPres after short incubation periods , whereas the two recipients of deleukocyted RBCC (only red cells and plasma) developed myelopathy. In addition, the deleukocytation of blood with low infectivity did not impair the transmission of myelopathy (macaques R8–R11).We combined information on the prion nature of the different neurological impairments, and then detailed the distribution of the different phenotypes within the animals exposed to brain or blood infectivity. The results of this analysis clearly showed that the emergence of atypical prion disease phenotypes correlated with the exposure to soluble infectivity derived from brain or plasma. Indeed, crude brain homogenates induced the expected vCJD phenotypes in all macaque recipients , whereas myelopathy occurred in only one recipient (10%) of the corresponding soluble extracts Fig. . Among mres negative phenotypes were higher in recipients of plasma than in the recipients of buffy coat, and also in recipients of supernatants than in those animals that received pellets of soluble brain homogenate, whereas all recipients of vCJD-infected crude brain homogenates developed PrPres positive prion disease  profiles  had detectable accumulation of PrPd in their lymphoid organs. We hypothesise that two distinct pathophysiological pathways may coexist  vCJD clinical cases is very low. This is in contrast to recent estimates suggesting, paradoxically, that there has been massive exposure of the human populationd and spongiform change in all or part of the CNS. Similar results were achieved by intravenous administration of soluble material derived from infectious brains, which is an acknowledged model of blood infectivity. Furthermore, it is important to note that the transmission of this atypical prion disease in non-human primates is apparently not prevented by the compulsory leukodepletion step for infected blood.However, in the same experiments, higher proportions (2- to 7-fold more) of recipients developed other fatal neurological impairments, some of them exclusively involving the spinal cord. These unexpected neurological impairments would escape the current criteria for the diagnosis of prion diseases; such criteria include the presence of abnormal PK-resistant PrP associated with spongiform change in the brain . Some conformations would correspond to forms of PrP detectable on the basis of their resistance to proteolysis, so called PrPres, whereas other conformations, that are oligomeric and until now undetectable by biochemical techniques, would constitute the most toxic and infectious entities. An even greater complexity may arise from the capacity of cellular PrP to interact naturally with several molecules including non-coding RNA, DNA or Aβ25.Interestingly, these atypical prion phenotypes occur after incubation periods similar to vCJD in mice, and even shorter in macaques, suggesting that they are due to different variants. According to our results, the onset of such atypical prion phenotypes, including spinal cord involvement, seemed to be preferentially promoted by the administration of soluble infectivity through the intravenous route, whereas the most commonly used method  is associated with the classical vCJD phenotype. So, we hypothesise that non-protease-resistant soluble infectivity in blood might follow an alternate pathophysiological pathway from the stage of peripheral replication; this pathway, that may even correspond to a direct neuroinvasion as recently describedres and infectivity/toxicity can be dissociated31. We previously reported the first obvious dissociation following primary transmission of BSE to mice11. Following intracerebral inoculation, all animals developed similar neurological signs but more than 50% had no detectable PrPres. On secondary and tertiary transmissions, however, the proportion of PrPres positive animals gradually increased to almost 100%. Recent communications suggest that a similar situation might exist in other models of experimental exposure to prions involving swine32 and cattle33. In the present study, we used two animal models and blood transfusion to demonstrate a complete dissociation that could be maintained on successive passages. The panel of incomplete syndromes that we have observed in both mice and non-human primates suggests that the phenotypic expression of vCJD prion infection may vary. The recent description of vCJD in codon 129 heterozygous patients34 may even expand this possible spectrum of clinical heterogeneity. Such variation ranges from the status of healthy carrier to classical vCJD and includes clinical-pathological presentations that would be excluded from the diagnosis of prion disease according to current criteria.In this hypothesis, PrPres, counterbalances the observations made using PrPres amplification techniques. Such PrPres amplification techniques generate large amounts of PrPres but little or none of the toxic and infectious form of PrP. RT-QuiC is reported to be able to amplify over 1012 the quantity of PrPres in scrapie samples by converting recombinant cellular PrP (PrPc) produced in bacteria, but almost no infectivity is generated35. PMCA, which uses normal brain as a substrate for the conversion of PrPc, is able to maintain infectivity during successive cycles despite serial dilutions and can even generate infectivity de novo from normal mouse and hamster brains37. However up to more than 99% of the PrPres generated, de novo is not infectious (dissociation of at least two logs between infectivity and de novo generated PrPres)38. None of these techniques, which are increasingly used for the diagnosis of human prion diseases, was able to generate PrPres from our infectious PrPres negative samples.The complete dissociation that we observed here, with toxicity in the absence of PrP39 reported a specific pattern of spinal cord involvement in cynomolgus macaques exposed orally to BSE. These animals remained asymptomatic, but they exhibited atypical deposition of PrPd that was transmissible to bovinized mice39. Necrotic myelopathies have been described in humans that share clinical and pathological features with the primates in the present study40. The neuropathological features in macaques  resembled focal forms of subacute necrotising encephalomyelopathy as previously reported in Leigh syndrome41 but their posterior columns showed tract degeneration. However, as in Leigh syndrome, dysfunction of energy metabolism may be suspected42. Moreover, the atypical myelopathy in macaques shows similarities to other human myelopathies that range from neuromyelitis optica spectrum disorders (NMOSD), for which inclusion criteria are wide43, to the more recently described FOSMN44 and to certain forms of amyotrophic lateral sclerosis like FLAIL arm syndrome45. This latter syndrome presents as a pure lower motor neuron disorder that then evolves after a number of years to include upper motor neuron lesions46, a feature that we did not observe in our primates. However, in the absence of suitable nursing facilities, these primates were killed relatively early for ethical reasons.The neurological impairment described here in macaques appears to be unique as it has not been reported previously. However, Holznagel et al.d in their lymphoid tissues. Moreover, should such atypical agents or their subsequent passages in humans lead to neurological impairment, there is a significant risk that they would not be diagnosed as related to prion infection in the absence of detectable PrPres and a quasi-exclusive spinal involvement. Our results enlarge the range of prion diseases that is already no longer restricted to PrPres positive diseases that target the brain49.This report strongly suggests that prions might remain hidden in the population and as the majority of healthy carriers may never develop a prion disease, an even greater proportion of contaminated individuals may never be diagnosed as healthy carriers, currently diagnosed according to the presence of PrP50 and our capacity to detect and prevent all infectious forms of prion disease.In conclusion, the range of incomplete syndromes that we observed between healthy carriers and typical vCJD indicates that multiple forms of prion variants can coexist and may emerge in different forms depending upon the conditions under which transmission occurred. This has obvious consequences for public health, and questions the uniqueness of the BSE/vCJD strainPrimates and mice were housed and handled in accordance with the European Directive 2010/63 related to animal protection and welfare in research, and were under constant internal surveillance by veterinarians. Social enrichment was a constant priority with individual activities and feeding controlled according to the risk of infection. Animals were handled under anaesthesia to limit stress, and killing was performed for ethical reasons when animals lost autonomy. CETEA ethical committee approved the present experiments . Samples derived from patients were obtained in accordance to the rules in force at that time. The sizes of experimental groups were defined in accordance with the reduction and refinement concepts of 3R’s, depending on the respective expected rates of transmission. According to the specificity of the macaque model, each case is to be considered as single clinical case.Macaca fascicularis) were provided by Noveprim (Mauritius), checked for the absence of common primate pathogens before importation, and handled in accordance with national guidelines. They were all Met/Met homozygous at codon 129 of the prion protein gene (PRNP). Six weeks old, female C57Bl/6N and Swiss ND4 mice were provided by Janvier Labs (France). Animals were housed in level-3 animal care facilities  and regularly examined at least once a week. No statistical randomisation method was used to allocate animals to experimental groups. Samples for biochemical and histological analyses were coded (two different codes) and respective investigators were blind during analyses.Captive-bred 2–5-year-old male cynomolgus macaques (All the brain inocula were 1 or 10% (weight/volume) homogenates in 5% glucose solution. Blood products were sampled in citrate buffer.51) and brain inoculum from a BSE-infected primate . Corresponding high-speed supernatant (SHS) and pellet (PHS) were prepared