In vivo systems incorporating human cells and tissues in a physiological setting will help bridge this gap. In this regard, humanized mice with engrafted human cells provide suitable tools to study human specific pathogens and cancer. With a transplanted human immune system, they also offer a dynamic setting for immune responses. Central to the preparation of new generation humanized mice is the availability of various strains of immunodeficient mice. Many new advances in this arena include derivation of mouse strains transgenic for human cytokines and HLA alleles, allowing improved human cell engraftment and immune responses. Transplantation of tissues such as human liver together with an autologous immune system paved the way for new studies not previously feasible. Human specific pathogens such as HIV, hepatitis viruses, and malaria parasites are being intensely studied in these systems and important data on pathogen life cycles, viral latency, and human specific immune responses are gathered. In the cancer field, patient derived xenograft models are facilitating testing of various chemo- and immunotherapies. Recent applications of these models expanded immensely to address host-parasite interactions involving more diverse agents and in studying viral-bacterial co-infections as well. Studies on novel gene, cellular and antibody therapies have also greatly expanded by the use of these mice.Many knowledge gaps exist in translating research results from conventional animal models such as mice to the human, especially in the clinical context. The current Research Topic incorporates a number of original research papers and review articles addressing a wide range of topics that include new model development, viruses, bacteria, parasites, cancer, and vaccine studies demonstrating the ever increasing versatility of humanized mice in biomedical research.Cheng et al. compared immune reconstitution and HIV-1 infection between NRG-huHSC and NRG-Hu Thy/HSC models. Interestingly, both models were found to support comparable levels of virus replication, immunopathology, and therapeutic responses to ART and immunotherapy approaches suggesting that hu-HSC mice can be effectively used in many HIV experimental settings with reduced cost and labor. Soper et al. reviewed the impact of type I IFN (IFN-I) in HIV-1 infection in vivo utilizing hu-HSC mouse models. They found that the effects of IFN-I in the in vivo context were much more complicated than previously predicted from in vitro studies, thus underscoring the advantage of using humanized mouse models in assessing the nuances of IFN-I effects for/against viral infections.Two humanized mouse models are widely used in HIV research, the simpler and less expensive hu-HSC mouse model and the BLT hu-mouse model requiring surgery to transplant human thymic tissue and hematopoietic stem cells (HSC). Schmitt and Akkina reviewed the current status of HIV latency detection assays and discussed the higher sensitivity achieved utilizing humanized mouse-based viral outgrowth assays (hmVOA) vs. in vitro VOAs.A major goal in the HIV/AIDS field is to achieve full viral eradication and a complete cure. However, this has been elusive due to the presence of minute levels of latently infected cells even in fully virus suppressed patients on long-term therapy. Ultra-sensitive assays are needed to verify when full HIV remission is achieved. Evering and Tsuji reviewed the current work on HIV-1 in the CNS using human immune system (HIS) mouse models with a focus on cells of myeloid lineage playing a major role. They predict that the new HIS mouse models in the current pipeline will further facilitate novel diagnostic, therapeutic, and viral eradication strategies in the CNS.In the context of HIV-1 viral persistence, the central nervous system (CNS) has come into the limelight as a unique, immunologically privileged compartment supporting infection and consequent immune-mediated damage. Carrillo et al. reviewed recent developments in CAR-T cell-based immunotherapies and their combination with antibody targeting of immune checkpoint inhibitors such as PD-1. Stem-cell based approaches using TCRs against HIV and cancer were discussed. Hyperimmune activation during HIV-1 infection appears to be driven by chronic IFN-I induction. Hu-mouse studies determined that blocking the IFN-I signaling by antibodies decreased immune activation and resulted in reversal of T cell exhaustion.Lentiviral gene transduction of human hematopoietic cells including HSC opened up many avenues of gene and cellular therapies. Humanized mice played an ever increasing role in modeling these new strategies and providing important pre-clinical data. Kim et al. described the use of humanized DRAG mice (HLA class II DR4 transgenic) for HIV-1 transmission via intravaginal route. Superior human cell engraftment in mucosal sites was noted. Viral spread from the point of entry were studied in detail with the results supporting the utility of this improved model to study viral pathogenesis, tissue distribution, viral persistence and establishment of latent viral reservoirs. Volk et al. described a multidimensional analysis approach integrating human T cell signatures in lymphatic tissues with the sex of humanized mice as a predictor of responses after dendritic cell-based immunization. This new modality of multidimensional analysis can be potentially used as a framework for assessing predictive signatures of immune responses.Schönrich and Raftery reviewed the impact of humanized mice in VHF vaccines and therapeutics research, also emphasizing their role as surrogate models for the discovery of newly emerging zoonotic agents.Viral hemorrhagic fevers (VHF) such as Ebola, Dengue, and Crimean-Congo hemorrhagic fever with high fatality rates constitute important public health concerns. While the natural hosts for these viruses in the wild are asymptomatic, humans are severely affected, incriminating a role for the human immune system in mediating severe pathology. Münz detailed the tumorigenesis by these viruses, interesting aspects of how KSHV infection is sustained longer during EBV coinfection in hu-mice, how the adaptive and innate immune responses play out and how this knowledge can be used to develop effective vaccines in the future. With regard to modeling anti-tumor responses in vivo in humanized mice, Zumwalde and Gumperz reviewed the use of humanized mice engrafted with human umbilical cord blood-derived HSC. They also discussed how T cells get suppressed during EBV tumorigenesis and how immunotherapy strategies can counteract this.Hu-mice provide excellent models to study tumorigenesis and immune responses. Among the virus-related human cancers, EBV and KSHV account for 10% of morbidity. Their epidemiology varies drastically in different geographic regions. The review by Hanazawa et al. described the generation of a functional human TAM population in their novel humanized IL-6 transgenic mouse strain, NOG-hIL-6 Tg. Development of novel cancer immune therapies targeting immunoregulatory/immunosuppressive myeloid cells is now possible using this model. The same research group led by Takahashi et al. reported the derivation of a new transgenic mouse strain, NOG-pRORγt-γc, in which the γc gene was expressed in a lymph-tissue inducer (LTi) lineage by the endogenous promoter of RORγt. Lymph node development was greatly improved, a major deficiency with previous HIS mouse models. Increased numbers of IL-21–producing CD4+ T cells were seen in LNs and there was enhanced antigen specific IgG response thus providing a vastly improved HIS mouse model.The tumor microenvironment contains unique immune cells, termed myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) that suppress host anti-tumor immunity and promote tumor angiogenesis and metastasis. Staphylococcus aureus is an important human pathogen responsible for many disease conditions including fatal pneumonia and septicemia. While conventional mice have been useful to study these conditions to an extent, it has become clear that some virulence factors/toxins display higher specificity to the human cells/factors leading to more severe disease. Parker's review highlights the value of humanized mice in dissecting the role of S. aureus virulence factors in a human surrogate setting and in vaccine testing.Two reports focused on bacterial studies. Orientia tsutsugamushi. Although standard mouse models provided a basic understanding, data is sparse on human immunopathogenesis and immune responses. Jiang et al. described the successful use of a humanized DRAGA (HLA-A2 and HLA-DR4-transgenic) mouse model capable of efficient human cellular and antibody responses. Footpad infection with O. tsutsugamushi resulted in disseminated lesions in various organs and invoked human immune responses including T cell activation, specific antibody and cytokine secretion mimicking human disease and responses. Vaccination with killed whole cell O. tsutsugamushi gave rise to both humoral and cellular responses thus providing a human relevant model for future vaccines and therapeutics testing.Over one million people worldwide are affected annually by Scrub typhus, a disease caused by an intracellular bacterium Minkah et al. reviewed the current status of malaria animal models and point to the need to develop humanized mouse models that can support both the hepatic and blood stages of infection to study pathogenesis and enable therapeutic testing. With these criteria as a background, the report by Foquet et al. described establishment of a FRGN huHep/hRBC humanized mouse model. This animal model enabled human malaria parasites to successfully undergo the liver stages and culminate with the blood stages of infection in vivo. Imaging techniques used to test the efficacy of an inhibitory monoclonal antibody demonstrated the utility of the model in evaluating interventions that target one or both phases of the parasite life cycle.Malaria continues to inflict high morbidity and mortality numbering in millions in many parts of the world. Transmitted by mosquitoes, the parasite has a complex life cycle with many stages of development. In summary, this Research Topic highlights the recent advancements in biomedical research using different models of humanized mice. As can be seen, these models have been substantially improved over the past decade increasing their breadth in utility not only in studying the infection process of the pathogens but also allowed evaluation of host immune responses thus laying a foundation to build upon for future vaccine and therapeutic testing.We thank all the authors of the manuscripts for their contributions to the humanized mouse field and reviewers for their constructive comments and input.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.


A 70-year-old woman underwent a right-sided mastectomy and axillary lymph node excision because of invasive adenocarcinoma in 2007. She received additional radiotherapy on the mastectomy site and the right axilla. Extensive lymphedema of the right arm developed and formed ecchymosis, which persisted despite a microsurgical lymphovenous derivation in 2009. On clinical examination in 2017, a painful nodular and purpuric transformation of the skin was noted. Because of increasing pain in the upper arm, a biopsy of the skin was performed, revealing a cutaneous epitheloid angiosarcoma. The patient was referred for magnetic resonance imaging (MRI) of the right upper arm to evaluate local extent.Axial Tau Inversion Recovery (TIR) and T1-weighted images of the right upper arm Figure  and 1b sThe patient was treated with an amputation of the affected arm. Follow-up CT examination revealed progressive disease with new subcutaneous and intramuscular metastases in the right hemithorax, and the additional follow up MRI revealed diffuse skeletal metastasis.Stewart Treves syndrome or angiosarcoma secondary to chronic lymphatic obstruction is a very rare and lethal disease. It is most commonly seen in the upper arm of female breast cancer patients who underwent mastectomy and axillary lymph node excision.  Besides Typically, these lesions have intermediate signal intensity on T1-weighted images and high signal on images obtained with long repetition times on MRI. They show aggressive infiltration of the surrounding tissues. They may also show serpentine vascular channels, most often in the lesion periphery, a feature rarely seen in other soft tissue sarcomas. These lesions have no fat overgrowth.Imaging features on CT are nonspecific, showing a soft tissue mass with attenuation similar to muscle that is typically markedly enhancing.US descriptions are rare, showing complex hypo- or hyperechoic lesions with cystic components (resulting from haemorrhage). Doppler US often shows arteriovenous shunting.The treatment of choice is surgical resection of the affected limb, though local recurrent and distant metastasis are frequent due to their aggressive nature, and despite radical treatment, prognosis is poor.


