Chronic hepatitis C (CHC) is a major cause of hepatocellular carcinoma (HCC) worldwide. While directly acting antiviral (DAA) drugs are now able to cure virtually all hepatitis C virus (HCV) infections, even in subjects with advanced liver disease, what happens to the liver and progression of the disease after DAA-induced cure of viremia is only beginning to emerge. Several large-scale clinical studies in different patient populations have shown that patients with advanced liver disease maintain a risk for developing HCC even when the original instigator, the virus, is eliminated by DAAs. Here we review emerging studies derived from multiple, complementary experimental systems involving patient liver tissues, human liver cell cultures, human liver slice cultures, and animal models, showing that HCV infection induces epigenetic, signaling, and gene expression changes in the liver associated with altered hepatic innate immunity and liver cancer risk. Of critical importance is the fact that these virus-induced abnormalities persist after DAA cure of HCV. These nascent findings portend the discovery of pathways involved in post-HCV immunopathogenesis, which may be clinically actionable targets for more comprehensive care of DAA-cured individuals. Hepatitis C virus (HCV) causes chronic infection and liver disease and infects an estimated 71 million people worldwide . The awaIFN-based cures can reverse liver damage . HoweverHCV is a highly successful pathogen that subverts both innate and adaptive immune mechanisms in infected hepatocytes, neighboring non-parenchymal cells, and circulating NK cells and T cells. A sure indication of the importance of a host defense mechanism is the precision with which successful pathogens have evolved to evade it. Thus, while human host resistance to HCV, as well as the responsiveness of an individual to DAA therapy, is closely tied to specific alleles of the interferon lamda 3 (IFNL3) gene for subjects with HCV genotype 3 infection , the impThe genome wide association studies (GWAS) implicating IFNL3 in the response to therapy ,33, the The spontaneous resolution of HCV infection in a minority of patients also sheds light on the mechanisms of host defense. In a study comparing over 900 subjects with evidence of spontaneous HCV clearance and over 1400 chronically infected controls, the same IFNL3 gene polymorphisms were associated with self-cure as with cure by IFNA plus ribavirin . The samThe innate immune features of chronic HCV that are sustained after self-cure, cure by IFNA plus ribavirin therapy, and cure by DAA therapy have not been extensively documented. A longitudinal study of innate immunity in spontaneous resolution of HCV in intravenous drug users suggested that most innate immune genes in peripheral blood cells returned to baseline by the time of follow-up . HoweverIn the face of so many mechanisms through which HCV disables innate immunity, the induction of innate immune genes and cytokines requires explanation. Our own work shows that semi-purified hepatocytes result in a much richer innate immune response to HCV than highly purified hepatocytes , implyinHCV also dysregulates innate immune function through effects on cells that are not readily susceptible to infection, i.e., myeloid cells and natural killer (NK) cells. The most abundant myeloid cells in the normal liver, Kupffer cells, are liver-resident macrophages; in mouse models there is strong evidence that many of these cells originate from the yolk sac and the fetal liver , while aWhile the HCV protease exerts immune effects in the infected cell, HCV core protein is well known as a disruptor of immunity ,53 and hMonocytes from HCV+ individuals contain detectable HCV RNA, but this is lost as they differentiate into dendritic cells . The HCVHCV also exerts powerful effects on, and appears to be controlled in part by, NK cells . A lowerMarkers of NK cell activation also correlated with strong T cell responses, whether or not the virus was eliminated . The lowThere is an emerging literature looking at immune restoration of T cell functions following DAA-mediated cure. For example, regulatory T cell (Treg) frequencies remain high post-DAA cure, and as such, may hinder immune surveillance of HCC . For mucUsing transcriptomic and epigenomic technologies ,76,77,78Two independent laboratories have shown that chemical or genetic modulation of histone-modifying enzymes causes reversal of HCV-induced epigenetic changes. For example, treatment of HCV-infected cells with the histone acetyl transferase (HAT) p300/CBP inhibitor C646, which inhibits acetylation of H3K9, or suberoylanilide hydroxamic acid (SAHA), which blocks histone deacetyltransferases (HDAC) of class I and II activity, or knockdown of bromodomain proteins BRD3, BRD4 and histone deacetylase 9 (HDAC9), reverted the HCV-induced changes in the HCV cell culture model ,84.The effects of these epigenetic changes on the status of “the prognostic liver signature” (PLS) were then analyzed. The PLS is a well-characterized, 186-gene liver expression signature that has been shown to predict survival and HCC risk in patients with advanced liver disease of all etiologies . The PLSBioinformatics analyses reveal that the PI3K pathway is epigenetically perturbed upon chronic HCV infection in patients, and that PI3K pathway activation persists after DAA eradication of the virus. The PI3Ks are associated with transmembrane receptors for cytokines and growth factors including G-protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). PI3Ks are found on the cytoplasmic side of the plasma membrane. Receptor activation by cytokines or growth factors activates PI3Ks to catalyze the phosphorylation of lipid substrate phosphatidylinositols (PIs) into phosphatidylinositol  tri-phosphates (PIP3). PIP3 molecules in turn activate downstream signaling kinases, including PKB and mammalian target of rapamycin (mTOR), which promote cell growth and survival . PI3K acp < 0.034 for HCV cirrhosis and p < 0.006 for sustained virologic response (SVR), i.e., cure cohorts) [As a specific example of a gene alteration, the protein sphingosine kinase 1 (SPHK1) level was induced by HCV infection and remained elevated after DAA cure . SPHK1 ccohorts) . Thus, Scohorts) . Thus, pViral cure by DAAs in the majority of infections in most people at all stages of liver disease is a breakthrough in the management of chronic hepatitis C. Nevertheless, the issue of how to manage cured patients with advanced liver disease and how to mitigate the risk of HCC is only beginning to be understood. Recent studies indicate that HCV-induced epigenetic, signaling, and gene expression changes persist after virus eradication and are strongly correlated with a risk of developing HCC. These virus-induced alterations that persist after virus eradication also extend to the level of hepatic innate immunity and hepatic non-parenchymal cells. Continued research is required to better define the molecular mechanisms of action by which HCV induces persistent epigenetic changes and how these changes confer the reprogramming of cellular gene expression and signaling pathways that leads to liver cancer. These questions require research structures that use multiple, complementary experimental systems that range from cells to tissues to in vivo models. The discovery of persisting epigenetic, transcriptomic, and signaling alterations involved in post-HCV immunopathogenesis may represent clinically actionable targets for urgently needed HCC chemoprevention, completing the comprehensive care of people who are cured of their HCV infection. In addition, defining markers for liver disease progression occurring in spite of the cure of HCV could provide valuable diagnostic tools for clinicians.


Actinic keratosis is considered a precancerous lesion, constituting a precursor to squamous cell carcinoma (SCC) formation. Perineural invasion has been observed in patients with cutaneous carcinoma due to local subcutaneous tissue destruction and primarily involves the trigeminal nerve due to rich innervation provided by the supraorbital nerve in addition to the facial nerve. An unusual case of perineural infiltration and orbital invasion of squamous cell carcinoma associated with actinic keratosis is presented. A 70-year-old Caucasian woman presented with complete left eye ophthalmoplegia, total left upper-eyelid ptosis, and facial pain with paresthesia. Computed tomography revealed a process of the soft tissues in the left cheek infiltrating the infraorbital canal, pterygopalatine fossa, inferior orbital fissure, and left cavernous sinus with periosteal adherence. Magnetic resonance imaging revealed pathological extension via the left infraorbital canal with a considerable area of necrosis. Treatment of facial actinic keratosis may not prevent malignant transformation and can delay diagnosis and treatment of SCC. A deep biopsy appears to be essential for a correct diagnosis. Perineural spread of cutaneous SCC may be characterized by insidious progression in the cranial trigeminal nerve, abnormal ocular motility, diplopia, or external ophthalmoplegia. Actinic keratosis (AK) is considered a precancerous lesion, constituting a precursor to squamous cell carcinoma (SCC) formation . The risA 70-year-old Caucasian woman presented with complete left eye ophthalmoplegia, total left upper-eyelid ptosis, and facial pain with paresthesia in the dermatome of the first (V1) and second (V2) branches of the trigeminal nerve that was established gradually in the duration of the previous 6 months. She also complained about visual deterioration in the left eye.Regarding her medical history, she underwent photodynamic laser treatment two years prior to the baseline visit for a left cheek actinic keratosis  and receClinical examination revealed complete left III, IV, and VI nerve palsy  and trigA computed tomography (CT) scan revealed an infiltrating process of the soft tissues in the left cheek infiltrating the infraorbital canal, the pterygopalatine fossa, the inferior orbital fissure, and the left cavernous sinus with a periosteal adherence. Magnetic resonance imaging (MRI) revealed pathological extension via the left infraorbital canal of the 30 × 27 mm diameter soft tissue jugal process with a considerable area of necrosis. It was located in the fat of the anterior area of the left maxillary sinus and was infiltrating toward the deep facial massif. A second homogenous infiltrating process in the deep facial massif was described, invading the left masticator space, the pterygopalatine fossa, the temporal anterior left meninx, the orbital apex, and the left cavernous sinus with perineural infiltration Figures .Ηistology revealed actinic keratosis with diffuse nonspecific inflammatory and fibrotic lesions located in the epidermis and the dermis. The patient underwent a later deep orbital floor biopsy with an infraorbital trigeminal V2 nerve biopsy under general anesthesia. The histopathologic examination demonstrated a poorly differentiated SCC invading the infraorbital nerve and the periosteum with perineural involvement. Histological markers for EMA, AE1, and AE3 were positive.A punch biopsy of the left cheek was performed. Positron emission tomography (PET)/computed tomography (CT) did not reveal any primary tumor of secondary metastatic localization but micronodular parenchymatous pulmonary lesions of the mid and inferior right lung. Subsequently, the patient underwent chemotherapy with paclitaxel/fosfamid/cisplatin, and 4 1-week cycles of 70 Gy wide-field radiation therapy of the left cheek and cervical area were performed. At the time, clinical examination revealed no signs of recurrence of the cheek SCC and on follow-up CT scans. The symptoms remained unchanged with a complete left eye ophtalmoplegia, altered sensation, and intermittent acute pain in the left trigeminal area.In the context of SCC, perineural invasion has been observed in 3-14% of patients with cutaneous carcinoma due to local subcutaneous tissue destruction . The mosAccording to Feasel et al., 60-70% of the patients with perineural invasion are asymptomatic in the early stages of the disease. Perineural invasion is not considered until orbital and cavernous sinus involvement is observed and implies an often delayed diagnosis –10.Local treatment of facial actinic keratosis may not prevent malignant transformation and can delay diagnosis and treatment of SCC, often diagnosed at a late stage. A deep biopsy appears to be essential for a correct diagnosis. Abnormal ocular motility, diplopia, and external ophthalmoplegia must be considered a red flag for perineural spread of cutaneous squamous cell carcinoma with insidious progression in the cranial trigeminal nerve.


