EYA4 is a member of the vertebrate eya gene family of transcriptional activators and plays several roles in both embryonic and inner ear development. The majority of EYA4 gene mutations are associated with autosomal dominant non-syndromic hearing loss (DFNA10). In addition, some mutations in this gene cause autosomal dominant syndromic hearing loss with dilated cardiomyopathy. EYA4 is a rare cause of sensorineural hearing loss, and only a limited number of papers regarding mutations in this gene have been published. Thus, detailed clinical features remain unclear.EYA4 pathogenic variant. Pure-tone audiometry revealed bilateral, nearly symmetric, moderate sensorineural hearing loss in the low and middle frequencies. Minor abnormalities were observed on the patient’s electrocardiogram and echocardiography without any apparent symptoms. Next-generation sequencing is effective in elucidating the etiology of hearing loss, and the present findings suggested the possible phenotypic expansion of deafness caused by EYA4 gene mutations.We conducted next-generation sequencing of a Japanese individual with progressive sensorineural hearing loss and identified an  To date, ~180 non-syndromic hereditary sensory hearing loss loci and more than 100 corresponding genes have been identifiedEYA4 is a member of the vertebrate eya gene family of transcriptional activators that interact with other proteins, such as the sine oculis homeobox (SIX)  protein family, to ensure normal embryologic development2. Moreover, EYA4 also plays several roles in the mature inner ear system3. The majority of EYA4 gene mutations are associated with autosomal dominant non-syndromic hearing loss (DFNA10) (OMIM:# 601316)6. In addition, some mutations in this gene cause autosomal dominant syndromic hearing loss with dilated cardiomyopathy (OMIM:# 605362)7. EYA4 is a rare causative gene of sensorineural hearing loss and only a limited number of papers regarding mutations in this gene have been published. Thus, the complete picture with regard to the clinical features associated with these gene mutations remains unclear.EYA4 pathogenic variant was identified.Remarkable recent advances in molecular genetics technology, particularly in the clinical applications of next-generation sequencing technology, have made it easier to identify disease-causing genes in a relatively short period and at low cost. Here, we report a Japanese individual with progressive sensorineural hearing loss (SNHL) in whom an A 43-year-old Japanese male from a non-inbred family presented to our hospital to clarify the cause of his hearing loss. His maternal grandmother, mother, maternal uncle, and sister also have late-onset hearing loss (after acquisition of language) Fig. . The proThe patient’s hearing loss was first noticed during a checkup at age 26. He began wearing a hearing aid at age 36. Further deterioration in his hearing was recognized subjectively at 40 years of age due to loud noises associated with his work as a carpenter. At the age of 41, his tinnitus and hearing loss further worsened after exposure to loud firework explosions. No noticeable effects were observed upon treatment with various medications, including steroids. Clinically, he has no accompanying disease or disorder, but abnormalities in his electrocardiograms have been observed since his second decade of life.Pure-tone audiometry revealed bilateral, nearly symmetric, moderate SNHL with hearing loss in the low and middle frequencies. The hearing loss was progressive, and an audiogram at age 43 showed hearing loss at all frequencies with a flat pattern Fig. . He has 8, no mutations were observed upon examination of 46 known mutations in 13 deafness genes. Secondary screening using comprehensive next-generation sequencing analysis for 63 genes reported to cause hearing impairment was performed as described previously9.There was insufficient power in the kindreds to perform linkage studies, and we could not obtain consent from any of his family members. Thus, molecular diagnosis was performed using genomic DNA extracted from the peripheral blood of the proband alone. In the primary screening using the Invader assayEYA4:NM_004100.4:c.1177 C > T:p.Q393X (in exon13) (hg19:chr6:g.133804239 C > T) was identified in the eyes absent 4 gene  in this patient. This result was confirmed by Sanger sequencing. This variation was previously reported as a pathogenic variant in a non-syndromic SNHL Korean pedigree10. This variant was absent in the 1,000 Genomes Project; 6,500 Exome Variant Server; 333 healthy controls in the Shinshu University Project; Human Genetic Variation Database and 2KJPN for the Japanese population databases; and the 1,000 controls of Iowa University. Only one case carrying this variant was present in the Exome Aggregation Consortium database . According to the pathogenicity classification based on the Association College of Medical Genetics (ACMG)11, we regard the p.Q393X variant as “pathogenic”.As a result of this genetic analysis, a heterozygous variation EYA4 mutations present with non-syndromic SNHL, but some also experience dilated cardiomyopathy and heart failure accompanied by SNHL7. We, therefore, performed a detailed cardiac examination to confirm whether the patient had a non-syndromic phenotype. His electrocardiogram revealed an abnormal Q wave at V1 and V2 , and eight are located in the variable region of the EYA4 transcripts. The affected individual in this study carrying p.Q393X showed a similar progression of hearing deterioration to that observed in DFNA10 patients15. Further, the audiometric configuration of the previously reported Korean SNHL pedigree with the same mutation resembles that of the patient in this study10. It is therefore possible that both a mutational hot spot and founder effect are involved.Allele variants in EYA4 gene mutations is a rare associated symptom. However, it should be noted that some cardiac abnormalities  were detected by echocardiography in three of nine cases with EYA4 mutations6. In terms of gene expression, EYA4 is expressed in the heart and the inner ear16. Normal cardiac gene expression is sensitive to the concentration of EYA4-SIX complexes;5 therefore, a partial EYA4 deficiency may induce small cardiac abnormalities despite a clinically normal status. Even in asymptomatic patients, it is possible to avoid overlooking potential cardiac degradation leading to more serious conditions, such as heart failure, by cardiac examination. The present data suggest that EYA4-associated hearing loss possibly exhibits an extensive spectrum of conditions ranging from minor abnormalities showing clinically normal heart function to dilated cardiomyopathy. It is hypothesized that the susceptibility to cardiomyopathy may be determined by the mutational position on the EYA4 transcripts;6 however, no information of any genotype–phenotype correlation is currently available. Any such correlation will become clearer as data are accumulated in the future.The dilated cardiomyopathy caused by EYA4 mutations.Next-generation sequencing is an effective strategy for elucidating the etiology of hearing loss with high genetic heterogeneity, and our findings will provide a basis for further exploration of the pathological mechanisms and appropriate counseling for patients with EYA4 gene mutation. Minor abnormalities were observed on the patient’s electrocardiogram and echocardiography without any apparent symptoms. The present findings suggested the possible phenotypic expansion of deafness caused by EYA4 gene mutations.In this paper, we report a Japanese patient with progressive sensorineural hearing loss carrying a rare autosomal dominant


When tumor cells disseminate to the skeleton, they are bathed in a rich milieu of hematopoietic cells. Bone marrow myeloid cells as resident macrophages appear poised to engulf apoptotic tumor cells in a manner similar to when they engulf normal apoptotic cells during development and homeostasis, a process termed efferocytosis. Intriguingly we identified a different program of events in a macrophage when it engulfs an apoptotic cancer cell versus a non-cancer cell . Upon efin vivo. Such an example was reported via inactivation of phosphatidylinositol 3-kinase-γ (PI3Kγ), which led to activation of cytotoxic CD8+ T-cells and tumor regression [in vivo. A recent study shows how hematopoietic cell kinase  accelerates the growth of colon cancer by inducing the tumor-promoting M2-like macrophage polarization [An attractive potential therapy is based on the hypothesis that a macrophage can be reprogrammed towards an anti-tumor phenotype rization , furtherin vivo delivery of siRNA that targets Stat3 linked to CpG oligonucleotide agonist of toll-like receptor 9 (TLR9) [In addition to NF-kB activation, the Stat3 activation state of macrophages deserves special attention since simultaneous NF-κB and Stat3 activation correlates with tumor promoting activity. Given the importance of Stat3 signaling in the pro-tumoral function of macrophages it is tempting to speculate that inhibition of PI3Kγ would induce tumor regression in correspondence with low Stat3 activity, and even more effective anti-tumor responses could be achieved with both PI3kγ and Stat3 inactivation. Both Stat3 and NF-κB are persistently activated in cancer-associated immune cells and are intimately related to resistance to targeted therapies. Direct inactivation of Stat3 signaling has been achieved in mice via 9 (TLR9) . This ap9 (TLR9) . The eluAnother intervention strategy would be to inhibit CXCL5-CXCR2 signaling, since CXCL5 increased with macrophage efferocytosis of apoptotic tumor cells and is increased in patients with prostate cancer metastasis . PharmacSince efferocytosis of prostate cancer cells induces the activation of an inflammatory profile in macrophages, the use of pro-resolving mediators including lipoxins, resolvins and others could represent important points of intervention in metastatic prostate cancer. These mediators are normally released by immune cells to resolve inflammation and restore tissue homeostasis, and hence could accelerate the clearance of dying cancer cells while activating anti-inflammatory signaling to decelerate tumor progression. A recent work showed that resolvins inhibited tumor growth via enhancing the clearance of dead cells , supportSince immunosuppression is a common denominator of tumor growth in different cancer types, novel therapies that reactivate the patient's immune system are emerging as potent strategies in the battle against cancer. Success in the clinical use of immune checkpoint inhibitors in different tumor types are great examples of their potential, and additional combinatorial therapies will enhance the effectiveness of these inhibitors. Our results present new insights to the mechanisms that provide tumor cell advantages in the skeletal microenvironment and open new possibilities to strategically deplete tumor cells while re-programming macrophages to clear away dead cells leaving an environment that allows for resolution.