The optical molecular imaging of inflammation is an emerging strategy for interventional medicine and diagnostics. The host's inflammatory response and adaptation to acute and chronic diseases provides unique signatures that have the potential to guide interventions. Thus, there are emerging a suite of molecular imaging and sensing approaches for a variety of targets in this area. This review will focus on two key cellular orchestrators that dominate this area, neutrophils and macrophages, with recent developments in molecular probes and approaches discussed. Recent developments in optical molecular imaging are enabling the identification and evaluation of inflammatory cells such as macrophages and neutrophils in a variety of imaging scenarios. This is important as these leukocytes are an important part of host defense and immune homeostasis. Their pivotal roles as professional phagocytes in acute and chronic inflammatory disease may allow their use as versatile interventional biomarkers and as such fluorescent imaging of these cells has the potential to advance disease treatment and management, by producing sub-cellular information in real-time.Intrinsic tissue autofluorescence adds further value when the structures of extracellular matrix or metabolic activities are of interest. The predominant modality of fluorescence intensity measurements, coupled with recent technological developments such as label-free non-linear optical images and Raman analyses , 2, havein vivo detection of bacteria  independently of neutrophils in altered activation states. The accumulation of neutrophils on the ocular surface in dry-eye disease (DED) is thought to result in pathological NET formation . With thAnother layer of insight may be accessible via measurement of neutrophil activity, as studies of patients with inflammatory bowel disease have demonstrated. Patient studies found changes in fecal lactoferrin, calprotectin and the protease HNE were significantly correlated with endoscopy and could be used to distinguish between mild disease, mucosal healing and clinical remission, and even predict flare onset . HNE is Neutrophils are the first leukocytes to be recruited to an inflammatory site. Their capacities to respond en masse and rapidly underscore their potential as biomarkers capable of producing large readouts in short time-frames—properties that are particularly desirable for surgical guidance. The relatively short neutrophil half-life acts to limit any off-target consequences of labeling to days or hours, whilst the persistence of the neutrophilic influx in inflamed tissues makes neutrophil readouts useful over the entire disease monitoring period, irrespective of its duration. Overall, neutrophil imaging creates an ideal paradigm for patient-specific disease monitoring and intervention. Recent advances in the fluorescent and Raman spectroscopic modes of optical imaging are, for the first time, enabling neutrophil burden and activity to be described non-invasively and dynamically with tissue-level resolution .Human neutrophil elastase (HNE) is a serprocidin stored at millimolar concentrations in the azurophil granules of neutrophils . AlthougCraven et al. recently reported a probe that revealed neutrophil activation. This was designed to detect the serine proteases (serprocidins) using a pan-serprocidin substrate. A tribranched probe was developed which maintains an optically super-silent ground state with a methyl red and fluorescein FRET pair on each of its three branches  40). Th. Th40). in vitro, structural differences between the murine and human NE active sites and functional differences between murine and human neutrophils mean the clinical utility of NE680 has yet to be demonstrated.The neutrophil elastase probe NE680 is a near-infrared multi-branched probe which is sensitive to cleavage by murine NE and HNE, amongst others  41). It. It41). in vivo studies, exciting developments in optical probe design provide discriminatory power between related proteases. Despite their concomitant release from degranulating neutrophils, the various serprocidins perform distinct molecular functions  was developed . Using san lungs . Craven an lungs . The abid = 2 nM) generates a sensitive readout of leukocyte distribution  or NIR  labels has also been demonstrated. However FPR1 is not cell-type specific and these probes bound macrophages . Th. Th43). ribution . The abirophages  and neutrophages , 68. cFLin vivo. Although neutrophil depletion was capable of removing signal, it also removes important cross-talk from the LPS inflammatory response.Pellico et al. created neutrophil-specific radiotracers with dual PET and MRI signals by coating gallium-doped nanoparticles with cFLFLF for FPR1 labeling . CoatingA truly neutrophil specific target has proved elusive. Instead neutrophil specificity has arisen from combinatorial strategies exploiting the unique confirmations of certain targets within neutrophils. For example, HNE reaches such high concentrations in neutrophils that most systems will fail to detect the minority of HNE positive monocytes which bares far lower quantities of the enzyme. Neutrophil specific functions can also be exploited by careful probe design. Phagolysosome alkalinisation does not occur in monocytes and enhance the fluorescence signal of some fluorophores. The super-silent FRET probe combines these structural and functional cellular characteristics to achieve neutrophil specificity. Label-free methods of cell identification have also sought neutrophils using targets found in other cell types but uniquely arranged in neutrophils.Neutrophil imaging in the context of inflammatory research has aimed at understanding the relevance of cellular components to cell function. Questions of molecular colocalization and characterization are well-served by fluorescent labels for elucidating the spatiotemporal relationships of cells and subcellular structures without altering the processes for investigation. As translational research influences surgical guidance questions of long-term cytotoxicity and ease-of-adoption are more important. Label free imaging, which may seem imprecise for research purposes, is often cheaper, safer and does not require the considerable translation effort of alternative techniques. Molecules such as NADH, elastin, and hemoglobin have been claimed to provide intracellular and extracellular autofluorescence, but signals from cells will always be an amalgamation of signatures of multiple and complex analytes. There is still much characterization to generate clinically meaningful autofluorescence imaging and techniques such as fluorescence lifetime imaging may become an important source of differentiation. Reflectance microscopy, which distinguishes between matter of different refractive indices, is now also capable of cellular resolution. For high-resolution imaging, individual molecules can be detected and quantified via Raman spectroscopy. Label free imaging is en route to becoming as informative as fluorescence image-guided surgery can be, a limit it may exceed in a multi-modal platform.By measuring the minute proportion of photons which interact with molecules inelastically, Raman spectroscopy can, non-destructively, describe the chemical composition of unlabeled matter. The first look at a leukocyte using Raman spectroscopy was when Puppels et al. compared eosinophils and Chinese hamster lung cells . The groIn vivo TPEM was performed laparoscopically with a “stick” lens which avoids GRIN lens associated spherical aberrations revealing ovarian cancer through a laparoscopic procedure, centimeters into the body  carries additional fiber optics challenges such as fiber non-linearity distorting the two-photon excitation light and the low quantum yields of intrinsic fluorophores . These athe body . Howeverchnology .E. coli (Using two-photon excited fluorescence (TPEF), Zeng et al. characterized blood cells by their endogenous fluorescence lifetime signals . TPEF flE. coli .Macrophages are readily found at the site of inflammation or infection, forming the first defense against pathogens. Although they play a key role in the initiation of defensive inflammation, recent literature suggests they are also responsible for the resolution of inflammation and repair processes . SeveralMacrophages have been broadly classified into two different phenotypes—M1 and M2, with further subsets M2a, M2b, M2c identified, and they display plasticity by moving between these phenotypes . General+ ovarian cancer (NCT03180307). For inflammatory cells, the β isoform (FR-β) is expressed on activated macrophages and has a high affinity for its ligand folic acid (Kd = 10−10 M) (Folate receptor (FR) targeting is a promising method for visualization of cells that overexpress the folate receptor. Recently, a demonstration of the capabilities of folate targeting during fluorescence guided surgery of cancer was described by Mahalingam . OTL-38 10−10 M) . Its ide10−10 M) . Studies10−10 M) . A numbe10−10 M)  and macr10−10 M) , 86, 96.+ macrophages was found to uptake folate-FITC, and competition experiments showed that this uptake could be inhibited with the non-conjugated folic acid—showing FR specificity. Finally, a technetium-radiolabelled folate (FolateScan: EC20) was utilized to perform SPECT imaging of human inflamed joints in a Phase II trial (NCT00588393).Xia et al. investigated the presence of the folate receptor on activated macrophages from arthritic patients . Fluoresex vivo lung macrophages with an M2 phenotype  bound the green fluorescent probe, folate-Oregon Green. Further, SPECT/CT imaging of 99mTc-EC20 showed uptake in ovalbumin induced asthmatic lungs, while there was no uptake in the lungs of healthy mice ..ex vivo + immune cells, specifically in mouse models of colitis and atherosclerosis (−/− mice fed a high fat diet) were injected via the tail vein with 2 mg/kg of NT-liposome-DiD or Fol-liposome-DiD, respectively. Fluorescence imaging showed selective uptake of Fol-liposomes in the atherosclerotic mice.Recent work by Poh et al. showed that a folate liposome could act as a specific method of targeting FRclerosis . This stTo further our understanding of the clinical applicability of folate targeted imaging agents, the field should aim to evaluate their performance in human inflammatory models. It is however also worth mentioning an observation from this field; the use of folate depleted media is necessary for any folate targeting cell studies due to high levels of folic acid in routine media formulations.