The short-term and long-term consequences of the most frequent painful procedures performed in the ICU are unclear. This study aimed to identify the risk factors associated with pain-related discomfort perceived by critically ill patients during the whole ICU stay as self-reported by patients at the end of their ICU stay.The study involved 34 ICUs. Adult patients who survived an ICU stay of 3 calendar days or more were eligible for inclusion. Discomforts, including the pain-related discomfort, were assessed using the French 18-item questionnaire on discomfort in ICU patients, the “Inconforts des Patients de REAnimation” (IPREA). Patients scored each item from 0  to 10 . Associations between patient characteristics at ICU admission, life support therapies and main potentially painful procedures performed during the ICU stay and pain-related discomfort scores assessed at the end of the ICU stay were analyzed.n = 2130) were included. The median pain-related discomfort score was 3 (IQR 0–5). From the univariate analysis, pain-related discomfort scores were negatively correlated with age and positively correlated with ICU stay duration; surgical patients reported significant higher pain-related discomfort scores than medical patients; chest drain insertion, chest drain removal, use of bladder catheter, central venous catheter (CVC) insertion, complex dressing change, and intra-hospital transport were associated with pain-related discomfort scores. From the multivariate analyses using generalized estimating equations models, only age, chest drain removal, use of a bladder catheter, CVC insertion, and intra-hospital transport were the main risk factors associated with pain-related discomfort scores.Patients with complete IPREA questionnaires  than patients who did not experience these events. This study may pave the way for further targeted studies aiming at investigating a causal link between these common procedures in the ICU and adult critically ill patients’ perceptions of their ICU stay regarding recalled pain.Trial Registration: Clinicaltrials.gov Identifier NCT02442934, retrospectively registered on May 13, 2015 Critically ill patients in intensive care units (ICUs) are exposed to stressful conditions and experience discomfort from multiple sources, such as the environment or the treatment being provided, which depends on the organization  and the patient’s health status –6. AmongSeveral hypotheses may explain why the program was ineffective for reducing pain scores. First, previously implemented health policies for pain management may have previously been be prioritized over managing other discomforts. Second, measures to reduce pain reported by critically ill patients may require more time to implement, because of complexity of pain management in adult critically ill patients due to highly individual pain patterns, and unique features in the ICU such as impaired communication and altered mental status, potentially painful procedures and use of invasive devices . MoreoveBetter identification of procedures that cause the pain reported at the end of ICU stays would enable developing more effective strategies against pain in the ICU. Frequent ICU-associated procedures that can cause pain include arterial blood gas sampling and tracheal suctioning in mechanically ventilated patients, which lead to more frequent arterial catheter insertions and greater care for tracheal suctioning , 11. PatThis study was an ancillary study of the IPREA3 study , a multiThe effectiveness of the program was assessed from the French questionnaire on ICU-related self-perceived discomforts Table  15, 16], 1615, 1The study included 34 centers, all in France: medical, surgical, and mixed medical–surgical ICUs at academic tertiary care hospitals or community hospitals.All patients aged 18 years or older who survived an ICU stay of 3 calendar days or more were eligible for inclusion. We excluded patients who died during the ICU stay, patients under trusteeship, patients whose diminished mental capacity (as assessed by the bedside ICU nurse at her discretion) prevented the administration of the IPREA questionnaire, patients who did not understand French sufficiently to answer the questions, and patients transferred to another ICU while mechanically ventilated. During the IPREA3 study, all 34 ICUs enrolled patients during three 1-month periods (at baseline and 6- and 12-month post-baseline), respectively, in October 2014, April 2015, and October 2015. The patient sample for this study comprised all patients included in the IPREA3 study during these three periods, regardless of the group, control or interventional, resulting from the randomization of the ICUs.“Have you had more pain than usual for you? For example from needles, catheters, tubes, being turned or washed”. As each of the discomfort items, the pain-related discomfort was scored from 0  to 10  [The pain-related discomfort score, used as the dependent variable, is derived from the IPREA questionnaire and corresponded to item 9 and the following question: comfort) . Each pacomfort) . The ranRegulatory monitoring was performed in accordance with the French law requiring the approval of the French Ethics Committee . All records and patient identities remained confidential in accordance with the regulations of the French National Committee of Informatics and Liberties  and the French Consultative Committee for data processing in health research . Consent was obtained from all participants.Demographic and clinical characteristics were collected from patients at admission or during their first day in the ICU, including age, sex, first 24-h Simplified Acute Physiology Score (SAPS II), scores exploring prior health status , reasonP value of ≤ 0.10, and with potential interest for adjustment (group and inclusion month). The center was used as within-subject effect. The final models produced beta coefficients and standard errors. Independent variables with higher beta coefficients are those with greater relative effect on pain-related discomfort score. Statistical analyses were performed in accordance with the statistical analysis plan using SPSS software . All tests were two-sided. P < 0.05 was considered statistically significant.Sample characteristics were described using numbers and percentages, mean and standard deviation (SD), and median and interquartile range (IQR). Pain-related discomfort scores were compared between subgroups , seven were medical ICUs, and 15 were mixed  ICUs. Twenty-two of the 34 ICUs were located at tertiary care hospitals; 12 were at community hospitals. During the three 1-month enrollment periods of the IPREA3 study, 2130 patients were included; 634, 758, and 738 were included in November 2014, April 2015, and November 2015, respectively. Table The median pain-related discomfort score was 3 IQR 0–5). From the univariate analysis, age was negatively correlated with pain-related discomfort score and positively correlated with the ICU stay duration. Medical patients reported less pain-related discomfort than did surgical patients, and patients treated with MV during their stay reported greater pain-related discomfort than did other patients Table . Patient. From thWe used a large sample of 2130 French ICU patients treated at academic tertiary care and community hospitals to determine factors modulating pain-related discomfort during ICU stays. All the patients were previously included in the IPREA3 study, whose primary endpoint was the overall discomfort score derived from the French 16-item IPREA questionnaire. This first version of the IPREA questionnaire was validated  through a process based on international guidelines using a large sample of critically ill patients hospitalized in different ICU types  at different institutions  . HoweverTo our knowledge, this was the first study exploring the weight of a large panel of potentially pain- or discomfort-inducing procedures or treatments used to treat critically ill patients.The four procedures undergone by patients during the ICU stay that were associated with greater pain-related discomfort were chest drain removal, bladder catheter use, CVC insertion, and intra-hospital transport.Chest drain-related procedures  seemed to more strongly affect pain perception during ICU stays than did most other procedures considered invasive or discomfort-inducing . It is also important to note that no specific mandatory measure to reduce pain associated with either chest drain insertion or chest drain removal has been implemented during the study period in the participating ICUs. Previous studies that found chest drain-related procedures to be painful , 22 ofteIntra-hospital transport during ICU stays was also associated with self-reported pain-related discomfort. Intra-hospital transport of critically ill patients is a well-known risk factor for unexpected adverse events and complications, such as pulmonary complications, hemodynamic complications, infection, interruption of critical drugs, etc. –31, but As previously reported, younger people were also independently associated with higher self-reported pain-related discomfort scores compared with those of older people .Sex, patient type , prior health status, and ICU stay duration were not independently associated with pain scores.We used a comprehensive approach because the patients interviewed at the end of the ICU stay were not specifically informed of precisely what happened to them during their ICU stay before the discomfort assessment. Moreover, the interviewed patients were not asked if they remembered having undergone a particular procedure but only to score each of the 18 discomfort items. For example, patients may be unaware of having a chest drain inserted or withdrawn, a bladder catheter, a CVC insertion or being transported outside the ICU at any time during their stay. The healthcare team did not control or influence patients’ memories, thus reducing any suggestive bias. Moreover, the patients were not interviewed immediately after the potentially painful event as is the case in most studies aiming to assess or prevent procedural pain .Our study had several limitations. First, even if the bedside nurse asked patients to rate the severity of each discomfort for the entire stay in the ICU, patients may have reported discomforts, including pain, experienced only during the final part of the ICU stay if they could not remember what they experienced at the beginning of their ICU stay. Thus, events occurring during the last days of their ICU stay, such as chest drain removal after the clinical situation improved, may have had more influence on the pain-related discomfort score than did events that occurred at the beginning of their ICU stay, such as arterial catheter insertion or bronchoscopies. Future studies should record and analyze the time course of all procedures and life support therapies undertaken during the ICU stay. Second, all discomfort item scores, particularly the pain-related discomfort scores, were obtained with the help of a nonblinded bedside nurse (or assistant nurse), which could introduce bias. We accepted that conducting a pragmatic trial to explore potential benefits in the ICU with such an easily reproducible program would require participation from bedside nurses. Third, we could not assess the link between potential painful or discomfort-inducing procedures or life support therapies and the overall ICU-induced pain burden experienced in nonsurvivors during their ICU stay because ICU nurses administered the IPREA only to survivors on the day of discharge. This limitation also applied to patients who could not be questioned such as those with diminished mental capacity, those with language barriers, those who were transferred to another ICU while mechanically ventilated, or those who were urgently discharged. The issue of representativeness of this sample of ICU survivors among the general population of critically ill patients remains uncertain. Fourth, we could not analyze the impact of analgesic or sedative drug dosages or nonpharmacological therapies used during the procedures or the implementation of life support therapies. Fifth, we did not record details concerning chest drain-related procedures , 36. DraHowever, despite these limitations and without being able to affirm the causal link between these events and the painful memorization of the ICU stay at discharge, these first results showing an association between the occurrence of such potentially painful procedures and the pain-related discomfort scoreshould encourage us to properly assess the balance of the benefits and risks of these common procedures such as maintenance of a bladder catheter, intra-hospital transport for an iterative CT scan, CVC insertion or thoracic drainage.Our study showed that adult ICU patients who underwent chest drain removal, bladder catheter, CVC insertion, and intra-hospital transport during their ICU stay reported higher pain-related discomfort scores (regarding the entire ICU stay and assessed at the end of the ICU stay) than patients who did not experience these events. This study may pave the way for further targeted studies aiming at investigating a causal link between these common procedures undergone by adult critically ill patients during their ICU stay and recalled pain at ICU discharge.