We report a very rare case of severe pelvic disruption with an absolutely normal computed tomography scan due to the effective placement of a pelvic binder.Pelvic binders are routinely used in the prehospital setting for stabilization of pelvic injuries in patients with trauma. Emergency department trauma management relies on primary and secondary survey assessment and imaging, most often computed tomography, in hemodynamically stable patients. Maintaining the pelvic binder We report the case of a 49-year-old Caucasian man referred to our Emergency Department after a high velocity motorcycle accident. Primary assessment revealed a left wrist deformation and pelvic pain, and a pelvic binder was applied by paramedics. A total body computed tomography scan was performed after arrival in our Emergency Department and did not reveal any pelvic injury. The pelvic binder was removed and because of persisting symphyseal pain, pelvic plain radiography was performed revealing a pelvic disruption with an opening of the pubic symphysis and of the left sacroiliac joint (“open book” type pelvic injury) requiring surgical stabilization.Pelvic binders may mask pelvic disruption in patients with trauma. Pelvic plain radiography should be repeated after pelvic binder removal in patients with high velocity trauma and pelvic symptoms or neurological alterations limiting the reliability of clinical examination.The online version of this article (10.1186/s13256-018-1808-7) contains supplementary material, which is available to authorized users. Pelvic fractures occur in approximately 20% of multiple trauma cases, with a mortality rate of up to 20% when associated with hemodynamic instability , 2. EarlCurrent Advanced Trauma Life Support (ATLS) guidelines recommend chest and pelvic X-ray for all patients with blunt trauma. However, different studies suggest that pelvic X-rays have limited value compared with computed tomography (CT) scanning , 5 and tin situ may allow stabilization of pelvic injuries during imaging but might also hinder the visualization of some pelvic lesions [Pelvic binders have become the preferred method of stabilization for the prehospital management of suspected pelvic ring injuries and are used widely in the management of exsanguinating pelvic trauma. Various reports suggest that they may reduce unstable pelvic fractures and improve hemodynamic status –10. Main lesions , 12. Pre lesions , 12 We rA 49-year-old, previously healthy, Caucasian man, a motorcyclist, was referred to our Emergency Department (ED) after a high velocity frontal collision with a car. He was married and worked as a car body repairer. He had no tobacco smoking or drinking history and was not taking any medication. A prehospital primary survey assessment according to ATLS protocols revealed a hemodynamically stable patient with blood pressure (BP) of 136/82 mmHg and heart rate (HR) of 65 beats per minute (bpm) without airway or breathing alterations. He was oriented and conscious and reported pain in the symphyseal region and left arm. Cervical spine immobilization and intravenous access were obtained and a pelvic binder was applied by paramedics  and liver and renal functions were normal. The chronologic timeline of patient management and investigations is provided in the Additional file No relevant pelvic anomaly was detected Figs.  and 3 inBecause of the high velocity of the crash and persisting symphyseal pain, plain anteroposterior pelvic radiography was ordered shortly after the CT. Pelvic radiography revealed a non-osseous pelvic disruption, with an opening of the pubic symphysis (more than 2.2 cm) and of the left sacroiliac joint  –15


Moxifloxacin, an FDA-approved antibiotic, was used for labeling cells and its fluorescence was strongly observed in Paneth cell granules by TPM. Moxifloxacin labeling of Paneth cell granules was confirmed by molecular counterstaining. Comparison of Paneth cells in wild type, genetically obese (ob/ob), and germ-free (GF) mice showed different granule distribution. Furthermore, Paneth cell degranulation was observed in vivo. Our study suggests that TPM with moxifloxacin labeling can serve as a useful tool for studying Paneth cell biology and related diseases.Paneth cells are one of the principal epithelial cell types in the small intestine, located at the base of intestinal crypts. Paneth cells play key roles in intestinal host-microbe homeostasis via granule secretion, and their dysfunction is implicated in pathogenesis of several diseases including Crohn’s disease. Despite their physiological importance, study of Paneth cells has been hampered by the limited accessibility and lack of labeling methods. In this study, we developed a simple  Paneth cells are terminally differentiated cells residing at the base of intestinal crypts, interspersed with intestinal stem cells. Paneth cells are found only in the small intestine, but not in the colon, of healthy individuals. Paneth cells have densely packed secretory granules containing antimicrobial factors such as lysozyme (Lyz) and antimicrobial peptides (AMPs) for mucosal defense, and play key roles in maintaining intestinal homeostasis against commensal and pathogenic microorganisms via degranulation4. Paneth cells also help to sustain and modulate intestinal stem cells by secreting essential growth factors and regulatory molecules5. Deficiency or dysfunction of Paneth cells, therefore, is associated with microbial dysbiosis, tissue regeneration failure, and various inflammatory diseases in the gut9. On the other hand, severe inflammation and microbial imbalance caused by environmental stressors or vulnerable genetic predisposition can lead to functional alteration or apoptosis of Paneth cells. Moreover, presence of Paneth cells outside of the small intestine, called Paneth cell metaplasia, is often observed in patients with colitis and other gastrointestinal inflammatory diseases11.The small intestine is where most of the digestion and absorption of food takes place in the body. The intestinal epithelium also serves as a protective barrier against numerous microorganisms in the intestinal lumen and is composed of several cell types such as absorptive enterocytes, goblet cells, enteroendocrine cells, intestinal stem cells, and Paneth cellsex vivo tissues, because isolated Paneth cells did not survive in culture conditions. With recent advances in the intestinal organoid culture, long-term studies of Paneth cells are now possible, rendering molecular and cell biological dissection of Paneth cell functions much more feasible5. Despite the numerous advantages, however, the intestinal organoid culture system comprised only of epithelial cells is short of recapitulating the intricate cross-talks among epithelial cells, immune cells, stromal cells, and nerve cells that are present in the intact small intestine. Thus, it is highly desirable to develop a reliable method to study Paneth cells in situ in live animals.Historically, studies of Paneth cells mainly focused on their antimicrobial functions. Their studies had to rely on a short-term intestinal crypt culture or the immunostaining of 15. TPM is a nonlinear fluorescence microscopic technique, capable of three-dimensional (3D) cellular imaging of live organs with its relatively high-imaging depths and reduced photodamage17. Distribution and behavior of immune cells in the small intestine were studied by TPM with either immunofluorescent staining or transgenic (Tg) mice expressing fluorescent proteins13. Label-free TPM based on the intrinsic contrasts such as autofluorescence (AF) and second harmonic generation (SHG) was also used to image the intestine15. Recently, we introduced moxifloxacin as a non-specific cell-labeling agent for TPM19. Moxifloxacin is an FDA-approved antibiotic for the treatment or prevention of ocular and pulmonary infections and has excellent tissue penetration characteristics21. Moxifloxacin has an intrinsic fluorescence property and its two-photon (TP) fluorescence was characterized22. TPM of biological tissues with topical application of moxifloxacin ophthalmic solution showed approximately 10-fold fluorescence enhancement of cells compared to AF19.With the advance of microscopic techniques such as two-photon microscopy (TPM), intravital imaging has been used to study various animal organs including the mouse small intestinein vivo imaging method of using moxifloxacin and TPM for observing Paneth cells and their granules in the intact mouse small intestine. Distinctive granular structures of Paneth cells were clearly visible at the base of intestinal crypts when moxifloxacin-based TPM was used to image the small intestine from the serosa. Moxifloxacin labeling of Paneth cell granules was verified by counterstaining with specific fluorescent markers. Paneth cells of various mouse types such as wild type mice and genetically obese (ob/ob) mice raised in conventional specific pathogen-free (SPF) condition, and wild type mice raised in germ-free (GF) condition were imaged and compared. Furthermore, Paneth cell degranulation was monitored by time-lapse TPM imaging in vivo.Herein, we demonstrate a new In vivo moxifloxacin-based TPM of the small intestine in wild type C57BL/6 specific-pathogen-free (SPF) mice was conducted by using an intestinal holder . UEA-1 specifically binds to glycoproteins containing α-linked fucose that are highly enriched in Paneth cell granules and also present in Paneth cell membrane. To ascertain that the moxifloxacin-labeled granular structures indeed reside inside Paneth cells, the intact small intestine was labeled with moxifloxacin, rhodamine-UAE-1, and Hoechst 33342  in TPM images. The spectrally unmixed blue, green, and red channel images showed cell nuclei labeled with Hoechst 33342, granular structures labeled with moxifloxacin, and Paneth cell membranes labeled with rhodamine-UAE-1, respectively. The cells intercalated in between Paneth cells containing moxifloxacin-labeled granules were considered to be intestinal stem cells, based on their well-understood spatial distribution. Further investigation with Tg mice having a fluorescent marker for Lgr5 would conclusively identify intestinal stem cells23.der Fig. . The mouder Fig. . Moxiflolli Fig. . Since rIn vivo moxifloxacin-based TPM could visualize Paneth cell granules in the small intestine of wild type SPF mice. Since Paneth cells play central roles in maintaining the intestinal host-microbial homeostasis, different intestinal environments might be associated with alterations of Paneth cell granules24. Previous immunohistochemical analysis of the small intestinal sections showed reduced lysozyme contents of Paneth cell granules in obese humans and animals compared to lean controls26. Therefore, we decided to test whether moxifloxacin-based TPM could be used for detecting the alterations of Paneth cell granules in live mice under different environmental and metabolic conditions.ob/ob) mice raised in a SPF condition were compared to Paneth cells in