Human immunodeficiency virus (HIV)-infected patients are at an increased risk for developing opportunistic infections, reactive conditions and neoplasms. As a result, a broad range of conditions are frequently included in the differential diagnosis of HIV-related lesions. The clinical history of HIV infection may, however, be misleading in some cases. Illustrative cases are presented in which knowledge of a patient's HIV status proved to be misleading and increased the degree of complexity of the cytologic evaluation. Case 1 involved the fine needle aspiration (FNA) of a painful 3 cm unilateral neck mass in a 38-year-old female with generalized lymphadenopathy. Her aspirate revealed a spindle cell proliferation devoid of mycobacteria that was immunoreactive for S-100 and macrophage markers . Multiple noncontributory repeat procedures were performed until a final excision revealed a schwannoma. Case 2 was a CT-guided FNA of a positron emission tomography positive lung mass in a 53-year-old man. The acellular aspirate in this case contained structures resembling fungal spore forms that were negative for mucicarmine and GMS stains, as well as cryptococcal antigen immunocytochemistry. A Von Kossa stain confirmed that these pseudo-fungal structures were calcified debris. Follow up revealed multiple calcified lung and hilar node based granulomata. Case 3 involved the cytologic evaluation of pleural fluid from a 47-year-old man with Kaposi sarcoma and recurrent chylous pleural effusions. Large atypical cells identified in his effusion were concerning for primary effusion lymphoma. Subsequent pleural biopsy revealed extramedullary hematopoiesis, documenting these atypical cells as megakaryocytes. These cases demonstrate that knowledge of a patient's HIV status can be misleading in the evaluation of cytology specimens, with potential for misdiagnosis and/or multiple procedures. To avoid this pitfall in the setting of HIV infection, common entities unrelated to HIV infection and artifacts should always be included in the differential diagnosis. Human immunodeficiency virus (HIV) causes acquired immunodeficiency syndrome (AIDS). As CD4 T-cell numbers decline, HIV-infected patients become progressively more susceptible to opportunistic infections  and the development of malignancies. AIDS-defining cancers include Kaposi sarcoma (KS), cervical cancer and high-grade non-Hodgkin lymphoma. Patients infected with HIV may also have unusual manifestations of common infections and neoplasms, such as spindle cell tumors associated with mycobacteria. As patients infected with HIV are living longer due to the benefits of antiretroviral therapy, they are now also more likely to develop certain non-AIDS-defining cancers such as lung cancer, hepatocellular carcinoma and Hodgkin lymphoma. HIV-infe3 and HIV viral load of 191,528 copies/mL presented with left neck pain. She was naïve to antiretroviral therapy. An ultrasound of her neck revealed four enlarged, hypoechoic, enhancing masses in the upper lateral neck interpreted to be lymph nodes. A fine needle aspiration (FNA) was performed that yielded scant material showing a morphologically bland spindle cell lesion [A 38-year-old HIV positive female with a CD4 cell count of 260 cells/mml lesion , thoughtl lesion  and the l lesion .Incidental nodules were found on a chest X-ray in a 53-year-old HIV positive male. A follow-up positron emission tomography (PET) and CT scan raised the possibility of a neoplastic process. A lung FNA was performed one month later, which yielded an acellular specimen that was comprised of granular debris with several ovoid bodies that resembled budding fungal spore forms measuring up to 15 μm or larger . MucicarA 47-year-old male patient with a past medical history significant for smoking, AIDS, anemia (hematocrit 35.3%) and KS presented with recurrent chylous pleural effusions. Multiple pleurocentesis procedures were performed with fluid submitted for cytological evaluation. The majority of cells in these effusions were reactive mesothelial cells and admixed granulocytes. Also identified in these pleural effusions Figures  and 7 weLack of pertinent clinical information provided with specimen samples that are submitted to the clinical and anatomic laboratories is a frequent complaint of pathologists. Inadequate knowledge of a patient's clinical history may hinder the pathologic interpretation. The three cases presented demonstrate how the knowledge of a patient's HIV status, in contrast, can occasionally be misleading in the evaluation of cytology specimens, with potential for misdiagnosis and/or multiple potentially unnecessary procedures. Taking into consideration the high prevalence of HIV and AIDS around the world, and limited resources for diagnosis in developing countries, cytology  has proven to be a useful method for diagnosis, reducing the necessity for surgical excision, and facilitating rapid triage for therapy.In case 1, the spindle cell lesion originally identified in the FNA material was subsequently histologically proven to represent a schwannoma. The differential diagnosis of a spindle cell proliferation/lesion in an HIV infected patient includes entities somewhat unique to HIV such as KS, mycobacterial spindle cell pseudotumor and Epstein Barr Virus (EBV)-associated smooth muscle tumors, as well as spindle cell lesions independent of HIV infection . Most FNA specimens of KS have a bloody background in which intact to loosely cohesive clusters of bland spindle-shaped cells are distributed.‐6 CloselHistoplasma capsulatum. The identification of hyphae or yeast forms on cytological smears takes into account morphological criteria  and additional staining characteristics.[H.capsulatum (size range 2–5 μm), Cryptococcus neoformans (size range 5–15 μm), and Blastomyces dermatidis (size range 8–20 μm). Several of these fungi may also demonstrate budding , a thick capsule  or a thick refractile wall . In case 2, fragmented calcific debris that formed round bodies was initially mistaken for potential fungal forms. However, unlike fungi these calcified structures did not stain with mucicarmine or GMS stains. Intrinsic and extrinsic contaminants [Patients infected with HIV are also at increased risk for fungal opportunistic infections. Several of these fungal infections may be dimorphic , such as pulmonary histoplasmosis caused by eristics. Round tod pollen) and evend pollen) may be m1/3 to 2/3 of patients with KS[Pleural disease encountered in the HIV-infected patient may be due to infection , malignancy , Castleman's disease, or may accompany systemic problems .‐20 In ths with KS‐22 and is with KS Cytologis with KS‐25 AlthoIn summary, we present three cases which illustrate how knowledge of a patient's HIV status was misleading in the evaluation of varying cytology specimens, with potential for misdiagnosis and/or multiple unnecessary procedures. In the setting of HIV infection, common entities unrelated to HIV infection must be maintained within the differential diagnosis. Contaminants and artifacts in these patients have the potential to masquerade and mimic significant infections. The use of ancillary studies including cytochemical stains and immunocytochemical studies are invaluable in such cases to establish an accurate diagnosis.No competing interest to declare by any of the authors.http://www.icmje.org/#author. Each author has participated sufficiently in the work and take public responsibility for appropriate portions of the content of this article.Each author acknowledges that this final version was read and approved. All authors of this article declare that we qualify for authorship as defined by ICMJE As this is case report without identifiers, our institution does not require approval from Institutional Review Board (IRB) To ensure integrity and highest quality of CytoJournal publications, the review process of this manuscript was conducted under a double blind model(authors are blinded for reviewers and reviewers are blinded for authors)through automatic online system..


Dear Editor,We report a rare complication of late posterior subluxation of rollable intraocular lens (IOL) after an uneventful microphacoemulsification cataract surgery. A 60-year-old non-diabetic, non-hypertensive male patient was operated for microphacoemulsification, phaco chop technique for senile cataract in the right eye using 0.9 mm limbal incision which was enlarged to 1.5 mm and an acrylic rollable IOL  was implanted in the bag. The patient regained 20/20 vision (on Snellen's chart) at the end of six weeks of follow-up and had no complaints. Then after three months the patient returned with decrease in vision in the right eye (20/200). The slit-lamp examination revealed posterior capsular opacification with shrinkage of anterior capsule. Patient underwent uneventful YAG capsulotomy (around 2 mm diameter). Patient regained 20/20 vision at two weeks follow-up. However, two months later, patient returned with painless progressive diminution of vision (finger counting at 2 meters) with glare and uniocular diplopia. There was no history of trauma. The slit-lamp examination  and ultrMicroincisional cataract surgery is a safe procedure with a very short learning period for an experienced cataract surgeon and rollable ultrathin IOLs eliminate the need for enlargement of corneal incision. However,3It has been postulated that the shrinkage of the anterior capsule exerts centripetal forces on the ends of the plate haptics, causing the optic to move posteriorly and to exert pressure against the posterior capsule. If either the posterior or anterior capsule is disrupted by Nd:YAG laser treatment, the forces created by capsular contraction against the flexible lens may cause extension of radial tears and appear to be a substantial risk for further capsule-tearing, releasing the IOL into the vitreous cavity, even months later. The forces of capsular contraction can impart a spring-loading effect on plate haptic silicone lenses. Due to inadequate capsular adherence, these lenses are at risk of posterior dislocation from capsular rents following Nd:YAG laser treatment.It is important to have a close follow-up of patients with plate haptic rollable IOLs undergoing YAG capsulotomy. Patients should be warned of this rare possible complication. Vitrectomy and IOL exchange with rigid PMMA lens implantation in the sulcus seems to be effective in tackling this complication.