Monteggia equivalent lesion represents a series of combined elbow and forearm injuries that resemble typical Monteggia fracture either in presentation or mechanism. The term has gradually evolved since its introduction, as sporadic case reports continued to complement it. The aim of this study was to present a furthermore type of that lesion which no previous study had reported and arouse pediatric orthopedists’ additional awareness of it.A 11-year-old girl, whose injury pattern initially appeared to be a mild radial neck fracture with undisplaced proximal ulnar fracture, and without radial head dislocation, was treated with closed reduction and long-arm splint immobilization. Acceptable results were acquired at first-week follow-up, yet dramatic changes turned up 2 weeks later when the dislocated radial head was found. A further reduction to the fracture and joint site only resulted in a subluxated and incongruous radiocapitellar joint on the three-dimensional computed tomography (3D-CT). Then a definitive operation was performed, which involved a Boyd incision, correction of radial head tilting, opening wedge osteotomy of the proximal ulna and proper fixation respectively. And acceptable results were achieved 1 year later.This case, with occult proximal ulna fracture, angulated radial neck fracture, subsequent radiocapitellar dislocation, and articular incongruity, was deemed as a rare Monteggia type-one equivalent fracture-dislocation variant rather than an ordinary radial neck fracture and it facilitates further understanding and management of the Monteggia fracture. Monteggia fracture, named after Giovanni Monteggia in the nineteenth century and well described and classified by Dr. Bado decades ago, involves ulnar fracture and concomitant dislocation of the radial head. The term “fracture” has gradually been replaced by those like “lesion”, “fracture-dislocation”, “injury” in literature, stressing the importance of radiocapitellar joint and reflecting an increasing awareness of the complexity regarding its manifestation and mechanism among pediatric orthopedists.Apart from the four distinctive patterns, the so-called concept of “Monteggia equivalent lesion/variant” has since been considerably expanded with decades of sporadic reports. A quite number of these types tend to be misdiagnosed or neglected due to the occult presentation of radiocapitellar joint or bowing ulna on radiographs, especially in pediatric patients when immature epiphysis interferes with judgment.The case we presented represents one of the tricky Monteggia equivalent fracture-dislocation variants that no previous study has ever reported. Pediatric orthopedists’ additional awareness of this variant facilitates further understanding and management of the Monteggia fracture.An 11-year-old right-hand dominated girl initially presented at our clinic with X-rays taken at a community clinic. It was 1 day after she fell off a chair with the affected upper limb stretched out for a book on a shelf. The X-rays Fig. . DramatiEarly in 1814, Monteggia  in 1967 Up to date, through literature in Scientific Citation Index, we found a few authors had referred to the terms of “equivalent lesion/injury/fracture-dislocation” –19, someMost of the cases demonstrated acute dislocation of radiocapitellar joint or acute radial shaft and neck separation but intact joint while only rest two showed some sort of chronic change of proximal radius region , 19. As Among those cases, the one Ruchelsman  presenteDue to the rarity, the mechanism for this lesion have to be inferred from those resembling cases. As for pediatric radial neck fracture, the classical mechanism was introduced by Jeffery as the compression force on the radial side and the associated traction lesions on the ulnar aspect . While tSpecifically in this case, the initial X-ray Fig.  would haOn considerations of the above, we might take some management notes for pediatric radial neck fracture with proximal ulna involved. Initial closed reduction should be properly handled with a sufficiently immobilized cast or double splints. The magnetic resonance imaging (MRI) or elbow arthrography are better ordered when plain X-ray reveals tricky manifestation. Finding potential ulnar bowing and annular ligament rupture will facilitate predicting the tardy dislocation of the radiocapitellar joint. Once a delayed radial head dislocation developed, especially combined with humeroradial joint incongruity and potential ulnar bowing, indication for surgery could be explicitly clear. Radial neck tilt ought to be corrected, to avoid incongruity of the radiocapitellar joint even if the joint was reduced. A process to use a K-wire to lever the radial neck and intramedullary nail to stabilize it was suggested by Ceroni  to acquiPediatric Monteggia equivalent lesion remains a tricky condition to distinguish due to occult presentation on plain radiographs of unclear epiphysis-comprised radiocapitellar joint or underlying hard-to-discern bowing ulna. Diagnostic and treating dilemma regarding it was abundant in the literature. However, this report presented a previously undescribed type-I Monteggia equivalent lesion with a prior radial neck fracture, late radiocapitellar dislocation, and proximal ulna crack. The inferred mechanism and managing procedure, though unable to be a guideline due to the low-evidence nature of this study, was still instructive for those dealing with pediatric elbows. The authors suggest ordering the MRI or elbow arthrography when plain X-ray reveals tricky manifestation in those radial neck fractures with proximal ulna involved. Finding potential ulnar bowing and annular ligament rupture will facilitate predicting the tardy dislocation of the radiocapitellar joint. Though initial closed reduction is still optimal, close follow-up is firmly required and proper surgery procedure is needed when dislocation of redial head turns chronic.