according to the protocol described by Berardi et al.17 to model soluble and microsomal blood infectivity respectively. Briefly, 10% brain homogenates were centrifuged (low speed) at 825×g for 15 min. The resulting supernatant was ultracentrifuged  after extensive sonication. Corresponding supernatant (SHS) and pellet (PHS) fractions were inoculated IV through the tail vein with the equivalent of 1.5 mg of brain (200 µl) per animal. As controls, mice were inoculated intracerebrally with a crude 10% (w/vol) homogenate derived from the same BSE-infected primate brain .The inocula used for the experiments using soluble brain infectivity were brain inoculum from BSE-infected cattle . The supernatants obtained were injected into recipient macaques 53.In the first group of animals exposed to crude brain homogenates, four primates were inoculated IV with serial dilutions of a crude brain homogenate derived from a BSE-infected primate (primate D8)g for 13 min. Deleukocyted blood samples were prepared by pooling RBCC and plasma after fractionation. Deleukocyted RBCC were obtained through filtration on a commercial device  and then re-suspended in corresponding plasmas in proportions used for paediatric transfusion53.Blood samples from three UK and one French vCJD patients were used in parallel with primate blood samples drawn into sodium citrate. Corresponding RBCC, plasma and buffy coat fractions were collected after centrifugation at 2000×g for 13 min. Buffy coat and plasma were recovered after fractionation.Sixty Swiss mice were inoculated intracerebrally with 2 mg of brain from Swiss mice infected with the vCJD-adapted strain. Their blood was collected 20 weeks post inoculation, pooled and centrifuged at 2000×d in their spinal cord, respectively.Transfusion experiments in primates are extensively described in the Supplementary Text C57Bl/6 and Swiss mice were inoculated intracerebrally (2 mg of tissue) or IV (0.2 mg of tissue) with brain, spinal cord or spleen homogenates from mice or primates.d were performed on brain sections as previously described using 3F4, Sha-31, SAF-60, POM-1, SAF-32 or 12F10 antibodies after a proteolysis step54. Immunohistochemical detection of GFAP was performed using polyclonal rabbit anti-GFAP antibody (Dako); apoptotic cells were detected using the In Situ Cell Death Fluorescence Kit (Roche)55.Tissues were fixed in 4% formalin for histological examination. Neuropathology and immunohistochemical detection of PrP53, or a specific ELISA using Saf-53 as the capture antibody and 11C6 as the detection antibody for mouse samples56), or processed for western blot analysis as previously described54 using 3F4, Saf-37 or Sha-31 anti-PrP monoclonal antibodies.PrP was purified according to the TeSeE purification protocol (Bio-Rad), in the presence (40 µg/mg protein) or absence of proteinase K. Purified samples were detected by ELISA (TeSeE Kit for primate samples136R154Q171 PrP variant PrPc (tgShXI) was used to prepare the PMCA substrate as previously described58. PMCA amplification was performed as previously described59. Each PMCA run included a reference vCJD sample (10% brain homogenate) as a control for the amplification efficiency. Unseeded controls (one unseeded control for eight seeded reactions) were also included in each run. PMCA reactions were seeded with 1/50 diluted tissues homogenates (10% weight/volume). For each sample, at least four replicates were tested in two independent runs. Control sample was serially diluted 10-fold (10−2–10−10) before being used to seed PMCA reactions.A transgenic mouse line that expresses ovine A59. Immunodetection was performed using two different monoclonal PrP-specific antibodies, Sha-31 (1 µg/ml)60, and 12B2 (4 µg/ml)61, which recognise the amino-acid sequences YEDRYYRE (145–152), and WGQGG (89–93), respectively.Samples were submitted to five successive amplification rounds, each composed of 96 cycles (10 s sonication-14 min and 50 s incubation at 39.5 °C) in a Qsonica700. After each round, (i) reaction products (one volume) were mixed with fresh substrate (nine volumes) to seed the following round while (ii) a part of the same product was analysed by western blot (WB) for the presence of PK-resistant PrP as previously described62. The buffer contained 400 mM NaCl and Syrian golden hamster recombinant PrP (aa 23–231) was used as substrate. Dilutions of brain and spinal cord homogenates were prepared in N2 buffer and each sample was run in triplicate. Reactions were seeded with 2 µl of dilution in a final volume of 100 µl. Plates were incubated in a BMG Fluostar Omega plate reader at 45 °C for 70 h, with cycles of 1 min shake  and 1 min rest throughout the incubation.RT-QuIC was performed as originally described63 using a dedicated suit to maintain level-3 confinement.Magnetic resonance imaging (MRI) was conducted on a 7 Tesla horizontal system (Varian). Animal positioning and monitoring was performed as previously describedThe data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary informationPeer review file


Horizontal gene transfer (HGT) is a main mechanism of bacterial evolution endowing bacteria with new genetic traits. The transfer of mobile genetic elements such as plasmids (conjugation) requires the close proximity of cells. HGT between genetically distinct bacteria largely depends on cell movement in water films, which are typically discontinuous in natural systems like soil. Using laboratory microcosms, a bacterial reporter system and flow cytometry, we here investigated if and to which degree mycelial networks facilitate contact of and HGT between spatially separated bacteria. Our study shows that the network structures of mycelia promote bacterial HGT by providing continuous liquid films in which bacterial migration and contacts are favoured. This finding was confirmed by individual-based simulations, revealing that the tendency of migrating bacteria to concentrate in the liquid film around hyphae is a key factor for improved HGT along mycelial networks. Given their ubiquity, we propose that hyphae can act as focal point for HGT and genetic adaptation in soil. Bacteria play a dominant role, with typical densities of 10Pseudomonas putida strains, revealing HGT events by an emerging green fluorescence signal. To test a possible bridging effect of mycelial networks, plasmid donor and recipient were inoculated on individual agar pieces separated by an air-filled gap. Environments allowing for low, medium and high mobility of bacteria were simulated using (semi-)solid media prepared with varying agar concentrationsWe used a reporter system for bacterial Horizontal Gene Transfer, based on motile Pseudomonas putida KT2442::dsRed-lacIq(pWW0::Plac-gfp), which expresses the red fluorescent protein DsRed and the repressor LacIq, inhibiting the expression of a plasmid-encoded GFP. Pseudomonas putida KT2440 acted as potential plasmid recipient; the resulting transconjugants Pseudomonas putida KT2440(pWW0::Plac-gfp) express GFP indicating successful plasmid transferPythium ultimum was inoculated on the donor side. Within 24 hours it formed a mycelium bridging the gap thereby establishing a viable transport path between both bacterial populations  solid ABC-media1920−5 transconjugant cells) were observed, even in high mobility systems that permitted bacterial swimming (−5 cells), a significantly higher amount of transconjugants was detected in mycelial systems permitting medium or low mobility . Thus, the beneficial effect of mycelia was especially pronounced in low and medium mobility systems, i.e. in environments where bacteria had to rely on the mycelial structures for migration and habitat colonization. The same trend was even more pronounced in systems supplemented with glass fibres as abiotic, i.e. metabolically inactive transport vectors. In presence of glass fibres, systems showed significantly higher amounts of transconjugants than without transport vectors in all three cases . Even in high mobility systems with glass fibres there was a notable increase in transconjugants, compared to systems with mycelia or without transport vectors. This increase is attributed to the fact that the glass fibres represent ‘ideal’ transport vectors: straight, unbranched transport pathways with a hydrophilic surface, creating wide continuous water films for bacterial movement, while at the same time providing a rigid surface for bacteria to settle and conjugate.In plain agar systems without transport vectors, only very few transconjugants , which determined both the bacterial fraction colonizing the hyphal surface and the number of transconjugants . Model parameters were chosen according to the laboratory microcosm or literature values except for the characterization of the exchange flux of bacterial cells between the agar phase and the liquid film on the hyphal surface. This flux, defined by the attachment and detachment parameters njugants . Interesnjugants .Pseudomonas putida2126In this study we give experimental and theoretical evidence that mycelia-based dispersal promotes HGT due to an elevated contact probability between bacteria. Conjugation requires close physical contact between bacterial donor and recipient cells with reported effective distances of <2 μm for t) and surpasses the probability in the higher-dimensional domain by two orders of magnitude already after a very short time (~1 s). This effect may become even larger if thin water films reduce the randomness of bacterial movementWe propose two driving mechanisms for the increased contact probabilities in the hyphosphere: Reduction of the space available for bacterial dispersal and, secondly, reduction of the degree of freedom of bacterial mobility. Such reasoning becomes clear by comparing a scenario in which bacterial cells populate a cube of water with a scenario in which cells only populate a water film surrounding hyphae forming a network within a cube of the same volume. Even in case of dense networks, the latter scenario results in a clearly reduced volume available for bacterial dispersal. In contrast to the water cube scenario, hyphae additionally represent defined corridors for dispersal, and cells migrating along a hypha can overcome longer linear distances than in the water cube in a given time span. Presuming bacterial mobility within