Osx knock-out mice lack bone completely and cartilage is normal. This opens a new window to the whole research field of bone formation. Osx inhibits Wnt pathway signaling, a possible mechanism for Osx to inhibit osteoblast proliferation. These reports demonstrate that Osx is the master gene that controls osteoblast lineage commitment and the subsequent osteoblast proliferation and differentiation. This review is to highlight recent progress in understanding the molecular mechanisms of transcriptional regulation of bone formation by Osx.Bone formation is a complex developmental process involving the differentiation of mesenchymal stem cells to osteoblasts. Osteoblast differentiation occurs through a multi-step molecular pathway regulated by different transcription factors and signaling proteins. Osx  is the only osteoblast-specific transcriptional factor identified so far which is required for osteoblast differentiation and bone formation.  Runx2-null mutants, no endochondral and no membranous bones form [Runx2 is required for the differentiation of mesenchymal cells into preosteoblasts. As a downstream gene of Runx2, Osx is required for the differentiation of preosteoblasts into mature osteoblasts. Osx is specifically expressed in all osteoblasts. In Osx-null embryos, cartilage is formed normally, but the embryos completely lack bone formation [β-catenin in either skeletal progenitor cells or at a later stage of osteoblast development in mouse embryos blocks osteoblast differentiation [Bone formation takes place through two distinct processes: endochondral ossification involving a cartilage model and intramembranous ossification by which bones form directly from condensations of mesenchymal cells without a cartilage intermediate. Bone formation is a highly regulated process involving the differentiation of mesenchymal stem cells to osteoblasts. Osteoblasts produce a characteristic extracellular collagenous matrix that subsequently becomes mineralized after hydroxyapatite crystals deposition. Much progress has been made in understanding the factors that control the gene expression program through the osteoblast induction, proliferation, differentiation, and maturation. Osteoblast differentiation occurs through a multistep molecular pathway regulated by different transcription factors and signaling proteins Table . Indian ormation . Wnt signtiation -7. Otherntiation -12. ThisOsx was discovered as a bone morphogenic protein-2 (BMP2) induced gene in mouse pluripotent mesenchymal cells, encoding a transcription factor that is highly specific to osteoblasts [Osx gene is located in chromosome 15 in mouse and in chromosome 12 in human. There are only two exons in the Osx gene. Exon 1 sequence encodes the seven N-terminal amino acids of Osx, and exon 2 contains the remaining open reading frame (ORF) and 3-prime UTR. The mouse Osx protein is a 428 amino acid polypeptide with a molecular mass of about 46 kDa as shown in Figureeoblasts . Osx is Osx transcripts are not detected before embryonic stage E13 [Osx first appears in differentiating chondrocytes, the surrounding perichondrium, and mesenchymal condensations of future membranous bones of E13.5 embryos. After E15.5, Osx is strongly expressed in cells that are associated with all bone trabeculae and bone collar formation. Weak expression of Osx is observed in the prehypertrophic zone. Osx is highly expressed in bone trabeculae and in secondary ossification centers after birth. Osx is only expressed in cells in the bone matrix and the inner (endosteum) and outer (periosteum) bone surfaces.During mouse embryogenesis, Osx gene was inactivated in the mouse embryonic stem (ES) cells using homologous recombination to understand Osx function. Most of the exon2 coding sequence was deleted. As a result, the Osx gene was inactivated. Heterozygous Osx mutant mice were normal and fertile. Homozygous Osx mutant mice were lethal and these mice had difficulty in breathing, rapidly became cyanotic, and died within 15 min of birth. Newborn homozygous mutant mice showed severe inward bending of forelimbs and hindlimbs [Osx-null embryos have normal cartilage development, they completely lack bone formation, so neither endochondral nor intramembranous bone formation occurs. The mesenchymal cells in Osx-null mice do not deposit bone matrix, and cells in the periosteum and the condensed mesenchyme of membranous skeletal elements cannot differentiate into osteoblasts. In the endochondral skeletal elements of Osx-null mutants, a dense mesenchyme emerges from the perichondrium/periosteum and invades the zone of hypertrophic chondrocytes along with blood vessels. However, cells in this mesenchyme are arrested during differentiation. A similar, dense mesenchyme is also found in the membranous skeletal elements. Bone trabeculae are completely absent in all skeletal elements. Although mineralization does not occur in membranous skeletal elements, it does in the endochondral skeleton because of the physiological mineralization of the zone of hypertrophic chondrocytes. No mineralization occurs in the periosteum, suggesting that bone collars do not form.It has been demonstrated that Osx is necessary for bone formation and mineralization in vivo . The Osxindlimbs . AlthougOsx-null mutant embryos, expression of type I collagen (Col1a1) in the condensed mesenchyme of the membranous skeleton and the periosteum and mesenchyme of the endochondral skeleton is severely reduced. Expressions of the osteoblast-specific markers such as osteonectin, osteopontin and bone sialoprotein (BSP) cannot be detected in these mesenchymes. In E18.5 Osx-null embryos, osteocalcin, a late, highly specific osteoblast marker, is not expressed in endochondral and membranous skeletal elements. Despite a lack of osteoblast markers expression, Runx2 expression in Osx-null mutants remains comparable to that of wild-type osteoblasts in the dense mesenchyme of both membranous and endochondral skeletal elements. Thus, osteoblast differentiation is completely arrested in Osx-null embryos, even though similar expression of Runx2 remains compared to wild-type embryos. On other hand, over-expressed Osx in vitro has been shown to induce expression of osteocalcin and collagen type 1a1.In Osx-null embryos the number of TRAP-positive cells appear to be reduced compared to wild-type embryos. In long bones of Osx-null embryos cells from the periosteum invade the zone of the hypertrophy chondrocyte as a wedge-shaped expansion of the periosteum in which osteoblast precursors are arrested in their differentiation. These observations are supported by the evidence that expressions of both OPG and RANKL are downregulated, but the ratio of OPG/RANKL increases in E18.5 Osx-null calvarial cells [In the skeletal elements of E18.5 al cells . ExpressLrp5-null mice, which phenocopy the osteoporosis-pseudoglioma syndrome in humans [Lrp5 lead to high bone mass syndrome in patients [Dkk1 mutant mice increase with an increased number of osteoblasts [Dkk1 in osteoblasts causes severe osteopenia with decreased osteoblast numbers [It has been demonstrated that canonical Wnt signaling is required for normal osteoblast proliferation. A marked increase in osteoblast proliferation occurs when β-catenin is stabilized in osteoblasts during mouse embryonic development . Moreoven humans , developn humans . In contpatients  and in mpatients . The Wnteoblasts . In cont numbers . These dOsx-null embryos, expression of the Wnt antagonist Dkk1 was abolished, and that of Wnt target genes c-Myc and cyclin D1 was increased. It has been demonstrated that Osx binds to and activates the Dkk1 promoter. Osx is shown to inhibit β-catenin-induced Topflash reporter activity and also inhibit β-catenin-induced secondary axis formation in Xenopus embryos. Moreover, this study showed that in calvaria of E18.5 Osx-null embryos harboring the TOPGAL reporter transgene, β-galactosidase activity was increased, suggesting that Osx inhibited the Wnt pathway in osteoblasts in vivo [Recent studies in our research group have provided evidences showing that the osteoblast-specific transcription factor Osx is able to inhibit Wnt pathway activity during osteoblast differentiation . In calv in vivo . Osx canThe PRR region of Osx is responsible for disruption of Tcf1 binding to DNA, and for inhibition of β-catenin transcriptional activity. These findings indicate that Osx negatively controls the activity of β-catenin in two different mechanisms shown in FigureOsx-null calvaria showed greater BrdU incorporation than wild-type calvaria, and primary calvarial cells from Osx-null E18.5 embryos also grew faster than wild-type cells. On the other hand, Osx over-expression in C2C12 mesenchymal cells inhibited cell growth. Because Wnt signaling has a major role in stimulating osteoblast proliferation, it is speculated that Osx-mediated inhibition of osteoblast proliferation is a consequence of the Osx-mediated control of Wnt/β-catenin activity. These results add a new layer of control to Wnt signaling in bone formation.We have shown that Osx decreases osteoblast proliferation . E18.5 OALP), bone sialoprotein (BSP) and type I collagen, as they commence to produce and mature osteogenic extracellular matrix. Finally, they express genes involved in mineralization of the extracellular matrix such as osteocalcin (OC), osteopontin. This highly regulated program of gene expression and cellular differentiation is governed by the expression and activity of different transcription factors. These factors do not act alone but interact with each other to integrate diverse signals and fine-tune gene expression.Osx is necessary for the osteoblast lineage ,13. FollOsx-null mutant phenotype and recent studies, the following brief model for osteoblast differentiation is proposed as shown in FigureRunx2-expressing biopotential progenitors can differentiate into either osteoblast or chondrocyte depending on cell signaling. Then cells differentiate into preosteoblasts, a process in which Runx2 play an essential role. At this stage, preosteoblasts express early osteoblast marker genes like ALP. Next step, preosteoblasts differentiate into mature osteoblast, a process in which Osx plays a critical role. Mature functioning osteoblasts strongly express characteristic later osteoblast marker genes such as OC and BSP. In the membranous and endochondral skeletons, Osx-null preosteoblasts are blocked from differentiating into osteoblasts, so there is no mature osteoblast without Osx. In Osx-null embryos, osteoblast differentiation markers, such as OC, BSP and osteonectin, are not expressed. Because the promoter regions of several osteoblast marker genes contain binding sites for Runx2 that are functional in DNA transfection experiments [in vivo and produce a bone-specific matrix.Based on the characterization of the eriments ,21, it iWnt/β-catenin signaling has an essential role in osteoblast differentiation during embryonic development and has a major role in stimulating osteoblast proliferation during both embryonic and postnatal development. Osx is an osteoblast-specific transcription factor, required for osteoblast differentiation. The inhibition of Wnt/β-catenin signaling activity by Osx, also constitutes a possible mechanism for the inhibition of osteoblast proliferation by Osx. Recent observations that Osx inhibits Wnt signaling pathway in vitro and in vivo provide novel concept of feedback control mechanisms involved in bone formation .Osx-null mice, while no Osx transcripts are detected in skeletal elements in Runx2-knockout mice [Osx gene promoter [Osx is believed