In this article we review the multiple effects of Hippo-YAP signaling in osseointegration of dental implants by regulating osteogenesis, angiogenesis and osteoimmunology in peri-implant tissue, as well as highlight prospective future directions of relevant investigation.The social demand for dental implantation is growing at a rapid rate, while dentists are faced with the dilemma of implantation failures associated with unfavorable osseointegration. Clinical-friendly osteogenesis, angiogenesis and osteoimmunology around dental implants play a pivotal role in a desirable osseointegration and it’s increasingly appreciated that Hippo-YAP signaling pathway is implicated in those biological processes both  With the increasing social burden of growing elderly population, some age-implicated diseases are changing people’s demand for medical services including tooth loss. American Association of Oral and Maxillofacial Surgeons illustrates that 70% adults between 35 and 44 years old have at least one permanent tooth lost and 26% adults lose all by 74 years old, which has significant impacts on general health physically and mentally through direct and indirect mechanisms . TherefoBrånemark originally proposed the concept of osseointegration to describe the direct and stable connection between bone tissue and titanium implants. Zarb proposed a clinical description that it was a clinically asymptomatic fixation of functional-loaded implants . A desirA favorable osseointegration of bone-implant interface is attributed to peri-implant osteogenesis, angiogenesis and osteoimmunology properties. On the one hand, these three factors play their respective important roles in regulating the process of osseointegration. Peri-implant bone osteogenesis is indispensable for stability and function of dental implants, which is regulated by the dynamic balance of osteoblasts, osteoclasts, osteocytes, etc. . AngiogeHippo-YAP, a highly implicated pathway, is known to be involved in regulating organ size, tissue regeneration and cancer development. Hippo signaling senses and responds to upstream cell biomechanical cues including cell contacts, cell polarity and other biomechanical signals. MST1/2 and SAV1 are phosphorylated and activate the complex of LATS1/2 and MOB1A/B, thus activating downstream reaction . NegativThe concept of osteoimmunology was first established by Arror et al., emphasizing the interaction between skeletal and immune system . On the Inflammatory response around dental implants is usually considered a pathological process. In fact, as a process of direct connection between foreign body and bone tissue, osseointegration would initially raise the foreign-body reaction to implants. The foreign-body reaction starts with layer of proteins that come from blood and interstitium immediately forming on the surface of biomaterial after implantation, which activates inflammatory reaction of related cells as a result . Researcin vivo the two extreme polarization states hardly exist since in most cases macrophages display both M1 and M2 characteristics phenotypes and exist as an intermediate state along the polarization spectrum , which are equally necessary in angiogenesis and vessel remodeling. Moreover, the process of angiogenesis involves several signals and pathways, mainly including growth factors , Notch signaling, MMPs and so on .Hippo-YAP responds to a variety of upstream signals in both intracellular and extracellular microenvironment. Hippo-YAP takes part in cell biomechanical respond mechanism to ECM, specifically, low ECM resistance leads to inhibiting activation of YAP/TAZ while high ECM resistance stimulates YAP/TAZ nuclear translocation . What’s After dephosphorylation and nucleus translocation, YAP bonds on the enhancer of the target gene with TEAD1-4 to regulate gene transcription. It has been revealed by ChIP-seq data that AP-1 is also widely recruited to transcription regulatory sequences with YAP/TAZ-TEAD complex . By reguThere is strong evidence indicates that BMSCs are regulated by some biological signals on differentiation into bone tissue via Hippo-YAP pathway. The knockout of GNAS activates Hippo signaling pathway and eventually suppresses osteogenic differentiation of BMSCs , consistHippo-YAP signaling pathway is also involved in osteoblast differentiation through multiple biological mechanisms. The suppression of YAP may induce lowering ligand bmp2a in MSCs, leading to a severe damage of Bmp signals in osteoblasts nearby, which downregulates osteoblast differentiation through a cell non-autonomous way . In a rein vitro that Polygonatum sibiricum polysaccharide (PSP) suppresses Hippo-YAP pathway to inhibit differentiation of osteoclasts from BMMs through downregulating expression of miRNA-1224, of which the target gene is Limd1  exhibits increased osteoclast differentiation and conversely MST2 overexpression inhibits it . Zhao etis Limd1 . Additiois Limd1 . Althougis Limd1 . Osteoclis Limd1 .Crosstalk between osteoblasts and osteoclasts can influence bone remodeling in a cell non-autonomous way. It is uncovered in a recent study that mechanical sensing protein PIEZO1 in osteoblastic cells promotes nuclear translocation of YAP and increases type II and IX collagens expression through PIEZO1-YAP axis mechanistically, causing inhibition of osteoclast resorption activity .in vivo, which was mediated by perilacunar/canalicular weakened remodeling, indicating a perspective target for future study  as well  are differentiated from monocytes with hematopoietic system origin, while as tissue−resident macrophages, peritoneal macrophages (PEMs) are indicated to be independent of hematopoietic progenitors and originated from yolk sac in recent works . However+ and TCR+ macrophages  and alternatively activated macrophages (M2) according to the existing classification model, with specific cytokine profile and biologic functions, respectively. In fact, as two extreme activated states, M1 and M2 rarely exist in microenvironment since macrophages are actually polarizing along the polarization spectrum and present some transitional state signatures with both M1 and M2 hallmarks . In recerophages . In concIn this review we suggest that a clinical-friendly osseointegration is essentially determined by favorable osteogenesis, angiogenesis and osteoimmunology, with a complex series of peri-implant cellular and molecular events happening during those procedures. Moreover, Hippo-YAP signaling pathway plays pivotal multiple regulatory roles in osteogenesis, angiogenesis and osteoimmunology. In short, the potential effects of Hippo-YAP on promoting osseointegration will contribute to the discovery of prospective clinical therapy.AZ and HY designed the outline and drafted and critically revised the manuscript. JL designed the figures. JZ, YJ, and BW contributed to drafting the manuscript. LX contributed to designing and critically revising the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The purpose of this narrative review was to examine the applicability of IOS procedures regarding single and multiple fixed implant restorations. Clinical outcomes for monolithic zirconia and lithium disilicate restorations produced through a direct digital workflow were reported.A MEDLINE (Pubmed) search of the relevant English-language literature spanning from January 1st 2015 until March 31st 2020 was conducted. In vitro studies comparing digital implant impression accuracy by different IOS devices or in vitro studies examining differences in accuracy between digital and conventional impression procedures were included. Also, RCTs, clinical trials and case series on the success and/or survival of monolithic zirconia and lithium disilicate restorations on implants, manufactured completely digitally were included. In vitro and in vivo studies reporting on restorations produced through an indirect digital workflow, case reports and non-English language articles were excluded. The aim was to investigate the accuracy of IOS for single and multiple fixed implant restorations compared to the conventional impression methods and report on the variables that influence it. Finally, this study aimed to report on the survival and success of fixed implant-retained restorations fabricated using the direct digital workflow.For the single and short-span implant sites, IOS accuracy was high and the deviations in the position of the virtual implant fell within the acceptable clinical limits. In the complete edentulous arch with multiple implants, no consensus regarding the superiority of the conventional, splinted, custom tray impression procedure compared to the IOS impression was identified. Moreover, complete-arch IOS impressions were more accurate than conventional, non-splinted, open or close tray impressions. Factors related to scanbody design as well as scanner generation, scanning range and interimplant distance were found to influence complete-arch scanning accuracy. Single implant-retained monolithic restorations exhibited high success and survival rates and minor complications for short to medium follow-up periods.The vast majority of identified studies were in vitro and this limited their clinical significance. Nevertheless, intraoral scanning exhibited high accuracy both for single and multiple implant restorations. Available literature on single-implant monolithic restorations manufactured through a complete digital workflow shows promising results for a follow-up of 3–5 years. The origins of intraoral scanning technology (IOS) can be traced back in the early 1970’s when Dr Francoise Duret and coworkers pioneered the first dental intraoral digitizer to obtain an optical impression . Since tThe implementation of the direct digital workflow in fixed implant prosthodontics is not without difficulties. Two main contributing reasons to this are identified in the literature, one being the variations in partial and complete-arch digital scanning accuracy of different IOS devices  and alsoNewer IOS hardware and software versions are constantly being introduced by the manufacturers that claim improved scanning accuracy, improved user interface and better patient experience. In addition, new monolithic materials with improved mechanical and physical properties are introduced to the dental market claiming better aesthetics and higher long-term success and survival , 19.The aim of this narrative review was to present an overview on the current evidence regarding the implementation of the direct digital workflow in partial and complete-arch edentulous patients rehabilitated with implant-supported prostheses. Moreover, this review attempted to compare IOS accuracy to conventional implant impression procedures, identify the main clinical factors that influence IOS accuracy and report on the success and survival of the monolithic zirconia and lithium disilicate restorations produced with this particular clinical workflow.An electronic search of publications from January 1st 2015 to March 31st 2020 was conducted. The cut-off point (2015) was selected because the rate of advancement in scanner hardware and software  has acceThis review included randomized control clinical trials (RCTs), prospective and retrospective clinical trials, case series and in vitro studies focusing on intraoral digital implant impression accuracy. In vitro and in vivo studies comparing different IOS devices in terms of scanning efficiency were included. Studies comparing intraoral digitization to conventional implant impressions in terms of accuracy were also included. Reports on the accuracy of fit as well as on the success and/or survival of monolithic zirconia and lithium disilicate restorations on implants, produced through an IOS impression procedure were also identified and included in this review. In vitro and in vivo studies looking into the fit accuracy of restorations produced through an indirect digital workflow (laboratory scanning) were excluded. Case reports were also excluded. The search included only English-language articles. To further identify any missed articles, the reference lists of the included papers were screened.What is the IOS accuracy in single implant sites.How does IOS accuracy compare to conventional impression accuracy in short-span and completely edentulous implant sites.What are the factors influencing IOS’s accuracy.What