the liquid film around hyphae resembles a two- rather than a three-dimensional movement, the calculated increase of contact probability of two cells amounts to a factor of approximately dry soil−1) with networks extending to 102 to 104 m length per g of topsoil33Filamentous fungi represent up to 75% of the subsurface microbial biomass  was used as plasmid donor, expressing DsRed while at the same time repressing plasmid-encoded GFP by the simultaneous expression of LacIq. Pseudomonas putida KT2440::yfp acted as potential plasmid recipient, constitutively expressing YFPPseudomonas putida KT2440::yfp(pWW0:: Plac-gfp) simultaneously expresses YFP and GFP, detectable with appropriate filter sets for yellow and green fluorescence, indicating successful plasmid transfer. Donor cells were grown on Brunner-Mediumm-toluic acid (mTol) to select for the pWW0 plasmid. Recipient and wildtype cells were grown on R2A mediumA bacterial reporter system consisting of two Transconjugant cells were created by cross-streaking donor and recipient cells on R2A media supplemented with 20 mM mTol and 18 g/L agar and cultivation for 24 hrs at 30 °C. After selection for plasmid harbouring cells on Minimal-Medium-Agar with 20 mM mTol, colonies were microscopically checked for their fluorescence signals. Transconjugant cells were selected based on the presence of yellow and green fluorescence and the absence of red fluorescence. Presence of the plasmid in the transconjugant cells was confirmed by plasmid isolation and subsequent gel electrophoresis, as described in the Pythium ultimum was grown at room temperature on plates of AB-mediumThe oomycete P. putida KT2442 donor strain was used in combination with the P. putida KT2440 wildtype-strain as potential plasmid recipient. To observe the influence of mycelial networks on spatially isolated donor and wildtype colonies, individual ABC agar pieces (18 g/L agar) were inoculated with 1 μL overnight cultures adjusted to an optical density of 0.05. As mycelial organism, P. ultimum was additionally inoculated on the donor side. The agar pieces were inverted and placed side by side in a 50-mm glass bottom dish  with an air gap of about 400 μm between them. Setups were incubated at room temperature for 3 days and examined with an AZ100M fluorescence microscope , using distinct filtersets for DsRed (F46-005) and GFP . Images were captured with the Nikon NIS-Elements software and annotated with Adobe Photoshop .For epifluorescence microscopy, the Depending on the agar concentration, the used media generally permitted three dimensional movements of motile bacteria. Liquid (3 g agar/L) and semi-solid agar (5 g agar/L) were assumed to allow nearly unhindered and hampered movement, respectively, whereas movement in solid agar (18 g agar/L) was regarded negligible13P. ultimum was inoculated on the side of each well and incubated at room temperature for 3 days until a complete, planar mycelial network had established on the agar in each well. Setups with abiotic transport networks were prepared in parallel by using glass fibres with diameter of ≈12 μm, cut to a length of 2 cm . Before covering the agar with approximately 300 fibres for each well, the glass fibres were cleaned by heating to 600 °C for 5 het al.To investigate the effect of alternative transport routes provided by mycelial networks, microcosms were set up in triplicates in 12-well plates  by filling each well with 2 mL AB-medium supplemented with 10 mM Na-citrate and the agar concentrations of choice. Solid and semi-solid agar were left to dry for 24 h, liquid agar for 1 h. P. putida donor and wildtype strain were examined after 3 days of incubation. Images were taken with an AZ100M fluorescence microscope , using the NIS-Elements software and distinct filtersets for DsRed (F46-005) and GFP . Adobe Photoshop  was used to annotate images and reduce noise. To reveal localization of transconjugants along mycelial network structures, a white overlay of the mycelial network was created by applying a signal intensity threshold to a transmission light image and modest manual corrections.To microscopically observe the influence of the mycelial network on transconjugant formation in the microcosms, parallel setups with a combination of Flow cytometric measurements were carried out using a MoFlo cell sorter  equipped with a water-cooled argon-ion laser . 488 nm light of 400 mW was used to analyze the forward light scatter (FSC) and the side light scatter (SSC), and to excite GFP, YFP, and dsRed. Fluorescences were detected after passage of the optical filters 510/10 (GFP), 542/27 (YFP), and 600/37 (dsRed), respectively . Data recording and subsequent data analyses were made applying Summit software v4.3 .For absolute quantification, cell numbers per mL were determined using reference microspheres  of known concentration as described previouslyThe identification and quantification of GFP/YFP fluorescent transconjugants within a cell suspension containing a multitude of YFP fluorescent recipient and dsRed fluorescent donor cells was done as follows: Pure cultures of recipient, donor, and transconjugant cells were analyzed by flow cytometry. On an FL1 (GFP fluorescence) vs. FL2 (YFP fluorescence) dot plot, three areas (gates) were defined with the premises that gate 1 exclusively includes transconjugant cells, gate 2 exclusively those of the recipient strain, and gate 3 those of the FL1/FL2 non-fluorescent donor strain. Based on this definition, all conjugational samples were analyzed. If a cell had displayed a gate 1 specific FL1/FL2 fluorescence it was classified as a transconjugant. The total number of transconjugants per sample was calculated by means of the above mentioned yellow-green fluorescent, 1 μm microspheres.The transconjugant cell numbers within each replicate series (n = 6) were tested for skewnessDagar and Dhyphae, and could switch layers at locations where a fungal grid cell was present on top the agar grid cell according toMicrobial cells populated a two-dimensional grid of two layers in the model. The bottom layer was continuous and represented the agar, while the top layer was obtained by digitizing a microscopic image of actual fungal hyphae to represent the hyphal network. Microbial cells performed random walks .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Sickle cell anemia causes severe complications and premature death. Five common β-globin gene cluster haplotypes are each associated with characteristic fetal hemoglobin (HbF) levels. As HbF is the major modulator of disease severity, classifying patients according to haplotype is useful. The first method of haplotype classification used restriction fragment length polymorphisms (RFLPs) to detect single nucleotide polymorphisms (SNPs) in the β-globin gene cluster. This is labor intensive, and error prone.We used genome-wide SNP data imputed to the 1000 Genomes reference panel to obtain phased data distinguishing parental alleles.We successfully haplotyped 813 sickle cell anemia patients previously classified by RFLPs with a concordance >98%. Four SNPs  marking four different restriction enzyme sites unequivocally defined most haplotypes. We were able to assign a haplotype to 86% of samples that were either partially or misclassified using RFLPs.Phased data using only four SNPs allowed unequivocal assignment of a haplotype that was not always possible using a larger number of RFLPs. Given the availability of genome-wide SNP data, our method is rapid and does not require high computational resources. HBB) cluster are associated with different levels of HbF . , 6. 5, 6Haplotypes among 55 Southwestern Province patients classified using the RFLP method included 39 BEN/BEN, 11 CAR/CAR, 2 BEN/SEN, 1 BEN/CAR, 1 SEN/SEN and one unknown. The distribution of haplotypes for these subjects derived using the SNP method was 48 BEN/BEN, 3 BEN/UNKNOWN, 2 BEN/CAM, and one AI/AI  between 4-SNP haplotypes derived from a phasing algorithm using GWAS data with 5-SNP haplotypes determined using restriction analysis. Haplotype assignment in a sickle cell anemia iPSC library also showed high concordance and demonstrated the efficiency of SNP-based method to classify samples that failed RFLP classification. The discordance between SNP-based and RFLP ascertainment most likely resulted from errors in the RFLP classification that is sensitive to the presence of other SNPs in the restriction sites and the vagaries of restriction enzyme analysis and Southern blotting that was used for haplotype analysis in the CSSCD.HBG2), rs3834466 (Hinc2 5′ to HBE1), and rs549964658 (5′ to HBD) we had 100% concordance.In 30 Saudi East patients where the AI haplotype was ascertained by genotyping rs7482144  and upstream of OR51B4 (3′ olfactory gene cluster) in both patient groups. The SNP genotypes of the 11 CAR/CAR that we reclassified as BEN/BEN or BEN heterozygous had the same SNP genotype of BEN/BEN patients that were classified as such with both methods. The genotype data, average HbF, and the imputation quality score of rs968857 suggest that the high discordance in Southwestern Saudi patients is due to RFLP errors.To investigate the discordance in Southwestern Province patients, we examined the genotype data of the A limitation of our method is the dependency on the availability of GWAS data for many SNPs in the β-globin gene region. However, many large patient cohorts have been genotyped using genome-wide SNP arrays. In these patients, haplotype information might be useful as a covariate in a genetic risk analysis. RFLP analysis might be suitable for a small number of patients but requires optimization of all of the individual assays. The main advantage of our haplotype determination method is the rapid classification and high accuracy. This method can also be used for whole genome sequence data classification after SNP calling and phasing. Moreover, it is not sensitive to SNPs that alter the restriction enzyme recognition sequence that can lead to error using RFLPs.Phased data using only four SNPs allowed unequivocal assignment of a β-globin gene cluster haplotype that was not always possible using a larger number of RFLPs, and was also more accurate. With the availability of genome-wide SNP data our method is rapid and does not require high computational resources.