to be downstream of Runx2 in the pathway of osteoblast differentiation because Runx2 expression is normal in out mice . This ispromoter . It is n2 vitamin D3, which have positive roles in osteoblast function, have also been shown to up-regulate Osx expression [The mechanism underlying the regulation of Osx expression in osteoblasts is still unclear. Several studies have reported that some factors can modulate Osx expression. Both BMP-2 and insulin-like growth factor-1 (IGF-1) can induce Osx expression in undifferentiated mesenchymal stem cells . IGF-I-mpression ,26. It wIn vivo studies provide genetic evidence that p53 tumor suppressor blocks osteoblast differentiation and bone development [Osx promoter.Some studies indicate that negative regulators of osteoblastogenesis can inhibit Osx expression. TNF inhibited Osx mRNA in pre-osteoblastic cells without affecting Osx mRNA half-life ,28. Inhielopment ,28. Prolelopment . PTH inhelopment . Recentlelopment . ATF4-reDespite these interesting findings, the details concerning the regulation and function of Osx are incompletely understood.Osteoporosis is characterized by reduced bone mass, alterations in the microarchitecture of bone tissue, reduced bone strength, and an increased risk of fracture . OsteopoOsx are associated with bone mineral density (BMD) in children and adults probably through primary effects on growth [Osx (SP7) locus. A meta-analysis of these existing studies revealed strong association between SNPs in the Osx region and adult lumbar spine BMD. In light of these findings, this research group genotyped a further 3692 individuals from ALSPAC who had whole body BMD and confirmed the association in children as well.Recent study has indicated that genetic variants in the chromosomal region of n growth . A genomAlthough Osx has been identified to be associated with osteoporosis-related phenotypes, further investigation needs to be done to determine whether Osx will represent a useful diagnostic index of osteoporosis or molecular target for therapeutic manipulation.Runx2 , which is an upstream of Osx, have been shown to be the cause of the human genetic disease cleidocranial dysplasia [Osx mutation leads to any clinical human disease.Osx is indispensable for the commitment of the osteoblast lineage and the expression of the osteoblast-specific matrix proteins, including type I collagen, bone sialoprotein, osteonectin, and osteocalcin. No pharmacological approach to target Osx in osteoblasts has been reported. Heterozygous mutations in ysplasia . There iThe extensive studies by many laboratories to explore how to control the Wnt signaling pathway in osteoblasts stems from the realization that this pathway has an essential role in bone mass determination in the adult skeleton. There is also an expectation that efforts to pharmacologically target this pathway should yield promising agents to treat bone diseases such as osteoporosis. Results in our group showing that Osx inhibits Wnt/β-catenin signaling add an important new layer of control to the complex regulation of the Wnt pathway in osteoblasts .Osx gene into the mouse osteosarcoma cells inhibited tumor cell growth in vitro and in vivo and significantly reduced tumor incidence, tumor volume, and lung metastasis following intratibial injection. Using an in vitro migration assay, Osx suppressed the migration of tumor cells to lung extracts. These results suggest that Osx expression may play a role in osteosarcoma tumor growth and metastasis, and that osteolytic activity of tumor cells may be regulated by Osx via down-regulation of interleukin-1 gene transcription [It was observed that the Osx expression was decreased in two mouse osteosarcoma cell lines and in three human osteosarcoma cell lines . Transfecription . It is rcription .Bone formation is essential for maintenance and healing of the skeleton following injury and operative interventions, such as osteotomies and limb lengthening. In numerous orthopedic conditions, such as congenital pseudoarthrosis of tibia, femoral head osteonecrosis, and large bone lengthening, bone healing and regeneration remain challenging goal to achieve. Most therapy for skeletal diseases with less bone such as osteoporosis and osteonecrosis is aimed at inhibiting bone resorption, but to cure these diseases, it is also critically important to stimulate new bone formation. Therefore, there is currently great interest in understanding the regulation of osteoblast differentiation and activity to guide the development of anabolic therapies. Although no pharmacological approach to target Osx in osteoblasts has identified yet, an interesting future research direction is to look for upstream genes or molecules which can selectively target Osx expression and activity. We speculate that Osx could become a therapeutic target in efforts to stimulate the anabolic pathway of bone synthesis.Bone formation is a complex process regulated by multiple factors and pathways; it is clearly shown that Osx is required for the final commitment of osteoblast lineage. Although recent molecular and genetic studies using gene targeting in mice have established Osx as a master regulator of osteoblast differentiation during bone formation, the mechanisms of Osx regulation of osteoblast differentiation and function are still under investigation. Future studies to decipher the Osx direct upstream or downstream molecular targets, Osx expression regulation and Osx functional partners are required to clarify the detailed mechanism of the temporal and spatial regulation of Osx for bone formation and homeostatic regulation of skeletal system. The need to develop novel drugs that stimulate bone formation and thereby elevate bone mass (anabolic agents) has opened new research areas for therapeutic intervention in the treatment of bone-related diseases.Col1a1: type I collagen; OC: osteocalcin; BSP: bone sialoprotein; ALP: alkaline phosphatase; PRR: proline-rich region; BMP2: bone morphogenic protein-2; BMD: bone mineral density.Osx: Osterix; OB: osteoblast; E18.5: embryonic day 18.5; Ihh: Indian hedgehog; The authors declare that they have no competing interests.The author contributed to the article. The author has read and approved the final manuscript.


Research on the neural correlates of risk-related behaviors and personality traits has provided insight into mechanisms underlying both normal and pathological decision-making. Task-based neuroimaging studies implicate a distributed network of brain regions in risky decision-making. What remains to be understood are the interactions between these regions and their relation to individual differences in personality variables associated with real-world risk-taking.We employed resting state functional magnetic resonance imaging (R-fMRI) and resting state functional connectivity (RSFC) methods to investigate differences in the brain's intrinsic functional architecture associated with beliefs about the consequences of risky behavior. We obtained an individual measure of expected benefit from engaging in risky behavior, indicating a risk seeking or risk-averse personality, for each of 21 participants from whom we also collected a series of R-fMRI scans. The expected benefit scores were entered in statistical models assessing the RSFC of brain regions consistently implicated in both the evaluation of risk and reward, and cognitive control . We specifically focused on significant brain-behavior relationships that were stable across R-fMRI scans collected one year apart. Two stable expected benefit-RSFC relationships were observed: decreased expected benefit (increased risk-aversion) was associated with 1) stronger positive functional connectivity between right inferior frontal gyrus (IFG) and right insula, and 2) weaker negative functional connectivity between left nucleus accumbens and right parieto-occipital cortex.Task-based activation in the IFG and insula has been associated with risk-aversion, while activation in the nucleus accumbens and parietal cortex has been associated with both risk seeking and risk-averse tendencies. Our results suggest that individual differences in attitudes toward risk-taking are reflected in the brain's functional architecture and may have implications for engaging in real-world risky behaviors. Risk seeking and risk-avoidance play a central role in both normal and pathological decision-making. As such, characterizing the neural correlates of these traits and behaviors is a central focus of cognitive neuroscience and psychiatric research. Individuals with anxiety disorders, and those with high but not pathological levels of anxiety, exhibit high levels of risk-aversion A distributed network of brain regions has been implicated in task-based neuroimaging studies of risky decision-making, including orbitofrontal cortex (OFC), ventral striatum (nucleus accumbens), insula, anterior cingulate, and lateral frontal and parietal cortices Individual differences in risk-related personality factors have also been linked to individual differences in activation in several of the aforementioned brain regions. Higher levels of behavioral risk-aversion and harm-avoidance have been associated with increased activation in the inferior frontal gyrus (IFG) and insula Together, existing findings suggest that interactions among brain regions supporting the evaluation of risk and reward  approaches characterize functional brain networks while avoiding some of the constraints of task-based approaches Here, we employed R-fMRI approaches to identify differences in RSFC predicted by individual differences in a trait measure of risk-related cognitions. Specifically, we investigated whether patterns of RSFC in brain regions previously implicated in risky decision-making were related to individuals' beliefs about the consequences of engaging in risky behaviors. Furthermore, we sought to identify brain-behavior relationships that were stable across time (∼1 year). In order to do this, we administered the Cognitive Appraisal of Risky Events (CARE) questionnaire cognitive control and risk-aversion . Namely, increased expected benefit, or increased risk seeking, would be associated with weaker RSFC between these regions. Similarly, decreased expected benefit, or increased risk-aversion, would be associated with stronger RSFC between these regions. For brain regions associated with reward and risk seeking , we predicted expected benefit scores to be positively related to RSFC. Namely, increased expected benefit (risk seeking) would be associated with stronger RSFC between these regions, while decreased expected benefit (risk-aversion) would be associated with weaker RSFC.We predicted that expected benefit scores would be negatively related to RSFC between brain regions associated with http://www.nitrc.org/frs/?group_id=274.R-fMRI data were acquired from 21 right-handed healthy adults . All participants were college-educated native English speakers with no history of neurological or psychiatric disorders as confirmed by psychiatric interview, and had no contraindications to MRI. The study was approved by the institutional review boards of New York University (NYU) and the NYU School of Medicine. Prior written informed consent was obtained from all participants in accordance with the principles expressed in the Declaration of Helsinki. R-fMRI data from these participants have been reported in several published studies All participants completed the Cognitive Appraisal of Risky Events (CARE) questionnaire For the purposes of this study, participants' expected benefit scores were calculated as an