is the survival and success rate of monolithic implant-supported restorations manufactured using the direct digital workflow.The following questions were formulated and addressed in this review:Initial search identified 483 references. After application of the exclusion criteria, 72 references were eligible to be included in this review. Data from these studies regarding the type of IOS used, type of conventional impression used, impression accuracy in μm, type of study, reference scanner used were extracted and are presented in Tables Several in vitro studies were identified examining the scanning accuracy of IOS in single-implant edentulous sites Table . IOS scaAdditional implant positional discrepancy can be expected when a physical master model is digitally produced. Revilla-Leon et al.  in an inRegarding implant-rehabilitated short-span edentulous sites, several studies have compared the IOS accuracy of various scanner devices to the conventional impression accuracy Table . DigitalComplete-arch IOS accuracy of multiple implant impressions has been studied intensively in the past 5 years. The conventional method, utilizing elastomeric impression materials and multiple implant impression post splinting, has been the gold standard against which the accuracy of various scanners was tested Table . CurrentThere appears to be scientific evidence in the available literature, however, regarding the superiority of the digital intraoral scanning method in relation to the conventional, non-splinted elastomeric impression technique using either an open or a closed custom tray for complete-arch impressions , 40. In Besides the conventional and the complete digital workflow for edentulous arches with multiple implants, there is always the option of producing a 3D-printed or milled cast from the IOS impression and using this as the master model. Research on the accuracy of such models produced using rapid prototyping techniques is scarce Table . PapaspySeveral in vitro studies have been conducted comparing different scanner devices regarding both partial –47 and cSeveral clinical factors contributing to the global deviations in complete-arch intraoral scanning have been identified and studied in the literature. Operator experience is one clinical parameter that has been reported to influence scanning accuracy in a study utilizing an Active Wavefront Sampling (AWS) technology scanner (Lava COS)  but moreImplant angulation is another clinical factor that has been extensively studied for its effect on both partial and complete-arch implant digital impression accuracy. In the vast majority of studies, where single-part all-PEEK scanbodies were used as scanning posts for both partial , 24, 25 Further-on, regarding the design characteristics of scanbodies, their influence in scan accuracy has been tested in both partial and complete-arch digital implant impressions. The refractory and reflective indexes of all-PEEK scanbodies have been reported to be beneficial for complete-arch scan accuracy , 61. AddLighting conditions during a scanning session have recently been reported to influence global scanning accuracy. Research has shown that each IOS device scans more accurately in specific lighting conditions , 72 thatClinical studies regarding implementation of the complete digital workflow have been reported in the literature Table  with theIn the available literature on monolithic zirconia crowns, success ranged from 92 to 100% and survival ranged from 97.5 to 100% for a follow-up of 1–3 years –81. TechAvailable literature on monolithic lithium disilicate CAD-CAM crowns also shows encouraging results. Short-term in vivo studies exhibited a success rate of 89–100% and a survival rate of 100% for a follow-up of 2–3 years , 81. TecJoda and coworkers reported on a cohort of patients rehabilitated with single-implant, porcelain-layered zirconia crowns following an IOS impression and a digital model fabrication process , 84. At A recent in vivo study reported on the fit of 2,3 and 4-units zirconia fixed partial dentures on 2 implants following a complete digital workflow  Table 77. The auWith regard to time efficiency of the digital workflow, several studies have reported statistically significant shorter clinical and laboratory working times for the complete digital, compared to a semi-digital or conventional workflow for single-implant rehabilitation , 86–88. Within the scope of this review, no studies on the complete direct digital workflow for rehabilitation of multiple implants in edentulous arches were identified. Systematic reviews on the success and survival of implant-supported, zirconia complete fixed dentures fabricated through a conventional impression workflow, suggest that the use of monolithic or minimally veneered zirconia frameworks may help eliminate frequent complications encountered with veneering porcelain chipping , 91. MinBased on this literature review, the following can be concluded:The vast majority of identified studies were in vitro and this limited their clinical significance. Important clinical factors such as scanning accuracy and prosthesis’s misfit and their effect on technical or biological complications can only be studied effectively in longitudinal in vivo studies. For the single and short span implant sites, the IOS accuracy was high and the deviations in the position of the virtual implant fell within the acceptable clinical limits. When a semi-digital approach was elected, higher deviations in the position of the implant platform could be expected due to accumulated discrepancies in the 3D printing or milling fabrication process of the master model.In the complete edentulous arch with multiple implants, there was no consensus regarding the superiority of the conventional, splinted, custom tray impression procedure compared to the IOS impression. On the contrary, digital complete-arch impressions were more accurate than conventional, non-splinted, open or close tray impressions. 3D-printing of the master model could induce further discrepancies in the digital workflow depending on the printing technology and materials used.Newer generation scanners exhibited complete-arch deviation levels below the current acceptable threshold. Operator experience was not an influencing factor for complete-arch accuracy with newer scanners but critical experience level is yet to be determined. Lighting conditions during scanning can influence IOS device accuracy.All-PEEK, one-part scanbodies with cylindrical shape, smooth surfaces and adequate length were preferred. Implant angulation did not influence IOS accuracy when scanbodies with the above features were used. Both scanbody manufacturing tolerances and congruence between scanbody IOS mesh and CAD file have been shown to influence scan trueness and precision.Increasing scanning range and inter-implant distance can influence scan accuracy. Using auxiliary removable devices and interconnecting the scanbodies making sure not to disrupt their shape and size for correct digital registration, showed promising results. Limited evidence also suggested that implant connection type did not influence scan accuracy.Regarding the complete digital workflow, for single implants cases, monolithic restorations exhibited high success and survival rates with minor technical complications for short to medium follow-up periods (3–5 years). Patient acceptance and total clinical and laboratory time efficiency has also been reported to be high. For multiple implants, this workflow has not yet been documented adequately for clinical use. Future studies on outcome measures such as patient acceptance, time efficiency, and technical and biological complications of multiple implant-supported prostheses should be conducted to draw clinical conclusions.


Phenotypes extracted from Electronic Health Records (EHRs) are increasingly prevalent in genetic studies. EHRs contain hundreds of distinct clinical laboratory test results, providing a trove of health data beyond diagnoses. Such lab data is complex and lacks a ubiquitous coding scheme, making it more challenging than diagnosis data. Here we describe the first large-scale cross-health system genome-wide association study (GWAS) of EHR-based quantitative laboratory-derived phenotypes. We meta-analyzed 70 lab traits matched between the BioVU cohort from the Vanderbilt University Health System and the Michigan Genomics Initiative (MGI) cohort from Michigan Medicine. We show high replication of known association for these traits, validating EHR-based measurements as high-quality phenotypes for genetic analysis. Notably, our analysis provides the first replication for 699 previous GWAS associations across 46 different traits. We discovered 31 novel associations at genome-wide significance for 22 distinct traits, including the first reported associations for two lab-based traits. We replicated 22 of these novel associations in an independent tranche of BioVU samples. The summary statistics for all association tests are freely available to benefit other researchers. Finally, we performed mirrored analyses in BioVU and MGI to assess competing analytic practices for EHR lab traits. We find that using the mean of all available lab measurements provides a robust summary value, but alternate summarizations can improve power in certain circumstances. This study provides a proof-of-principle for cross health system GWAS and is a framework for future studies of quantitative EHR lab traits. Electronic Health Records (EHRs) have emerged as an abundant data source for deriving phenotypes used in genetic association studies. EHRs provide a broad range of clinical data in large health system cohorts and are readily incorporated into large-scale meta-analyses. The abundance of available data in EHRs introduces unique technical challenges, particularly longitudinal clinical lab measurements which lack the structure of more commonly used disease diagnosis codes. Conflicting strategies exist in the literature and it is not clear how portable these strategies are across health systems. In this study we performed a proof-of-principle meta-analysis of 70 clinical lab traits in two large-scale health systems: BioVU from Vanderbilt University and the Michigan Genomics Initiative from Michigan Medicine. Despite the challenges of matching labs across the two health systems, we observed a high replication rate for known genetic variants. Further, we identified 31 novel associations, 22 of which replicated in an independent BioVU cohort, indicating the potential for future meta-analyses. Finally, we explored the impact of various analytic strategies, looking for consistent effects between our two cohorts, to determine optimal strategies for future genetic analysis of EHR-derived lab traits. Laboratory testing is a key component of modern medicine. Laboratory measurements provide a glimpse into the functioning of the human body, allowing clinicians to diagnose and monitor disease. In most health systems, lab measurements are routinely captured in patient Electronic Health Records (EHRs) alongside disease diagnoses, free text notes and medical procedures to provide a detailed, longitudinal health history . EHRs prPSCK9 was initially discovered based on quantitative LDL measurement rather than clinical diagnosis [Several large-scale genetic studies have already leveraged biobanks linked to EHRs, such as the UK Biobank , Japan Biagnosis . In contiagnosis –13. HereThe rich source of quantitative lab measurements in EHR cohorts comes with unique concerns. Quantitative traits collected specifically for research purposes typically use a controlled experimental design to ensure consistency among samples. In contrast, lab measurements contained in EHRs are a historical record of medical care. As such, patients may have hundreds of lab measurements for some traits and none for others, depending on their specific health history and utilization of the health system. The measurements can be collected in times of sickness or good health leading to substantial variation in measurements for the same lab. Lab measurements can also be artificially modified by prescription medication, such as statin use for lowering LDL cholesterol. Moreover, recruitment mechanisms and health system demographics can dramatically shape the overall health of the biobank cohort, which