lean, wild type C57BL/6 mice raised in a SPF condition (SPF). TPM with and without moxifloxacin labeling was conducted in these mice. TPM without moxifloxacin labeling was based on the intrinsic AF of cells and the higher excitation power was used than the one for TPM with moxifloxacin labeling  and DAPI (blue) for labeling Paneth cell granules and the cell nucleus, respectively  mice showed almost no granular structures. Instead, relatively high moxifloxacin fluorescence was detected in some cells (Supplementary Video ob/ob) mice also revealed comparable results with the moxifloxacin-based TPM images, showing few or no granules at the base of intestinal crypts. Our in vivo moxifloxacin-based TPM imaging results correlated well with the previous findings that lysozyme-containing Paneth cell granules are significantly reduced in obese individuals25. Collectively, in vivo moxifloxacin-based TPM of obese (ob/ob) mice showed quite different states of Paneth cells from those in lean SPF mice. In GF mice, moxifloxacin-based TPM images clearly showed apically clustered Paneth cell granules at the base of intestinal crypts , and GF mice, probably because they do not express AF. However, TPM without moxifloxacin showed different AF expression of cells at the base of intestinal crypts in these mouse conditions. In wild type SPF mice, some cells at the base of intestinal crypts were visible by expressing relatively strong AF. However, in both obese (ob/ob) and wild type GF mice, cells were difficult to visualize based on AF. Different AF expression in cells might be associated with either different environmental and metabolic conditions. Although moxifloxacin-based TPM visualized Paneth cell granules in both wild type SPF and GF mice, TPM without moxifloxacin showed different AF expression in the cells at the base of intestinal crypts. TPM both with and without moxifloxacin showed different states of Paneth cells and their granules in these mouse conditions.Paneth cells in wild type C57BL/6 mice raised in a GF condition (GF) and genetically obese  solution in the equal amount was injected using the identical protocol. 3D TPM imaging was conducted longitudinally at the same sites of the mouse small intestine in 10 min, 30 min, and 60 min after the injection. The longitudinal representative single x-y plane TPM images of Paneth cells at the bottom of the intestinal crypts after the injection are shown  into mice either intraluminally or intraperitoneally. CpG-ODNs mimic bacterial DNA and trigger exocytosis of large secretory granules in Paneth cells via TLR9 activationown Fig. . A schemown Fig. . In 10 mown Fig. . The conin vivo imaging method of Paneth cell granules in the intact mouse small intestine was developed and verified. TPM was used to assess Paneth cells in the small intestine of live mice. Moxifloxacin was used to label Paneth cell granules as well as other cells by topical administration. Although moxifloxacin labeled cells non-specifically, it labeled Paneth cell granules especially well with strong fluorescence in wild type SPF mice. Moxifloxacin labeling of Paneth cell granules was confirmed by counterstaining Paneth cells with an immunofluorescent marker, rhodamine-UEA-1  and GF mice. In obese (ob/ob) mice, moxifloxacin-based TPM showed much less Paneth cell granules at the base of intestinal crypts. In vivo TPM results of obese (ob/ob) mice were confirmed by fluorescence images of the small intestine section stained with DAPI (blue) and rhodamine-UEA-1 (red), and was consistent with a previous study25. In GF mice, moxifloxacin-based TPM showed similar distribution of Paneth cell granules at the base of intestinal crypts to those of SPF mice. Moxifloxacin labeling of Paneth cell granules was examined further by counterstaining with LysoTracker. While LysoTracker labeled Paneth cell granules similarly to moxifloxacin in the case of SPF mice, it labeled only a small portion of the moxifloxacin-labeled Paneth cell granules in GF mice. LysoTracker labeling results of these two mouse conditions were consistent with the previous studies, which showed alteration of Paneth cell granule contents in GF mice compared with SPF mice26. Since moxifloxacin labeled more Paneth cell granules than LysoTracker in GF mice, moxifloxacin labeling might be less sensitive to granule compositions or conditions.In this study, a new 29. Moxifloxacin can pass through the membrane of cells and granules owing to its relatively high lipophilicity. Once Moxifloxacin enters the granules, its lipophilicity could decrease in the acidic microenvironment, leading to its accumulation within the granules. However, moxifloxacin and LysoTracker counterstaining results showed that moxifloxacin labeling of Paneth cell granules could be less dependent on pH, suggesting that there might be environmental factors other than pH contributing to the retention of moxifloxacin inside Paneth cell granules.Although moxifloxacin seemed to label most cells in the mouse small intestine with varying degrees and no clear specificity in TPM, it labeled Paneth cell granules especially well compared to other cellular structures. Moxifloxacin labeling mechanism of Paneth cell granules might be explained at least partially by a decrease in moxifloxacin’s lipophilicity in acidic conditionsMoxifloxacin-based TPM of Paneth cells showed slightly different fluorescence spectrum of moxifloxacin in granules compared to other parts such as the cell cytoplasm. Moxifloxacin fluorescence in Paneth cell granules was slightly red-shifted, and this might be related with the pH variation in various intracellular compartments. The single-photon fluorescence spectrum of moxifloxacin was measured in various pH conditions from 3.76 to 8.20  region of the small intestine. It is possible that CpG-ODN-induced Paneth cell degranulation might be a prolonged process and occur in a non-synchronized fashion among the cells in different regions of the small intestine. Interestingly, CpG-ODNs was unable to induce Paneth cell degranulation in an 31. A recent study showed that Paneth cell dysfunction could be used as an early indicator of some diseases33. Notably, the potency, safety, and pharmacology of the moxifloxacin administration have been studied in various tissues35, and it was confirmed that moxifloxacin penetrates and accumulates in the gut36. Moxifloxacin-based TPM could assess Paneth cells and granules in the intact mouse small intestine by 3D imaging from the serosa. In mice, Paneth cells and granules were located at relatively shallow depth, approximately 80 µm deep from the serosa. However, Paneth cells in larger animal models might not be assessable due to increased tissue thickness. Multiphoton imaging methods using longer excitation wavelengths such as three-photon microscopy might be useful for assessing Paneth cells in large animal models with higher imaging depths than TPM38. Longitudinal TPM of the small intestine in the current setup was limited to approximately 8 hours, which may not be enough for some other in vivo applications. Longitudinal in vivo TPM for the longer time duration could be possible by using an abdominal window chamber40.Moxifloxacin-based TPM could be applied to functional imaging of Paneth cells, aiding the study of intestinal homeostasis and various gut diseases such as Crohn’s disease and ulcerative colitisin vivo by TPM with moxifloxacin labeling. In vivo TPM with and without moxifloxacin labeling showed different states of Paneth cell granules at the base of intestinal crypts in SPF, obese (ob/ob), and GF mice. Moxifloxacin-based TPM would be useful for studying the interplay of small intestinal Paneth cells and mucosal immune defense system in health43, inflammatory bowel disease (IBD) pathogenesis48, and metabolic syndrome49. Perhaps, it could also be developed as a diagnostic or prognostic method including endoscopy to examine the abnormalities of Paneth cell granules in IBD animal models and human patients in vivo46. Therefore, TPM with moxifloxacin labeling might be useful to study Paneth cell functions and related diseases in preclinical animal models.In this study, Paneth cells and granules in the intact mouse small intestine were visualized Vigamox, Alcon Laboratories, Fort Worth, US) was used during the experiment to label cells including Paneth cells in the small intestine of live mice. This solution contains 0.5% (5 mg/mL) moxifloxacin hydrochloride  as the active ingredient. Moxifloxacin ophthalmic solution was always kept enclosed to prevent possible photo-bleaching. Moxifloxacin solution was topically administered to label Paneth cells in the small intestine.Moxifloxacin ophthalmic solution (green (300–495 nm), and red (495–680 nm). In case of triple labeling with Hoechst 33342, moxifloxacin, and rhodamine-UEA-1, emission light was spectrally resolved by 4 NDD channels using the same filter sets as the above. The acquired images were displayed in 3 pseudo-colors of blue (300–495 nm), green (495–560 nm), and red (560–680 nm). In case of moxifloxacin and Lysotracker double labeling and TPM with the commercial system, the excitation wavelength was set at 800 nm, and emission fluorescence was spectrally resolved and displayed in 2 pseudo-colors . Moxifloxacin-based imaging with the custom TPM was conducted with a single emission detection channel covering 300–680 nm and was displayed in green pseudo-color. Autofluorescence based imaging with the custom TPM used 2 detection channels and was displayed in different pseudo-colors . 