average of their ratings on 4 factors: risky sexual behavior, heavy drinking, illicit drug use, and aggressive and illegal behaviors. Motivated conceptually by a desire to focus on those risky behaviors most likely to lead to adverse personal consequences, and following both previously published studies We therefore obtained one score per participant that indicated an individual's level of expected benefit from engaging in a wide range of risky behaviors. Scores could range from 1  to 7 (extremely likely to experience positive consequences). We interpreted lower expected benefit scores as indicating a more risk-averse personality, with higher expected benefit scores indicating a more risk seeking personality. These individual scores were entered as covariates of interest in group-level R-fMRI analyses to identify areas between which RSFC varied as a function of one's perceptions of the positive consequences associated with risk-taking.3; duration  = 6.5 min). During each scan, participants were instructed to rest with eyes open while the word “Relax” was projected onto the center of the display screen. A high-resolution T1-weighted magnetization prepared gradient echo sequence  was also collected for spatial normalization and localization.All MRI scans were collected on a Siemens Allegra 3.0-T scanner at the NYU Center for Brain Imaging. Resting state fMRI scans consisted of 197 contiguous echo planar imaging whole-brain volumes  4 to 16 months (mean  = 10.0 [SD 4.1]) prior to the Primary Scan (or “Scan 2”). Since we were interested in brain-behavior relationships that were stable across time, we treated Scan 1 as our “Replication Scan,” with which we investigated the stability of results emerging from the Primary Scan data analyses. Finally, 18 participants provided a third R-fMRI scan (“Scan 3”) collected ∼45 minutes after Scan 2, during the same session. In total, data from all 3 R-fMRI scans  were available for 16 participants, data from two scans 5 months apart (Scans 1 and 2) for one participant, data from two scans 45 minutes apart (Scans 2 and 3) for two participants, and data from one scan only (Scan 2) for two participants.Twenty-one participants provided a resting state scan during a session in which the CARE questionnaire was also administered. We treated the scan collected during this session as our “www.fmrib.ox.ac.uk) and included spatial smoothing using a Gaussian kernel  and mean-based intensity normalization of all volumes by the same factor . Temporal bandpass filtering (0.009–0.1 Hz) and linear and quadratic detrending were then carried out in AFNI. Registration of each participant's high-resolution anatomical image to a common stereotaxic space  was accomplished using a two-step process FLIRT in FSL FNIRTSlice timing correction for interleaved acquisition (using Fourier-space time-series phase-shifting), motion correction , and despiking (detection and reduction of extreme time series outliers) were carried out using Analysis of Functional NeuroImages (AFNI) FAST), the mean global signal, and 6 motion parameters  as described in previously published studies In order to control for the potential confounding effects of motion and physiological processes , signals from 9 nuisance covariates were removed from each participant's preprocessed data. These were: white matter and cerebrospinal fluid signals  were selected as seed regions for RSFC analyses. Four of these regions were selected based on their association with the assessment of risk and reward . Another six regions were selected based on their association with executive function and cognitive control . All 10 of these seed ROIs were created using parcellation units derived from the Harvard-Oxford Cortical Structural Atlas probability thresholded at 75%. A high tissue probability was employed to maximize the likelihood of including only voxels that were classified as unique to each seed ROI. This conservative approach ensured that our atlas-based ROI definition minimized error due to normalization and smoothing across anatomical regions and across participants.Two additional seeds located in the left and right dorsal anterior cingulate cortex (ACC) were also included. This region of ACC was previously shown to be functionally connected with a higher order cognitive network including lateral prefrontal regions implicated in cognitive control 3dfim+, carried out in individuals' native space. This analysis produced participant-level correlation maps of all voxels in the brain that were positively and/or negatively correlated with the seed time series. Finally, these correlation maps were converted to Z-value maps using Fisher's r-to-z transformation. Participant-level correlation maps were computed for each scan separately , and “Multi-Scan” mean correlation maps were created for participants with more than one scan. All maps were then transformed to MNI152 2 mm standard space.Each participant's 4-D residuals volume was spatially normalized by applying the previously computed transformation to MNI152 2 mm standard space. We then extracted the mean time series from each seed by averaging across all voxels in each seed ROI. Using these mean time series, we performed a correlation analysis for each participant and each ROI using the AFNI program http://www.nitrc.org/projects/fcon_1000).Scripts containing a similar sequence of the processing commands employed here to compute seed-based RSFC have been released as part of the ‘1000 Functional Connectomes Project’ flameo (ordinary least squares). In addition to the group mean vector, demeaned expected benefit scores and two nuisance variables (demeaned age and sex) were included in the model. Cluster-based statistical correction for multiple comparisons was performed using Gaussian random field theory  to identify regions whose connectivity with a seed ROI was significantly predicted by expected benefit scores. We then tested for the presence of the same relationship in our Replication Scan (Scan 1) by extracting the mean correlation (with the same seed ROI) for significant clusters identified using the Primary Scan, and correlating the resultant values with expected benefit scores. Scan 2 was chosen as the Primary, or “base,” scan because its acquisition was closest in time to the collection of the questionnaire. This allowed us to investigate whether significant relationships between expected benefit and RSFC observed in the Primary Scan (at the time the CARE was administered) were also observed in the Replication Scan (collected 4 to 16 months earlier). Given the presence of FEAT in FSL. In addition to the two group mean vectors, two nuisance variables per group  were included in the model. Cluster-based statistical correction for multiple comparisons was performed using Gaussian random field theory . This group-level analysis produced thresholded Z-statistic maps comparing the functional connectivity of each seed ROI between groups  as well as maps of the significant positive and negative functional connectivity of each seed ROI within each group . These analyses allowed us to determine whether the same significant relationships between RSFC and expected benefit observed in the previously described covariate analyses could also be observed in a direct comparison of the RSFC maps between participants who expected more or less benefit from engaging in risky behaviors. As such, these group comparisons are a replication of the correlation analyses and have been included to provide a complementary, alternative view (not an independent test) of the observed brain-behavior relationships.To further investigate the relationship between RSFC and expected benefit (EB) scores, participants were divided into two groups  based on a median split. Twenty participants were included – participants with the 10 lowest scores were included in the Lower EB group, and participants with the 10 highest scores were included in the Higher EB group. One participant with intermediate scores was excluded. For each seed ROI whose significant relationships with expected benefit replicated across Scans 1 and 2 , a two-sample t-test was carried out on participants' Multi-Scan mean correlation maps  using Expected benefit ratings from participants in the current study ranged from 1.00 to 4.57 on a 7-point Likert scale (mean = 2.54 [SD 0.88]). Although this mean score suggests that, on average, participants did not expect positive consequences from engaging in risky behaviors, across participants there was sufficient range in the ratings to provide an estimate of the effect of expected benefit on RSFC in subsequent neuroimaging analyses.Significant relationships between expected benefit scores and RSFC were detected for several seed regions   = −0.49, p<0.05 (r(17)  = −0.45, p = 0.06.Increased expected benefit from engaging in risky behaviors was associated with weaker positive connectivity between the IFG and insula. In other words, the less benefit expected from risk-taking , the stronger the positive connectivity between IFG and insula. In the Primary Scan, we observed a significant voxel-wise inverse relationship between expected benefit and RSFC between the right IFG pars opercularis seed and right insular cortex , top row, p<0.05 , top rowr(17)  = −0.41, p = 0.09, but was significant after controlling for age and sex, r(17)  = −0.51, p<0.05 , the weaker their negative connectivity between nucleus accumbens and parieto-occipital cortex. In the Primary Scan, we observed a significant voxel-wise inverse relationship between expected benefit scores and RSFC between the left nucleus accumbens seed and right parieto-occipital cortex   RSFC maps for each participant, and focused on those seeds whose RSFC patterns were consistently negatively associated with expected benefit scores across scans .nd and 3rd columns), regions of insula positively correlated with the IFG were more extensive in the Lower than the Higher EB group. This finding was consistent with the results of the expected benefit covariate analysis, demonstrating stronger positive functional connectivity between these regions with lower expected benefit scores.Participants in the Lower EB group  had an average expected benefit score of 1.82 (SD 0.39), while participants in the Higher EB group  had an average score of 3.26 (SD 0.65). In order to account for any differences between groups, age and sex were entered as nuisance covariates in the analysis. As expected, direct comparisons between groups revealed significantly greater positive functional connectivity between the right IFG pars opercularis seed and bilateral insular cortex in the Lower EB, relative to the Higher EB, group (th column). However, as illustrated in nd and 3rd columns), the region of parieto-occipital cortex whose activity was negatively correlated with nucleus accumbens was more extensive in the Higher than the Lower EB group. Although the Higher and Lower EB groups did not differ significantly in RSFC, the overall patterns of RSFC were consistent with the finding of weaker negative functional connectivity with decreased expected benefit scores  observed in previous analyses.In contrast, no significant group differences in RSFC were observed for the left nucleus accumbens seed . Moreover, they demonstrate that information about individuals' beliefs about the consequences of real-world risk-taking can elicit predictable brain-behavior relationships without the requirement to perform a specific task , which necessarily limits the type of risky behavior interrogated.We observed a stable (over 4–16 months) brain-behavior