RUNX1 is a recurrently mutated gene in sporadic myelodysplastic syndrome and leukemia. Inherited mutations in RUNX1 cause familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). In sporadic AML, mutations in RUNX1 are usually secondary events, whereas in FPD/AML they are initiating events. Here we will describe mutations in RUNX1 in sporadic AML and in FPD/AML, discuss the mechanisms by which inherited mutations in RUNX1 could elevate the risk of AML in FPD/AML individuals, and speculate on why mutations in RUNX1 are rarely, if ever, the first event in sporadic AML. DNMT3A, ASXL1, IDH2, and TET2 , signaling molecules , splicing factors , and proteins with other functions   that promote their clonal expansion and impair downstream differentiation. The earliest initiating step in leukemia is a mutation that generates a hematopoietic stem cell (HSC) that is, in some poorly understood way, primed for leukemic transformation by secondary mutations. The mutations in HSCs that initiate sporadic leukemia in adults generally occur in genes encoding epigenetic regulatory proteins such as RUNX1 mutations; however, the lifetime risk approaches nearly 100% for germ line CEBPA mutations  are also frequently found in sporadic acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) , acute lymphocytic leukemia (ALL), atypical chronic myeloid leukemia (aCML), and secondary AML  in de novo AML and t in acute lymphocytic leukemia (B-ALL). Both of these translocations are initiating events in sporadic leukemia and can be acquired in utero as evidenced by their presence in Guthrie card samples from newborns diagnosed in childhood with AML or B-ALL  to reflect the possible worse prognosis as compared to other AML types  is a clonal hematopoietic disorder characterized by ineffective hematopoiesis and is a harbinger of AML in approximately 25–30% of patients   were also rare or undetected.The paucity of initiating RUNX1 mutations are more common in clonal cytopenia of undetermined significance (CCUS)  , inv(16)/t(16), and t, presumably because the mutations are either functionally redundant or antagonistic. Other common MDS or AML mutations  were neither enriched nor inversely correlated with RUNX1 mutations.The ability to monitor HSC expansion by changes in variant allele frequencies in FPD/AML individuals by next generation sequencing may offer the opportunity to intervene at the pre-leukemic stage, prior to the appearance of overt MDS or leukemia. Knowing when to intervene and treat a healthy individual at an early stage of disease, however, requires knowing which secondary mutations are likely to lead to disease progression. In sporadic leukemia, it is assumed that the most potent cooperating mutations are those that most frequently co-occur due to selection. For CDC25C in 4 patients, concluding that somatic mutations in CDC25C (which are rarely observed in sporadic AML) were the most common genetic event in AML arising from FPD/AML  from the United States analyzed somatic mutations in 7 FPD/AML patients with MDS or AML and found no mutations in either CDC25C or in the second RUNX1 allele, but instead, identified mutations in a collection of other genes including BCOR, PHF6, DNMT3A, TET2, CREBBP, U2AF1, NUP214, SMC3, and PDS5B , base excision (BER), nucleotide excision (NER), double-strand break repair , and interstrand DNA crosslink repair by the Fanconi Anemia (FA) / BReast CAncer susceptibility (BRCA) pathway  protein. Several studies showed that expression of RUNX1-RUNX1T1 in primary HSPCs or cell lines resulted in down regulation of a number of genes involved in BER, including in vitro gradually acquired more γ-H2AX foci than normal cells over time, indicating that they more rapidly accumulated unrepaired double-strand breaks  polymerase (PARP) inhibitors  to promote leukemogenesis was greatly enhanced by transplantation into old recipient mice, whereas when transplanted into young hosts, leukemogenesis was largely suppressed  linked et al. showed that RUNX1 interacted with the IκB kinase (IKK) complex in the cytoplasm, and deletion of Runx1 activated NF-κB signaling  and committed myeloid progenitors in the bone marrow  in the bone marrow of both young and aged mice , Runx1+/−, and wild type HSCs performed equivalently well in serial transplantation experiments, in that by the fourth round of transplantation, HSCs of all three genotypes were essentially exhausted, and hence had similar self-renewal potential  and normal bone marrow cells followed by transplantation into mice resulted in a greater selective expansion of Runx1 deficient HSPCs (lineage negative Sca1+ Kit+ cells) and myeloid lineage cells in the bone marrow compared to irradiated wild type or non-irradiated Runx1 deficient cells (Cai et al., Runx1 deficient HSPCs to endogenous and genotoxic stress (Cai et al., Tp53 mutation, consistent with this interpretation (Marusyk et al., Runx1 deficient HSPCs were less apoptotic, grew more slowly than normal HSCs, had a lower rate of protein translation, and had markedly decreased ribosome biogenesis. Consistent with decreased ribosome biogenesis, Runx1 deficient HSPCs were smaller than normal HSPCs, and preliminary data suggested that this small cell phenotype was shared with HSPCs from FPD/AML individuals (Cai et al., RUNX1 mutations confer a relatively poor prognosis in AML. It may also explain the observation that RUNX1 mutations occurred at a higher frequency in MDS patients previously exposed to low-level radiation as compared to all MDS patients (Harada et al., An interesting and potentially clinically consequential feature of RUNX1 mutant HSPCs is that they exhibit a greater selective advantage over normal HSPCs following exposure to radiation. Irradiation of a mixture of RUNX1 mutations cause leukemia predisposition because every HSC in an FPD/AML individual is already one mutation along the path to AML. However, it is likely that inherited RUNX1 mutations play a more active role in promoting leukemia progression. The dysregulation of DNA repair and decreased apoptosis of RUNX1 mutant HSCs along with an increased inflammatory microenvironment may contribute to the markedly increased incidence of MDS, AML, and T-ALL in FPD/AML individuals. We envision a model whereby RUNX1 mutations increase the rate at which secondary mutations are acquired; moreover, increased inflammatory signals delivered by RUNX1 mutant myeloid lineage cells or other cells in the RUNX1 mutant bone marrow microenvironment provide a selective pressure that confers a competitive advantage to FPD/AML HSCs that have acquired secondary mutations (Figure RUNX1 mutation does not have a competitive advantage over normal HSCs and hence does not expand and initiate leukemia.It was originally thought that inherited DNMT3A mutations have been reported in two asymptomatic FPD/AML individuals (Churpek et al., DNMT3A mutation, which in a normal individual only modestly increases the risk of leukemia, confer a much greater risk in the context of an inherited RUNX1 mutation? More data and longitudinal studies correlating the stepwise accumulation of mutations in individuals with FPD/AML with clinical outcome are needed to predict which mutations most likely portend a rapid progression to MDS or AML. Mechanistic studies in animal and cell models will also be necessary to understand what is driving leukemia progression in FPD/AML and to identify strategies that can avert, delay, or reverse this progression.The initiation and evolution of leukemia in FPD/AML individuals can be carefully followed by next generation sequencing. Individuals diagnosed with FPD/AML could be monitored on a regular basis for evidence of clonal HSC expansion driven by acquired secondary mutations and treated before they progress to frank AML. However, we still do not know which secondary mutations are the most deleterious and whether they are the same as or different than the co-occurring mutations in sporadic AML. For example, All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Large-scale genomic studies have made major progress in identifying genetic risk variants for schizophrenia. A key finding from these studies is that there is an increased burden of genomic copy number variants (CNVs) in schizophrenia cases compared with controls. The mechanism through which these CNVs confer risk for the symptoms of schizophrenia, however, remains unclear. One possibility is that schizophrenia risk CNVs impact basic associative learning processes, abnormalities of which have long been associated with the disorder. To investigate whether genes in schizophrenia CNVs impact on specific phases of associative learning we combined human genetics with experimental gene expression studies in animals. In a sample of 11 917 schizophrenia cases and 16 416 controls, we investigated whether CNVs from patients with schizophrenia are enriched for genes expressed during the consolidation, retrieval or extinction of associative memories. We show that CNVs from cases are enriched for genes expressed during fear extinction in the hippocampus, but not genes expressed following consolidation or retrieval. These results suggest that CNVs act to impair inhibitory learning in schizophrenia, potentially contributing to the development of core symptoms of the disorder. Specifically, we investigated whether CNVs in schizophrenia cases from three large cohorts are selectively enriched for genes expressed during the consolidation, retrieval or extinction of fear associations in the hippocampus. Identifying which specific cognitive processes are impacted by schizophrenia risk genes is an important step towards determining how genetic risk can lead to the development of the complex symptoms seen in schizophrenia and related disorders.28 together, giving a total of 11 917 case and 16 416 control subjects. These CNV data sets have been previously validated and full genotyping, CNV calling and quality control information can be found in the original publications.28 Approval by the local ethics committee was granted for the use of these samples in genetic association studies. All CNVs from the ISC, MGS and CLOZUK data sets were annotated with their overlapping genes from corresponding genome Builds 35, 36 and 37, respectively. Analyses were performed on CNVs at least 100 kb in size and covered by at least 15 probes, to optimise CNV calling reliability. For comparison analyses, CNVs were compared between melanoma patients and additional non-psychiatric phenotypes. These CNVs were obtained from a separate study29 and underwent the same filtering process .CNV data were compiled from three European case-control data sets: the International Schizophrenia Consortium , the Molecular Genetics of Schizophrenia  study and a UK study of schizophrenic patients taking clozapine combined with the Cardiff Cognition in Schizophrenia sample ,19 Conditioning was characterised by robust conditioned freezing (immobility) responses upon re-exposure to the training environment indicating robust contextual fear conditioning and recall of contextual fear memory.21 For a full description of