3D TPM was conducted by acquiring multiple x-y plane images with a stepwise increment of 2 μm in the z direction. TP images consisted of either 512 × 512 pixels or 1024 × 1024 pixels and had the field of view of either 300 μm × 300 μm, 150 μm × 150 μm, or 75 μm × 75 μm. Imaging speed was approximately 0.8 frames/s and 0.2 frames/s for the commercial and custom-built TPM systems, respectively.Both a commercial TPM  and a custom-built TPM were used in this study. The commercial TPM used a Ti-Sapphire laser  with specifications of 140 fs pulse width and 80 MHz repetition rate and a 25× objective lens . The custom-built TPM used another Ti-Sapphire laser  and a 20× objective lens . Laser power was measured at the back aperture of the objective lens using a power meter , and the laser power at the sample was calculated by accounting the beam clipping at the back aperture of the objective lens and transmission efficiency of the objective lens. Excitation laser was tuned to 780 nm for both moxifloxacin and autofluorescence based TPM. Excitation laser power was approximately 5–15 mW and 50–120 mW depending on the imaging depth for moxifloxacin and autofluorescence based imaging, respectively. The commercial TPM had 4 non-descanned detection (NDD) PMT channels and was used for spectral imaging with either moxifloxacin only or additional fluorescence labeling. The custom TPM had 2 detection channels and was used for either autofluorescence or moxifloxacin-based imaging. In moxifloxacin-based TPM using the commercial TPM, emission light was spectrally resolved by 4 NDD channels consisting of a set of dichroic mirrors of 495 nm, 560 nm, 620 nm. The acquired images were displayed in 2 pseudo-colors of ob/ob) mice (n = 8) in a C57BL/6 background were purchased , and kept in a SPF condition with a normal chow diet, and subjected to TPM experiments when the weight reached 40~50 g. All animal experimental procedures were conducted in accordance with institutional guidelines and regulations and approved by the Institutional Animal Care & Use Committee at Pohang University of Science and Technology (POSTECH-2017-0027). After each experiment, the mice were humanely euthanized.All mice were kept at the animal facility of POSTECH Biotech Center under a SPF or a GF condition. Wild type C57BL/6 mice (8–10 weeks-old) bred in a SPF condition  or a GF condition  were used. Obese  and medical grade oxygen. To perform in vivo TPM of the small intestine, a custom-built intestinal holder  was used  of live mice by gently pulling the small intestine out of the surgically opened abdominal cavity and by mounting it on the intestinal holder. Gentle pressing of the pulled small intestine with the cover glass helped to reduce the motion artifacts .Mice were anesthetized via a face mask administrating a gas mixture of 1.5%/vol isoflurane , Hoechst 33342 , rhodamine–conjugated Ulex Europaeus agglutinin 1 , and a lysosomal probe  were topically administered on the surface of the intact small intestine 10 minutes prior to TPM. In case of TPM imaging from the luminal side . To monitor the in vivo degranulation process of the Paneth cell, CpG-ODNs  was careful injected into the lumen of the small intestine. To detected the results of the Paneth cell degranulation, CpG-ODNs  was intraperitoneally injected 5 hours prior to TPM in vivo27. As the negative control, PBS solution of the same amount as CPG-ODNs was injected using the same protocol.For TPM of Paneth cell degranulation process in the intact small intestine of live mice The quantitative analysis of Paneth cell degranulation was performed by counting the total number of pixels of Paneth cells granules in the 3D volumetric TP images after intraperitoneal injection. Several image processing algorithms were applied to increase the contrast of Paneth cell granules in TP images (Supplementary Fig. Supplementary InformationSupplementary Video 1Supplementary Video 2Supplementary Video 3Supplementary Video 4Supplementary Video 5Supplementary Video 6Supplementary Video 7Supplementary Video 8Supplementary Video 9Supplementary Video 10Supplementary Video 11


Innovative chemotherapeutic agents that are active against Mycobacterium tuberculosis (Mtb) are urgently required to control the tuberculosis (TB) epidemic. Mycobacterium tuberculosis (Mtb) are urgently required to control the tuberculosis (TB) epidemic. The Mtb cell envelope has distinct (lipo)polysaccharides and glycolipids that play a critical role in Mtb survival and pathogenesis and disruption of pathways involved in the assembly of the Mtb cell envelope are the primary target of anti-tubercular agents. Here we introduce a previously unexplored approach whereby chemical agents directly target the extracellular glycans within the unique Mtb cell envelope, rather than the intracellular biosynthetic machinery. We designed and synthesised multimeric boronic acids that are selectively lethal to Mtb and function by targeting these structurally unique and essential Mtb cell envelope glycans. By tuning the number of, and distance between, boronic acid units high selectivity to Mtb, low cytotoxicity against mammalian cells and no observable resistance was achieved. This non-conventional approach may prevent the development of drug-resistance and will act as a platform for the design of improved, pathogen-specific, next generation antibiotics.Innovative chemotherapeutic agents that are active against  Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is the world's leading cause of death from a single infectious agent claiming the lives of 1.7 million people annually.Mtb are increasing at an alarming rate and include the emergence of Mtb strains that are not treatable with any of the current antibiotic regimens.Mtb possesses a distinctive cell envelope that is uniquely complex and rich in a diverse range of unusual carbohydrates and lipids.Mtb and provides a highly efficient permeability barrier that prevents intracellular access to many antibiotics and severely complicates anti-tubercular treatment regimens. The core of the Mtb cell wall is comprised of three main components: a cross-linked peptidoglycan (PG) network, a highly branched arabinogalactan (AG) with both arabinose and galactose found in the furanose form, and long chain (C60–90) mycolic acids.Mtb envelope is an outer capsule composed of polysaccharides, predominantly α-glucan, and proteins.Mycobacterium genus and are found in unusual conformations with distinct glycosidic linkages. Molecular pathways directly involved in the biosynthesis of the Mtb cell envelope have proven to be especially vulnerable to chemotherapeutic agents and include the front-line drugs isoniazidd-cycloserine.Mtb by inhibition of arabinan biosynthesis.g chain C0–90 mycoMtb cell wall glycans are essential for its survival and pathogenesisMtb cell envelope barrier. To evaluate this idea further  to evaluate the effect of the number and separation of boronic acids units to selectively target Mtb glycans and kill Mtb. A range of compounds bearing the glycan-targeting unit, 3-carboxy-phenyl boronic acid (3-CPBA), were synthesised (4–8) using acid chloride or carbodiimide coupling with the appropriate di- or tetra-PEG (poly(ethylene glycol)) amine to provide flexibility of the spacer group between the relative position of the boronic acid functional groups (ESI Schemes 1–35–7). Compound 4 had an ethyl linker as a further control, but longer alkyl chains were not soluble. For 8, a first generation PAMAM (polyamidoamine) dendrimer core was synthesised and used to generate a tetrameric boronic acid . To ensure the boronic acids were accessible for glycan binding the Alizarin red (ARS) assay was employed to map the selectivity of the dimeric boronic acids against a panel of carbohydrates and were found to retain the same affinity trends as the monomeric boronic acids  displayed low antibacterial potency with no selective preference for Gram-negative or mycobacterial strains. Remarkably, exposure of the same strains to the dimeric boronic acids 4–6, which vary in distance between the boronic acids from 12–23 atoms (1.6–3 nm), resulted in a dramatic decrease in the MICs  was ineffective, with higher MICs and loss of specificity for mycobacteria. Multivalent presentation of boronic acids has been reported to lead to increased binding affinities,8, based on a 1st generation poly(amidoamine) dendrimer, also showed mycobacterial selectivity with similar MIC values to the dimers 4–6 . These results indicate that an optimal spacer length of 1.6–3 nm (compounds 4–6) between the two boronic acid moieties is optimal whereas longer lengths >9 nm (7) reduce potency.This library of glycan-targeting compounds was evaluated for antibacterial activity using the resazurin-reduction assayMtb for 4–6. The MBC data demonstrates that these boronic acid dimers are bactericidal against actively growing Mtb at concentrations of 6.25 mM  were also determined against  Table S1.99 values above 25 mM, and no lysis or agglutination of red blood cells was observed at concentrations as high as 25 mM supporting a unique mode of action against mycobacteria and a window of activity  mediated agglutination,  and tetramer 8 therefore show higher selectivity for mycobacteria compared to the mammalian cells tested that have distinct cell-surface glycans.The panel of boronic acids showed no significant cytotoxicity against human lung A549 cells, human T-lymphocyte Jurkat cells and human epithelial Caco-2 cells with MIC Table S2. To dete4–6) interacted with the front-line anti-tubercular agent rifampicin a checkerboard assay was used. No synergistic or antagonistic effects were noted with the sum of the fractional inhibitory index (ΣFIC) calculated as 2. Mono-boronic acids have been reported as having β-lactamase inhibitory activity4–6 for compound interactions with the β-lactam meropenem on Mtb. The ΣFIC for each combination was calculated4 and 0.6 for compounds 5–6 indicating no synergetic and importantly no antagonistic action on the growth inhibition, compared to the synergistic activity of meropenem in combination with the β-lactamase inhibitor sulbactam with a ΣFIC 0.3  was employed , 4 μM (TDM), 12 μM (TMM) and 41.5 μg mL–1 (LAM)  was observed, and very weak binding for PIM 1 + 2 (which have correspondingly lower degrees of glycosylation) and notably, no detectable binding for isolated mycolic acid methyl esters (MAMEs) and sulpholipid I which contains a sulphated trehalose moiety. This is consistent with a multivalent interaction between cell wall components that contain cis-diols and the boronic acid and clearly demonstrates that boronic acids have the necessary capacity to engage with these essential Mtb cell envelope constituents. To further determine the origin of the specificity of the multimeric boronic acids for mycobacterial glycans, we probed the interaction of the boronic acid functionalised sensor with mammalian specific glycans using GM3 , which contains a galactose residue in the pyranose conformation linked to a terminal sialic acid moiety, and no detectable binding was observed  Mtb cells were evaluated by BLI for binding to the boronic acid functionalised sensors. Mtb gave significantly faster rates and extent of binding compared to E. coli  . Despited Fig. S6. Whole E E. coli . To evald Fig. S6, consistM. bovis BCG was exposed to 2× MIC of boronic acid dimer 6 and analysis of the whole cell protein expression profile at 3 hours, 24 hours and 48 hours was performed. A list for all the identified proteins, annotations and fold changes compared to controls at each time point are in ESI List S1  showing almost identical activity to the dimers. Biolayer interferometry revealed strong and selective interactions with isolated Mtb glycans and whole intact Mtb cells and whole cell proteomics identified a broad stress response rather than a single target, which may contribute to the lack of resistance observed. The multimeric display of boronic acids was crucial to their mechanism of action and distinct function compared to analogous monovalent boronic acids. This concept of inhibiting the extracellular glycans on Mtb presents a unique opportunity to develop pathogen specific agents and represents an important step in the identification of new TB drug targets.In conclusion, here we have introduced a new approach to selectively kill The authors  are named inventors on a patent application relating to this work.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.