relationship for regions typically implicated in cognitive control and risk-aversion. Specifically, decreased expected benefit from risky behavior  was associated with stronger positive intrinsic connectivity between the IFG and anterior insula. A group comparison further demonstrated increased insular participation in the IFG intrinsic connectivity network in individuals endorsing lower relative to higher expected benefit from risky behavior. These observations are congruent with the consistent implication of these two brain regions in risky decision-making, most notably when individuals respond in a more risk-averse manner.In the context of cognitive or behavioral control, the IFG has been repeatedly implicated in inhibitory control Perhaps due to its implication in addictive disorders Disruption of insula function via lesions Our findings draw further attention to a fundamental role for both the IFG and insula in the cognitive evaluation of risk. Functional interactions between these two brain regions may contribute to the avoidance of risky behaviors.different functional networks . Although there is controversy regarding the interpretation of negative RSFC networks, one based in the nucleus accumbens and the other based in parietal cortex. This finding differs from our findings for the IFG and insula, where the cognitive evaluation of risk was associated with the spatial extent of the IFG-based functional connectivity network (which included the insula).We observed a second stable brain-behavior relationship for the nucleus accumbens, a region long implicated in reward and risky decision-making. Decreased expected benefit from risky behavior  was associated with weaker negative intrinsic connectivity between the left nucleus accumbens and right parieto-occipital cortex. In general, positive and negative functional connectivity provide different types of information about the brain's intrinsic functional organization. Specifically, negative functional connectivity is most often observed between brain regions that are members of The nucleus accumbens is commonly implicated in the processing of risk and reward. Increased activation in the ventral striatum, including the nucleus accumbens, has been associated with risk seeking decisions Although perhaps less commonly discussed, activation in regions of lateral parieto-occipital cortex  has been reported in several studies of risky decision-making. Right parietal activation has been associated with risk-related processing especially in situations involving choice With regard to interactions between nucleus accumbens and parietal cortex, co-activation of the ventral striatum and right inferior parietal cortex has been associated with loss aversion. Loss-related decreases in activation in these areas were greater than gain-related increases in activation. This differential neural response to loss predicted individual differences in behavioral loss aversion during risky decision-making Our findings add to a growing literature demonstrating that individual differences in behavior or behavioral tendencies are reflected in the brain's intrinsic functional architecture Beyond brain-behavior relationships, previous work has also shown that individual differences in RSFC predict task-evoked fMRI activation We used the CARE questionnaire to quantify participants' beliefs about the consequences of engaging in risky behavior. This required the assumption that individuals' ratings of the expected benefit from risky behavior were indicative of their level of risk seeking (higher ratings) or risk-aversion (lower ratings). Our use of the CARE questionnaire was motivated by a desire to capture individual differences in risk-related personality traits associated with real-world risk-taking behaviors. Fromme and colleagues Even though beliefs about the potential benefits of engaging in risky behaviors have been shown to be more predictive of actual risk-taking than beliefs about their negative consequences, it could be argued that our participants' expected benefit scores reflect a preoccupation with the positive, and an under-appreciation of the negative consequences of these behaviors. Though not a central focus of the brain-behavior relationships examined in the current study, in order to address this consideration, we examined participants' ratings of expected risk  in relation to their ratings of expected benefit. For all 4 dimensions of risky behavior included in our analyses, participants indicated that the likelihood of negative consequences was greater than the likelihood of positive consequences . This pattern of results suggests that participants were well aware of the potential negative consequences associated with these behaviors, and that our results, although focused on ratings of expected benefit, are not merely due to an undervaluation of possible adverse outcomes.a priori regions of interest. This lack of stability could reflect a number of factors such as limited power, variable fMRI signal, or signal loss. However, an examination of the group-level masks for each analysis indicated good signal in all seed ROIs for all scans, ruling out signal loss as an explanation. Further, the demonstration of stable relationships for the IFG/insula and nucleus accumbens/parietal cortex mitigates concerns about signal variability. Another explanation is that relationships observed for areas such as the middle frontal gyrus reflect transient, state effects, rather than enduring behavioral tendencies. Activation in some of these regions may be most reliably elicited during the evaluation of immediate risks and rewards. These possibilities merit further study with both task-based and resting state approaches.One critical aspect of the current study was the identification of brain-behavior relationships that were stable across time. This limited our focus to those results that remained significant across scans occurring roughly one year apart. Since the CARE questionnaire was not administered at both time points, we cannot be sure that participants' beliefs about risk-taking did not change in the time between scans. This could be one possible explanation for why we did not observe stable relationships between expected benefit and RSFC in several of our If replicated, results from the current study will have implications for the understanding, characterization, and possible early identification of abnormal functional connectivity between brain regions associated with risk-related processing. It is well established that a wide range of psychiatric disorders are associated with abnormal behavior and neural activity during risky decision-making Table S1Regions exhibiting a significant relationship between expected benefit from engaging in risky behaviors and resting state functional connectivity in the Primary Scan.(0.04 MB DOC)Click here for additional data file.


The zebrafish is a powerful model for investigating the immune system in vivo, as it is genetically tractable, and optically transparent. To visualise MPO activity within zebrafish neutrophils, we created a genetic construct that expresses human MPO as a fusion protein with a C-terminal fluorescent tag, driven by the neutrophil-specific promoter lyz. After introducing the construct into the zebrafish genome by Tol2 transgenesis, we established the Tgsh496 line, and confirmed transgene expression in zebrafish neutrophils. We observed localisation of MPO-mEmerald within a subcellular location resembling neutrophil granules, mirroring MPO in human neutrophils. In Spotless (mpxNL144) larvae—which express a non-functional zebrafish myeloperoxidase—the MPO-mEmerald transgene does not disrupt neutrophil migration to sites of infection or inflammation, suggesting that it is a suitable line for the study of neutrophil granule function. We present a new transgenic line that can be used to investigate neutrophil granule dynamics in vivo without disrupting neutrophil behaviour, with potential applications in studying processing and maturation of MPO during development.The neutrophil enzyme myeloperoxidase (MPO) is a major enzyme made by neutrophils to generate antimicrobial and immunomodulatory compounds, notably hypochlorous acid (HOCl), amplifying their capacity for destroying pathogens and regulating inflammation. Despite its roles in innate immunity, the importance of MPO in preventing infection is unclear, as individuals with MPO deficiency are asymptomatic with the exception of an increased risk of candidiasis. Dysregulation of MPO activity is also linked with inflammatory conditions such as atherosclerosis, emphasising a need to understand the roles of the enzyme in greater detail. Consequently, new tools for investigating granular dynamics  Th. Thlyz sh496. To verify whether the lyz:MPO-mEmerald transgene was expressed in neutrophils of stably transgenic fish, they were crossed to the red neutrophil reporter line Tg(lyz:nfsB-mCherry)sh260, and screened for any co-expression of fluorescent proteins. Both transgenes were expressed in neutrophils throughout the CHT sh496 fish were outcrossed to Tg(lyz:nfsB-mCherry)sh260 fish, and at 3dpf the double-transgenic larvae were imaged in high detail using an Airyscan confocal microscope. Both transgenes are expressed in the same cells, with MPO-mEmerald localising with a granular subcellular distribution sh496; Tg(lyz:nfsB-mCherry)sh260 larva shows that the intracellular MPO-mEmerald signal is highly dynamic sh496 fish exhibit disrupted neutrophil migration to inflammatory and infectious stimuli. Tgsh496 fish were crossed to Tg(lyz:nfsB-mCherry)sh260 and at 3dpf their larvae were separated into two groups: non-humanised (lyz:nfsB-mCherry only) and humanised  to determine how expression of MPO-mEmerald affects these responses.In addition to its role in potentiating ROS generation in neutrophils, MPO also influences neutrophil migration to inflammatory stimuli . Accordilyz:MPO-mEmerald does not interfere with neutrophil recruitment to sites of injury.To assess neutrophil migration to inflammatory stimuli, we used a tailfin-transection model that initiates neutrophil recruitment to a vertically transected tailfin injury in zebrafish larvae . Non-humlyz:MPO-mEmerald, we used an otic vesicle infection model to investigate neutrophil recruitment [S. aureus USA300 at 3dpf. The larvae were then fixed in paraformaldehyde at 4 hours post infection (hpi) and stained with Sudan Black B to detect neutrophils. Injection of S. aureus USA300 induces robust recruitment of neutrophils to the otic vesicle, with comparable numbers recruited between non-humanised and humanised larvae  from the Tgsh496 line. This was achieved using an existing zebrafish line known as Spotless (mpxNL144), which possesses a premature stop codon in the first exon of the mpx gene [Tgsh496 line to create a line that expresses only human MPO. Before we could create this line, it was necessary to develop a genotyping protocol that could accurately identify Spotless mpxNL144 fish.While the mpx gene . Once acmpxNL144 allele can be identified by PCR amplification of the mutated gene from genomic DNA, followed by restriction digest of the PCR product. The restriction enzyme BtsCI recognises 5’ GG ATG NN 3’ sites in DNA, one of which is present within the mutated mpxNL144 gene (GGA TGA) but not the wild-type mpxwt gene (GGA CGA), allowing the enzyme to determine the presence of a mpxNL144 allele sh496 line was crossed to the Spotless line to create zebrafish that express the lyz:MPO-mEmerald transgene and do not produce functional endogenous mpx. Once created, Tgsh496; mpxNL144 