in turn dictates lab measurements available for analysis. The broad impact of using such “real world” measurements for genetic association studies is unclear. Questions remain over the effect and robustness of analytic choices made when analyzing EHR-based lab traits including how best to summarize complicated, longitudinal lab measurements and whether comorbid diseases highly correlated with lab measurements must be considered. Prior studies are not consistent in addressing these concerns. For example, GWAS of EHR-derived quantitative traits in Biobank Japan enrolled patients with at least 1 of 47 diagnoses and controlled for all 47 diagnoses while testing each lab . In contIn this paper we explore strategies for analyzing quantitative lab measurements extracted from EHRs and describe the first large-scale meta-analysis of EHR-derived lab traits across independent health systems. We used lab measurements and genetic data from two academic health systems: the BioVU cohort from Vanderbilt University  and the Our initial challenge was identifying which labs to meta-analyze between the health systems. Accurately matching labs is complicated by the fact that no standardized coding scheme exists for lab measurements. Dichotomous disease traits are readily matched between health systems using the ubiquitous ICD coding system for disease diagnoses . AlthougThe meta-analysis required several strategic choices regarding data preparation and statistical analysis. We explored the consequences of various analytic choices using a series of mirrored analyses performed in MGI and BioVU. In particular, we varied the summary statistic for lab measurements and the inclusion of covariates to control for comorbid diseases in the GWAS. We compared the results between the independent biobank cohorts to assess consistency of effects. We hypothesized that alternative summary statistics to the basic mean could provide more powerful genetic analyses. We considered: the median lab measurement due to robustness against data recording errors and extreme measurements, the first available lab measurement to mitigate the effects of prescription drugs on modifiable lab traits, and the maximum recorded measurement to magnify variation in extreme measurements. The comorbidity analysis compared GWAS results from models that included indicator covariates for a wide array of diseases to models that did not.PheWeb web browser [http://pheweb.sph.umich.edu/mgi-biovu-labs and available for bulk download at https://phewascatalog.org/labwas and ftp://share.sph.umich.edu/mgi_biovu_labwas/.The complete set of GWAS summary statistics from this analysis are broadly available to the research community. We encourage others to use this data to replicate their own GWAS findings and perform hypothesis-driven lookups on specific SNPs or lab traits of interest. Our results are viewable through an interactive  browser  at http:We analyzed data from two university hospital biobanks that link electronic health records with genetic data: BioVU from Vanderbilt University and the Michigan Genomics Initiative (MGI) from Michigan Medicine. We restricted our analysis to unrelated patients of European ancestry because of insufficient patient sample sizes and a paucity of known variants in non-European populations.The BioVU cohort has been described previously . BrieflyThe MGI cohort has also been described previously . BrieflyWe extracted all available clinical lab measurements and metadata from the electronic health records of MGI samples and BioVU samples. We collapsed distinct labs when obvious duplications were present . Available metadata differed slightly between the health systems but included brief text descriptions, unit of measurements, and range for normal values. We excluded individual lab measurements taken outside the health system labelled as “External.” In cases where multiple tests examined the same analyte, e.g. blood glucose, we removed point of care (POC) tests which are more susceptible to technical artifacts and tend to be deployed in intensive care or emergency settings where acute disease or treatment effects supervene determinants of the underlying baseline ,23. Lab In order to study the effect of underlying health conditions we extracted ICD9 and ICD10 diagnosis codes from the EHR of the BioVU and MGI cohorts. We searched for diagnosis for 42 diseases with the potential to alter a clinical lab measurement . We starWe first performed GWAS analysis of each lab trait separately in the MGI and BioVU cohorts. We performed multiple GWAS for each lab, varying the statistic used to summarize the longitudinal lab measurements for each sample  and the inclusion of binary indicators for diagnosis comorbid diseases in the GWAS regression.epacts 3.3.0 [For each GWAS, the distribution of lab summary statistics was inverse normalized separately within the MGI and BioVU cohorts prior to regression analysis. In a separate analysis of the BioVU cohort, we determined that inverse normalization of lab values performed better than applying no transformation, or a log or square root transformation for controlling GWAS type I error. Genome-wide association tests were performed on the inverse normalized traits using additive linear regression models containing age, sex and four principal components as covariates. The comorbidity model controlled for disease status by inclusion of an additional 42 covariates for the binary disease phenotypes. The regression analyses were performed in the BioVU cohort using PLINK  and in tts 3.3.0 .p indicates a SNP that increases in significance  for the alternate summary statistic. A negative value of Δp indicates a decrease in significance for the alternate analysis. Scatterplots of Δp computed in MGI and BioVU summarize the magnitude and consistency of change in p-value significance between the cohorts  on a set of LD-pruned SNPs for each meta-analyzed lab.We meta-analyzed the GWAS results from the MGI and BioVU default analysis . The meta-analysis was performed using d errors . We compWe created a list of previously identified genetic associations for each analyzed lab trait using the GWAS catalog   or arithmetically-dependent traits , and therefore likely to indicate the same biological association. We report the “peak” or most significant SNP when a group of novel SNPs are in linkage disequilibrium.We performed a replication analysis of novel associations identified in the meta-analysis using an independent cohort of BioVU samples that became available after the original meta-analysis was performed. This replication cohort consisted of 29,043 European ancestry adult individuals with extant lab data recruited using the same procedure as the initial BioVU cohort, genotyped on the same MEGA genotyping array, and subjected to the same data QC procedure. We declared a novel association to be replicated if the replication p-value was <0.05 and the direction of effect was consistent with that from the meta-analysis.Data were collected according to Declaration of Helsinki principles. MGI study participants’ consent forms and protocols were reviewed and approved by the University of Michigan Medical School Institutional Review Board (IRB ID HUM00099605 and HUM00155849). Opt-in written informed consent was obtained for each MGI participant. BioVU is Vanderbilt University's biobank of DNA extracted from leftover and otherwise discarded clinical blood specimens. BioVU operates as a consented biorepository; all individuals must sign the BioVU consent form in order to donate future specimens.We extracted all available clinical lab measurements from the electronic health records (EHRs) for genotyped samples in two academic biobank cohorts: the Michigan Genomics Initiative  (MGI) atWe analyzed 70 labs matched with high confidence between the health systems and having at least 1,000 samples with the lab measured in each health system . We searλGC) confirmed the meta-analyses were well-controlled [λGC across all labs was 1.035, ranging between 0.995 and 1.103. Consistent with polygenicity [λGC values. The mean λGC for labs with zero testable catalog SNPs was 1.020. Labs with one to twenty testable Catalog SNPs had mean λGC of 1.028 and labs with greater than 20 testable Catalog SNPs had mean λGC of 1.066.The 70 EHR-derived lab traits were first analyzed separately in the cohorts using the mean measurement as the individual-level outcome. The meta-analysis sample size differed between labs, ranging from 7,429 for uric acid to 46,382 for hematocrit , reflectntrolled . The meagenicity , traits We first performed a replication analysis of the 1,313 GWAS catalog SNPs to validate the EHR-derived lab traits. Overall, we replicated 982 of the GWAS catalog SNPs, giving an overall replication rate of 74.8% . ReplicaSeveral factors influenced our ability to replicate individual catalog SNPs , each coReplication rate was also dependent on both the best previously reported p-value for the association and the sample size of the study reporting the association . Our repWe identified 264 SNP-lab trait pairs representing potentially novel associations. Based on visual inspection, these SNPs corresponded to 31 distinct peaks for which we report the lead SNP having the strongest association signal at each peak .We performed a replication analysis of the 31 lead SNPs using an independent cohort of 29,043 BioVU patients that became available after the initiation of our primary analysis. One SNP potentially novel for both immature granulocytes measures failed QC filtering in the replication cohort and could not be tested for replication. In total, we replicated 22 of the 31 (71%) novel associations . Among tTMPRSS6 (transmembrane serine protease 6), and rs8022180, an intronic variant in TRAF3, were shown to be associated with bilirubin and serum total protein level, respectively, in a Japanese population [GPT, a gene associated with alanine aminotransferase levels in the Korea Biobank [Several of our novel findings have biological or existing evidence that support the association. Three of the associations have recently been identified for the same lab in non-European cohorts. rs855791, a missense variant in pulation . rs11257 Biobank . Our resCTRB1 was associated with both amylase and lipase, clinical assays of pancreas function used to diagnose pancreatitis. While the SNP itself has previously been linked to blood protein measurements [CTRB1 gene encodes chymotrypsin, a component of digestive enzyme secreted by the pancreas, and was previously shown to be associated with alcoholic chronic pancreatitis [FUT9, a gene that showed association with diabetic neuropathy in a trans-ethnic meta-analysis [The intronic variant rs8051363 in urements , the CTRreatitis . A seconanalysis .AMY2B, AMY2A and AMY1) on chromosome 1, each of which encodes enzymes that digest starch into sugar [AMY2A, a pancreatic amylase enzyme, most common in populations historically lacking starch rich diets [The amylase-associated SNP rs1930212 resides near three amylase genes , a signaling protein involved in response to various hormones. Variation in GNAQ is associated with Sturge-Weber syndrome [One of our novel results for calcium, rs2839899, is an intronic variant in syndrome , a heredCOBLL1 previously linked to fasting blood insulin and Type 2 diabetes [NDUFAF6 and TP53INP1, has recent associations with T2D [Three SNPs showed associations with glucose. rs7607980 is a missense variant in diabetes –41. rs92diabetes . And, alwith T2D  and eosiwith T2D  among UKWe note that several associations occurred within the HLA region on chromosome 6, notably for glucose, hemoglobin A1C, and TSH. These variants are likely segregating with HLA types, which are strongly associated with various autoimmune diseases including diabetes and autoimmune thyroiditis, which have strong effects in these particular labs.We computed the genetic correlation between pairs of labs using LD score regression  to learnA prominent cluster of labs (top right corner of the heatmap) contains primarily white blood cell traits including measures of immature granulocytes, lymphocytes, monocytes and neutrophils. The immature granulocytes also showed a strong correlation with ferritin (ferrit), an iron storage and acute phase protein. Ferritin and immature granulocytes can both be elevated during severe acute