A NaErF4@NaYF4 core−shell nanoparticle is constructed to host the upconversion process for minimizing non-radiative dissipation of excitation energy by surface quenchers. Furthermore, an integrated optical microring resonator is designed to promote absorption of excitation light by the nanoparticles, which alleviates quenching of excited states due to cross-relaxation and phonon-assisted energy transfer. As a result, multiphoton upconversion emission with a large anti-Stokes shift (greater than 1150 nm) and a high energy conversion efficiency (over 5.0%) is achieved under excitation at 1550 nm. These advances in controlling photon upconversion offer exciting opportunities for important photonics applications.The applications of lanthanide-doped upconversion nanomaterials are limited by unsatisfactory brightness currently. Herein, a general strategy is proposed for boosting the upconversion efficiency in Er Here, the authors control concentration quenching in a stoichiometric Er3+ compound using a core−shell nanostructured host and an integrated optical waveguide circuit excitation platform. The efficient telecom to ultraviolet upconversion allows for exploration of photonic applications involving upconversion nanomaterials. Nevertheless, despite of the unique optical properties, wider application of upconversion processes has been hindered by limited emission intensity due to low concentration of optical centers (or dopants) in most nanoparticles. When dopant concentration increases, interactions of optical centers become significant as a result of reduced inter-dopant distance, causing depopulation of excited states and thus attenuation in the upconversion emission  emissions can be generated upon near-infrared (NIR) excitation. The effect has enabled precise and remote control of biochemical reactions as well as the creation of full-color 3D displaysion Fig. .Fig. 1Ge6 W cm−2) and dye sensitization, depopulations of excited states are compensated and lead to elevated quenching concentrations of Tm3+, Er3+, and Nd3+ activators10. Core−shell nanostructural engineering has also been exploited to mitigating concentration quenching by eliminating dissipation of energy to lattice and surface defects. Using this method, concentration quenching in both sensitizer (Yb3+) and activator (Er3+) ions is greatly suppressed17. Despite these research efforts, photon upconversion at a high concentration of activators is usually dominated by low-order processes featuring emission in the spectral region of long wavelengths. Challenges remain for achieving highly efficient multiphoton upconversion with emissions in the short-wavelength regime by using low power excitation. Particularly, there are no known effective approaches to achieve efficient UV emission under excitation in the wavelength range for optical communication, where many inexpensive and high-performance lasers and optical components can be readily acquired from the mature telecommunications industry.Attempts have been made to alleviate concentration quenching so that unusually high dopant concentrations can be used to boost upconversion emission intensity. By promoting the excitation process through the use of high excitation irradiance  nanoparticles with and without a protection layer of NaYF4. The inert NaYF4 shell is able to selectively suppress energy loss to lattice defects and surface quenching sites that arises from energy migration through the dopant sublattice19. Assessment of the core−shell nanoparticles thereby allows us to probe the quenching mechanism. The nanoparticles were synthesized by a layer-by-layer epitaxial growth protocol  scanning TEM image of a representative sample shows distinguished Z-contrast between the NaErF4 and NaYF4 layers  was considerably enhanced at high Er3+ concentrations. By contrast, emissions at short wavelengths are only marginally intensified, resulting in decreased relative intensity ratio of green to red emissions at elevated Er3+ concentrations. The observations are ascribed to quenching of the 4S3/2, 4F9/2, and 4I9/2 states by cross-relaxation and phonon-assisted energy transfer  also accounts for the weak emissions at the short-wavelength end  is attributed to the enhancement of energy transfer upconversion that depletes these two states  of each excited state as a function of Er50% Fig. , which intration 0% is att3+. In order to achieve efficient multiphoton upconversion luminescence, joint use of high excitation power is also crucial to counteract the depopulation of the high-lying intermediate states. In an upconversion process, an intermediate state is depopulated by being excited to a higher-lying state in addition to linear decay to lower-lying states. The excitation process is pump power dependent and thus upconversion emission dominates over luminescence quenching under high excitation powers24.The mechanistic investigation reveals that the use of core−shell nanostructure alone is unable to alleviate concentration quenching in all the energy states of Er−21 cm2)25, conventional approaches for enhancing the upconversion rates are focused on increasing the excitation intensity and/or localizing the illumination field. Attempts have been made to modulate excitation field around upconversion nanoparticles by surface-plasmon coupling and photonic-crystal engineering27. However, these methods need tedious processing of upconversion nanoparticles and the amplification of optical field may prove to be limited. Here we propose a more effective approach to enhance the upconversion by using an optical microring resonator excitation platform   that is slightly higher than the SU-8 index (n = 1.57)  by the FEM method  were examined by a series of TD-DFT calculations. The electrical field in the microring resonator was simulated by three-dimensional finite-difference time domain (3D-FDTD) method. Finite element method (FEM) was used to calculate the modal propagation constants and field profiles of E-field.The oscillator strengths were derived from calculated electric-dipole transitions by time-dependent density functional theory (TD-DFT). To perform the excited state calculations, we chose the two-electron based Tamm-Dancoff approximation imported from self-consistently corrected ground state wavefunctionsTEM and HAADF scanning TEM were carried out on a JEM-2100F transmission electron microscope at an acceleration voltage of 200 kV. The upconversion emission spectra were recorded with a Hitachi F-4600 spectrophotometer. The decay curves were measured by an Edinburgh FLSP920 spectrometer. Optical micrographs were recorded with an advanced research microscope . All measurements were performed at room temperature.Supplementary Information


Overexpression approaches and fluorescence microscopy techniques allow investigating important spatiotemporal aspects of gene regulation as well as quantifying gene function. Consequently, fluorescence microscopy techniques help answer important questions on gene regulation such as addressing the role of a specific gene product for neuronal survival under different treatments. Here, we describe a versatile tool to measure effects of a transfected gene of interest on neuronal survival upon metabolic stress. We focus on nutrient starvation of cultured rodent primary neurons as a model of metabolic stress but our approach can easily be generalized and adapted to other cell types or to investigate single gene function in regulating neuronal survival under various conditions. The regulation of gene expression provides a critical link between the genome and the phenotype of a cell in general and for neuronal function and survival in particular. However, understanding this link in all its complexity has not yet been accomplished. Especially transcriptomic and proteomic methods are used to unravel this elaborate and multi-layered system of interactions between genes, RNA molecules and proteins. However, important spatiotemporal information is lost when genetic, epigenetic or proteomic approaches are used as the sole determinant of gene function. In contrast, the combination of overexpression approaches and fluorescence microscopy techniques allows addressing the important “where and when” aspects of gene regulation as well as quantifying gene function. Indeed, fluorescence microscopy techniques help answer important questions on gene function such as addressing the role of a specific gene product for neuronal survival under different treatments. As such, fully automated microscopes enable the acquisition of the large number of images needed to quantitatively analyze these important gene expression questions  protein family consists of pro- and anti-apoptotic master regulators of cell death  and Bcl-XL containing vector each as a positive control (PC) or an empty vector as a negative control (NC). In principle, plasmid vectors expressing the gene of interest could be cotransfected with H2B-GFB to investigate its function compared to the PCs and NCs. After 10 days in culture, images of H2B-GFP-expressing neuronal nuclei are captured employing semi-automated fluorescence microscopy and subsequently analyzed using CellProfiler to quantify viable neurons by automatically identifying and quantifying H2B-GFP expressing nuclei. Ultimately, the number of counted nuclei after treatment divided by the number of nuclei counted before treatment provides a measure for the anti- or proapoptotic effect of a gene of interest.Landesamt für Gesundheit und Soziales (LAGeSo), Berlin. Brains of day 17 rat embryos (E17) or day 15 old mouse embryos (E15) were isolated and cortices were dissected as described  and transfected using a Nucleofector-II  device using program O-003 for rat neurons or O-005 for mouse neurons. Immediately after transfection, neurons were resuspended in 500 μl DMEM (Biochrom) supplemented with 3.7 g/l NaHCO3, 4.5 g/l d-glucose, 10% FCS. Next, 4 × 104 neurons per well (~2.6 × 105 cells/cm2) were plated in poly-D-lysine  and collagen pre-coated half area 96-well imaging plates . Medium was changed 4 h after transfection to remove dead cells and debris. Twenty-four hours after transfection cells were gently washed with 1× PBS and Neurobasal-A  supplemented with B27 (Thermo Fisher Scientific) was added. One week after transfection, 50 μl of culture medium was removed and replaced with fresh NBM-A + B27. Cells were cultivated for 10 days. Note that the desired cell density may differ depending on the experimental setting and may need to be determined empirically. The optimal cell number for plating and therefore cell density depends on the number of dead cells accumulating during preparation. Moreover, electroporation itself influences the viability of neurons. 50%–70% of neurons die within 12 h after electroporation but the remaining cells grow well in culture. Cotransfection rate was approximately 90% as previously described  at subsequent time points. Using a 96-well template, 16 images per well from 10 wells per transfection were collected, adding up to a total of 160 images per condition. Likewise, on day 10 after transfection culture medium was removed, cells were washed once with 1× PBS and the same FOV were imaged 24 h after treatment. Images of transfected mouse cultures were used in Figure On day 9 after transfection, culture medium was collected in 96-well plates and rat primary neuronal cultures were washed once with PBS. Cells were imaged in Live Cell Imaging Solution  containing 140 mM NaCL, 2.5 mM KCl, 1.8 mM CaCl2 as previously described  for 6 h at 37°C and 5% COCellProfiler 2.2.0 was downloaded from the CellProfiler webpageNeuronal nuclei are scored as live or dead depending on the parameters mentioned above. Next, the total number of “live” nuclei after GD was divided by the number of live nuclei before GD and the percentage plotted and statistically analyzed.Data were analyzed and plotted in GraphPad Prism. Details are indicated in the figure legend.Counting fluorescent nuclei is a straightforward phenotype. Various tools, including overexpression of fluorescent cellular markers, , immunofluorescence staining of transcriptions factors or counterstains , are available to sufficiently stain nuclei. However, detecting nuclei can be challenging due to segmentation problems caused by clumped nuclei. Therefore, cell density observation and evaluation are crucial to obtain meaningful nuclei counts.The neuronal survival assay described herein implements different experimental procedures Figure . For optThe functionality and applicability of the developed microscopy-based neuronal survival assay and subsequent image analysis using CellProfiler software is best illustrated using a simple experimental set-up. Thus, here we will only focus on a PC- and NC-transfected neurons subjected to GD as an example of metabolic stress. For this, a Bcl-XL containing plasmid was cotransfected with a nuclear marker H2B-GFP) as the PC or an empty vector as the NC. The strong anti-apoptotic and neuroprotective function of Bcl-XL has been widely recognized and therefore constitutes a suitable PC to measure neuronal survival or cell death in similar assays  and 24 h after glucose deprivation (post GD). However, neuronal cell numbers in these types of assays are notoriously variable Figures . Thus, sWe demonstrated that the assay described herein functions as a versatile tool to gain important insights into the functional effects of a gene of interest on neuronal survival after metabolic stress . While many methods to measure cell survival or cell death parameters exist  for deriving causal protein-signal networks (Sachs et al., Our approach is simple on purpose as it can be rapidly adapted even by novice users. Even though the relatively simple task of manually counting fluorescent neuronal nuclei is feasible in principle , performIn summary, using the microscopy-based neuronal survival assay described here in combination with automated image analysis employing the open source tool CellProfiler, it is possible to investigate protective or detrimental effects of a specific gene of interest on neuronal survival after functional stimulation such as nutrient starvation. Furthermore, this method can easily be adapted to different cell types, cell markers and different stressors or treatments, thereby making it a powerful and versatile tool for the broad neuroscience community.KW conceived and performed the neuronal survival assay experiments, developed the CellProfiler pipeline, analyzed the data and wrote the manuscript. AM discussed the data and allocated funding support to the project. PM supervised all aspects of this work, analyzed and discussed the data and edited the manuscript. All authors have read and approved the final version of the manuscript.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.