larvae were stained with the myeloperoxidase-dependent stain Sudan Black B to determine whether this conferred staining; these larvae were compared against three sibling control groups–mpxNL144, mpxwt/NL144 and Tgsh496; mpxwt/NL144. All groups tested containing a functioning mpx allele sh496; mpxwt/NL144) stained with Sudan Black B, indicating that the stain identifies functional endogenous myeloperoxidase (mpxNL144), but surprisingly, neither did the Tgsh496; mpxNL144 human MPO-only larvae, indicating that the lyz:MPO-mEmerald transgene does not produce a functional MPO enzyme sh260, which expresses mCherry in the cytoplasm of zebrafish neutrophils. Both transgenes were expressed within the same cells sh260 line. This was observed in several instances; in double transgenic neutrophils, distinct areas of the cell remain unlabelled with mEmerald sh496 transgenic line may be used to study granule dynamics in vivo without disrupting neutrophil function.In addition to the role of MPO in antibacterial defence, it is also an important enzyme regulating the migration of neutrophils to sites of infection and inflammation, primarily by mediating H2O2 flux . Using aammation . One disammation –5. Curreproaches . TherefompxNL144) fish, an mpx-null mutant line created in a separate study [mpx gene, followed by restriction digest with BtsCI sh496 line to generate a line that expresses human MPO, and does not express zebrafish Mpx sh496; mpxNL144). By comparing staining with Sudan Black B with sibling controls, we found that MPO-mEmerald is not produced as a functional enzyme, as lyz:MPO-mEmerald expression does not complement staining in the mpxNL144 background. It is unclear why the Tgsh496 line does not produce functional MPO, however it is important to note that MPO is a complex glycoprotein enzyme that undergoes numerous tightly regulated post-translational modifications. Before mature MPO is produced, the peptide associates with calreticulin and calnexin in the endoplasmic reticulum before undergoing a series of proteolytic events leading to insertion of a haem group and dimerisation of the enzyme, followed by glycosylation and ending with granule targeting [in vitro studies show that dimerisation is not required for enzyme function [Tgsh496 line may also be useful in investigating how MPO is processed and targeted to the granules during development.Once a robust method for identifying Spotless fish was established, the Spotless line was crossed to the argeting . The impargeting ,48–50, afunction ,51,52. Afunction . Differein vivo without disrupting neutrophil behaviour. Additionally, the Tgsh496 line may be used to investigate processing and targeting of MPO during development, which is currently uncharacterised in vivo. Lastly, we provide a protocol for genotyping endogenous myeloperoxidase-null Spotless (mpxNL144) fish, which will prove useful in future studies investigating myeloperoxidase in the zebrafish.We have generated a transgenic zebrafish line expressing fluorescently labelled human MPO within its neutrophils. The enzyme is non-functional and does not interfere with neutrophil recruitment to sites of infection or inflammation, suggesting that it may be used to study granule dynamics S1 MovieTgsh496; Tg(lyz:nfsB-mCherry)sh260 larva. Maximum intensity projection of 60 slices with a total width of 10μm, 1 minute per timepoint, 10 frames per second. The timelapse shows numerous neutrophils in the caudal haematopoietic tissue of a 3dpf larva.An airyscanner confocal timelapse of a double-transgenic (AVI)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Acute Myeloid Leukemia (AML) is the most common form of leukemia in adults with an incidence of 4.3 per 100,000 cases per year. Historically, the identification of genetic alterations in AML focused on protein-coding genes to provide biomarkers and to understand the molecular complexity of AML. Despite these findings and because of the heterogeneity of this disease, questions as to the molecular mechanisms underlying AML development and progression remained unsolved. Recently, transcriptome-wide profiling approaches have uncovered a large family of long noncoding RNAs (lncRNAs). Larger than 200 nucleotides and with no apparent protein coding potential, lncRNAs could unveil a new set of players in AML development. Originally considered as dark matter, lncRNAs have critical roles to play in the different steps of gene expression and thus affect cellular homeostasis including proliferation, survival, differentiation, migration or genomic stability. Consequently, lncRNAs are found to be differentially expressed in tumors, notably in AML, and linked to the transformation of healthy cells into leukemic cells. In this review, we aim to summarize the knowledge concerning lncRNAs functions and implications in AML, with a particular emphasis on their prognostic and therapeutic potential.  Acute myeloid leukemia (AML) is an aggressive malignancy of the hematopoietic system caused by malignant transformation of hematopoietic stem- or progenitor cells. AML is the most common form of acute leukemia in adults with an incidence of 4/100,000 cases per year . HistoriArbitrary defined as transcripts of over 200 nucleotides without coding potential, lncRNAs were originally considered as dark matter due to their low expression and poor conservation across species compared to their messenger RNAs counterparts ,14,15,16Because of their heterogeneity, much debate remains to find the best classification system for lncRNAs that are currently defined according to their genomic location. Indeed, lncRNAs can be divided into sense  or antisAs illustrated in Nuclear lncRNAs are implicated in epigenetic and transcriptional regulations by recruiting activator or repressor chromatin-modifying complexes and transcription factors onto target genes ,31. MostRegulation of gene expression is crucial during hematopoietic differentiation . LncRNAsConcerning the potential role of lncRNAs as diagnostic biomarkers, the most inspiring example is PCA3 (Prostate Cancer Antigen 3) for prostate cancer diagnosis. It is a prostate specific lncRNA overexpressed in prostate cancer patients. Found in the urine of most cancer patients compared to healthy patients, it is undetectable in other tumor types ,48. AlthIn AML, several lncRNAs have been characterized and described as oncogenes  or tumorThe best described function of lncRNAs is their involvement in epigenetic and transcriptional regulation. They are known to act as guides, leading to the recruitment of proteins or complexes to chromatin sites to regulate downstream gene expression ,31. INK4bp15 locus . P. P145]. Amongst the top upregulated LSC-associated lncRNAs, Bill et al. identified the promising lncRNA DANCR whose knock-down in LSCs resulted in decreased stem-cell renewal and quiescence, suggesting its oncogenic role. The delivery of nanoparticles containing siRNAs against DANCR prolonged the survival of AML mouse model even after serial transplantation . Another supportive case of targeting lncRNAs by siRNAs is UCA1, where molecular evidence has established that there is a close relationship between UCA1 and Adriamycin resistance in pediatric AML cases . By usinAnother synthetic oligonucleotide-based molecular product used to knock-down lncRNA expression has been developed: the Antisense oligonucleotide (ASO). ASO (as GapmeRs) are single-stranded DNA oligonucleotides duplexing by base complementarity their target lncRNAs to promote their degradation by RNase H ,153. For example, ASO has been used in De Clara et al. study to knock-down the newly identified XLOC_109948 lncRNA. In this study, they showed that low XLOC_109948 expression is correlated with a good prognosis especially for NPM1-mutated AML patients and that its downregulation using ASO in NPM1-mutated AML cells treated with Ara-C or ATRA enhances apoptosis suggesting a role for XLOC_109948 in drug sensitivity . Targeting lncRNAs implicated in chemoresistance with interference strategies could improve drug response and outcome. Curna Inc., MiNA Therapeutics, RaNA Therapeutics Inc. and others are taking steps towards the development of oligonucleotide-based strategies . The greCatalytic degradation is another therapeutic strategy. Ribozymes are single-stranded RNA able to target and cleave RNA in a site-specific manner ,157. AntAptamers could also provide greater specificity. These single stranded DNA folded into secondary and tertiary structures are able to bind a wide range of molecules, hiding specific-binding structures to limit activity. The chemical structure of aptamers could be modified to enhance their stability and half-life. Aptamer-based -therapeutics are undergoing clinical trials for different indications  . In 2004Through their ability to bind proteins to regulate chromatin organization, transcription, and translation, Fatemi et al. proposed innovative therapeutic strategies based on Small molecule Inhibitors able to disrupt lncRNA-protein interactions ,161. A sRe-expression of the specific tumor suppressor lncRNA may be induced by common gene therapy strategies, packaging the whole transcript into viral or non-viral delivery tools. Recombinant viral systems used in gene therapy have been extensively reviewed previously . Howeverα oncoprotein in APL cells and restores the process of myeloid differentiation in those cells [Even though its implication as AML clinic target was not proposed yet, HOTAIRM1 lncRNA seems to be a promising candidate in APL treatment. Indeed, as aforementioned, enhanced HOTAIRM1 expression induces degradation of PML-RARse cells . RestoriNew Genome editing strategies such as CRISPR/Cas9 are developing very fast and seem to be a powerful tool to knock-in or knock-out lncRNA candidates . ModifieA great challenge in cancer therapy is to selectively kill tumor cells without harming healthy cells. As H19 lncRNA is highly upregulated in several cancers, a plasmid containing diphtheria toxin under the control of H19 regulatory sequences has been designed to selectively target tumor cells which overexpressed H19 compared to normal cells. Diphtheria Toxin is massively produced and kills H19-overexpressing tumor cells selectively. This construct leads to an overall reduction in tumor size and is under clinical trials for ovarian (NCT00826150), pancreatic (NCT00711997) and bladder cancers (NCT00595088) . This coFinally, given their tissue specific expression, using the same strategy with the promoter of a myeloid specific lncRNA could lead to the development of a new tool to target selectively myeloid lineage to treat AML patients.There is no doubt as to the role of lncRNAs in leukemia development, progression, and drug resistance. However, due to the limited number of studies in AML, their application as therapeutic agents in clinical routine is still at its beginnings. Few results have been replicated across cohorts, probably due to no-standardized sample collection and quantification techniques, but also as a consequence of AML biological complexity, characterized by spatio-temporal relationships between the coding and non-coding genome. Adequate sample sizes, well-designed cohort studies, and validation of the results in independent cohorts are needed to confirm their clinical usefulness. The functional and molecular characterization of lncRNAs is also needed to select the most promising lncRNA targets and to design the best IncRNA therapeutic tool with appropriate efficacy and safety profile. Translating this knowledge into clinical practice still represents a tremendous challenge.