the conditioning procedures and tissue collection see Barnes et al. (2012)20 and Scholz et al. (2016).21 In all experiments adult male Lister Hooded rats were placed into a context for 3 min during which they received a single delivery of a single-scrambled foot shock . We have previously shown that this context-shock pairing is sufficient to produce reliable contextual fear conditioning.31 Rats were then killed 2 h after either consolidation, retrieval or extinction of the fear association. For the consolidation group, animals were killed direct from their home cages 2 h after conditioning. For the retrieval condition, animals were re-exposed to the conditioned context 48 h after conditioning for 2 min only, a manipulation we have shown produces a fear response but is not sufficient to engage extinction.30 For the extinction condition, animals were re-exposed to the conditioned context 48 h later for 10 min, a manipulation that is sufficient to develop robust behavioural extinction of the original context-fear association.30 To generate a control group for the conditioning procedure, balanced for overall stimulus exposure, we utilised dorsal hippocampal brain-derived neurotrophic factor (BDNF) antisense infusions to block contextual fear conditioning.31 Control rats (n=8) received bilateral microinfusions of 2 nmol μl−1 BDNF antisense oligonucleotides (5′-TCTTCCCCTTTTAATGGT-3′) into the dorsal hippocampus, sufficient to block fear conditioning, whereas rats in the consolidation group (n=8) received BDNF missense oligonucleotides (5′ -ATACTTTCTGTTCTTGCC-3′) at 1 μl per hemisphere into the dorsal hippocampus . Infusions were given 90 min before placement into the conditioning context. To generate a control condition for the retrieval group balanced for stimulus exposure, we similarly used antisense infusions previously shown to block processes that support the maintenance of the contextual fear memory, but not conditioning.31 Specifically, control rats (n=8) received dorsal hippocampal microinfusions of 2 nmol μl−1 Zif268 antisense oligonucleotides (5′- GGTAGTTGTCCATGGTGG-3′), whereas the retrieval group (n=8) received infusions of Zif268 missense oligonucleotides (5′-GTGTTCGGTAGGGTGTCA-3′). All infusions were 90 min before re-exposure to the context. In the extinction paradigm, to generate a control group matched for exposure, rats were initially trained to distinguish two contexts before contextual fear conditioning in one of the two contexts. After 48 h of conditioning, rats in the extinction group (n=6) were re-exposed for 10 min to the conditioned context whereas control animals (n=6) were re-exposed to the non-conditioned context. To assess contextual fear in the rats, freezing behaviour was quantified during all context exposures, as reported previously.21 These behavioural data conformed to the expected patterns of conditioning and within session reductions of freezing responses to the conditioning context, indicative of extinction. Rats were killed by CO2 inhalation 2 h after conditioning (consolidation group) or 2 h after context exposure  for tissue extraction. Experiments were conducted in accordance with the United Kingdom 1986 Animals (Scientific Procedures) Act (Project licenses PPL 30/2236 and PPL 30/2722).Associative learning was assessed in a previously published set of experiments using hippocampus-dependent contextual fear conditioning in rats.21 and are accessible through GEO Series accession number GSE66153. The dorsal CA1 region of the hippocampus was dissected and snap frozen before microarray analysis. Samples from individual rats were hybridised to separate microarrays  as described previously.21 There was no amplification of cDNA before hybridisation. Raw intensity data from each of the three Affymetrix data sets  were normalised using RMA and MAS5.0 procedures.21A full description of the microarray procedure and results can be found in the original studiest-test P-values, representing the significance of differential expression (increased or decreased), were combined using Fisher's method. In cases where several Affymetrix probe sets targeted the same gene, their combined P-values were further combined using Simes P-value correction procedure, giving one P-value per gene. For gene set selection, genes were ranked by this P-value. In primary analyses, the top 5% learning-related genes were taken as the gene set. For secondary analyses, the top 1, 2, 5, 10, 15, 20 and 25% genes from each data set were used. For permutation correction, the entire gene list was permuted 2000 times, each time taking the top 1–25% genes as randomised gene sets.Rat genes targeted by Affymetrix probe sets were identified from the Affymetrix Rat Genome Array 230.2 resources. Probe sets that do not target a unique gene were filtered from the data sets. Rat Entrez gene IDs were converted to human homologues using their shared HomoloGene ID (accessed through the MGI Vertebrate Homology table 3 March 2015). Genes that did not have a unique human homologue were excluded from the data sets. For each independent consolidation, retrieval and extinction microarray data set, MAS5.0 and RMA moderate 8 For each gene set, case-control status was regressed against the number of overlapping genes and the following covariates: CNV size, number of genes per CNV, chip type and CNV study. All analyses were two-tailed. Primary analyses were corrected for multiple testing using Bonferroni's correction. Secondary analyses were corrected by calculating the fraction of randomly permuted gene sets of the same size that yielded a case enrichment as or more significant, to remove the effect of background enrichment , indicating that this enrichment is a general property of schizophrenia CNVs and is not driven by one population or cohort (P=3.0 × 10−4), but not in CNVs from MGS (P=0.18) or ISC (P=0.11), which reflects the lower power of analyses in individual, smaller cohorts.Consolidation, retrieval and extinction of contextual fear memory each affected the expression of a distinct group of genes in the hippocampal CA1 region, with little overlap across the learning conditions21 . We ther21  are statr cohort . The topP=0.0034, Bonferroni corrected) and duplications (P=0.042, Bonferroni corrected; These results indicate that people with schizophrenia are enriched for CNVs affecting genes involved in extinction learning. To further investigate the origin of this effect, we examined the enrichment of learning-related genes in deletion CNVs and duplication CNVs separately. This analysis revealed that the top 5% extinction-related genes were enriched in both deletions (P=0.59; retrieval P=1.0; extinction P=1.0; To test whether the enrichment of extinction-related genes is specific to schizophrenia-associated CNVs, we conducted a control analysis using CNVs from an independent sample of 2416 patients with melanoma vs 6335 control subjects. No association was identified between consolidation-, retrieval- or extinction-related genes and melanoma case CNVs (consolidation CLDN5, MFI2, ATP10A, SNRPN and CGNL1 (28 We also identified a number of overlapping genes whose biological function remains to be well characterised, illustrating that our combined bioinformatics and functional analysis can identify the contribution to risk of genes that are not annotated sufficiently for traditional pathway analysis approaches.We finally sought to investigate which specific CNV-associated genes drive the enrichment for genes differentially regulated during extinction learning. The full gene set is listed in nd CGNL1 .4, 28 We9 and studies of individual risk genes and familial risk,34 and provide evidence that CNVs confer vulnerability to the disorder by impacting on specific cognitive processes.Our results demonstrate a selective impact of genes in schizophrenia-associated CNVs on the molecular pathways regulated by extinction learning. By using gene expression data sets generated from functional experiments in animals we extend previous results from pathway analyses of schizophrenia-associated CNVs35 Indeed, an impairment in extinction learning could explain core symptoms of the disorder, such as the characteristic persistence of delusional beliefs in schizophrenia in the face of conflicting evidence.15 Our findings are also consistent with previous work showing impaired extinction of fear associations in schizophrenia as well as persistent activation of the hippocampus to fearful stimuli in the disorder.37 The observation that genes in schizophrenia-associated CNVs are enriched for targets engaged during extinction learning in the CA1 region of the hippocampus also suggests a potential mechanistic explanation for the heightened activation of the CA1 region seen in both prodromal cases and individuals with established schizophrenia.39Our results are consistent with previous studies suggesting that impaired extinction and inhibitory learning contribute to the development of psychotic symptoms in schizophrenia.40 raising the possibility that the link between CNVs and associative learning is of wider relevance to those psychiatric disorders in which CNVs have a role. We also note that although we show strong evidence of an association of schizophrenia-related CNVs with extinction learning, the experimental design makes it more difficult to conclusively reject any association with consolidation and retrieval phases of associative learning.Extinction and inhibitory learning are implicated in a number of other psychiatric and neurodevelopmental disorders. Is it notable that CNVs conferring risk to schizophrenia also confer risk for other neurodevelopmental disorders,The present results highlight the potential of using bioinformatics to integrate human genetic studies with experimental studies in model organisms in order to investigate the functional processes impacted by genetic variation. This approach overcomes some of the limitations of traditional pathway analyses, which are constrained by the prior annotation of genes and gene variants to functional pathways. The method used here, in contrast, allows all genes to be included in the analysis, regardless of their degree of prior functional annotation. The integration of human genetic data with experiments in animal models is also highly complementary to human post mortem gene expression studies, allowing additional hypothesis testing about the functional effects of risk-associated genetic variation.Overall, these findings are consistent with the hypothesis that altered associative learning contributes to the pathogenesis of schizophrenia. Furthermore, they suggest that schizophrenia-associated CNVs particularly impact on inhibitory forms of learning such as extinction. Deficits in extinction learning may contribute to the development of both the cognitive and psychotic symptoms seen in the condition. The present results also highlight a novel approach to investigate the functional processes impacted by complex genetic risk factors in neuropsychiatric disorders by using bioinformatics approaches to integrate functional studies in animals with human genetic data.