Superparamagnetic iron oxide (SPIO)-labeled human MSCs were injected intravenously into mice treated with CCl4 and subjected to hepatic macrophage-depletion. Liver tissues were collected at different intervals post transplantation for subsequent histopathological, morphometric, immunohistochemical, gene expression and ultrastructural studies. The homing of the transplanted MSCs was evidenced by tracing them within the niche by iron staining and immunohistochemical studies. MSCs differentiated into hepatocyte-like cells and intimal smooth muscle cells as evidenced by their expression of human albumin and α-smooth muscle actin with a concomitant increase in the level of mouse hepatocyte growth factor. A post transplantation reduction in the liver fibro-inflammatory reaction was found and was promoted by liver macrophages depletion. Thus, it could be concluded from the present study that prior manipulation of the microenvironment is required to improve the outcome of the transplanted cells.Mesenchymal stem cells (MSCs) therapy show different levels of effectiveness in the context of different types of liver damage, suggesting that the microenvironment of the injured liver is a key determinant for effective stem cell therapy. The objective was to assess the modulatory effect of hepatic stem cell niche components on the transplanted MSCs during liver injury induced by carbon tetrachloride (CCl Stem cell interacts with various inputs from the microenvironment such as soluble factors, ECM and intercellular contacts acting together to influence stem cell fate3; endothelial cells, which are capable of new vessels formation and other recruited inflammatory cells. NPCs produce cytokines and growth factors, like transforming growth factor β (TGF β), that have a major effect on hepatic progenitor cells (HPCs) and hepatocyte proliferation4.Non-parenchymal cells (NPCs) in the liver include stellate cells/myofibroblasts, which are the principle source of collagen; macrophages, which are involved in tissue remodelling and fibrosis resolution after tissue damage3.Macrophages act at different sites of inflammation, participating in the process of inflammation and repair, demonstrating different activities such as regulation of inflammatory cells, tissue debridement, recruiting and activating myofibroblasts, and regulating spontaneous recovery of fibrosis in wound healing. These functions of macrophage are mediated through secretion of cytokines, like TGF-β and tumour necrosis factor- α TNF-α5. Furthermore, evidences from other studies suggesting a role of the inflammatory cytokine TGF-β in regulating stem cell behaviour7.Recent studies demonstrate a role of macrophage in modulating the hematopoietic stem cell niche, in which reductions in bone marrow (BM) mononuclear phagocytes led to reduced BM stromal derived factor-1(SDF-1) levels, the selective down-regulation of hemopoietic stem cell retention genes in niche cells, and mobilization of hemopoietic stem cells to the blood stream4)-induced liver injury and in rodent hepatic oval cell regeneration models, suggesting its involvement in stem cell proliferation, migration, and differentiation8.Increased expression of hepatocyte growth factor (HGF) has been demonstrated in carbon tetrachloride . The control group comprised 30 age-matched and sex-matched healthy mice. All mice were aged 4 weeks and weighed an average of 20 ± 5 g. The animals were obtained from the Schistosoma Biological Material Supply Center of the Theodor Bilharz Research Institute (TBRI), Giza, Egypt. They were housed under conventional conditions and fed a standard diet with free accessibility to water. All animal work was conducted in accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals and was approved by the Research Ethics Committee of the Theodor Bilharz Research Institute, Giza, Egypt.The present study was conducted on 90 Swiss albino male mice with liver injury induced by carbon tetrachloride -labeled MSCs, Group III: A group with mild liver fibrosis induced by CCl4, Group IV: A group with mild liver fibrosis induced by CCl4 with hepatic macrophage depletion and transplanted with SPIO-labeled MSCs, Group V: A group with mild liver fibrosis induced by CCl4 that was nondepleted and transplanted with SPIO-labeled MSCs, Group VI: A group with marked liver fibrosis (Cirrhosis) induced by CCl4,Group VII: A group with cirrhosis induced by CCl4 with hepatic macrophage depletion and transplanted with SPIO-labeled MSCs, and Group VIII: A group with cirrhosis induced by CCl4 that was nondepleted and transplanted with SPIO-labeled MSCs.4 (in corn oil) three times/week for 5 weeks.Intra-peritoneal (IP) injection of 10 µl/g body weight (BW) of 10% CCl4 (in corn oil) three times/week for 12 weeks .IP injection of 10 µl/g BW of 20% CClet al.9.Grading of liver damage was done using the fibrosis-scoring system of Ishak 10. Depletion was confirmed by immunohistochemical analysis using the Anti f4/80 antibody for mouse macrophages.After induction of liver fibrosis, mice were administered an intravenous (IV) injection of 0.1 ml/mouse (30 g BW) of either clodronate-containing liposomes (for the fibrosis and cirrhosis groups) or empty liposomes (for the control group)  approximately 48 hr before MSC transplantation. After 2 days, the liver macrophages were depleted as previously describedThe MSCs used in the present study were originally isolated and expanded from a donated human umbilical cord with the donor’s consent after a full-term cesarean delivery. The cells obtained were cultured in complete culture medium comprising low-glucose Dulbecco’s modified Eagle medium  supplemented with 1% L-glutamine, 30% fetal calf serum  and 1% penicillin-streptomycin . The cells were collected between the third and fourth passages for further transfusion into mice.11. Briefly, cultured MSCs were incubated with the labeling medium (DMEM containing 50 μg/ml iron and 0.75 μg/ml PLL) for 48 h at 37 °C with 5% CO2 at a density of 3 × 105 cells/ml. Control MSCs were cultured in DMEM without iron or PLL. After cell recovery, the cells were either harvested by trypsin digestion or examined in situ for viability and iron staining. As outlined below, Perls’ Prussian blue staining  and transmission electron microscopy were both used to assess iron staining.Isolated MSCs from the third passage were labeled with a combination of superparamagnetic iron oxide (SPIO)  and poly-L-lysine , as previously describedImmediately following the labeling procedure, an aliquot of the labeled cells was fixed for 30 min in 2.5% glutaraldehyde in PBS buffer at room temperature. The cells were then washed three times in PBS and postfixed for 30 min in 2% osmium tetroxide in PBS buffer. After dehydration in 4 changes of graded ethanol  with a 5 min gap, they were embedded in Epon 812 substitute  and polymerized at 60 °C for 48 h. Semi-thin sections were cut, stained with methylene blue-azure II, and examined by light microscopy to choose the region of interest for ultrathin sectioning. The ultrathin sections were then prepared using an Ultracut R ultramicrotome , double stained with uranyl acetate and lead citrate and examined with a Philips EM 208 S electron microscope .6 SPIO-labeled MSCs per mouse were injected intravenously in the tail vein (1 × 106 cells/100 µl).After 5 weeks and 12 weeks of CCl4 injection, 1 × 10Mice were sacrificed at different time intervals post transplantation. Liver samples obtained at 24 h were used for the assessment of homing, whereas those obtained at 12 weeks were used to assess the engraftment of transplanted MSCs. The liver samples were subsequently processed using histopathologic, ultrastructural, immunohistochemical and quantitative real-time polymerase chain reaction (RT-PCR) techniques.Excised livers were immediately fixed in a 10% formalin solution and embedded in paraffin. They were then processed and stained with hematoxylin and eosin (H&E) stain to examine histopathologic changes. Masson’s trichrome stain was used to qualitatively assess the grade of fibrosis.Hepatic tissue sections (5 μm in thickness) were prepared and stained with Sirius red (SR) to quantify collagen content using a Zeiss Scope A1 microscope equipped with a CCD camera (Axiocam MRC5). Image analysis was performed using the Axiovision Rel.4.8 computer software.Immunohistochemistry was performed using an avidin–biotin complex immunoperoxidase technique. Human-specific antibodies with no cross-reactivity to mouse antigens were used to label the following markers: α smooth muscle actin ; albumin ; and Von Willebrand factor . The anti-OxPhos complex IV subunit I monoclonal antibody  and the rabbit monoclonal Anti-f4/80 mouse antibody  were also used. We used a preformed streptavidin–biotin–peroxidase complex and peroxidase-DAB   according to the manufacturer’s instructions. Sections were counterstained with Mayer’s hematoxylin and mounted using DPX medium. Positive and negative control slides for each marker were included in each run. As a negative control, a liver tissue section was processed as described but with the primary antibody omitted.et al.12. Briefly, IHC staining using the anti-OxPhos complex IV subunit I monoclonal antibody (Invitrogen) (dilution 1:50) for detecting patches of cytochrome c oxidase-negative hepatocytes in liver invariably connected to the portal areas suggested an origin from a long-lived cell, presumably a stem cell.A technique for lineage tracing in liver tissue was used following Fellous 13. The homing of the transplanted MSCs into the recipient liver was estimated semi-quantitatively by Prussian blue staining and counting the number of periportal regions (within the same cytochrome c oxidase–negative patches) by considering the bluish coloration in 5 consecutive positive high-power fields.At 24 h after MSC transplantation, following location of the niche by IHC, serial sections from the same paraffin block were used for Prussian blue staining as previously described14. The level of expression was normalized to the reference gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cat. No. QT01658692).Gene expression analysis was performed using quantitative real-time polymerase chain reaction (qPCR). The total RNA was extracted and purified from liver tissue using the RNeasy Protect Mini Kit  according to the manufacturer’s instructions. Complementary DNA was synthesized by reverse transcription (RT) of the RNA using the QuantiTect RT Kit . Specific bioinformatically validated primers were provided by Qiagen for the quantification of mouse HGF (Cat. No. QT00158046), mouse TGFB1 (Cat. No. QT00145250) and mouse SDF1 (CXCL12) (Cat. No. QT00161112). The expression of mRNA was assessed by qPCR using the QuantiTect SYBR Green PCR kit  and an ABI PRISM 7500 sequence detector . The quantification was performed using the comparative Cq method as previously describedThe data were analyzed using version 22 of the SPSS statistic software . The data were expressed as the median and percentiles for quantitative non-parametric measures. The following tests were performed: the Wilcoxon Rank Sum test for comparisons between two independent groups for non-parametric data; the ranked Spearman correlation test to study the possible association between two variables among each group for non-parametric data; and Kruskal–Wallis test for comparisons between more than two groups for non-parametric data. A probability of error  less than 0.05 was considered significant.MSCs isolated and propagated in cultured media formed adherent cell layers of spindle-shaped cells with a confluency of 70–90%. Viability of these cells was 90–95%. The cells were assessed for efficient labeling by SPIO using Perls’ Prussian blue staining and ultrastructural studies where the presence of bluish iron particles was visualized by an inverted microscope. Most of the MSCs treated with SPIO displayed cytoplasmic staining when compared to the control (unlabelled) MSCs Fig. .Figure 1The electron microscopic examination of the labeled MSCs confirmed the internalization of SPIO nanoparticles by the cells rather than simply their bonding to the cell surface. The SPIO nanoparticles appeared as black aggregates in numerous round endosomes in the cytoplasm and perinuclear area, while cell membrane-bound nanoparticle clusters were not detected Fig. .IHC staining using the anti-OxPhos complex IV subunit I monoclonal antibody detected patches of cytochrome C oxidase-negative areas located in close proximity to or in direct contact with the portal tract region. The patches varied in size but had clearly defined boundaries, seemingly always extending to the peri-venous regions Fig. . TracingSuccessful hepatic macrophage depletion was confirmed by immunohistochemical analysis using the Anti f4/80 antibody for mouse macrophages Fig. To determine whether MSC transplantation reduces liver fibrosis in macrophage-depleted mice, we administered MSCs intravenously to cirrhotic or fibrotic mice and assessed liver fibrosis in untreated mice. Liver fibrosis assessed by H&E, SR and Masson’s trichrome staining Fig. .Figure 2Histopathological examination of the recipient mouse liver during different stages of liver damage (both mild fibrosis and cirrhosis) revealed a statistically significant reduction in the percentage of the assessed fibrotic area after MSC transplantation and macrophage depletion compared to the nontransplanted group, as shown by SR staining, and that the reduction in the percentage of the assessed fibrotic area was favored by the depletion of liver macrophages Fig. . MoreoveMSCs differentiated into hepatocyte-like cells as evidenced by their expression of human albumin. The expression of human albumin in small clusters of polygonal cells (as newly formed hepatocyte-like cells) was cytoplasmic, distributed in small clusters near the portal areas, perivascular and scattered intralobularly in the hepatic parenchyma, as shown in Fig. The lack of endothelial differentiation of MSCs was reported in our study as evidenced by absence of expression of human VWF in both fibrotic and cirrhotic livers.The expression of human αSMA and albumin after MSC transplantation was statistically significantly higher in macrophage-depleted animal groups compared to the nondepleted groups, as shown in Table The results showed that the relative mRNA expression level of mouse TGFb1 was increased after CCl4-induced liver fibrosis compared to the baseline value in the control group and was not changed significantly in cirrhosis compared to the control Table . HoweverThe relative mRNA expression levels of mouse HGF and SDF1 did not change significantly among all groups of mild fibrosis before and after transplantation compared to each other  - labelled MSCs were injected into mice in which liver fibrosis by carbon tetrachloride was induced. To identify the role of macrophages as one cellular component of the niche, selective hepatic macrophage-depleted animal model subgroup was used using clodronate liposome. Transplanted labelled stem cells are then traced for their homing into their niche and were studied for their differentiation into hepatocytes, neovasculature or myofibroblasts and these activities were correlated with various levels of soluble factors released in the stem cell niche compartment at early and late stages of liver fibrosis.On the basis of the recently recognized potential of MSCs to contribute to tissue repair after injury in different diseases including heart and liver diseasesOur findings demonstrated efficient homing of transplanted MSCs into the recipient liver of injured mice, with a higher effeciency in cirrhotic livers than in the mildly fibrosed livers compared to normal mice transplanted with the same MSCs, which did not show homing of these cells.et al.21 and Sato et al.22. These researchers reported that labelled human MSCs were retrieved from the liver of CCl4-injured mice after intravenous and intrahepatic injection and they had differentiated into hepatocyte-like cells.Our findings are in agreement with those of both Seo et al.23. These investigators found that transplanted hematopoietic stem cells differentiate into hepatocytes at a very low level in mice without liver injury, suggesting that both liver injury and the surrounding environment could be essential for transplanted cells to recruit to the liver and to differentiate into hepatocytes. These factors were explained later in studies suggesting that stromal cell-derived factor 1 (SDF1) and its receptor CXCR4 might be important for stem-cell recruitment to the liver24.The absence of human cells in the liver of control mice following IV transplantation of MSCs observed in our study is in agreement with the findings of Wagers 25.Our results revealed that SDF1 level was statistically significantly higher in all cirrhosis groups post transplantation (week 12) with more increase in depleted transplanted group compared to the non-transplanted counterparts, however no statistically significant difference was found in the mild fibrosis groups. Similarly, recent study demonstrated that the SDF-1/CXCR4 axis play an important role in the chemotaxis, homing, and differentiation of MSCs to the injured liveret al., who attributed the lack of migration of circulating stem cells from the bone marrow to the liver in patients with a variety of chronic hepatic injuries to the fact that there could be a balance between injury and endogenous repair mechanisms, excluding the need for extra hepatic progenitors, in a mildly damaged liver26. While the extrahepatic BM-derived stem cells could only come into action when the body’s repair mechanisms are overwhelmed, as occurs in severe cirrhosis27. This could be supported by our finding of a concomitant increase in the number of engrafted and differentiated cells in the cirrhotic transplanted group in relation to the mild fibrosis group.Several possibilities could explain the lack of increase of SDF1 levels in all of the mild fibrosis groups and its marked increase in the cirrhotic depleted transplanted group. These explanations are in line with the view of Di Campli 29. Additionally, other studies suggested that MSCs could exert their anti-fibrotic effects through the secretion of matrix metalloproteinases30. However, some studies failed to show the beneficial effect of MSCs in reducing the fibro-inflammatory reaction, as they conversely found that MSCs might contribute to fibrosis production. They suggested that this might be related to either the various subpopulations of MSCs with opposing effects or the type of liver injury and extent of fibrosis31.Several studies have shown both antifibrotic and anti-inflammatory effects of MSCs derived from several sources. They related this protective effect to the ability of MSCs to decrease the level of inflammatory cytokines, such as IL-1β, IL-6, TNF-α and TGF-βvs. 7.20 for mild fibrosis without transplantation, p = 0.005) and . In spite of the observed reduction in the liver fibro inflammatory reaction after MSC transplantation in both mild fibrosis and cirrhosis groups without macrophage depletion, yet this reduction did not reach statistical significance  Table  from the stellate cell32. This is supported in our study by the substantial reduction in the TGFb1 level in the mild fibrosis depleted transplanted vs. mild fibrosis nondepleted transplanted group .In our study, the histopathological picture of early mild fibrosis animal group revealed a mild fibro-inflammatory picture evidenced by MNCs infiltration and mild perivascular fibrosis in the portal area and around central veins, and more fibrosis with loss of hepatic lobular architecture was present in the cirrhotic group as represented by Masson trichrome stain and Sirus red stain analysis. Twelve weeks later, we correlated the changes that took place after MSCs transplantation in comparison to the corresponding group without transplantation. Our findings revealed that there was a statistical significant reduction in liver fibro-inflammatory reaction after MSC transplantation , which frequently occupy this location. This pattern of expression suggested neointimal formation rather than transdifferentiation into activated myofibroblast-like cells. This finding was supported by both the reduction in fibrosis level which was found after MSC transplantation and the absence of any correlation between αSMA expression and fibrosis level. These results were supported by previous studies that demonstrated that bone marrow MSCs are prone to participating in neointimal growth by differentiating into vascular smooth muscle cells expressing the contractile αSMA marker36.In addition to the beneficial reparative effect of MSCs in liver disease, unwanted effects such as myofibroblastic transdifferentiation was reportedIn conclusion, MSC transplantation is a beneficial therapeutic strategy in chronic liver diseases on behalf of its anti-inflammmatory, antifibrogenic and transdifferentiation potentials. However, macrophages, as a key determinant component in the stem cell niche, were found to interfere with these functions, necessitating prior manipulation of the microenvironment in which stem cells are engrafted to improve the outcome.