Voluntary medical male circumcision has been promoted in high HIV prevalence settings to prevent HIV acquisition in males. However, the uptake of circumcision in many sub-Saharan African settings remains low. While many studies have measured circumcision prevalence, understanding circumcision incidence and its predictors is vital to achieving ambitious circumcision prevalence targets.Rural KwaZulu-Natal, South Africa.We measured circumcision incidence over the period 2009–2014 in a longitudinal population-based cohort with high HIV prevalence and low circumcision prevalence. Multivariable survival models with Weibull distributions were used to assess socio-demographic, behavioral and biological predictors of circumcision incidence.Between 2009 and 2014, circumcision prevalence among males 15–49 years in the cohort increased from 3% to 24%. Among 6,203 males 15–49 years, 873 new circumcisions occurred over 13,678 person-years . Circumcision incidence was substantially higher amongst young males: 15–19 year olds were twice as likely to circumcise as older males. In the survival model, shorter household distance to the nearest healthcare facility, knowledge of HIV status and biological HIV-negative status were associated with an increased likelihood of circumcision incidence.Circumcision prevalence among males in rural KwaZulu-Natal remains well below South Africa’s national 80% coverage target across age groups. In this population, distance to the nearest healthcare facility and knowledge of HIV status were important independent predictors of circumcision incidence. Mobile circumcision clinics and innovative HIV testing services may be important tools to help achieve circumcision targets. Global voluntary medical male circumcision (VMMC) prevalence and incidence remains low despite efforts to scale the HIV prevention intervention.,2 VMMC iSouth Africa, one of WHO’s 14 VMMC priority countries,  began exth century, the predominant culture in the KwaZulu-Natal region, the Zulu culture, stopped practicing traditional circumcision to retain warrior-aged males; [Despite these efforts, national VMMC coverage in South Africa remains far below the ambitious 80% coverage target. In latest estimates of national circumcision coverage from 2012, 47% of males 15 years and older in South Africa reported circumcision and the majority of these reported traditional circumcision (53%). There was, however, significant demographic and regional variations in circumcision coverage. Circumcid males;  in 2012,A number of studies in sub-Saharan Africa have examined factors associated with circumcision prevalence and found younger age, Muslim faith, urban residence, higher education, and wealth to be positively associated with the prevalence of circumcision.–25 A majWe used longitudinal, population-based surveillance data from KwaZulu-Natal, South Africa to measure VMMC prevalence, incidence and predictors of VMMC incidence in a high HIV prevalence, real-world population. Such knowledge is crucial for tailoring existing VMMC campaigns and informing future VMMC campaigns in South Africa.2) in the rural uMkhanyakude district of KwaZulu-Natal, South Africa has been collecting longitudinal surveillance data since 2003.[The Africa Health Research Institute (AHRI) demographic surveillance site , participants were not given their HIV test results and were instead referred to facility-based HIV testing and counseling services if they wanted to learn their HIV status. AHRI does not promote or provide VMMC as part of their surveillance. Circumcision-specific questions were included in the initial 2003 survey and then re-introduced in 2009.Ethical approval for AHRI surveillance was granted by the Biomedical Research Ethics Committee, University of KwaZulu-Natal. This analysis was exempted from additional review by the Harvard T.H. Chan School of Public Health’s Institutional Review Board since it used anonymized secondary data.We included data collected between January 2009 and November 2014 for our analysis. To be included in our analyses, respondents had to be: (i) male; (ii) 15–49 years old (target circumcision age group); and (iii) a resident in the surveillance area. We constructed an open circumcision incidence cohort. Eligible respondents entered the cohort when they first reported their circumcision status. To be included in our circumcision incidence cohort, respondents had to additionally: (iv) self-report their circumcision status at least twice ; and (v) self-report not being circumcised when they first appeared in the surveillance data.We assumed that participants remained circumcised in all years following their first report of being circumcised and additionally assumed that respondents’ date of circumcision occurred midway between their last report of not being circumcised and first report of being circumcised. AHRI research assistants did not define circumcision to surveillance participants, thus circumcision status was determined by the participants themselves and may have included traditional circumcision. Respondents who reported being circumcised were asked why they got circumcised  and where the circumcision was conducted . In KwaZulu-Natal, VMMC camps are two day events that often take place at schools during the holidays; here young males are taught life skills, undergo HIV counseling and testing, and are circumcised.We considered a range of potential predictors of VMMC incidence.,19 At thWe calculated circumcision prevalence by dividing the number of males who self-reported being circumcised by all males in the surveillance data who reported their circumcision status for each year and age group; we calculated confidence intervals using binomial confidence limits. We calculated annualized percentage change in circumcision prevalence over time by dividing the difference in circumcision prevalence from 2014 to 2009 by circumcision prevalence in 2014 and the number of years of observation, then multiplying by 100. We calculated circumcision incidence for a smaller subset of the sample that met the inclusion criteria for the open circumcision incidence cohort. We computed circumcision incidence for all participants and for specific age groups (based on cohort entry) using males that we identified as newly circumcised and person-years of follow-up; we used Poisson confidence limits to calculate confidence intervals.We used multivariable survival models with Weibull distributions to identify significant predictors of circumcision incidence, using covariate values from the year participants entered the data  and robust standard errors. We explored using Cox proportional hazard models, but our data violated the proportional hazards assumption see  Figs.3838 We begWe conducted four sensitivity analyses to test the robustness of the results from the multivariable model: (1) a logistic regression model, where we used baseline characteristics to predict circumcision incidence (no person-years); (2) a survival model, where we assumed that all males with inconsistent self-reported circumcision status (i.e. reported being uncircumcised after reporting being circumcised) remained uncircumcised; (3) a survival model, where we used multiple imputation chained equations (MICE) for missing data; and (4) a survival model, where we dropped all missing data. We used MICE to impute the predictor variables that remained missing after the adjustments for missing data described above; we used predictive mean matching for continuous variables (distance to the nearest clinic), logistic regression for binary variables , ordered regression for ordered categorical variables (asset index), and multinomial regression for non-ordered categorical variables . We performed MICE using the ‘mi impute’ procedure in Stata 13.1 . We used We performed all statistical analyses using Stata 13.1.Between January 2009 and November 2014, 13,182 unique males 15–49 years were sampled, 13,022 reported their circumcision status, and 1,759 of these males  self-reported ever being circumcised. As shown in Of all the 13,022 males who reported their circumcision status, 6,203 meet the inclusion criteria for our open circumcision incidence cohort, The bivariate and multivariable associations with circumcision incidence are shown in All significant predictors of circumcision incidence remained significant in the sensitivity analyses with the exception of biologically confirmed HIV-negative status see . In the In a real-world, high HIV prevalence population in KwaZulu-Natal, South Africa during an era of known circumcision benefits on reduced HIV transmission 2009–2014), we found that younger age, proximity to the nearest healthcare facility, biologically confirmed HIV-negative status, and knowledge of HIV status were significant predictors of circumcision incidence. Within our circumcision incidence cohort, circumcision incidence rates were substantially higher for males 15 to 19 years compared to males of other age groups. The majority of circumcised males self-reported getting circumcised for health reasons and going to a government hospital for circumcision. Despite impressive gains in circumcision prevalence over this study period, in 2014 circumcision remained far below South Africa’s 80% national VMMC prevalence target: prevalence was below 30% in all age groups, even in the younger age groups where the gains in circumcision incidence were largest.–2014, weCircumcision prevalence in this rural population in KwaZulu-Natal could be increasing for a number of reasons including changing government policies and social norms. We have discussed the South African government’s efforts to promote VMMC uptake through communication campaigns, equipping healthcare facilities with tools for VMMC expansion, and targeting youth with VMMC camps. However,While younger males appear to be circumcising at a higher rate compared to older males in our study population, the incidence of the best known, one-time HIV prevention intervention among males 20 years and over remains low and their risk of HIV infection remains high. In this longitudinal, population-based surveillance data, the rate of circumcision incidence for all males aged 15 to 49 years in this population (6.4/100 PY from 2009 to 2014) is not much higher than the estimated HIV incidence rate in the same population (4.3/100 PY from 2009 to 2011). CircumciTo achieve national circumcision targets, policy makers need to understand both whom circumcision campaigns are reaching and what factors predict circumcision incidence. Our bivariate analyses provide evidence on the first question, showing that in this population, circumcision incidence was greatest among males who were: younger; have some education; are from wealthier households; live in rural (compared to peri-urban or urban) areas; live closer to clinics; have never had sex; and are biologically confirmed HIV-negative. These findings highlight the need to expand circumcision opportunities for low-socioeconomic status, sexually experienced, somewhat older males.Our multivariable analysis provides evidence to the question: what interventions are needed in low-circumcising groups? Our finding that living further from a healthcare facility remained a risk factor for not getting circumcised, after adjusting for other factors, suggests that mobile circumcision clinics might play an important role in increasing circumcision incidence–especially since the majority of new circumcisions in this population are self-reported to be in government hospitals. Biologically-confirmed HIV-negative status was also associated with increased circumcision incidence in our multivariable analysis. This reflects South African circumcision guidelines, which recommend the procedure only for males not living with HIV. While circumcision does not reduce onward transmission for HIV-positive males, careful messaging around additional circumcision health benefits  for males living with HIV may be important in raising circumcision rates in this setting.,42A key policy-relevant finding of our multivariable analysis is that self-reported knowledge of HIV status is a significant predictor of circumcision incidence. It is possible that males who know their HIV status have better access to health services or take advantage of health services at the point of circumcision. Alternatively, knowledge of HIV-negative status may persuade males in KwaZulu-Natal to undertake VMMC. Only 44.4% of males reported knowing their HIV status in AHRI surveillance in 2014, which presents an opportunity to increase VMMC by increasing knowledge of HIV status. Knowledge of HIV status might be increased through improved HIV testing and counseling services, including door-to-door home-based testing ,44 or HIThis study has considerable strengths. This study is one of few to measure circumcision incidence in a sub-Saharan African settings,  and the Our findings, however, should be interpreted in the context of some limitations. First, given the open nature of this cohort, there were missing data. This missing data were notable for sexual behavior and biological HIV status data because participants could opt out of survey sections that collected this data. The robustness of results in the MICE and complete case