Numerous studies have documented the incidence and nature of injuries in professional rugby union, but few have identified specific risk factors for injury in this population using appropriate statistical methods. In particular, little is known about the role of previous short-term or longer-term match exposures in current injury risk in this setting.Our objective was to investigate the influence that match exposure has upon injury risk in rugby union.We conducted a seven-season (2006/7–2012/13) prospective cohort study of time-loss injuries in 1253 English premiership professional players. Players’ 12-month match exposure (number of matches a player was involved in for ≥20 min in the preceding 12 months) and 1-month match exposure  were assessed as risk factors for injury using a nested frailty model and magnitude-based inferences.The 12-month match exposure was associated with injury risk in a non-linear fashion; players who had been involved in fewer than ≈15 or more than ≈35 matches over the preceding 12-month period were more susceptible to injury. Monthly match exposure was linearly associated with injury risk , although this effect was substantially attenuated for players in the upper quartile for 12-month match exposures (>28 matches).A player’s accumulated (12-month) and recent (1-month) match exposure substantially influences their current injury risk. Careful attention should be paid to planning the workloads and monitoring the responses of players involved in: (1) a high (>≈35) number of matches in the previous year, (2) a low (<≈15) number of matches in the previous year, and (3) a low-moderate number of matches in previous year but who have played intensively in the recent past. These findings make a major contribution to evidence-based policy decisions regarding match workload limits in professional rugby union.The online version of this article (doi:10.1007/s40279-017-0721-3) contains supplementary material, which is available to authorized users. Injury incidence and the resulting absence from match play and training in elite rugby union are high in comparison with most team sports , and theThe introduction of professional full-time training, advancements in sports science, and law changes in rugby union have resulted in marked changes in players’ physical characteristics  and matcAlthough sports injury data often contain repeated events within individuals , few published studies have considered how these repeated measurements impact upon the statistical assumptions made in their analyses, leading to potentially spurious conclusions . As seveA seven-season prospective cohort design was used to record all match and training injuries sustained by professional rugby union players in the English premiership. Data collected from the 12 league teams in each of the seven seasons between 2006/07 and 2012/13 were included in the analysis, giving rise to a total of 15 teams because of promotions and relegations during this period. All consenting players who were members of the first team squad were eligible for inclusion. Data pertaining to 1253 professional rugby union players were included in the analysis . The study was approved by the research ethics committee of the academic host institution where the project was based for each season, and written informed consent was obtained from each participant. All data were anonymized, and all procedures were performed in accordance with the Declaration of Helsinki ) for rugby union matches has been reported to be trivial , externaThis study demonstrates that players who have been exposed to low (<15) or exceptionally high (>35) 12-month match exposures have a substantially higher current injury risk. Month match exposures are linearly and positively associated with injury risk, although this effect is attenuated for players in the upper quartile for 12-month match exposures (>28 matches). These data make a major contribution to the support decisions relating to player workload management at individual clubs as well as to decisions regarding fixture scheduling and policies relating to player match exposure limits for sport administrators.Supplementary material 1 (PDF 10 kb)Below is the link to the electronic supplementary material.


Constitutional ring chromosomes are rare orphan chromosomal disorders. Ring chromosome syndrome featuring growth retardation and mild to intermediate intellectual disability is likely caused by the dynamic behavior of ring chromosome through cell cycles. Chromosomal and regional specific phenotypes likely result from segmental losses and gains during the ring formation. Although recent applications of genomic copy number and sequencing analyses revealed various ring chromosome structures from an increasing number of case studies, there was no organized effort for compilating and curating cytogenomic and clinical finding for ring chromosomes.A web-based interactive ‘Human Ring Chromosome Registry’ using Microsoft Access based relational database was developed to present genetic and phenotypic findings of ring chromosome cases. Chinese ring chromosome cases reported in the literature was reviewed and compiled as a testing data set to validate this registry.A total of 113 cases of ring chromosomes were retrieved in all chromosomes except for chromosomes 16, 17 and 19. The most frequently seen ring chromosomes by a decreasing order of relative frequencies were ring 13 (14%), X (12%), 22 (10%), 15 (9%), 14 (7%), and 18 (7%). Genomic imbalances were detected in 18 out of 19 cases analyzed by microarray or sequencing. Variable clinical manifestations of developmental delay, dysmorphic facial features, intellectual disability, microcephaly, and hypotonia were noted in most autosomal rings. Chromosomal specific syndromic phenotypes included Wolf-Hirschhorn syndrome in a ring chromosome 4, cri-du-chat syndrome in a ring chromosome 5, epilepsy in ring chromosomes 14 and 20, Turner syndrome in ring chromosome X, and infertility in ring chromosomes 13, 21, 22 and Y. Effective growth hormone supplemental treatment for growth retardation in a ring chromosome 18 was noted.Based on findings from these Chinese ring chromosome cases, guidelines for cytogenomic diagnosis and criteria for case registration were proposed. Further research to define underlying mechanisms of ring chromosome formation and dynamic mosaicism, to delineate the genotype-phenotype correlations, and to develop chromosome therapy for ring chromosomes were discussed.The online version of this article (10.1186/s13039-018-0367-3) contains supplementary material, which is available to authorized users. Constitutional ring chromosomes are a rare type of intra-chromosome structural abnormality with an estimated occurrence of 1 in 50,000 newborns –4. ThrouPatients carrying an autosomal ring chromosome shared certain clinical manifestations including proportional growth retardation, developmental delay, mild to severe intellectual disability, microcephaly, and mild to intermediate dysmorphic facial features. These common manifestations were classified as ‘ring chromosome syndrome’ and were thought to be caused by ring chromosome instability from recognized cases of complete ring chromosome , 12. HowDespite an increase volume of case reports of ring chromosomes from prenatal to postnatal clinics and more detailed cytogenetic and genomic findings with the introduction of genomic technologies, it is still a challenge to interpret the compound effects and to predict the clinical consequences from the dynamic mosaicism and genomic imbalances of ring chromosomes. The rarity of ring chromosome cases demands organized efforts to compile and curate findings from ring chromosome cases for better diagnostic practice and disease classification. This report presented the design and implementation of an online interactive human ring chromosome registry and a comprehensive review of ring chromosome cases in the Chinese population. This initial effort could lead to future collaboration toward the construction of a human ring chromosome atlas as a clinical, research and educational resource for this rare type of chromosomal abnormality.http://web.gxmu.edu.cn/shengwu/HRC/home.asp”. To validate the modular relations and functions of this human ring chromosome registry, ring chromosome cases in the Chinese population was collected as the initial testing data set.A Microsoft Access based relational laboratory information management system had been developed and implemented for a clinical cytogenetics laboratory . Followihttp://www.sinomed.ac.cn/), VIP (http://qikan.cqvip.com/), CNKI  and WANFANG DATA (http://www.wanfangdata.com) as well as English journals archived in the PubMed (https://www.ncbi.nlm.nih.gov/pubmed/) for a period from 1979 to June 2017. For each case, the gender, age, cytogenetic and genomic results, clinical findings, and family history were retrieved, reviewed and registered into the ring chromosome registry. The relative frequencies of ring chromosomes were calculated by the case number of a specific chromosome divided by the total number of cases. The presence and penetrance of clinical features was shown in a heat map by the observed frequency among cases of a chromosome or all autosomes. Cases with supernumerary small marker chromosomes (sSMC) in the form of a small ring chromosome were excluded because these cases and their cytogenomic and clinical findings were summarized in an online sSMC database (http://ssmc-tl.com/sSMC.html).Case reports and original articles of constitutional ring chromosomes were searched from Chinese medical journals archived in the SinoMed , indicating no statistically significant gender bias. However, more female cases in ring chromosomes 13, 18 and 22 were noted. There is a need of more cases to evaluate the sex ratio for individual chromosome. Compound chromosomal abnormalities were noted in one physically normal adult male who was referred by a perinatal infant death and detected with a derivative chromosome 6 from a 6p/13q translocation and a ring chromosome 13 [A systematic search of Chinese ring chromosome cases found a total of 113 cases from 94 case reports and four original articles. The age, gender, study materials