inflammation, explaining this correlation.As expected, HgbA1C and glucose were strongly correlated. More interestingly, they also clustered with Red cell Distribution Width (RDW) and Erythrocyte Sedimentation Rate (SedRat). This cluster of labs showed negative associations with high density lipoprotein (HDL), mean cell hemoglobin concentration (MCHC), and mean cell hemoglobin (MCH). This supports a pathophysiology where the metabolic syndrome  is linked by complex mechanisms to persistent low-level inflammation (elevated SedRat), and anemia of chronic disease .We identified a cluster containing red cell indices–mean cell hemoglobin concentration (MCHC), mean cell hemoglobin (MCH), and mean cell volume (MCV)–with total bilirubin (Bili) and transferrin saturation (%SAT). This reflects the biology of hemoglobin–iron is carried to red cell precursors by transferrin and incorporated into heme and thence hemoglobin, red cells are filled with hemoglobin, and at the end of a red cell lifecycle, heme is broken down into bilirubin.Additional clusters include (1) calcium (Ca), albumin (Alb) and total protein in blood (TProt), (2) thyroid stimulating hormone (TSH) and lactate dehydrogenase (LDH), and (3) hematocrit (HCT), red blood cell count (RBC) and hemoglobin (Hgb) with free tetraiodothyronine (FT4). Albumin (Alb) is the major blood protein, so Alb levels are unsurprisingly correlated with total blood protein (TProt). Calcium homeostasis is driven by free calcium, while albumin acts as a calcium sink, therefore calcium (Ca) levels would reasonably be expected to correlate with Alb .Hematocrit (HCT), red blood cell count (RBC) and hemoglobin (Hgb) are interrelated measures of oxygen carrying capacity in blood and unsurprisingly correlated. In our study, they are also correlated with free tetraiodothyronine (FT4). Anemia  may be a feature of hypothyroidism (low FT4), and tetraiodothyronine—thyroid hormone—has been reported to play a role in red cell maturation ,48.A final cluster was identified linking thyroid stimulating hormone (TSH) to lactate dehydrogenase (LDH). Muscle breakdown, manifesting as weakness, is a feature of hypothyroidism, and therefore other laboratory anomalies seen in hypothyroidism include release of muscle enzymes including LDH ,49.We explored the impact of analytic choices on downstream analysis by performing parallel GWAS analyses in the MGI and BioVU cohorts with one of the analytic steps perturbed from our original analysis: either the individual-level statistic used to summarize longitudinal lab measurements  or the inclusion of covariates for underlying comorbid health conditions. We performed these analyses on the 22 lab traits for which there were least 20 testable GWAS catalog SNPs, using the catalog SNPs to interpret the effect of each analytic strategy on true risk variants.Overall, 13.3% of testable catalog SNPs showed a major change in significance when using the median as opposed to mean value for the summary statistic . The medIn comparison, the first available measurement and the maximum measurement had a greater impact on association p-values for catalog SNPs. In both cases, the alternate summary statistic was most likely to cause a concordant decrease in significance. Using the first available measurement resulted in concordant increase for only 3.1% of catalog SNPs, whereas 16.9% of catalog SNPs had a concordant decrease and 4.5% had discordant changes in significance. Using the maximum available measure had similar performance .Despite an overall trend of reducing significance of known risk variants, several related labs for blood oxygen carrying capacity did benefit from using the first available or maximum measurements. Red blood cell count (RBC), hematocrit (HCT) and hemoglobin (Hgb) each showed concordant increase in significance for several of their respective catalog SNPs without negatively impacting remaining catalog SNPs. This likely reflects red cell biology. Conditions that decrease oxygen carrying capacity, such as blood loss or iron deficiency are far more common than those that increase it, polycythemia vera or severe obstructive sleep apnea, for example. Thus, maximum measurement of an individual’s oxygen carrying capacity more likely represents the genetically determined set point.The comorbidity model, containing binary covariates for 42 comorbid diseases with the potential to alter lab values, produced the largest proportion of catalog SNPs (6.2%) with concordant increased significance in MGI and BioVU among the alternate analysis strategies considered . DespiteThe clearest example of a substantial and consistent effect on catalog SNPs between MGI and BioVU was for HDL and Mean platelet volume (MPV). Interestingly, in contrast to this result for HDL, LDL had no catalog SNPs with concordant increase in significance and seven catalog SNPs with concordant decrease.This study represents the first cross-health system study of EHR-derived lab traits at large scale. We performed meta-analysis GWAS of 70 lab traits and have made these association results easily accessible to the research community. Thoroughly dissecting each lab-SNP combination is a daunting task. Here, we focused on replication of GWAS catalog variants to validate our data and highlighted novel genetic associations. We anticipate that our full results, including those which do not reach genome-wide significance will be useful in replicating future novel results, in studies which synthesize findings across multiple SNPs, or in hypothesis-driven studies which require less stringent thresholds.Our study serves as a proof-of-principle for performing cross-health-system genetic analysis of EHR-derived lab traits. The high replication rate for known GWAS variants indicates that EHR lab traits can be well-matched between discordant health systems and that measurements taken during real-life medical interactions sufficiently reflect those taken under more idealized experimental conditions. Moreover, this implies that mechanisms underlying variation in lab traits among healthy populations also act in a health system population with diseased individuals, strengthening their clinical relevance. By comparing various analytic strategies, we show that there is no optimal strategy that holds across all lab traits. In fact, we observed many instances in which the alternate analysis simultaneously increased significance for some risk variants and decreased significance for others. Thus, even within a given lab trait, an optimal strategy for variant discovery might not exist. We also considered a summary statistic based on Area Under the Curve for the longitudinal lab data ,51. AnalThe primary strength of our study was the access to two independent biobank cohorts. Using two cohorts provides an increase in sample size and power over analyzing and reporting on each cohort separately. In addition, the two-cohort design adds a built-in internal consistency check to our results by requiring effect sizes to be in the same direction in both cohorts. This additional requirement reduced the potential for unknown biases in the health system cohorts to create spurious results when replicating GWAS catalog SNPs or novel association discovery. Further, the independent cohorts provided the means to rigorously examine the portability of analytic strategies between biobanks. A similar analysis performed in a single cohort could produce recommendations over fitted to one specific context. Use of multiple sites increases the generalizability of our recommendations. This study was further strengthened by the fortuitous availability of an independent tranche of BioVU samples that provided an immediate replication cohort for the novel findings of our meta-analysis.Our study has implications for the design and analysis of similar studies in the future. Matching and analyzing lab data between health systems is difficult and requires substantial content knowledge. This study benefited from a multi-disciplinary team consisting of clinical experts to lead the categorization of the raw lab data extracts and statistical geneticists to guide analytic strategies. We leaned heavily on GWAS catalog SNPs to serve as positive controls. We recommend researchers to incorporate an explicit replication step to validate lab data prior to testing novel hypotheses. Summarizing the longitudinal measurements simply using the mean proved relatively robust across labs but was by no means optimal in all scenarios. Future studies can benefit from considering a summary statistic suited to the specific lab trait being evaluated. Our analysis also highlights that close attention must be paid to differences in the preparation and analysis of EHR phenotypes, particularly longitudinal lab measurements. Failing to replicate a prior finding can be due to lack of a true effect but also a variety of differences between biobank cohorts and analytic procedures.We were motivated to examine the effect of controlling for disease status because of its use in the analysis of lab traits in BioBank Japan . ControlA limitation of studying clinical labs in real-life cohorts is that some measurements will be affected by medication. We were unable to formally address the effect of medication because of unreliable measurements of medication. However, it remains an important consideration for future EHR-based lab studies and requires further study. There was indication that in situations where a disease diagnosis is likely to be accompanied by medication, for example a diagnosis of dyslipidemia with lipid labs, controlling for disease status diagnosis serves as a reasonable proxy to treatment status. As research interest in EHR phenotypes increases, we anticipate that improved capture of prescription data will facilitate the identification of medication effects.A further limitation of this study is the number of analyzed genetic variants. The study was restricted to ~800K SNPs because BioVU imputed genotypes were unavailable at time of analysis. Although this limited our ability to discover novel variation, the number of SNPs was more than sufficient to perform the primary purpose of the paper, a proof-of-principle replication analysis across a broad range of clinical labs and analytic strategies. However, there are likely many loci remaining to be discovered for these labs, particularly the understudied traits.In conclusion, we report the first lab-wide genome-wide association study linking data between two independent EHR-based cohorts. We achieved a high degree of replication of prior associations and report a modest number of new associations. In melding these data sets, we addressed key questions in design and analysis of ‘real world’ data that are increasingly relevant.S1 Table(XLSX)Click here for additional data file.S2 TableThese associations have been reported at least once in a mixed-sex, adult, European-predominant population not selected for the presence of any disease.(XLSX)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S1 FigPlease refer to the Methods section for a complete description. The x-axis corresponds the fold changes for the SNP in MGI and the y-axis corresponds to the fold changes for BioVU. Positive log-fold changes indicate that the alternative statistic yielded a smaller (more significant) p-value than using the mean as a summary statistic. The upper-right (green) quadrant plots SNPs that decreased in p-value in both cohorts for the alternative statistic. The lower-left (blue) quadrant plots SNPs that increased in p-value in both cohorts. The two remaining quadrants indicate SNPs with discordant changes in p-value between the cohorts. GWAS catalog SNPs are plotted in red, novel SNPs for a given lab (if applicable) are plotted in purple, and the remaining SNPs are LD-pruned (for plotting convenience) and plotted in black. The white diamond displays an empirical null distribution of fold changes for non-associated SNPs. The first 22 pages display the three alternative summary statistics  for a single lab. The following six pages contain the analogous plots showing log fold change in p-values for the comorbidity model, which includes binary covariates for various comorbid diseases with the potential to impact lab measures, to a default analysis that does not account for comorbidities.(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.