Heart failure (HF) with preserved ejection fraction (HFpEF) comprises nearly half of prevalent HF, yet is challenging to curate in a large database of electronic medical records (EMR) since it requires both accurate HF diagnosis and left ventricular ejection fraction (EF) values to be consistently ≥50%.We used the national Veterans Affairs EMR to curate a cohort of HFpEF patients from 2002 to 2014. EF values were extracted from clinical documents utilizing natural language processing and an iterative approach was used to refine the algorithm for verification of clinical HFpEF. The final algorithm utilized the following inclusion criteria: any International Classification of Diseases-9 (ICD-9) code of HF (428.xx); all recorded EF ≥50%; and either B-type natriuretic peptide (BNP) or aminoterminal pro-BNP (NT-proBNP) values recorded OR diuretic use within one month of diagnosis of HF. Validation of the algorithm was performed by 3 independent reviewers doing manual chart review of 100 HFpEF cases and 100 controls.We established a HFpEF cohort of 80,248 patients . Mean age was 72 years; 96% were males and 12% were African-Americans. Validation analysis of the HFpEF algorithm had a sensitivity of 88%, specificity of 96%, positive predictive value of 96%, and a negative predictive value of 87% to identify HFpEF cases.We developed a sensitive, highly specific algorithm for detecting HFpEF in a large national database. This approach may be applicable to other large EMR databases to identify HFpEF patients. Heart failure with preserved ejection fraction (HFpEF) is increasing in prevalence , and alrThe study was approved by the VA Boston Healthcare System Human Subjects Subcommittee IRB #2868). We used the national VA database to develop a HFpEF cohort utilizing EMR data recorded between 1/1/2002 to 12/31/2014. The VA uses Veterans Information Systems and Technology Architecture (VistA), which is a national integrated healthcare system with a comprehensive EMR across all the VA healthcare facilities. The VA has provided healthcare to millions of Veterans over the last two decades in more than 1700 hospitals, clinics, and nursing homes, with the majority of care recorded through VistA. A NLP tool which has been well established in VA database , 6, was 8. We useInternational Classification of Disease-Ninth Revision (ICD-9) codes diagnosis of HF (any 428.xx), (2) either B-type natriuretic peptide (BNP) or aminoterminal pro-BNP (NT-proBNP) values recorded OR diuretic usage within one month of HF diagnosis, (3) availability of echocardiogram in our system, and (4) having all recorded EF values ≥50%. We excluded patients with constrictive pericarditis (ICD9 code 425.1) and hypertrophic cardiomyopathy (ICD9 code 423.2) and any previous history of HF. We specified the criteria of all EF values ≥50% to ensure a cohort of definite HFpEF through the period of study. As shown in fig. HFpEF algorithm inclusion crtieria consisted of 1)  InternatFigure We used clinical trials definition to validate definite and probable HF cases . DefinitSymptoms of HF included non-exertional and exertional dyspnea, orthopnea, paroxysmal nocturnal dyspnea, fatigue, decreased exercise capacity, and rapid increase in weight, edema, or abdominal girth. Signs included rapid weight loss on diuretics, elevation of jugular venous pressure, edema, elevation of BNP/NT-proBNP, pulmonary edema/rales, any radiologic evidence of HF , hemodynamic measurements showing elevated filling pressures, and presence of ascites or hepatomegaly.Cases for chart review were randomly selected from the algorithm derived HFpEF cohort. Controls were randomly selected from a cohort who had a procedure code for echocardiogram in the medical records between 2002 and 2014 but no ICD-9 code for HF. Three independent reviewers  completed the chart review of 100 cases and 100 controls in a blinded fashion, with each chart reviewed by two reviewers. Any discrepancies between the reviewers were addressed by further discussion and a consensus was achieved.After manually reviewing the medical records for the 200 randomly selected patients, sensitivity, specificity, positive and negative predictive values were calculated to determine the ability of the algorithm to identify cases and controls relative to identification through manual chart review, which was considered the gold standard , 9. Our Comparison between algorithm-defined and manually reviewed cases and controls is shown in Table Table In a national cohort of VA patients, we demonstrated the feasibility of using NLP and EMR data to curate a HFpEF cohort with high specificity. Combining structured elements  captured from EMR with a NLP tool to capture LVEF from unstructured data, our algorithm was able to rapidly and accurately curate HFpEF from a large EMR database. Our approach led to the creation of a cohort of veterans with HFpEF which allows for investigation of clinical predictors and outcomes in HFpEF.Since HFpEF diagnosis requires clinical symptoms and signs inclusive of laboratory data and radiographic images and documentation of EF, correctly identifying HFpEF patients in a large cohort could be extremely challenging. This is a major limitation for population-based HFpEF research. The recent American College of Cardiology (ACC)/American Heart Association (AHA) guidelines suggested various cutpoints of EF values to diagnose different types of HF; including HFpEF with EF > 50%, heart failure with reduced ejection fraction (HFrEF) with EF < 40%, HF with intermediate EF with EF 40–50%, and HF with recovered EF whose values were < 40% in past but has now become > 50% . To our Similar approaches have been used to curate HF cohorts from EMR databases. For example, Bielinski and colleagues developed a tool to identify HF cases differentiated as HFpEF and HFrEF in an EMR database . In thisSimilar to ours, other algorithms have been developed for various other clinical variables. Electronic Medical Records and Genomics (eMERGE) ePhenotyping algorithms were developed using a similar iterative approach, where each clinical disorder algorithm goes through a series of iterations until the algorithm performs to a desired criteria. These algorithms have been successfully utilized to define phenotypes in various clinical disorders including cataract , dementiOur cohort is similar to other published epidemiological and clinical trial cohorts like Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist (TOPCAT) , OrganizThere were limitations in our algorithm. We may not have included all patients with HFpEF as we required stringent inclusion criteria, but this approach allowed us to more confidently identify true HFpEF patients. We did not have detailed echocardiographic parameters except EF values to characterize diastolic HF and its severity, although recent ACC/AHA guidelines  do not rIn conclusion, using a combination of structured, clinical parameters, and unstructured data, we have curated a well-refined algorithm to identify HFpEF patients to facilitate population-based research in HFpEF.


Thoracic aortic aneurysm is a typically silent disease characterized by a lethal natural history. Since the discovery of the familial nature of thoracic aortic aneurysm and dissection (TAAD) almost 2 decades ago, our understanding of the genetics of this disorder has undergone a transformative amplification. To date, at least 37 TAAD-causing genes have been identified and an estimated 30% of the patients with familial nonsyndromic TAAD harbor a pathogenic mutation in one of these genes. In this review, we present our yearly update summarizing the genes associated with TAAD and the ensuing clinical implications for surgical intervention. Molecular genetics will continue to bolster this burgeoning catalog of culprit genes, enabling the provision of personalized aortic care. This review presents an annual update to the article “Genes Associated with Thoracic Aortic Aneurysm and Dissection: Update and Clinical Implications” originally published in 2017 and updated in 2018 in AORTA.Thoracic aortic aneurysm (TAA) affects 1% of the general populationReport of inherited TAAD in the 1990sROBO4mutation was identified, including two variants that segregated with disease among two affected families.ROBO4is well expressed in vascular endothelial cells and plays a role in endothelial barrier function.ROBO4variants lead to the disruption of endothelial performance at a cellular level, thus altering vascular permeability, the authors cultured human aortic endothelial cells and either silencedROBO4or expressedROBO4variants. They confirmed thatROBO4abnormalities did indeed induce endothelial barrier dysfunction. Lastly, the authors created homozygousROBO4knockout mice and a knock-in mouse with anROBO4splice donor site mutation; the affected mice presented with a mix of aortic valve dysfunction (BAV and/or aortic regurgitation or stenosis) and AscAA, confirming their suspicion that a heterozygous mutation inROBO4can lead to a nonsyndromic presentation of BAV/AscAA.During 2018, several important studies were published that have enhanced our understanding of the pathogenesis of TAAD. Gould et alLTBP1, amongst other genes.LTBP1was considered the likely culprit given its relationship to TGF-β. In addition to TAA, the affected individuals displayed additional features of Marfan syndrome (MFS) and Loeys-Dietz syndrome, even though none of them met the criteria for diagnosis.Latent transforming growth factor binding proteins (LTBP), a family of extracellular matrix glycoproteins, have been shown to play a significant role in TGF-β regulation.LTBP3) gene have been associated with TAAD in a WES study of 271 individuals from unrelated families with heritable thoracic aortic disease (multiple affected family members) without a known genetic etiology for aortopathy.LTBP3in a second family were identified. Sequencing of 338 additional individuals with non-syndromic TAAD found nine additional heterozygousLTBP3rare variants. The authors also demonstrated thatLTBP3knockout mice manifested enlarged aortic roots and ascending aortas compared with wild type mice. These findings demonstrate that individuals withLTPB3are at increased risk for TAAD, in addition to the already established risk for skeletal and dental abnormalities.Mutations of the latent TGF-β binding protein-3 (ARIH1) have been observed in patients with early-onset or familial TAAD.AR1H1encodes a protein of the LINC (linker of nucleoskeleton and cytoskeleton), a protein complex essential for anchoring myocyte nuclei to the cytoskeleton.Rare mutations in the Parkin-like E3 ubiquitin ligase Ariadne-1  gene have been associated with TAAD. Shalata et al have identified an additionalMYLKmissense mutation in a single pedigree.Numerous mutations of the myosin light chain kinase (RAP1GDS1gene and known to be involved in the contraction of vascular smooth muscle cell (VSMC).+/−mouse model, since the complete knockout (SmgGDS−/−) was embryologically lethal, they observed that the downregulation of SmgGDS was causing “pathological phenotype changes in VSMC” via the angiotensin-II pathway.+/−, the mice had less aortic growth and fewer aortic ruptures, suggesting that SmgGDS could be used as a biomarker or a therapeutic agent.Insights to the pathogenesis of TAAD are as important as identifying TAAD variants. Nogi et alAdvances in 2018 have increased our understanding of the pathogenesis of TAAD. The number of genes with genetic variants or mutations associated with TAAD has increased from 29 in our original 2017 reportWe will continue to report annual updates regarding the “TAA genetic dictionary” with updates to the Table and Figure below and provide suggested surgical intervention criteria for each identified mutation.