and methods, cytogenetic and genomic results, major clinical findings, family history, and references are summarized in the Additional file osome 13 , and in osome 13 .Regional specific assays (RSA) such as FISH, multiple ligation probe amplification (MLPA), and short-tandem repeats (STR) were applied to 24 cases. Genome-wide aCGH and SNP-chip were used on 17 cases and genomic sequencing was used on two cases. Three out of 13 cases by RSA alone were noted with distal deletions while 18 out of 19 cases by genomic analysis were noted with distal deletions ranging from 160 Kb (kilo base-pair) to 34 Mb (mega base-pair). The latter result suggested that most cases of ring chromosome were incomplete rings although reporting bias of selected cases should be taken into consideration. The relative frequencies, the genomic imbalances, age and gender distribution of reported Chinese ring chromosome cases are shown in Fig. Dynamic mosaicism was detected in 63 cases by karyotype analysis of 20 to 250 metaphase cells. For cases with autosomal ring chromosomes, ring chromosome variants including ring size changes, interlocked rings, an extra copy of ring, or a derivative chromosome from ring breakage were noted in 2%–17% of cells with an average of 11%; monosomy due to the loss of the ring chromosome was noted in 3%–31% of cells with an average of 13%, and normal karyotype was noted in 1%–84% of cells with an average of 32%. For cases with sex chromosome rings, ring chromosome variants were not seen but losses of ring chromosome X or Y were noted in 9%–93% of cells with an average of 62%. These results indicated that only a small portion of cells can tolerate autosomal ring instability but a relatively larger portion of cells can survive the loss of a sex chromosome ring. However, the lack of diagnostic guidelines specific for analyzing dynamic mosaicism has made it difficult for reliable case-by-case and chromosome-to-chromosome comparisons of ring chromosome instability. In a prenatal case with a ring chromosome 13, cytogenetic analysis showed similar dynamic mosaicism of ring chromosome 13, dicentric ring 13, and monosomy 13 in cultured amniocytes and cord blood leukocytes but a small marker chromosome 13 and monosomy 13 in placenta tissue; aCGH analysis revealed a 4.22 Mb deletion at 13q34 in the ring chromosome 13 and a 91.8 Mb deletion of 13q11-q34 in the marker chromosome. This fetoplacental chromosomal discrepancy suggested a tissue-specific karyotype evolution and the marker chromosome as a residual fragment from ring chromosome rescue . The finOf the 113 cases with a ring chromosome, 89 cases  were assessed in perinatal or pediatric clinics, 14 cases  were diagnosed prenatally, and 10 cases  were noted in infertility or reproductive clinics. Major clinical findings compiled from 82 postnatal cases with autosomal ring chromosomes are shown as a heat map in Fig. Chromosome-specific syndromic phenotypes resulting from segmental imbalances were noted. Features of Wolf-Hirschhorn syndrome (OMIM#194190) known to 4p16.3 deletions was reported in a case with a ring chromosome 4. Cri-du-chat syndrome OMIM#123450) related with 5p deletions was reported in one case with a ring chromosome 5 and another case with a ring chromosome 2. Wilms tumor caused by heterozygous mutations in the WT1 gene at 11p13 (OMIM#194070) was noted in one case with a ring chromosome 11. Retinal defect was noted in one case with a ring chromosome 13, inferring an increased risk for retinoblastoma (OMIM#180200) caused by deletions and mutations in the RB1 gene at 13q14.2. Epilepsy as a highly penetrant phenotype was noted in all seven cases with a ring chromosome 14 and two cases with a ring chromosome 20. Epilepsy and other symptoms in ring chromosome 14 are referred as ring chromosome 14 syndrome (OMIM#616606). Growth hormone deficiency was reported in one case with a ring chromosome 18; periodic growth hormone supplement treatment was effective . Growth 450 relatThe clinical indications for prenatal diagnosis of ring chromosome cases included ultrasound findings of intrauterine growth restriction, oligohydramnios, microcephaly, lissencephaly, anencephaly, ventricular septal defect, and increased nuchal fold as well as abnormal prenatal screening results for increased risk of Down syndrome. Termination of pregnancy had been an option for fetus with severe anomalies and defined ring chromosome abnormalities. Excluding cases with parental denial or no follow up tests, parental studies performed in 60 families with a proband carrying a ring chromosome documented normal karyotypes for both parents. This result indicated that almost all ring chromosomes are de novo in origin. The clinical presentations from prenatal and postnatal cases with a ring chromosome and their family history could be helpful for genetic counseling.With annual births of 17.86 million in China for 2016 and the occurrence of 1/50,000 for a constitutional ring chromosome, there are an approximately 350 newborns with this group of rare orphan chromosomal disorders each year in the Chinese population. A comprehensive review of medical literature found only 113 reported Chinese cases with a ring chromosome which probably represented about 1% of total cases for the past four decades in the Chinese population. Even though only a small percentage of ring chromosome cases were published, this case series was by far the largest in an ethnic group. The findings from these cases provided insights into developing practice guidelines and research collaboration for genetic diagnosis, clinical management, and disease treatment.RPGRIP1, the neural retina leucine zipper gene NRL, and the FOXG1 gene. It was speculated that the formation of the ring could induce the spreading of heterochromatinization and dysregulate the gene expression [FOXG1 gene by an acentric ring chromosome 14 was noted in a patient with epilepsy [Routine chromosome analysis on a peripheral blood specimen should be performed on 100 metaphases for percentages of cells with typical ring chromosome, ring variants, and ring loss. FISH tests should be performed on 300 directly prepared interphase cells using chromosome-specific subtelomeric, centromeric, and targeted locus-specific probes to access distal deletions/duplications, ring variants and ring loss. This analysis of 100 to 300 cells will allow an accurate detection of dynamic mosaicism in 2%–3% of cells with a 95% confidence level . If possGenomic analysis by aCGH, SNP chip, or genomic sequencing should be performed to characterize the distal or interstitial copy number imbalances in the ring chromosomes. The result from this DNA-based genomic analysis should be correlated with findings from cell-based chromosome and FISH tests.http://genome.ucsc.edu/).Evidence-based interpretation should be provided regarding clinical significance for detected dynamic mosaicism and genomic imbalances of ring chromosomes. The implications on physical and mental development as well as risks for cancer and reproduction should be addressed . EvidencFollow-up parental study should be considered to determine de novo or familial transmission of the ring chromosome. It was estimated that inherited ring chromosomes exist in approximately 1% of ring chromosome cases; familial cases usually presented relatively mild clinical manifestations but one third of the transmitted offspring were more severely affected .Integrated analyses by karyotype, FISH and SNP chip on a series of 14 cases of autosomal ring chromosomes detected the dynamic mosaicism of ring chromosome variants in 5%–16.3% of the 300 metaphase cells examined and distal deletions in the range of 364 Kb to 18 Mb in 12 cases . The ranpression . Dysreguepilepsy . Based oThere is an urgent need for consensus on defining level of ring chromosome instability by observed dynamic mosaicism. This report and a recent case series documented a range of 2%–17% for autosomal ring chromosome variants and ring loss . A studyCases of ring chromosome presents a spectrum of clinical features caused by ring chromosome instability and additional features compounded by dosage effect from genomic imbalances, position effect from structural rearrangement, or changes in epigenetic modification , 36. Of An ad hoc task force proposed guideline recommendations for clinical diagnosis and management of ring chromosome 14 syndrome . These gFor registering cases into the implemented human ring chromosome registry, a task force by clinical cytogeneticists and geneticists will be organized to review cytogenomic findings following the diagnostic guidelines and to curate clinical findings. Accumulation of more ring chromosome cases with defined genomic structures and dynamic mosaicism and detailed clinical manifestations will provide better genotype-phenotype correlation for predicting clinical consequences and planning treatments.It has been thought that human ring chromosomes are formed by a starting breakage-fusion event at the distal or telomeric ends of both arms  and thenIt was suggested that larger ring chromosomes showed significantly more instability than small rings . This coReprograming human fibroblasts containing ring chromosomes 13 and 17 to induced pluripotent stem cells (iPSC) discovered the correction of ring chromosome through a compensatory uniparental disomy (UPD) mechanism . This ceThe implemented human ring chromosome registry can play important roles in facilitating genetic diagnosis and developing translational research. With the formation of a task force by clinical cytogeneticists and geneticists, criteria and procedures for registering, compiling and curating cases into this human ring chromosome registry will be formulated; evidence-based diagnostic guidelines and management recommendations for ring chromosome patients will be proposed. Easy access to compiled and curated diagnostic and clinical data as well as properly preserved residual specimens are the key prerequisites for promoting collaborative research. Clinical cytogenetic laboratories should consider registering cases into the human ring chromosome registry and saving residual specimens. Standard operating procedures for proper biobanking of residual specimens should be developed and ethical and legal considerations for research applications should be resolved .In summary, an online human ring chromosome registry was designed and implemented to review cytogenomic results and clinical manifestations from a set of ring chromosome cases reported in the Chinese population. Relative frequencies, age and gender distributions, and various phenotypes of all ring chromosomes except for chromosomes 16, 17 and 19 were revealed. Cytogenomic heterogeneity was noted in variable genomic imbalances in ring chromosomes, different levels of dynamic mosaicism, and possibly tissue-related karyotype evolution. Human ring chromosomes showed heterogeneous clinical findings ranging from intrauterine growth restriction, postnatal developmental delay and multiple congenital anomalies, adult reproduction failure, and risk for cancer. A framework of organized efforts for translational and basic research collaboration to define underlying mechanisms of ring chromosome formation and dynamic mosaicism, to delineate the genotype-phenotype correlations, and to develop chromosome therapy for ring chromosomes is under consideration.Additional file 1:Table S1. Chinese ring chromosome cases reported in the literature (XLSX 69 kb)