+ macrophages, from antibodies to a more recent report of a target-binding peptide. The Manocept concept of nanomolecule imaging agents targeting the lectin domain of CD206 has resulted in the FDA approval of γ-Tilmanocept, a 99mTc-labeled radiolabelled tracer for the imaging of sentinel lymph nodes , is a well-reported marker of M2 differentiated macrophages . Its funph nodes . A similph nodes . Therefo99mTc-labeled nanobody against MMR with an affinity of 2 nM determined by SPR and validated its use for SPECT/micro-CT imaging of tumor-associated macrophages in preclinical models  labeled nanobody (anti-MMR 3.49) showed that it had a high affinity for human MMR (KD = 1.8 nM), while in vivo biodistribution studies showed fast renal clearance and specific retention in the tumor and MMR-expressing tissue. In a small animal PET imaging study, the nanobody was specifically recognized by MMR in 3LL-R tumor bearing mice, when compared with the uptake in MMR-deficient mice  are a family of proteases that play a key role in ECM structure, function and remodeling. In the tumor microenvironment macrophages have been found to be potent producers of MMP 2 and 9 . It is iWhile there are many non-selective imaging agents for MMPs, there is a lack of selective MMP tracers and this may hamper the understanding of the role of a specific MMP in disease. For example MMP-12, macrophage elastase, is responsible for the breakdown of elastin and it is associated with a number of inflammatory pathologies such as aortic aneurysm, emphysema, and rheumatoid arthritis . It has in vivo  which had previously been shown to inhibit atherosclerotic plaque growth in vivo . This stin vivo . The selrophages . The prorophages . Althougin vivo and ex vivo models of colon cancer they showed that infiltrating TAMs initiate tissue remodeling at the tumor margins by secreting cathepsins. ProSense signal at the tumor margin was shown to be due to cathepsin B+ macrophage infiltration in a rat colon model (TAMs remodel the extracellular matrix (ECM) through MMPs and cathepsins, specifically during tumor invasion . Onda eton model . HoweverA number of alternative cathepsin activatable probes are under development , 113. WiThe nascent field of optical molecular imaging of inflammatory cells appears to be a vibrantly innovative arena full of early-stage biomarkers that offer promise of patient benefits. Targeting neutrophils and macrophages may deliver previously inaccessible measures of disease activity across common and life-threatening diseases. Whilst, in this review, the current stage of neutrophil and macrophage imaging has been discussed, clearly other inflammatory cells such as T cells play major role in interventional medicine. With many non-redundant targets and readouts available, probe multiplexing seems a particular advantage of this field with the potential to matching monitoring and treatment with immunological precision. As well as being informative in its own right, parallel label-free imaging may become a rapid means of interpreting labeled techniques across multiple contexts and machine learning data-reduction stands to push the power of OMI even further. An exciting therapeutic potential lies in the how imaging agents can impact the drug discovery process—with immune cells. These new technologies stand to enrich assay outputs, accelerate drug development decisions and clinical outputs, and enable better direct drug response metrics in trials.in vitro investigations very few have begun the translational journey. This inertia is perhaps not surprising as the technical demands of translating novel imaging methods can become overwhelming, especially within an academic environment. Probes must be innovative enough to meet clinical needs, yet synthetically feasible and complementary to imaging systems. This is a fiercely interdisciplinary pursuit from start to finish and nearly impossible without optical imaging device standards, as all technologies must meet safety and efficacy standards. Without standardization, investigators risk underestimating translation-ready reagents with “sub-standard” apparatus, or unnecessarily pursue doomed reagents with poor selectivity and specificity (Future studies will demonstrate the translatability of imaging agents into clinically useful optical probes. Despite the plethora of novel reagents advancing with cificity .GB and TC wrote the manuscript. KD and MB assisted with proofreading and preparation for publication.MB and KD are shareholders of Edinburgh Molecular Imaging. The remaining 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.


In mammalian olfaction, inhalation drives the temporal patterning of neural activity that underlies early olfactory processing. It remains poorly understood how the neural circuits that process incoming olfactory information are engaged in the context of inhalation-linked dynamics. Here, we used artificial inhalation and two-photon calcium imaging to compare the dynamics of activity evoked by odorant inhalation across major cell types of the mouse olfactory bulb (OB). We expressed GCaMP6f or jRGECO1a in mitral and tufted cell (MTC) subpopulations, olfactory sensory neurons (OSNs), and two major juxtaglomerular interneuron classes and imaged responses to a single inhalation of odorant. Activity in all cell types was strongly linked to inhalation, and all cell types showed some variance in the latency, rise times, and durations of their inhalation-linked response. Juxtaglomerular interneuron dynamics closely matched that of sensory inputs, while MTCs showed the highest diversity in responses, with a range of latencies and durations that could not be accounted for by heterogeneity in sensory input dynamics. Diversity was apparent even among “sister” tufted cells innervating the same glomerulus. Surprisingly, inhalation-linked responses of MTCs were highly overlapping and could not be distinguished on the basis of their inhalation-linked dynamics, with the exception of a subpopulation of superficial tufted cells expressing cholecystokinin (CCK). Our results are consistent with a model in which diversity in inhalation-linked patterning of OB output arises first at the level of sensory input and is enhanced by feedforward inhibition from juxtaglomerular interneurons which differentially impact different subpopulations of OB output neurons. Inhalation drives the temporal patterning of neural activity that underlies olfactory processing and rapid odor perception, yet the dynamics of the neural circuit elements mediating this processing are poorly understood. By comparing inhalation-linked dynamics of major olfactory bulb (OB) subpopulations, we find that diversity in the timing of neural activation arises at the level of sensory input, which is then mirrored by inhibitory interneurons in the glomerular layer. Temporal diversity is higher among OB output neurons, with different subpopulations showing distinct but nonetheless highly overlapping ranges of inhalation-linked dynamics. These results implicate feedforward inhibition by glomerular-layer interneurons in diversifying temporal responses among output neurons, which may be important for generating and shaping timing-based odor representations during natural odor sampling.Temporal patterning of neural activity is a fundamental aspect of information coding and processing by neural circuits. In the mammalian olfactory system, the primary driver of temporally patterned activity is inhalation of air through the nasal cavity. Inhalation delivers transient pulses of odorant to the olfactory epithelium, and so determines the initial temporal structure of olfactory sensory input to the brain and drives the temporal patterning of activity at subsequent processing stages . BehavioNeural circuits in the olfactory bulb (OB) mediate the first steps in processing olfactory inputs: here, olfactory sensory neurons (OSNs) drive excitation of mitral and tufted cells (MTCs), the principal output neurons of the OB, as well as activate multiple inhibitory circuits within and between OB glomeruli . This juin vivo. We used cell type-specific imaging with GCaMP-based reporters to record from different subpopulations of MTCs as well as presumptive PG cells, SA cells, and OSN inputs. We used an artificial inhalation paradigm to examine responses to a single inhalation of odorant with high fidelity and to precisely compare inhalation-linked response patterns across experiments and between cell types. We found distinct differences in the inhalation-linked temporal patterns of activity among different cell types, with PG and SA cell populations showing faster responses to inhalation than either mitral or tufted cells, and a range of excitatory MTC response dynamics that could not be accounted for by diversity in the dynamics of OSN inputs. At the same time, we observed a great deal of overlap in the inhalation-linked temporal patterns of activity among superficial tufted and mitral cells. Overall, these results support circuit models in which juxtaglomerular interneurons mediate rapid feedforward inhibition that contributes to diverse inhalation-linked temporal patterning in both MTCs.Here, we sought to better characterize the temporal dynamics of inhalation-driven activity among major circuit elements in the OB to refine models of OB circuit function during naturalistic odorant sampling Genetically-engineered mice expressing Cre recombinase (Cre) targeted to specific neuronal populations were used for experiments. Mice were either crossed to the Ai95 GCaMP6f reporter line (The Jackson Laboratory (JAX), stock #024105) or injected with a viral vector. The mouse strains using included GAD2-IRES-Cre mice , TH-Cre , DAT-IRES-Cre mice , OMP-Cre , PCdh21-Cre . Where applicable, mice used for virus injection were homozygous for the allele driving Cre expression. Using a stereotaxic head holder and drill, a small craniotomy (0.5–1 mm) was made on the dorsal surface of the OB. A glass pipette was lowered to a depth 50–150 μm to target PG or SA interneurons or 200–400 μm to target MTC populations. Mice were single housed following the surgery and imaged 14–28 d after injection.Cre-dependent expression of GCaMP6f  or jRGECO1a (AAV2/9 or AAV2/5 hSyn.Flex.NES.jRGECO1a.WPRE.SV40) was achieved with the injection of viral vectors. All viruses were obtained from the University of Pennsylvania Viral Vector Core. Injections were made into the dorsal OB under isoflurane anesthesia (0.5–2% in O2) was delivered directly to the tracheotomy tube, bypassing the nose. By controlling negative pressure through the sniff tube, each inhalation lasted exactly 150 ms and had a flow rate of 150 ml/min (300 ml/min for two nostrils). The onset of each inhalation was set to occur every 4 s (0.25 Hz), which enabled individual inhalation evoked calcium transients to be averaged. Next, a custom head bar was implanted, a craniotomy was made, and a coverslip was implanted using 2.5% low-melting-point agarose over the dorsal OB for imaging  was used during the implantation of a double tracheotomy , after w imaging . ThroughImaging data were collected using either a Moveable Optical Microscope (Sutter Instruments) coupled to a Mai Tai HP pulsed Ti:Sapphire laser (Newport Corp.) and controlled by Scanimage 5.1 , or a Neo)/Fo), with Fo being the mean fluorescence before the inhalation, averaged for each inhalation. However, for population level analyses, signals were averaged across three trials of 20-s odorant presentations  were generated by choosing 15 frames before and after odorant inhalation. Imaging trials consisted of three periods of odorant presentation, each lasting 20 s , with a 32-s interval between presentations. Responses to inhalation of clean air were taken from the last 10 inhalations from the interstimulus period to ensure that all odorant had cleared from the nose following the stimulus presentation. Note, small transients that immediately followed odor stimulation . Peak response amplitudes and time to peak values were calculated from ITAs that were filtered using a Gaussian-weighted moving average filter with a window length of 270 ms. From this filtered trace, response duration was calculated as the time from 50% of peak response on the rising slope of the signal to 50% of peak on the decaying slope of the signal. Time to peak was calculated from the maximum value of the filtered trace. For pseudocolor plots of ITA responses C, the meU test was used for unpaired comparisons across two groups , mitral cell (MC), OSN, PG cell, and SA cell populations) rejected the null hypothesis for one-sample Kolmogorov–Smirnov test for normality, therefore non parametric statistical tests were performed as stated throughout the methods. A Wilcoxon signed-rank test tested was used for paired comparisons across two groups  were made with a Vibratome and mounted onto a glass coverslip before imaging with a FluoView FV1000 Olympus confocal microscope at 10×, 20×, and 40× magnification.To compare the dynamics of inhalation-driven activity across different OB neuron populations, we used an artificial inhalation paradigm that allowed for precise comparison of temporal response dynamics across experiments, as described previously ; Fig. 1A+) neurons , however, their response dynamics were highly overlapping  imaged within the same preparation during the same odorant presentation B. When crlapping C,D. MCs rlapping E. Anothen = 3 fields of view from two mice; n = 3 paired imaging planes from two mice). Even with this within-preparation comparison, sTC and MC response dynamics were still highly overlapping  indeed showed onset latencies that were, as a population, significantly shorter than those of MCs (p = 3.7e–5) or the general sTC population (p = 0.0333) defined by Tbx21, PCdh21, or Thy1 expression . CCK+ sTCs showed the largest difference in their response durations, which were