As one of the most densely populated microbial communities on Earth, the gut microbiota serves as an important reservoir of antibiotic resistance genes (ARGs), referred to as the gut resistome. Here, we investigated the association of dietary nutritional content with gut ARG diversity and composition, using publicly available shotgun metagenomic sequence data generated from canine and feline fecal samples. Also, based on network theory, we explored ARG-sharing patterns between gut bacterial genera by identifying the linkage structure between metagenomic assemblies and their functional genes obtained from the same data.tet(W) was shared among the largest number of genera, predominantly among Firmicutes genera. Bifidobacterium, a genus extensively used in the fermentation of dairy products and as probiotics, shared tet(W) with a wide variety of other genera. Finally, genera from the same phylum were more likely to share ARGs than with those from different phyla.In both canine and feline gut microbiota, an increase in protein and a reduction in carbohydrate in the diet were associated with increased ARG diversity. ARG diversity of the canine gut microbiota also increased, but less strongly, after a reduction in protein and an increase in carbohydrate in the diet. The association between ARG and taxonomic composition suggests that diet-induced changes in the gut microbiota may be responsible for changes in ARG composition, supporting the links between protein metabolism and antibiotic resistance in gut microbes. In the analysis of the ARG-sharing patterns, 22 ARGs were shared among 46 genera in the canine gut microbiota, and 11 ARGs among 28 genera in the feline gut microbiota. Of these ARGs, the tetracycline resistance gene Our findings show that dietary nutritional content, especially protein content, is associated with the gut resistome and suggest future research to explore the impact of dietary intervention on the development of antibiotic resistance in clinically-relevant gut microbes. Our network analysis also reveals that the genetic composition of bacteria acts as an important barrier to the horizontal transfer of ARGs. By capturing the underlying gene-sharing relationships between different bacterial taxa from metagenomes, our network approach improves our understanding of horizontal gene transfer dynamics. The widespread use of antibiotics in human medicine, veterinary medicine, and agriculture has created unremitting selection pressure for antibiotic resistance since antibiotics were first introduced in the 1940s , 3 and tIn this study, we aimed to examine two different aspects of the gut microbiota, using publicly available shotgun metagenomic sequence data generated from canine and feline fecal samples. The first objective was to assess whether dietary nutritional content was associated with gut ARG diversity and composition by comparing these across different diet groups. Diet is one of the most influential factors shaping the gut microbiota –10. HoweThe second objective was to understand ARG-sharing relationships between gut bacterial genera by constructing ARG-sharing networks between genera, identifying genera that may play a key role in the horizontal transfer of ARGs, and assessing the extent to which ARG sharing between genera is constrained by bacterial taxonomic classification. We defined ARG sharing as the presence of a given ARG in different bacterial taxa. The recognition that horizontal gene transfer (HGT) plays a significant role in microbial evolution has encouraged us to consider a microbial community as a network of actors sharing genes. Recent studies have explored gene-sharing relationships between microbial genomes by applying network approaches to whole-genome sequence data –15. Howetet(W), tet(O), tet (44) (tetracycline resistance genes), mefA and mel (macrolide resistance genes), but a lower abundance of CfxA6 (a beta-lactam antibiotic resistance gene), compared with dogs with the baseline diet  . Likewise, the mean Shannon diversity index of ARGs was 19.8% higher in HPLC-fed kittens than MPMC-fed kittens  . Also, the mean Shannon diversity index of bacterial genera was 26.2% higher in HPLC-fed kittens than MPMC-fed kittens .A total of 23 ARGs were identified in ≥50% of the samples in both canine and feline data, with tetracycline and aminoglycoside resistance genes being the most frequent ARGs Fig.   test). In particular, HPLC-fed dogs showed a more distinct separation from those with a baseline diet than LPHC-fed dogs, as visualized in nonmetric multidimensional scaling (NMDS) ordinations , suggesting that samples with a similar taxonomic composition were more likely to show similar patterns of ARG composition than samples exhibiting different taxonomic composition.When ARG composition was assessed between the diet groups based on Bray-Curtis dissimilarity values, there was a statistically significant association between ARG composition and diet type in both canine and feline data  being detected in at least two genera in 93.8% (n = 60/64) of dogs and 75.0% (n = 9/12) of cats  global networks including all ARGs identified, and (ii) ARG-specific networks for which only one specific ARG was accounted for. A total of 46 and 28 bacterial genera were connected through the sharing of 22 and 11 ARGs in the canine and feline global networks, respectively Fig.   permutation test), and 2.3 times as high in the feline network , than the odds of sharing ≥1 ARG with genera belonging to different phyla  and feline  networks  networks were the largest, consisting of 21 and 12 bacterial genera belonging to four and two different phyla, respectively  network, Clostridium and Veillonella had the highest centrality measures in the feline tet(W) network, followed by Bifidobacterium. Macrolide resistance genes  and other tetracycline resistance genes, such as tet(O), tet(Q), and tet(44), formed relatively large canine and feline ARG-specific networks , tet(44), mefA, and mel were shared mostly or exclusively among Firmicutes genera, and tet(Q) among Bacteroidetes genera  or to those classified as critically important by the World Health Organization  [Some of the ARGs whose abundance was altered with dietary intervention also deserve special attention because they are known to confer resistance to antibiotics used frequently in primary care small animal veterinary practices  , 23. Therolides) . TherefoOur study also investigated the sharing of ARGs between bacterial taxa by identifying the linkage structure between metagenomic assemblies and their functional genes obtained from canine and feline fecal samples. Although gene sharing does not necessarily provide direct evidence for HGT, network approaches can provide new insights into microbial evolution because HGT inevitably creates networks of microbes over a wide range of evolutionary distances , 25. Sevtet(Q) was predominantly shared between Bacteroidetes genera in our study. tet(Q) has been associated with plasmids and conjugative transposons generally found in Bacteroides and close relatives, such as Prevotella and Porphyromonas (27–30). If these transmissible elements have been adapted to Bacteroidetes bacteria, they might have limited capacity to transfer genes to non-Bacteroidetes bacteria.Our networks show the extensive sharing of ARGs between a wide variety of genera in the canine and feline gut microbiota. The findings that genera from the same phylum tended to share ARGs and be classified into the same network community suggest that differences in the genetic composition of bacteria may limit the transfer and survival of ARGs in the new host genome. In particular, most ARGs tended to be shared exclusively by specific phyla. For example, tet(W) and lnuC, were shared extensively between different phyla, suggesting that transmissible elements involved in the transfer of these ARGs may have broad host ranges. In particular, tet(W) networks comprised the largest ARG-specific networks, consistent with the fact that tet(W) is one of the most prevalent tetracycline resistance genes in mammalian gut bacteria [Bifidobacterium had the highest centrality in both canine and feline tet(W) networks, suggesting that this genus has the potential to modulate the HGT dynamics of tet(W). Its high centrality could be explained by the flanking of tet(W) by transposase genes in Bifidobacterium [tet(W) from Bifidobacterium to other bacteria in the canine and feline gut microbiota. Considering the widespread use of Bifidobacterium in the fermentation of dairy products and as probiotics [tet(W) should be closely monitored when Bifidobacterium is used in food products.However, it should also be noted that certain ARGs, such as bacteria . Bifidobacterium . Transpoacterium . Thus, iobiotics , 30, ourOur study has some limitations. First, although MyTaxa, a homology-based taxonomy classifier used to annotate contigs to bacterial genera and phyla, has relatively high accuracy at the phylum and genus levels and is considered to be superior to other annotation tools , it is sOur study shows that dietary nutritional content alters the antibiotic resistance potential of the gut microbiota, supporting the hypothesis that there are intrinsic links between protein metabolism and antibiotic resistance. Future research should investigate whether such alteration in the gut resistome is indeed linked to the development of antibiotic resistance in clinically-relevant gut microbes. Our network approach shows the extensive sharing of ARGs across a wide range of canine and feline gut bacteria, suggesting that the gut microbiota serves as an important ARG reservoir and HGT hotspot. The modular network structure reflects the barriers to ARG spreading between bacterial genera, with phylum membership playing a significant role.We analyzed publicly available shotgun metagenomic sequence data generated by two previous studies , 3. Thes− 5 [After removing paired-end reads with low-quality bases , reads < 30 bases, and PCR duplicates from the data using the pipeline we described before , 34, we − 5 . We clas− 5  and comp− 5 and with more than 90% identity and 50% coverage. We obtained the normalized abundance of ARGs by summing the RPKM values of the genes aligned with the same ARG.For ARG annotation, we performed de novo assembly of paired-end short reads from each animal into contigs using IDBA-UD , 39. AftWe analyzed the canine and feline studies separately because their study designs were different. First, we identified the core ARGs, defined as the ARGs present in ≥50% of the samples. Second, we assessed the diversity of ARGs by computing the Shannon diversity index, which accounts for both richness  and evenness  . We hypoWe then assessed whether ARG composition was associated with dietary nutritional content in the following way. We computed Bray-Curtis dissimilarity values for all possible pairs of samples based on the normalized ARG abundance data. Bray-Curtis dissimilarity values range from 0 to 1, with higher values indicating more dissimilar ARG composition between two given samples. Based on these values, we ordinated samples in reduced space using NMDS  and perfFinally, we performed a Procrustes analysis to test the hypothesis that ARG composition is associated with taxonomic composition in the gut microbiota. Briefly, two NMDS ordinations by ARG and taxonomic composition were uniformly scaled and rotated until the squared differences between them were minimized . We thenCi and Cj annotated to bacterial genera Bi and Bj, respectively, contained predicted genes annotated to a specific ARG, Bi and Bj were assumed to share that ARG in their genomes. The predicted genes were assumed to represent the same ARG if their BLASTn sequence identity was 100%, to assess ARG-sharing relationships within the most recent molecular timescale. Networks were constructed for each animal species. They were unweighted and undirected, with nodes representing bacterial genera found to share ARGs in the sampled canine or feline gut microbiota. Two bacterial genera were linked by an edge if at least one ARG was found on contigs belonging to these two genera and originating from the same animal. For each animal species, we constructed two different types of network: (i) global networks including all ARGs identified in the gut microbiota, and (ii) ARG-specific networks for which only one specific ARG was accounted for. For example, while an edge represented the sharing of ≥1 ARG of any kind in the global networks, in a network specific to the tetracycline resistance gene tet(W), an edge represented the sharing of ≥1 tet(W) genes between two bacterial genera. The global networks showed the overall distribution of ARGs across microbial taxa, whereas ARG-specific networks revealed patterns specific to individual ARGs.We constructed networks that described ARG-sharing patterns between gut bacterial genera based on taxonomic and ARG annotation of shotgun metagenomic sequence data Fig. . For taxFor both network types, we assessed the centrality of each genus by computing the degree, eigenvector, and betweenness using the igraph package  in R 4747 to ideThe structure of each global network was then characterized. First, we performed a QAP logistic regression to assess whether genera from the same phylum were more likely to share ARGs than with those from different phyla , 52. We p-values in this study were adjusted by the false discovery rate [All ery rate .Additional file 1:Table S1. Summary statistics of de novo assembly.Additional file 2:Table S2. The Kendall rank correlation between centrality metrics in the canine and feline global network.Additional file 3:Table S3. Quadratic Approximation Procedure (QAP) logistic regression results.Additional file 4:Table S4. The edge density within and between structurally equivalent groups in the canine and feline global network.Additional file 5: The ARG-specific network of the canine and feline gut microbiota. (1) ARG-specific network of the canine gut microbiota: Figure S1 and Tables S5 and S7. (2) ARG-specific network of the feline gut microbiota: Figure S2 and Tables S6 and S8.