Zika virus (ZIKV) infection in pregnant women has been established as a cause of microcephaly in newborns. Here we test the hypothesis that neurodevelopmental stages when the brain is undergoing rapid growth are particularly vulnerable to the effects of ZIKV infection. We injected ZIKV intracranially into wild type C57BL/6 mice at two different time points: early postnatal development, when the brain is growing at its maximal rate, and at weaning, when the brain has largely reached adult size. Both time points showed widespread immunoreactivity for ZIKV and cleaved caspase 3  throughout the brain. However, in early postnatal ZIKV injected mice, some brain areas and cell types display particularly large increases in apoptosis that we did not observe in older animals. Corticospinal pyramidal neurons, a cell type implicated in human microcephaly associated with ZIKV infection, are an example of one such cell type. Proliferating cells in the ventricular zone stem cell compartment are also depleted. These findings are consistent with the hypothesis that periods of rapid brain growth are especially susceptible to neurodevelopmental effects of ZIKV infection, and establish a valuable model to investigate mechanisms underlying neurodevelopmental effects of ZIKV infection and explore candidate therapeutics. Transmitted by mosquito, it is related to several other human pathogens, including West Nile virus, dengue, Japanese encephalitis virus and yellow fever. A ZIKV epidemic in Brazil in 2015 coincided with a dramatic increase in cases of microcephalic newborns. Several lines of evidence from human cases and animal studies indicate an association between ZIKV infection during pregnancy and abnormal fetal development123456in vitro and in vivo models have recently been developed. Common themes have emerged across models of ZIKV infection in cultured human neural progenitor cells and brain organoids, as well as in immunocompromised and immunocompetent mouse models: efficient infection and replication of ZIKV in neural progenitor cells (NPCs)891011128910111278910111491112141517To meet the urgent need for models to investigate the link between ZIKV infection and abnormal brain development, several Here we make use of a complementary animal model of ZIKV infection—intracranial injection of ZIKV in wild type C57BL/6 mice—to test the hypothesis that neurodevelopmental stages when the brain is undergoing rapid growth are particularly vulnerable to the effects of ZIKV infection. The peak of the brain growth spurt in rodents occurs during early postnatal life, with adult brain mass being largely attained by weaning at three weeks of age2021To test the effects of ZIKV infection at the peak of growth rate in the developing mouse brain, we performed intracranial injection of either saline or ZIKV (strain MR766) into postnatal day 7 (P7) wild type C57BL/6 mice. Infected mice stopped gaining weight from 2 days post-inoculation (2dpi) . By 4dpiAt 4dpi, we collected the brains of infected animals and controls for histological analysis. Immunostaining with anti-ZIKV antibody revealed evidence of widespread infection throughout the brain of ZIKV injected animals . Axl, a Given the evidence linking ZIKV infection to microcephaly, we measured the mass of brains harvested from ZIKV infected mice at P7, 4dpi. We found that after only four days post-injection, brain mass was significantly decreased in ZIKV injected mice as compared to controls , demonst+ cells, including the occipital cortex and hippocampus , a marker of apoptosis. We found evidence of widespread apoptosis, as indicated by anti-CC3 staining, throughout the brain, with some brain regions having particularly high density of CC3pocampus . Quantifl cortex . Coronall layers . This inA previous study using intracranial injection of ZIKV in newborn mice found hippocampal degeneration at 7dpi+ or Sox2+ cells that were co-labeled with CC3  stem cell compartmentted mice . This re+ cells are positive for ZIKV , a time when the mouse brain has largely attained adult size, has similar or different effects on apoptosis in the brain as compared to ZIKV infection at P7. We performed intracranial injection of either ZIKV or saline in P21 wild type C57BL/6 mice. ZIKV infected mice stopped gaining weight from 3dpi . By 4dpiAt 4dpi, we collected the brains of infected animals and controls for histological analysis. ZIKV immunoreactivity was broadly distributed throughout the cerebral cortex and hippocampus , althoug+ cells throughout the brain as compared to controls  are susceptible to ZIKV-induced apoptosis. This extends our knowledge of cell types that are vulnerable to the effects of ZIKV infection to include not only proliferating neural progenitor cells, but also post-mitotic neurons that are undergoing rapid increases in cell size. This P7 intracranial infection model recapitulates aspects of human neuropathology associated with ZIKV infection, including degeneration of corticospinal neurons, and provides relatively easy to score phenotypes to study neurodevelopmental mechanisms of ZIKV infection. Furthermore, the robust apoptosis phenotype present in these animals provides a useful model for testing candidate therapeutics and for investigating whether some genetic backgrounds may confer enhanced susceptibility or resilience to the neurodevelopmental effects of ZIKV infection.In summary, we have reported the neurodevelopmental effects of intracranial injection of ZIKV in wild type C57BL/6 mice, a strain commonly used for neurobiological applications. Consistent with our initial hypothesis, there is a more severe apoptotic response to ZIKV infection at P7, when the brain growth rate is maximal, as compared to P21, when the brain has largely attained adult size. While ZIKV infection at P7 causes altered proliferation in cortical stem cells similar to what has been reported in other models, we also find that neurons that are post-mitotic but still actively increasing in size  was obtained from World Reference Center for Emerging Viruses and Arboviruses at University of Texas Medical Branch. Virus was grown in Vero 76 cells (ATCC # CRL 1587) maintained in high glucose DMEM with 10% fetal bovine serum (FBS). The infectious virus titers were determined by plaque assays on Vero cells (ATCC # CCL 81) incubated for 4 days with 1% low melting point agarose overlay.7 pfu/ml) or with saline following hypodermic anesthesia (one week old mice) and isoflurane anestheisa (three week old mice). Animals were observed daily for clinical illness. Necropsy of both the ZIKV infected and control animals were carried out at 4 day post inoculation (dpi). Mice injected at one week were given hydrothermic anesthesia 4dpi, and mice injected at three weeks were anesthetized with isoflurane and bled via cardiac puncture, followed by perfusion with ice cold phosphate bufferd saline (PBS) and 4% paraformaldehyde (PFA) at 4dpi. The brains were collected and placed in ice-cold PBS followed by overnight fixation in ice cold 4% PFA in PBS.The timed pregnant C57Bl/6 females were purchased from Charles River Laboratories  and maintained at the Animal Resources Center, TSRI Florida. One week old and three week old mice were intracranially (i.c.) inoculated, using 0.3 ml 31G syringes (one week old mice), and 1 ml 27½ G (three week old mice), with 30 μl of ZIKV  by experimenter who was blind to the experimental condition. Three ROIs were measured in each animal.Brain sections were immunostained with anti-CTIP2 antibody (1:2000 dilution). Regions of interest (ROIs) were cropped from primary somatosensory cortex barrel field  using Photoshop (Adobe), and then the layer V cortex, as indicated by high levels of CTIP2 staining, was further cropped for density analysis. CTIP2+ cells were counted in a 200 μm wide radial column of the primary somatosensory cortex barrel field . For hippocampus, CC3+ cells were counted in a 200 m2 of CA1 region in hippocampus. VS-DESKTOP software (Olympus) was used to quantify CC3+ cells.Brain sections were immunostained with anti-cleaved caspase 3 (CC3) antibody (1:2000 dilution). For cerebral cortex, CC3post hoc tests were used to assess the body weight between saline and ZIKV injected mice over 4 days post injections. Two-way ANOVA and Tukey’s post hoc tests were used to assess the density of CC3 positive cells between saline and ZIKV infected mice in different brain regions. In all cases, normality was assessed using KS normality tests to ensure appropriate statistical tests were used. All statistics were performed using Graph Pad Prism 6, with significance set at p < 0.05.Planned comparisons between treatments  were performed for all assays using independent sample t-tests. Two-way ANOVA and Sidak’s How to cite this article: Huang, W.-C. et al. Zika virus infection during the period of maximal brain growth causes microcephaly and corticospinal neuron apoptosis in wild type mice. Sci. Rep.6, 34793; doi: 10.1038/srep34793 (2016).