uniformly short  and significantly shorter than the general sTC population . Thus, CCK+ sTCs appear to constitute a distinct subpopulation of sTCs with more rapid inhalation-triggered response patterns.Finally, we measured inhalation-linked responses in sTCs defined by their expression of the peptide transmitter CCK, which likely constitute a subset of Tbx21+, Thy1+, or PCdh21+ sTCs  and havepulation E. LikewiWe also examined inhalation-linked suppression in MTCs. Previous reports indicate that about a third of MTCs show odorant-evoked suppression of ongoing activity . We asseOverall, these data suggest that the chief difference in the inhalation-triggered dynamics of MCs versus sTCs is that MCs show a greater range of excitatory response latencies and durations than do sTCs. At the same time, they also suggest that sTC and MC responses do not unambiguously map to any single parameter of the inhalation-linked response, including response latency, response duration, or even response polarity. This finding does not contradict recent work which found that inhalation of clean air evokes distinct temporal dynamics across MC and TC populations , 2014, aOne determinant of diverse MT cell inhalation-linked response dynamics could be diversity in the temporal patterns of sensory input to the OB . To assen = 88 glomerulus-odor pairs, five mice; + sTCs  and significantly faster than that of MCs and other sTCs , while SA cell response durations were significantly longer than all three of these populations . Interestingly, SA and sTC response durations were statistically indistinguishable . The similar inhalation-linked dynamics among these groups, which encompasses axon terminals, small-sized PG somata and larger-sized sTC somata suggests that variation in calcium dynamics, as a function of cellular compartment or soma size is not a major determinant of the response dynamics seen after odorant inhalation. Overall, these results suggest that inhalation-linked PG and SA responses largely follow those of OSN inputs, while a subset of SA cells exhibit longer-lasting responses. Their short onset latencies are consistent with both cell types mediating rapid feedforward inhibition of MCs and sTCs during inhalation.SA cell responses differed from those of PG cells mainly in their response durations  can show distinct temporal response patterns , while sInitially, we directly compared PG and sTC response dynamics during the same odor stimulus and within the same glomerulus using two-color imaging. We used Thy1-GCaMP6f: GAD2-Cre crosses, expressing the red shifted calcium indicator jRGECO1a  in PG cen = 14 glomeruli from six mice) compared with 19 ± 0.01 ms (n = 16 glomeruli from four mice) for PG cells  were not different in mean onset latencies, and the mode of their latency distribution was identical for the two cell types. Some earlier studies have reported clear differences in the latencies of mitral versus tufted cell responses to inhalation of air, although these timing differences disappear during odorant stimulation , 2014; tRespiratory patterning of ongoing activity in the absence of odorant stimulation is well-documented and has been hypothesized, among other functions, to serve as a reference for a timing-based code for odor identity . Here, wSurprisingly, a small fraction of clean air-driven responses were suppressive, with such suppression observed in sTCs, MCs, and even some OSN inputs. To our knowledge, this is the first report of inhalation alone driving suppression of activity in these cell types. One explanation for these results could lie in the low frequency (0.25 Hz) of artificial inhalation used in our experiments: if some OSN populations are sensitive to odor components arising from within the animal’s own nasal cavity, e.g., from metabolic processes, these components could drive basal activity of OSNs and in sTCs of their target glomeruli, which would be transiently removed by each inhalation of clean air. This effect could be even more pronounced in the intact, awake mouse where OSNs are exposed to exhaled air containing metabolic odorants .What can we infer from these comparisons about the primary synaptic interactions shaping inhalation-linked patterning of OB output? First, the data suggest that PG and SA cell excitation largely follows OSN input dynamics, consistent with evidence from slice studies that these cells are highly sensitive to OSN stimulation mediated either by monosynaptic or disynaptic excitation . We saw Overall, these results establish a basic framework for how glomerular circuits are engaged to shape inhalation-linked patterning of OB output. Our findings support the hypothesis that feedforward inhibitory circuits can add to the initial diversity of temporal patterns of input relayed by OSNs. additional experiments are necessary to integrate other OB cell types into this framework, e.g., granule cells, deep SA cells, external plexiform layer interneurons and centrifugal inputs from the olfactory cortex may also contribute to shaping respiratory patterning of OB output. Understanding the response dynamics of each of these cell types with respect to a single inhalation of odorant should allow for a dynamic model of OB network function across the fundamental unit of information sampling in the olfactory system. Such a model may be used to yield insights into olfactory processing across the full range of sampling frequencies used in the behaving animal.


Mycobacterium tuberculosis (Mtb) to adopt a slowly growing or nongrowing state within the host plays a critical role for the bacilli to persist in the face of a prolonged multidrug therapy, establish latency and sustain chronic infection. In our previous study, we revealed that genome maintenance via MazG-mediated elimination of oxidized dCTP contributes to the antibiotic tolerance of nongrowing Mtb. Here, we provide evidence that housecleaning of pyrimidine nucleotide pool via MazG coordinates metabolic adaptation of Mtb to nongrowing state. We found that the ΔmazG mutant fails to maintain a nongrowing and metabolic quiescence state under dormancy models in vitro. To investigate bacterial metabolic changes during infection, we employed RNA-seq to compare the global transcriptional response of wild-type Mtb and the ΔmazG mutant after infection of macrophages. Pathway enrichment analyses of the differentially regulated genes indicate that the deletion of mazG in Mtb not only results in DNA instability, but also perturbs pyrimidine metabolism, iron and carbon source uptake, catabolism of propionate and TCA cycle. Moreover, these transcriptional signatures reflect anticipatory metabolism and regulatory activities observed during cell cycle re-entry in the ΔmazG mutant. Taken together, these results provide evidence that pyrimidine metabolism is a metabolic checkpoint during mycobacterial adaptation to nongrowing state.The ability of  Mycobacterium tuberculosis  to adopt a nongrowing state within the host plays a critical role for the bacilli to persist in the face of a prolonged multidrug therapy, establish latency and sustain chronic infection. Therefore, understanding the molecular events underlying growth control and metabolic adaptation of nongrowing Mtb is believed to be particularly important for the development of new therapeutic strategies [Mtb during chronic infection of mouse and contributed to antibiotic tolerance of stationary-phase culture and intracellular Mtb [Mtb genome maintenance under stress environments. Given that all of these phenotypes are akin to a nongrowing state, we speculate that mazG may implicate in mycobacterial adaptation to growth-limiting environments. In this preliminary data report, we provide evidence that housecleaning of pyrimidine nucleotide pool coordinates metabolic adaptation of nongrowing Mtb.Despite the availability of chemotherapy, tuberculosis remains a leading cause of death due to a single bacterium infection. Increasing evidence shows that the ability of rategies ,2. In ouular Mtb ,4. ThesemazG is required for mycobacterial adaptation to nongrowing state, we measured the survival of wild-type Mtb and the ΔmazG mutant under hypoxic and nutrient-starvation conditions, i.e. the two in vitro models of mycobacterial dormancy. Under both conditions, while the wild-type Mtb and the complemented ΔmazG mutant maintained slightly decreased level , which was when the ΔmazG mutant begins to show reduced survival compared with that of wild type [mazG mutant compared with wild-type Mtb (Dataset S1). For a global view of key regulational changes, differentially regulated genes were grouped into functional categories according to TubercuList server classification (p = .0005). This transcriptional signature is an indication of DNA instability in the ΔmazG mutant, given that the induction of transposase was shown to be a common response to DNA damage in Mtb and Escherichia coli [cas1/2, mrr, ung, ligA and mutY) also reflects increased DNA damage in the ΔmazG mutant (Dataset S1). Significantly upregulated category was also observed for detoxification and adaptation processes, such as mazEF, vapC and ephE (involves in detoxification of oxidatively damaged lipids). Together, these transcriptional signatures corroborate the genome safeguarding and antioxidative roles of MazG [A recent study demonstrated that the stress environment of macrophages could induce nongrowing or slow-growing forms of Mtb . To inveild type . Totallyhia coli ,8. Moreo of MazG ,4.p = .021), intermediary metabolism and respiration (p = .042) and lipid metabolism (p = .003) , dcd (dCTP deaminase) and pyrR (repressor of pyrimidine synthesis) in the ΔmazG mutant [Among the differentially regulated genes, functional categories relative to metabolism were also significantly enriched, including cell wall and cell processes  and synthesis of methyl-branched lipids , were upregulated in the ΔmazG mutant (uspB), glycerol-3-phosphate (ugpA) and peptides (drrBC and dppB), as well as catabolism of disaccharide  were significantly upregulated in the ΔmazG mutant , which is implicated in the resuscitation of mycobacteria from dormancy via a mechanism of cell wall remodeling [malQ reflects an increased usage of maltose derived from trehalose , was also upregulated (1.7-fold) in the ΔmazG mutant (Dataset S1). Expression of whiB5, which encodes a transcriptional regulator involved in the resume of growth after reactivation from chronic infection and maintenance of metabolic activity following prolonged starvation [mazG mutant as well. In sum, these transcriptional changes thus reflect a metabolic state observed during cell cycle re-entry [mbtCDEFGH and irtB) were significantly upregulated  in the ΔmazG mutant may result in increased anaerobic respiration via fermentation of fumarate to succinate .Compared with wild-type modeling , was incwing Mtb . In linearvation , was incegulated .7-fold iecursors . In lineMtb remains unclear  resulted in failure to arrest growth under hypoxic dormancy model [Mtb and evaluate whether pyrimidine metabolism serves as a potential target for intervention of bacterial persistence.Increasing evidence is revealing the strong impact of bacterial metabolism on the mycobacterial life cycle, pathogenicity and immunoreactivity in the host . Howevercy model , providehttp://www.nature.com/emiSupplementary Information accompanies the manuscript on the Emerging Microbes & Infections website