sensitivity analyses, however, suggest that missing data were unlikely to have influenced our findings.Second, our analyses relied on self-reported circumcision status and the definition of circumcision was not clarified to surveillance participants. Due to the rarity of circumcision within this population, some males might have been confused as to what circumcision was or may have confused partial traditional circumcision with medical circumcision; as indicated by some participants reporting not being circumcised in survey years following ones where they reported being circumcised. Self-reported circumcision status may have also been subject to social desirability bias in later years when circumcision promotion campaigns were more prevalent throughout KwaZulu-Natal. While self-reported circumcision status has not been validated in the AHRI data, a validation study conducted in Kenya found the accuracy of self-reported circumcision status to be 99.0%. To accouThird, our sample covered only those aged 15 years and over and as a result, these findings cannot be generalized to younger age groups. This is a limitation because South African national statistics suggest that 40% of all new circumcisions occur among males 10 to 14 years. Finally,Increasing VMMC in a traditionally non-circumcision population is a challenge; the challenge is particularly daunting in the light of South Africa’s target to achieve national 80% circumcision coverage among males 15 to 49 years. Since 20S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S4 Fig(TIFF)Click here for additional data file.S5 Fig(TIFF)Click here for additional data file.S6 Fig(TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Neuroendocrine tumors represent an expansive group of neoplasms that share an etiology of epithelial origin with neuroendocrine differentiation. Poorly-differentiated neuroendocrine carcinomas behave similarly to their aggressive pulmonary counterpart, small cell lung carcinoma. Most patients with gastroenteropancreatic neuroendocrine tumors present with symptoms of metastasis, most commonly to the liver. There have been no case reports, to our knowledge, until now that demonstrate metastasis to the central nervous system.A 72-year-old male with poorly-differentiated stage IIIB neuroendocrine carcinoma of the colon presented with acute altered mental status and right facial droop. Head CT was negative for an acute hemorrhagic process without evidence of suspicious lesions. Several days later, the patient developed fever and neck stiffness suspicious for bacterial meningitis. A lumbar puncture procedure was performed. Cytology of the CSF demonstrated metastatic disease to the central nervous system and the final diagnosis of carcinomatous meningitis secondary to metastatic neuroendocrine carcinoma of the colon was made.High-grade gastroenteropancreatic neuroendocrine carcinomas most commonly metastasize to the liver, which often corresponds with the patient’s initial presentation. When neuroendocrine tumors do metastasize to the central nervous system, the primaries are most commonly of pulmonary origin. When meningeal metastasis does occur, it commonly presents as neurologic deficits or cerebrovascular events, rarely does meningeal metastasis mimic bacterial meningitis with symptoms of fever, photophobia and meningismus. As neuroendocrine carcinomas have been increasing in incidence over the past several decades, it is important to consider varying metastatic presentations when working up a patient with a diagnosis of neuroendocrine tumor. Neuroendocrine tumors represent an expansive group of neoplasms that share an etiology of epithelial origin with neuroendocrine differentiation and potential for hormone release. In the past two decades, the World Health Organization has re-classified neuroendocrine tumors with emphasis on their morphology and proliferation rates. The neuroendocrine tumors of the gastrointestinal tract, pancreas and liver are now recognized as either well-differentiated neuroendocrine tumors (NETs) or poorly-differentiated neuroendocrine carcinomas (NECs) . Poorly-A 72-year-old right handed male diagnosed with poorly-differentiated, stage IIIB neuroendocrine carcinoma of the colon s/p hemicolectomy, small bowel resection and carboplatin-etoposide × 3 cycles presented to the emergency department with acute altered mental status and right facial droop.Four months prior, he presented with constipation and anemia. Colonoscopy revealed a large raised flat lesion in the transverse colon and CT abdomen demonstrated RLQ mesenteric lymphadenopathy. He underwent right hemicolectomy and small bowel resection weeks later. Pathology was significant for poorly-differentiated grade 3, neuroendocrine carcinoma with focal lymphovascular invasion and tumor invasion through the muscularis propria into the subserosa. Margins were negative, no perineural invasion and 1/33 lymph nodes were positive for carcinoma. There was an absence of non-neuroendocrine component. Immunohistochemical stains were positive for: AE1/AE3, CD56, chromogranin, and synaptophysin; Ki-67 of 60% proliferative index. He was staged as pathologic T3N1a, stage IIIB.In the emergency department, head CT was negative for an acute hemorrhagic process and did not demonstrate any suspicious lesions. Within one day of admission, the facial droop resolved. Further imaging, CT chest abdomen pelvis, revealed stable enlarged mediastinal lymphadenopathy and a subcentimeter retroperitoneal lymph node but no progression was evident. Two days into the hospital stay, the patient developed fever and subsequently neck stiffness. His chest x-ray and urinalysis were non-diagnostic; EEG showed diffuse slowing but no seizure activity. A lumbar puncture was performed with cytopathology of the CSF suggesting metastatic disease to the central nervous system Fig. , along wMedical oncology and radiation oncology were consulted. No further chemotherapy was recommended as he had progressed after completing 3 of 6 cycles of carboplatin-etoposide. Enrollment in an erlotinib trial was discussed vs palliative therapy. While radiation was considered to be potentially palliative for his symptoms, radiation oncology deemed it would be unlikely to change his overall survival. He was started on palliative high-dose steroids and ultimately transferred to inpatient hospice care. The patient passed away within a week of entering hospice.This case depicts the rapid progression of a high-grade neuroendocrine carcinoma of the colon with metastases to the meninges causing atypical symptoms of carcinomatous meningitis.n = 24) developed brain metastasis, while the most common sites of metastasis were lymph nodes (52%) and liver (39%) [n = 24/31) were of pulmonary origin with other primaries located in the breast, esophagus, lymph nodes and uterus. Though case reports of metastatic brain neuroendocrine tumors originating from the pancreas and cervix have been reported, there are none to our knowledge of primaries originating in the large intestine [The most common site for metastasis of NECs of the GI tract is the liver, with up to 57% of tumors presenting with metastasis upon initial presentation . Other ser 39%) . Regardi9% . Regantestine .In addition to the uncommon primary and metastatic location of this neuroendocrine carcinoma, the case is also intriguing due to its atypical clinical presentation. When meningeal metastasis occurs, most commonly from breast cancer, lung cancer, or melanoma, it often presents with symptoms of neurologic dysfunction related to the occupied area of metastases . Rhun anWhile the patient did experience a facial droop that may be attributable to facial nerve involvement or a posterior circulation transient ischemic attack, the progression of his illness to include fever and nuchal rigidity is extremely unusual for the presentation of carcinomatous meningitis. Few reports have been documented in patients with meningeal carcinomatosis that demonstrate symptoms such as meningismus, fever, and photophobia mimicking acute bacterial meningitis. Due to this historically uncommon presentation, literature is limited to case reports surrounding acute meningitis in relation to recurrent NETs.Moreover, a unique presentation of carcinomatous meningitis from NEC is exhibited in this patient with the uncommon primary location in the colon. To our knowledge, this is the first report of meningeal metastasis from a neuroendocrine tumor of the large intestine. While still a relatively rare tumor, incidence of gastrointestinal neuroendocrine tumors has significantly risen since the 1970s; it is therefore important to be aware of the common presenting symptoms of neuroendocrine tumors . Though


Transcatheter aortic valve implantation (TAVI) can be challenging in case of complex anatomy such as bicuspid aortic valve stenosis or situs inversus. This report illustrates a successful procedure in a patient with both conditions after careful pre-operative planning and per-operative guidance by means of a novel software.We report the case of a 71-year-old man that presented a type 0 bicuspid aortic valve stenosis and situs inversus. We performed transfemoral TAVI using the Edwards Sapien 3 transcatheter heart valve (THV) and a novel software that provides insight for patient anatomy through computed tomography (CT) extracted three-dimensional reconstruction before the procedure aiming at improving THV positioning during the procedure through fusion of a CT-extracted virtual aortic annulus on the fluoroscopy screen and enhancing of calcifications that can be considered as anatomical landmarks. The Edwards Sapien 3 THV was accurately implanted in a supra-annular fashion. Post-operative echocardiography showed an excellent result without any para-prosthetic leak, and the patient reported symptom improvement.During TAVI the angiographic appearance of the cusps of a bicuspid aortic valve is irregular and asymmetric, which can lead to malpositioning, paravalvular regurgitation, and increased rates of pacemaker implantation after TAVI. Furthermore, usual anatomical landmarks can be even more disturbed by the situs inversus totalis. We believe that pre-operative three-dimensional reconstruction and per-operative fluoroscopy image processing, provided by software such as the one presented in this case report, can provide precious guidance for TAVI in patients with complex and unusual anatomy. Optimal result can be achieved with the transcatheter aortic valve implantation technique when treating complex anatomy such as bicuspid aortic stenosis and situs inversus totalis.Pre-operative planning and per-operative fluoroscopy image processing can provide valuable guidance when pursuing optimal technical result in complex and unusual anatomy.,Transcatheter aortic valve implantation (TAVI) can be challenging in case of complex anatomy. Transcatheter aortic valve implantation using the Edwards Sapien 3  valve has been recently reported to be an effective treatment of severe bicuspid aortic valve stenosis,Figure 1). On physical examination the patient had a systolic murmur predominantly located in the second left intercostal space on cardiac auscultation and bilateral crackles regarding the inferior parts of the lungs on pulmonary auscultation. Because of his medical history and a high Society of Thoracic Surgeons (STS) score of 11.9%, the Heart Team recommended TAVI rather than open surgery. The pre-operative multi-slice computed tomography (MSCT) evaluation of the aorta, and its branches confirmed patency of femoral arteries that allowed transfemoral approach  and absence of significant thoracic aortic aneurysm.A 71-year-old man was admitted to our hospital with dyspnoea due to severe aortic stenosis complicating a calcified type 0 bicuspid aortic valve. His medical history included diabetes mellitus, atrial fibrillation, chronic respiratory failure, and coronary artery bypass grafting surgery for left main artery stenosis, which uncovered a situs inversus totalis . A Sapien 3  26 mm was directly implanted in a high position , without post-dilatation, prosthesis constriction and no angiographic leak. The patient did not require permanent pacemaker implantation. A transthoracic echocardiography one week later confirmed an excellent result with no intra or paravalvular regurgitation, and the patient reported improved symptoms. In follow-up consultation 3 months after the procedure the patient reported improved symptoms and had no periprosthetic leak on transthoracic echocardiography.Pre-operative sizing was performed with the new ValveAssist 2  image processing software. Comparatively to the standard fluoroscopy, the new software allows the projection of the MSCT-extracted, manually drawn virtual aortic annulus on the live fluoroscopy screen, and the enhancement of the aortic valve calcifications and aorta calcifications that are used as anatomical landmarks for operator guiding for the positioning of the THV during the procedure  and associated text has been obtained from the patient in line with COPE guidance.Conflict of interest: T.M. is proctor and consultant for Medtronic and Microport. A.S. has served as a consultant for Edwards and Medtronic. All other author declared no conflict of interest.