Summary. Contrary to recent reports, this case demonstrated no devastating consequences of intravitreal injection of adipose tissue-derived stem cells during an observation period of up to 18 months. After vitrectomy, the cells cleared without evidence of either harm or integration.This study reports findings from a 56-year-old patient, who had received bilateral intravitreal injection of putative adipose tissue-derived stem cells at a private clinic in India with the promise of treatment of NAION. During an observation period of 8, respectively, 18 months, the intravitreally injected cells remained silent in the vitreous bodies without either therapeutic effects or complications. The cells cleared with vitrectomy without evidence of integration in the optic nerve or retina. Contrary to recent reports on patients receiving intravitreal injections of similar putative stem cells with the aim of treating AMD, our patient suffered no devastating ocular consequences.  Stem cells have emerged as a promising therapeutic modality for multiple diseases –3. BesidRecent reports have described four cases of putative adipose tissue-derived “stem cells” injected intravitreally in patients (aged 72 to 88 years) with age-related macular degeneration in two different private clinics , 5. WhilA 56-year-old male with vision loss from sequential nonarteritic anterior ischemic optic neuropathy (NAION) with a complete visual field defect and a visual acuity of 1/50 Snellen in each eye  was seenThe patient revealed that two months earlier he received intravitreal injections in both eyes of putative stem cells prepared from his abdominal subcutaneous adipose tissue. The full procedure from harvesting of adipose tissue to intravitreal injection had a total duration of about two hours and was carried out at a private clinic in New Delhi, India. Further details about the procedure were not known.The patient was observed and demonstrated a stable condition for several months . Eight mThis case demonstrates that cell suspensions of putative stem cells may be injected intravitreally without severe adverse effects. Whether this was due to entrapment in the vitreous body or simply lack of function of putative stem cell population is not known. The posterior hyaloid was very adherent in both eyes, which may be an effect from or a response to the intravitreally placed cell population. We did, however, not note any signs of cellular integration in the retina or the optic nerve and certainly no therapeutic effect was noted.The cell suspensions remained intravitreally for 8-18 months. Besides unchanged vitreal clouding, no serious adverse effects were noted in the observation period. In agreement with the immune privilege of the eye including the vitreous, this demonstrates that heterogeneously transplanted cells can remain silent without inducing an inflammatory response in the vitreous. This is unlike previous reports where intravitreal injections of similar adipose tissue-derived cell populations resulted in early and serious complications in all treated eyes. This included high intraocular pressure, anterior uveitis, lens subluxation, vitreal and intraretinal hemorrhage, and retinal detachment with aggressive PVR , 5. ThisThe stem cell technique holds great promise for treatment and cure of a wide range of diseases; however, more research is needed. Currently, some private clinics, with a promise of cure, are exploiting patients with untreatable diseases. This case demonstrates that patients should be discouraged from such unethical and costly treatments.


Objective: Descemet membrane endothelial keratoplasty (DMEK) is the gold standard procedure for treatment of primary corneal endothelial disorders. Graft detachment is a frequent complication of DMEK, which often requires re-operation or re-bubbling. However, several cases of spontaneous corneal clearance despite graft detachment after DMEK have been reported. The underlying mechanisms of this phenomenon are poorly understood. We report three cases of corneal clearance after graft detachment in patients with Fuchs endothelial dystrophy and provide a review of the literature. Methods: An 81-year-old and a 69-year-old phakic patient as well as a 56-year-old pseudophakic patient with Fuchs endothelial dystrophy underwent Triple-DMEK and DMEK, respectively. All three patients presented postoperatively with blurred vision due to an almost complete detachment of the graft, as shown by slit-lamp photography and anterior segment optical coherence tomography (OCT). Results: Without additional intervention, gradual corneal clearance and presence of endothelial cells on the posterior recipient’s stroma were observed in all patients three months postoperatively. Increase in endothelial cell density, decrease in central corneal thickness (CCT), recovery of corneal transparency, and improvement of visual acuity were documented in all patients.Conclusions: Our findings support the theory of corneal clearance after Descemet membrane endothelial transfer (DMET) (“free-floating” donor Descemet graft in the recipient anterior chamber after descemetorhexis). Further understanding on endothelial homeostasis might lead to innovative approaches in handling endothelial disorders.  Descemet membrane endothelial keratoplasty (DMEK) is a standardized technique, which is utilized for treatment of primary or/and secondary corneal endothelial disorders  such as DMEK is described as a superior technique over other lamellar techniques like Descemet’s stripping automated endothelial keratoplasty (DSAEK) for treatment of corneal endothelial disorders , due to A successful outcome of DMEK has been strongly associated with the adherence of the donor graft to the posterior surface of the corneal stroma , 5]. Th. Th5]. TRemarkably, successful outcomes after DMEK, despite graft detachment, have also been reported. In this report, we present three cases of positive outcome, following late graft detachment, left without re-intervention and we attempt a comprehensive discussion of this condition by providing a review of the literature. An 81-year-old phakic female, a 69-years-old phakic male, and a 56-year-old pseudophakic female presented in our clinic with progressive deterioration of visual acuity, epiphora and photophobia of both eyes. ® Scheimpflug camera.In all patients, preoperative slit-lamp examination revealed corneal edema with extensive cornea guttata in both eyes, caused by Fuchs endothelial dystrophy. A best-corrected visual acuity (BCVA) of 4/20, 6/20 and 2/20 was measured in the worst eye of each patient, respectively. In all patients, reliable measurements of endothelial cell density by corneal specular microscopy could not be obtained due to the advanced stage of endothelial dysfunction. Central corneal thickness (CCT) was 687 µm, 606 µm, and 832 µm, respectively, as measured by Oculus PentacamThe first two patients underwent Triple-DMEK (phacoemulsification and intraocular lens implantation combined with DMEK) while the third one underwent single DMEK as described by Melles and colleagues . In all The first and the third patient were discharged on the fifth postoperative day without presentation of any complications and with attached graft as documented by anterior segment OCT. In the second patient, re-bubbling was necessary due to incomplete attachment of the graft. During the re-bubbling procedure, the graft was accidentally partially folded. This patient was discharged after absorption of the air bubble. Approximately one week following discharge, all three patients were referred to our clinic again with photophobia and increased blurry vision. Slit-lamp examination demonstrated diffuse corneal edema and corneal decompensation, highly likely as a result of graft detachment. Anterior segment OCT Figure 1  confirmeWe followed a “wait & watch” approach in all three patients and close follow-up visits were undertaken. Remarkably, gradual corneal clearance was observed within three months in all patients. Upon slit lamp examination three months postoperatively, the corneal stroma was more transparent in all three patients, despite the fact that it was covered neither from donor tissue nor from the patient’s Descemet’s membrane. Central corneal thickness demonstrated a significant decrease from 687 to 572 µm (17%) in the first patient, a moderate decrease from 606 to 556 µm (8%) in the second one while in the third patient a more significant decrease from 832 to 580 µm (30%) was documented Figure 2 . Those changes in corneal pachymetry were accompanied by an increase of visual acuity from 4/20 to 8/20 in the first patient, from 6/20 to 12/20 in the second and from 2/20 to 4/20 in the third one within the same time period. Although a marked improvement of the CCT was seen in all three cases, BCVA has not been a reliable outcome parameter. The reduced visual acuity despite the more advanced corneal clearance in the second and third patient was attributed to a concomitant retinal disorder and central retinal vein occlusion, respectively. Furthermore, the “free-floating” Descemet graft was positioned within the visual axis, causing blurry vision. However, despite the detached donor tissue, enlarged and irregularly shaped endothelial cells were found by noncontact corneal specular microscopy on the recipient’s posterior corneal stroma in all patients Figure 3 .The above described positive findings were maintained over one-year follow-up, despite the still detached grafts. However, re-DMEK was performed after 14 months in the first patient, with successful graft attachment and a final visual acuity of 20/20. The second and third patient refused further interventions.In this case report, we present three cases of spontaneous corneal clearance, improvement of BCVA and corneal re-endothelialization after almost complete graft detachment following DMEK, in three patients with Fuchs endothelial dystrophy. A decrease in central corneal thickness together with a presence of endothelial cells on the recipient’s posterior stroma was observed three months after the diagnosis of graft detachment without any additional operative intervention.Similar to our observations, corneal transparency and improvement of BCVA along with presence of functional endothelial cells on the posterior stroma, despite graft detachment, have been previously reported. Of note, such positive outcomes were observed independently of the time of diagnosis and the extent of graft detachment. Dirisamer et al. described the involved patterns of corneal re-endothelialization following complicated DMEK, categorizing them in 4 groups depending on the extent of detachment and the positive or negative outcome . CornealEndothelial cell regeneration has also been discussed as a possible mechanism of re-endothelialization, found after a solely mini central stripping of Descemet’s membrane in patients with focal corneal edema without keratoplasty as well as in eyes with corneal graft detachment after DSAEK . in vivo confocal laser scanning microscopy. Comparison of the morphology of the endothelial cells between the denuded areas and the opposite or adjacent corneal quadrants revealed that the endothelial cells covering the patient’s denuded corneal stroma resembled those of the donor graft. This observation means that endothelial cells might migrate from the donor graft to the denuded corneal recipient’s stroma. However, this theory fails to explain the different response after DMET of patients with Fuchs endothelial dystrophy and bullous keratopathy.Jacobi et al.  addresseBased on the above theories, we assume that the healthy endothelial cells seen in our patients are the result of either cell migration from the detached donor graft onto the posterior recipient’s stroma, regeneration of the remaining endothelial host’s cells, or a combination of both. The concept of regeneration allows the assumption that the endothelial cells themselves may not be primarily dystrophic but their dysfunction may be the result of specific conditions . PresencIn conclusion, spontaneous recovery of corneal transparency along with improvement of visual acuity and increase in endothelial cell density is possible after graft detachment following DMEK, even without any further intervention. Such positive outcomes can be attributed to the increased endothelial cell repopulation by migration or/and by regeneration. These findings merit further study in regard with the corneal endothelial homeostasis in order to develop innovative approaches in handling endothelial disorders.BCVA: best-corrected visual acuityDMEK: Descemet membrane endothelial keratoplastyDMET: Descemet membrane endothelial transferDSAEK: Descemet’s stripping automated endothelial keratoplastyCCT: central corneal thicknessre-DMEK: repeat Descemet membrane endothelial keratoplastyTriple-DMEK: phacoemulsification and intraocular lens implantation combined with DMEKOCT: optical coherence tomographyThe authors declare that they have no competing interests.


The most recent genome-wide association study in amyotrophic lateral sclerosis (ALS) demonstrates a disproportionate contribution from low-frequency variants to genetic susceptibility to disease. We have therefore begun Project MinE, an international collaboration that seeks to analyze whole-genome sequence data of at least 15 000 ALS patients and 7500 controls. Here, we report on the design of Project MinE and pilot analyses of successfully sequenced 1169 ALS patients and 608 controls drawn from the Netherlands. As has become characteristic of sequencing studies, we find an abundance of rare genetic variation , the vast majority of which is absent in public datasets. Principal component analysis reveals local geographical clustering of these variants within The Netherlands. We use the whole-genome sequence data to explore the implications of poor geographical matching of cases and controls in a sequence-based disease study and to investigate how ancestry-matched, externally sequenced controls can induce false positive associations. Also, we have publicly released genome-wide minor allele counts in cases and controls, as well as results from genic burden tests. SOD1 SOD1 [TAR[C9orf72 . In so-c[C21orf2 , common  [UNC13A , SARM1 [A [SARM1 , MOBP [1M1 [MOBP , and SCFP [SCFD1  loci andhttp://www.datafairport.org).Despite these recent advances in ALS genetics, the bulk of risk loci in ALS remain undiscovered. The most recent and largest ALS GWAS, performed in 12,577 cases and 23,475 controls, showed a disproportionate contribution of low-frequency variants to the overall risk of ALS . Genome-While common variant association studies have become mostly standardized over the last decade, rare variant association studies such as Project MinE face an array of new challenges. Sequencing studies demand large sample sizes to detect small effects at rare variants, thus making large-scale collaborative consortia a necessity. Sequence data itself, measuring into the terabytes, poses a substantive data storage, processing, and management challenge. The analytic effects of (tight) spatial clustering of rare variants is not yet well understood , 14, makwww.projectmine.com, Fig. The Project MinE consortium includes ALS research groups from 16 different countries collaborating in a “franchise” design . Analysis teams  can submit analysis proposals to the consortium and then gain access to all genomes and make use of the SURFsara compute facilities Fig. . Access Patients were diagnosed with definite, probable, and probable lab-supported ALS according to the revised El Escorial Criteria . For thehttps://euromotor.umcutrecht.nl) from the genetic data in a clinical database called Progeny. The phenotypic and genetic data can be connected through sample identifiers. Phenotypic data storage is organized similarly to the sequencing data: every contributing group has default full access to their own phenotypic data, but data can easily be jointly shared analysed if desired. This core clinical dataset includes date of birth, sex, site of onset, date of disease onset, diagnostic category, Forced Vital Capacity at time of diagnosis, cognitive status, and a revised ALS functional rating scale score [For all participants in Project MinE, we have defined a core clinical dataset, meant to be collected and made available for all cases  on the Illumina HiSeq 2000 platform. Sequencing was 100 bp paired-end performed using PCR-free library preparation, and yielded ~40× coverage across each sample. The Isaac pipeline [QC of the data included QC at an individuals and variant level. Full details of QC are provided in the supplement.Principal components were calculated for all individuals including variants at different allele frequency thresholds using GCTA . The eigWe phased all non-singleton variants using SHAPEIT2  and thenGenotypes at common variants (MAF > 0.5%) were tested for an association with case/control status using logistic regression (PLINK v1.9) , 25 assup-value from each simulation was extracted ; the fifth-most extreme p-value represented the p-value threshold necessary to maintain study-wise type I error at 5%. Subsequently, we assessed the impact of including ancestry-matched, externally sequenced controls  insteadAll participants gave written informed consent and the institutional review board of the University Medical Center Utrecht approved this study. Additional approval was obtained to access the Dutch Municipal Personal Records Database.http://databrowser.projectmine.com/.The summary statistics generated in this study are available at the Project MinE databrowser: N = 498) and the 1000 Genomes Project . This observation reflects population-specific variants and the growing number of rare variants that will continue to be discovered as sequencing is performed in increasingly larger samples around the globe. As expected, most common variants (MAF > 1%) have been observed in these two datasets (97.5% in GoNL and 98% in the 1000 Genomes Project) reflecting global sharing of common variation.After QC, 1,169 unrelated Dutch-ancestry cases and 608 ancestrally-matched controls were available for analysis. Their baseline characteristics are displayed in Table p = 8.4 × 10−284) and the second principal component explained 24% of the variance in longitude (p = 1.7 × 10−107) of the geographical distribution of the samples across the Netherlands. Prediction models including the first twenty principal components predicted the birthplace of individuals with high accuracy . The eigenvectors of the first principal component explained 55% of the variance in latitude  IBD segments  with ALS in our sample. We looked up known associated single-variants, three of which showed nominal association to the trait  created perfectly geographically matched case-control sets and 2) introduced population structure between patients and controls . Similarly, burden testing comparing ALS cases and GoNL controls revealed a strongly inflated QQ plot and genomic inflation factor (λ = 2.49), demonstrating the challenges of using externally sequenced controls to identify disease genes in a separately-sequenced set of cases and controls.To test the analytic implications of a study design that uses its resources solely for the sequencing of cases and collects controls from an external source, we merged genotypes from the ALS cases with Dutch-ancestry samples whole-genome sequenced as part of the Genome of the Netherlands (GoNL) project . PrincipFamily-based and population-level genetic studies have revealed ALS as a complex disease with a distinct role for rarer genetic variation. Although numerous genetic variants have been identified as conferring ALS risk, the genetic basis in the vast majority of cases is not yet understood. Here, we have described the design and pilot analyses of a large-scale whole-genome sequencing study aimed at discovering new genetic risk factors and further elucidating the genetic basis of ALS.http://databrowser.projectmine.com/). This browser will be continuously updated as the project expands, and will provide more detailed data integration in future releases. To further enhance the transparency within the consortium itself and with external collaborators and researchers, the project has begun a GitHub repository, to share and maintain scripts and data processing pipelines.Data sharing and transparency in scientific research is advantageous for a host of reasons: it allows for analyses across large sets of samples, particularly for lower-prevalence diseases such as ALS; ensures rigorous experiments that can be reproduced by external groups; and allows for publicly-funded research to be made available to the public itself. Project MinE cannot publicly release individual-level genotype data due to consent. However, genotype frequency information and genic burden testing results are publicly available at the project’s online browser  is easily handled by a high-performance compute cluster, whole-genome BAM files (approximately 80 gigabytes per genome) demand a compute infrastructure that can handle terabytes and even petabytes of data. This includes direct delivery of the genomes that are currently being sequenced via a direct high-speed connection between the sequencing provider and a computer cluster with a GRID architecture (SURFsara). Though some research institutions are already prepared for such a deluge of data, others will need to carefully consider compute infrastructure and the technical ramifications of assembling big data before embarking on large-scale sequencing analyses. Further, having all data stored in a single location enables analysis across the full dataset, including on BAM-level data.Second, initial QC of the data suggests that the high-depth sequencing and downstream genotype calling is yielding high-quality data, reflected in our ability to reconstruct, through principal component analysis and identity-by-descent analyses, demographic observations that have been made in separate large-scale sequencing efforts in the Dutch population . FurtherThird, the high-resolution geographical clustering of rare variants as observed in these analyses could pose challenges in finding rare variants that contribute to ALS risk. The commonly applied burden test approach, however, does not seem to suffer from increased type I error rates induced by modest imperfections in geographically matching samples, as was shown by the burden test simulations. While it is possible that aggregating rare variants  in these tests makes the approach more robust to population structure, these results should be interpreted with caution. The modest power in our pilot sample likely limited our ability to detect the increasing type I error rates induced by imperfect geographic sample matching. With the increasing number of ancestrally diverse samples that are being included in Project MinE, false positive associations due to population stratification remain a serious concern, especially when our aim becomes identifying the causal single variant(s) that drive the genic burden signal. Correcting for principal components can help to control type I error, though potentially not sufficiently so in burden testing. More sophisticated analytic approaches, such as employing a linear mixed model in single variant and genic burden testing, will likely be helpful in controlling for population stratification across the full set of Project MinE samples .We further demonstrate that inclusion of externally-sequenced samples can pose analytical challenges. Specifically, we show that using ancestry-matched controls from publicly-available datasets can strongly inflate association test statistics if the data is simply combined at VCF-level. Sequence data can contain underlying structure due to sequencing platforms, coverage, alignment and calling algorithms. Inclusion of externally sequenced cases and controls (sequenced either separately or together) is non-trivial must be approached with extreme rigor. Methods for handling the merging and calling of heterogeneous sequencing datasets at a BAM-level are currently being applied in large consortia –33 and iWith the veritable explosion in methods and approaches being developed for whole-genome sequence data analysis, Project MinE will seek to leverage new and powerful methods for uncovering ALS risk variants. Current plans include more sophisticated functional annotation techniques, such as unsupervised-learning approaches to discriminate between functionally relevant and benign variation in the genome , 36; impFinally, in addition to testing SNVs and indels, we will explore the role of more complex genetic variation such as structural variants and repeat expansions, which are known to play an important role in ALS susceptibility, yet have never been studied at genome-wide scale. With a global collaboration in place, a wealth of genetic variation being generated, and new methods for sequence data constantly in development, Project MinE will be the largest and most complete study of ALS genetics to date, poised to reveal novel risk loci, fine-map known disease genes, and shed light on the biological drivers of disease.Supplementary material


Blattabacterium – providing nutritional augmentation to its host in the form of amino acid synthesis – displays radical genome alterations when compared to its most recent free-living relative Flavobacterium. To date, eight Blattabacterium genomes have been published, affording an unparalleled opportunity to examine the direction and magnitude of selective forces acting upon this group of symbionts. Here, we find that the Blattabacterium genome is experiencing a 10-fold increase in selection rate compared to Flavobacteria. Additionally, the proportion of selection events is largely negative in direction, with only a handful of loci exhibiting signatures of positive selection. These findings suggest that the Blattabacterium genome will continue to erode, potentially resulting in an endosymbiont with an even further reduced genome, as seen in other insect groups such as Hemiptera.Estimates suggest that at least half of all extant insect genera harbor obligate bacterial mutualists. Whereas an endosymbiotic relationship imparts many benefits upon host and symbiont alike, the intracellular lifestyle has profound effects on the bacterial genome. The obligate endosymbiont genome is a product of opposing forces: genes important to host survival are maintained through physiological constraint, contrasted by the fixation of deleterious mutations and genome erosion through random genetic drift. The obligate cockroach endosymbiont,  Insecta is the most speciose group among animals; at least half of extant genera are estimated to harbor obligate bacterial mutualists3. While some intracellular bacteria can be harmful or even lethal to their insect host, many others play an important role in host survival and fecundity8. These primary bacterial symbionts exist obligately within the cells of the insect, and are often required for the survival and reproduction of their host organism9. An intercellular lifestyle affords endosymbiotic bacteria relative safety from competition and exploitation, in exchange for increased ecological flexibility imparted onto the host species. In many cases, these obligate bacterial mutualists function in the provisioning, recycling, or degradation of essential nutrients, and are vital to those insect species that subsist on nutritionally narrow diets, such as those composed primarily of woody material, plant sap, mammalian blood, or decaying organic material11. However, within some insect species primary bacterial endosymbionts also function in non-nutritional roles such as parasitoid defense12.Comprised of over one million species, Class Noticola , all cockroach species contain endosymbiotic bacteria within their fat bodies14. These obligate endosymbionts belong to the genus Blattabacterium 16. Phylogenetic reconstruction suggests that cockroaches acquired these endosymbionts in a single infection event, dating between 300 million years ago - the approximate age of the first fossil roaches from the Carboniferous - and 140 million years ago, when currently extant families last shared a common ancestor18. Initially, the function of these endosymbionts was subject to speculation, owing to their recalcitrance to culture outside their host. However, modern DNA-sequencing techniques have allowed for the study of a number of Blattabacterium genomes. From these genomes, it was discovered that the function of Blattabacterium is primarily the synthesis of amino acids and vitamins from the nitrogenous waste products of the cockroach host19. Cockroaches store excess nitrogen as uric acid within their fat body cells20. The decaying organic matter on which cockroaches typically feed is poor in nitrogen content. Thus, a mechanism for recycling nitrogenous waste would be beneficial to any organism whose diet is nitrogen-deficient. Unlike most insects, which excrete waste nitrogen as uric acid, cockroaches excrete ammonia21. Blattabacterium are capable of utilizing both urea and ammonia because they contain an active urease as well as a functioning urea cycle that converts host urea to ammonia24. In addition, increases in dietary nitrogen intake by host cockroaches correlates with increases in uric acid buildup within that host’s fat bodies23.With the exception of a single cave-dwelling genus, Blattabacterium genomes have been sequenced from the following cockroach host species: Periplaneta americana19, Blatta germanica25, Cryptocercus punctulatus26, Blaberus giganteus27, Blatta orientalis28, Panesthia angustipennis29, Nauphoeta cinerea30 and the termite, Mastotermes darwiniensis31. While these genomes share similar gene composition and genome architecture, each also displays unique capacities for metabolic and physiological function. Thus, while the results of phylogenetic analysis support the hypothesis of co-cladogenesis between the endosymbionts and hosts18, gene composition of Blattabacterium is not directly congruent with host phylogeny; rather it varies likely as a function of host nutrition, its relative importance in the mutualism, and the interaction between phenotypic constraint, environmental natural selection, and genetic drift.Cockroaches represent an evolutionary lineage consisting of diverse and ancient taxa that have adapted to many habitats and exhibit broad nutritional ecology; their endosymbionts, therefore, represent an excellent system in which to assess relationships between these traits. To date, eight 32. Evolution of the bacterial endosymbiont genome is characterized by elevated mutation rates and biases resulting from the combined effects of physiological constraint preserving symbiont-critical genes, random genetic drift – driven by frequent population bottlenecks, bacterial asexuality, and lack of genetic recombination – and environmental selection acting to reduce genome size47. Endosymbionts have been shown to have higher substitution rates and values of non-synonymous to synonymous substitution rates a result attributed to small Ne.49. Acting through Muller’s Ratchet, asexual reproduction can prevent the recovery of wild-type genotypes through recombination50. Loss of recombination is a result of lost DNA repair, uptake, and recombination genes; which is a common pattern of all sequenced bacterial endosymbionts [56 reviewed in ref.57].An intracellular lifestyle strongly influences the selective pressures and evolutionary trajectories of bacterial endosymbionts59. Described genomes from endosymbionts suggest that physiological constraint acts to maintain a gene set that retains its functionality for the host; though selection might also be driving the erosion of bacterial endosymbiont genomes. Certain metabolites ordinarily produced by the bacteria itself may now be obtained directly from the host; under such circumstances, these genes become superfluous and are necessary for neither bacterial survival nor continued host fecundity. As such, a smaller genome results in a cell that is faster and more efficient to reproduce. Particularly at the beginning of endosymbiosis, rapid loss of unnecessary genes32 may be advantageous [Reviewed in ref.60]. Thus, while random genetic drift does act to reduce the bacterial endosymbiont genome through Muller’s Ratchet, physiological constraint acts to preserve genes crucial to symbiosis while environmental selection favors a reduced, more energy-efficient genome.However, selection in the form of physiological constraint acts to maintain genes important to the bacterial-insect symbiosis, although its role in the continued erosion of non-essential genes in the bacterial genome is largely unknownN/dS ratios, indicating an increase in positive selective pressures and rapid protein evolution63. Thus, we may conclude that the endosymbiont genome is the result of interplay between random genetic drift and the reduction of genes through relaxed selection within large portions of the genome, and physiological constraint acting to preserve those genes vital to host survival and fecundity.When compared to free-living bacteria, endosymbionts exhibit increased levels of mutation at synonymous and non-synonymous sites, as well as higher dBuchnera aphidicola64 from aphids, Wigglesworthia66 from the tsetse fly, Blochmannia67 from carpenter ants, and Blattabacterium19, from cockroaches. However, comparatively few of these genomes have been examined for signals of positive selection. Eight fully sequenced Blattabacterium genomes, in addition to the five fully-annotated free-living Flavobacterium genomes for comparison, offers a unique opportunity to investigate the patterns and processes that drive endosymbiotic genome evolution.Genome evolution in insect endosymbionts has been the topic of a number of studies. Full genomes from several endosymbionts have been published, including Blattabacterium strains, and compared them to those present within the closely-related68 but free-living Flavobacterium species , to examine the similarities and differences between these two evolutionarily related, but divergent, groups. We hypothesized that patterns of selection acting upon the Blattabacterium genome will manifest as an elevation in both non-synonymous and synonymous mutation events, as well as a higher dN/dS ratio at sites under significant levels of selection than the free-living Flavobacterium - indicating increased positive selection pressures and an elevated rate of protein evolution70. Additionally, we sought to determine whether patterns of selection observed in previous studies across limited numbers of genes are effective at predicting patterns of selection across an entire endosymbiont genome.We estimated the positive and negative selection events in the genomes of all sequenced Blattabacterium and five Flavobacterium species were manually compiled from genomes available in GenBank  we manually BLASTed each loci against all other Blattabacterium genomes, as well as against existing Flavobacterium genomes. Resulting BLAST hits were then manually compiled into single homologue files in nucleotide fasta format. We applied Clusters of Orthologous Groups (COGs) to categorize the function of genes in our dataset. Given that genome-wide COG composition is very similar among Blattabacterium30, we assessed composition genome-wide as well as in the subset of homologous genes found in all taxa using the Bacterial Annotation System (BASys71).Homologous genes for eight nk Table . With Blhttps://github.com/k8hertweck/Blattabacteria. Phylogenetic reconstruction of the evolutionary relationships of the eight Blattabacterium and five Flavobacterium species (with eight Escherichia coli strains as outgroup) was carried out using PhyloPhlan72 under default parameters and whole genomes obtained from GenBank 76 and assessed using 100 bootstrap replicates . The maximum likelihood tree for each gene  was used to create a reconciled species tree using ASTRAL v4.7.8 (github.com/smirarab/ASTRAL/)77 and 100 bootstrap replicates.All scripts developed for this analysis  can be found at nk Table . For eac78 suite of programs. For this analysis, three different selection tests were used: HyPhy’s BUSTED79, Quick Selection Detection (implementing MEME [Mixed Effects Model of Evolution])80, and Branch Site REL81. Each of these programs used the same sets of input data; namely the HyPhy alignment combined with the PhyML tree for each gene. Parameters used for these tests may be found here: github.com/k8hertweck/Blattabacteria/blob/master/blattabacteriaBUSTED.bf, github.com/k8hertweck/Blattabacteria/blob/master/blattabacteriaQSD.bf, github.com/k8hertweck/Blattabacteria/blob/master/blattabacteriaBranchSiteREL.bf.Selection analysis was performed using the HyPhy v2.2.1 (github.com/veg/hyphy)Summary statistics for all selection analyses performed were produced using an in-house script that may be found here: github.com/k8hertweck/Blattabacteria/blob/master/blattabacteriaSelectionSummary.sh. Summary statistics and input and output files for selection analysis may be found at: github.com/k8hertweck/Blattabacteria/tree/master/analysis. Statistics of particular import are those referencing branches under positive selection. This information was drawn from the BUSTED and Branch Sire REL output. Additional statistics were obtained from the output of blattabacteriaGeneSummary.sh .82. Within these models, number of positive and negative selection sites were log transformed. A handful of outliers were noted but retained, as their inclusion had a non-significant impact upon the resulting models.For analyzing the number of positive and negative sites of selection per gene length, a Linear Model was implemented using R version 3.4.4Number of positive and negative selection events vs. individual COG size were carried out using Generalized Linear Models with Poisson distribution with number of selection sites as the response variable and total number of nucleotides in the genome associated with a specific COG as the explanatory variable. As COG and gene length analyses used different datasets - number of selection events per total number of nucleotides of all genes associated with a given COG and number of selection events by gene length, respectively - we found that differing models better fit each type of analysis.83. Building upon this on a functional level, however, we also noted that signatures of both positive and negative selection (response variable) correlated strongly with the total number of nucleotides assigned to a specific COG (explanatory variable) across the Blattabacterium genome  as well as similar pairwise percent identities  when aligning the ribosomal 16S rRNA gene. Thus, extant Blattabacterium display signs of elevated rates of genome evolution in the form of increased levels of selection events. The increase in the number of sites experiencing negative or positive selection when compared to the free-living Flavobacterium suggests elevated levels of functional protein evolution in the endosymbionts. Only a limited number of loci display similar selection profiles between Blattabacterium and Flavobacterium  show signs of positive selection. These observations are in accordance with our previous understanding of the evolutionary history of Blattabacterium, as well as its function within its cockroach host as a nutritional endosymbiont aiding in the recycling of nitrogenous waste and the production of both essential and nonessential amino acids.Our findings indicate that the Blattabacterium. Using a parsimony approach, the ancestral genome of another primary insect endosymbiont, Buchnera-Ap, was reconstructed by Moran and Mira32. The results of Moran and Mira’s analysis indicated that much of the ancestral Bucnhera genome was lost during a relatively small number of large deletion events shortly after this bacteria’s transition to an intracellular lifestyle. While it is likely that that the Blattabacterium genome was reduced through similar mechanisms, a similar reconstruction within this group would offer us a more complete picture of the evolutionary origins of this unique cockroach endosymbiont.The analysis presented here could be augmented through a robust analysis of genome reduction within


PRKAR1A mutations in a subset of cyclical Cushing’s syndrome due to primary pigmented nodular adrenocortical disease, the molecular basis of cyclical Cushing’s syndrome has not been investigated. We speculated that cyclical Cushing’s syndrome may be due to mutations in the clock genes that govern circadian rhythms, including the hypothalamic-pituitary-adrenal axis.Apart from AHR) gene, which has previously been indirectly linked to pituitary tumorigenesis and clock system disruption. The AHR variant was found in a highly conserved site involved in phosphorylation. It was predicted to be damaging by multiple in silico tools and AHR tumour immunohistochemistry demonstrated loss of the normal nuclear staining pattern, suggestive of an inactivating mutation. We also found a novel, damaging germline missense variant in the retinoid X receptor gamma (RXRG) gene, multiple somatic chromosomal gains (including AHR), and a somatic mutational signature consistent with oncogenesis that may have acted synergistically with the AHR variant.A 47-year-old man presented with mass effects from a sellar lesion. He was ultimately diagnosed with cyclical Cushing’s disease due to a giant corticotrophinoma. We performed whole exome sequencing of germline and tumour DNA, SNP array of tumour DNA and tumour immunohistochemistry in order to detect variants in candidate circadian/pituitary-associated genes. We identified a rare germline missense variant in the aryl hydrocarbon receptor (AHR variant with predicted pathogenicity in the pituitary adenoma setting. Our preliminary data suggest that the highly conserved AHR gene may represent a link between pituitary tumorigenesis, the hypothalamic-pituitary-adrenal axis and the clock system. Further research may indicate a role for the gene in the development of cyclical Cushing’s disease.This is the first report of an  Cyclical Cushing’s syndrome (CCS), characterised by intermittent biochemical hypercortisolism, accounts for approximately 20% of endogenous Cushing’s syndrome (CS) . Cycles PRKAR1A mutations causing Carney’s complex, including the common manifestation of ACTH-independent CCS due to primary pigmented nodular adrenocortical disease (PPNAD) [PRKAR1A. The molecular basis of CCS has not otherwise been investigated. We performed whole exome sequencing (WES) in a man with CCD to investigate the possibility that CCS may be due to perturbation in the clock genes responsible for circadian rhythms, including the hypothalamic-pituitary-adrenal (HPA) axis.The only known genetic cause of CCS is germline  (PPNAD) . However (PPNAD) , and cyc2 but he had no supraclavicular fat pads, Cushingoid striae, facial plethora, ecchymoses or proximal weakness. He had a right-sided oculomotor nerve palsy and right-sided proptosis and conjunctival injection suggesting ophthalmic vein compression. He was eupituitary apart from fluctuating ACTH-dependent cortisol production ranging from normal to 35-fold ULN  array of tumour DNA to identify sequencing variants and copy number variation in circadian/pituitary-associated genes. Tumour immunohistochemistry was performed to further evaluate the leading genetic variant of interest. Further details are provided in Additional file AHR variant  was present in the heterozygous state in both germline DNA (30/65 reads) and tumour DNA (86/177 reads). The variant is located in a site that is highly conserved and likely to be involved in phosphorylation [cancer.sanger.ac.uk) in oesophageal adenocarcinoma [AHR variants on WES  situated in the ligand binding domain  [AHR variant in a man with CCD. We speculate that this likely inactivating variant may have contributed to CCD development via a loss of tumour suppressor function in the pituitary and disruption of circadian/infradian rhythms.The aryl hydrocarbon receptor (AHR) exists in a cytoplasmic complex with aryl hydrocarbon interacting protein (AIP), heat shock protein 90 and p23 protein . Amongst dioxin) –11. In tAHR mutations have not been reported in patients with pituitary adenomas, including recent WES cohort studies of patients with Cushing’s disease [AHR in the pituitary. AHR is the major binding partner of AIP, which is an established pituitary tumorigenesis gene [AIP-associated pituitary tumorigenesis, with somatotrophinomas from patients with germline AIP mutations typically showing decreased cytoplasmic and absent nuclear AHR staining [AHR may also have AIP-independent roles in pituitary tumorigenesis as AHR but not AIP expression is reduced in GNAS-mutated somatotrophinomas, and AHR activation increases the transcription of CDKN1B, which is another tumour suppressor gene implicated in pituitary and other endocrine tumours [To the best of our knowledge,  disease , 13. Howsis gene , 11. Los tumours .AIP germline mutations are most classically associated with somatotrophinomas, AHR may be more relevant to corticotrophinomas as in our patient. AHR immunostaining is found in the corticotroph-rich pars intermedia and normal corticotrophs demonstrate nuclear AHR immunostaining representative of activated AHR [Whereas ated AHR , althougated AHR .AHR variant at a highly conserved phosphorylation site [AHR may have proto-oncogenic effects. Recapitulating animal models, an excess of non-functioning pituitary adenomas (NFPA) and prolactinomas followed dioxin exposure from the 1976 Seveso accident in Italy [AHR SNPs, rs2066853  and rs4986826 , are over-represented in acromegalic patients in these regions with prevalences of 22.4 and 2.9%, respectively, compared to Caucasian ExAC allele prevalences of 9.9 and 0.3%, respectively [AHR SNPs and our patient’s variant all reside in exon 10, encoding the transactivation domain . Furthermore, RXRG belongs to the retinoid X nuclear receptor family that mediates the antiproliferative effects of retinoic acid, which has shown some efficacy in the treatment of Cushing’s disease [RXRG variant (p.R317H) in the ligand binding domain segregated upon WES of a familial prolactinoma kindred [RXRG has not previously been studied in Cushing’s disease. Our patient also had a somatic mutational signature typical of various cancer types [AHR and RXRG variants and somatic driver mutations.Other variants might have acted synergistically with the  disease . Another kindred , but RXRant p.R31H in the er types , 22 and AHR and CCD raised by this case study.A limitation of this case study is that the patient is currently in a prolonged state of normocortisolism, precluding additional investigations to demonstrate ongoing cyclicity. This is despite a significant tumour remnant, highlighting the discordance between structural and functional status in patients with CCD. Given the rarity of Cushing’s syndrome in general and CCD in particular, collaborative research is required to further examine the potential relationship between AHR gene may represent a link between pituitary tumorigenesis, the HPA axis and the clock system, implicating it in the development of CCD. With AHR known to be expressed in the pituitary, CCD may occur because of the combination of AHR-mediated pituitary tumorigenesis and disordered clock control of the HPA axis. Our patient’s somatic Chr 7 trisomy and germline RXRG variant might have been additive to his germline AHR variant, explaining how this variant can be seen in up to 1/10,000 individuals in population data despite the rarity of CCD. Alternatively, AHR variants might lead to cyclicity in individuals who happen to develop CS. Future research is required to determine whether AHR is a true pituitary tumorigenesis gene or a phenotypic modifier gene accounting for cyclicity in CS of various aetiologies.Preliminary data from this case study suggest that the highly conserved Additional file 1. Supplementary Methods, Figures and Table.