Choroidal neovascularization (CNV) is a sight-threatening disease and is characterized by the formation of pathological neovascularization in the choroid which extends into the subretinal space. Exudative age-related macular degeneration (AMD) is the formation of CNV in the macular area which leads to irreversible blindness. Continuous leakage and hemorrhage of the CNV lesion may eventually result in scarring or later fibrosis, which could result in photoreceptor cell atrophy. The current strategy for treating CNV is the use of antivascular endothelial growth factor (VEGF) agents. Many studies have demonstrated the efficacy of intravitreal anti-VEGF therapy. Other studies have also reported the side effects of single anti-VEGF treatment. And long-term inhibition of a single system may result in collateral damage to other visual elements. Pigment epithelium-derived factor (PEDF) is a 50 kDa protein that was first isolated from the conditioned medium of human RPE cells. PEDF has both antiangiogenesis and neuroprotective functions for photoreceptor cells. It may be a potential ocular antiangiogenic agent. This review outlines the distribution of PEDF in the eye, the mechanism of antiangiogenesis, the protective effect on the retina, and the relationship between PEDF and VEGF. Choroidal neovascularization (CNV), particularly when associated with age-related macular degeneration (AMD), is one of the leading causes of severe vision loss in the elderly. The reason for the formation of CNV is still not clear. It is recognized that the abnormal increase of the vascular endothelial growth factor (VEGF) plays a central role in the development of CNV . In animThe pigment epithelium-derived factor (PEDF) is a member of the serine protease inhibitor supergene family . It was This review describes the expression and distribution of PEDF in the eye, research status, and possible mechanisms of its anti-CNV functions. The possibility of combining PEDF with common anti-VEGF drugs is also introduced.Expression of PEDF protein in the eye has been investigated in both rats and humans. Different studies have different findings. Ogata et al. found that mRNA of PEDF was expressed in different cell types of normal rat eyes, such as corneal epithelial cells, corneal endothelial cells, lens epithelial cells, ciliary epithelial cells, the ganglion cell layer, and RPE cells . Renno eKarakousis et al. used immunocytochemistry to localize the PEDF in fetal and adult eyes with a polyclonal antibody (pAb) against amino acids 327-343 of PEDF. They found that pAb anti-PEDF labeled retinal RPE granules, developing cones, some neuroblasts, and many cells in the ganglion cell layer (GCL) in developing retinas ). In adult retinas, immunolabeling with pAb anti-PEDF demonstrated the protein in the rod and cone cytoplasm and nuclei of rods but not cones. Cells in the INL and GCL, choroid, corneal epithelium and endothelium, and ciliary body were also pAb PEDF-positive . Their rThere are also reports on PEDF level changes in the eyes of human CNV and AMD patients. Bhutto et al. examined the localization and relative levels of VEGF and PEDF in aged human choroid and AMD by immunohistochemical staining. They found that the most prominent sites of VEGF and PEDF localization in an aged control choroid were RPE Bruch's membrane-choriocapillaris complex including the RPE basal lamina, intercapillary septa, and choroidal stroma . The PEDHow PEDF controls the growth of new blood vessels is not well understood. Recent treatments for ocular neovascularization are mainly anti-VEGF. Members of this family include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and VEGF-F. VEGF-A is typically referred to as VEGF. The abnormal expression of VEGF-A has become a focal point of current research in retinal and choroidal neovascular diseases .The interactions between PEDF, a potent angiogenic inhibitor, and VEGF, an angiogenic stimulator, are largely unclear. Zhang et al. demonstrated that PEDF significantly decreased VEGF expression in both retinal capillary endothelial cells (RCEC) and Müller cells in oxygen-induced retinopathy rats. Also, PEDF decreased VEGF promoter activity under hypoxic conditions but not under normoxic conditions . There mγ-secretase-dependent cleavage of the C-terminus of VEGFR-1, which in turn inhibits VEGFR-2-induced angiogenesis. In addition, PEDF was also able to regulate the phosphorylation of VEGFR-1, which itself can regulate VEGFR-2 signaling [γ-secretase activities leading to cleavage of VEGF receptor-1 and VEGF receptor-2 [PEDF downregulating VEGF expression might partially be via inhibition of mitogen-activated protein kinase- (MAPK-) mediated hypoxia-inducible factor 1 (HIF-1) activation and competes with VEGF binding to RCEC and VEGFR-2. They suggested that the competitive blockade of the VEGF-RCEC and VEGF-VEGFR-2 binding may be responsible for the PEDF effects on VEGF-induced permeability and angiogenesis . Cai et ignaling . Howeverignaling . Kanemurceptor-2 .in vitro effects of PEDF on primary cultured human choroidal endothelial cells (CECs). They found that PEDF had little effect on normal CEC proliferation and migration. PEDF suppressed the proliferation and migration of VEGF-induced choroidal capillary endothelial cells [Wang et al. investigated the al cells . PEDF apal cells .The formation of blood vessels is an intricate process. The process needs participation of endothelial junction proteins, tube formation factors, and vessel stabilization molecules. Targeting these molecules during the formation of neovascularization may have advantages in antiangiogenic strategies . PEDF's Chen et al. examined the molecular actions of PEDF on Human Umbilical Vein Endothelial Cells (HUVEC). They found that induction of apoptosis in human endothelial cells by PEDF is through the activation of both the receptor-mediated and mitochondria-mediated pathways with activation of p38 followed by cleavage of caspases 3, 8, and 9. The result suggests that the apoptotic effects of PEDF are through multiple pathways and caspase dependent . The nonFas ligand (FasL) was induced by thrombospondin-1 (TSP1) and PEDF on endothelial cells, which destruct newly formed vessels marked by angiogenic stimuli that induced Fas. Volpert et al. suggested that the antiangiogenic activity of TSP1 and PEDF was dependent on the dual induction of the Fas and FasL pathways. They found that TSP1 and PEDF increased the surface display of Fas by using flow cytometry, and the dynamics of FasL expression were consistent with the kinetics of endothelial cell killing by TSP1 and PEDF . HaribalMacrophages have been demonstrated to play a proangiogenic role in retinal pathological vascular growth. The macrophages express high levels of VEGF and increase VEGFR expression in HUVEC. The macrophages might stimulate endothelial proliferation, thus contributing to the generation of neovascular tufts (NVTs). PEDF works as a powerful endogenous angiogenesis inhibitor, but its role in macrophage recruitment and polarization is largely unknown. Gao et al.  found thVascular permeability plays a key role in the sight-threatening diseases. VEGF promoted angiogenesis and vascular permeability. PEDF is not only an antiangiogenic agent; it also inhibits pathologically increased vascular permeability. Liu et al.  found thγ-secretase-dependent mechanism. They suggested that targeting downstream effectors of PEDF, such as VEGFR translocation and/or γ-secretase activity, may represent a promising therapeutic strategy for the treatment of diabetic vascular complications in the eye [Studies have shown that p38 MAP kinase and ERK play a role in VEGF-induced vascular hyperpermeability , 43. Yan the eye .Yamagishi et al. investigated how PEDF could inhibit the advanced glycation end product (AGE) signaling to vascular hyperpermeability. They demonstrated that the central mechanism for PEDF inhibition of the AGE signaling to vascular permeability is by suppression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species (ROS) generation and subsequent VEGF expression . Also, PPEDF has antiangiogenic and neuroprotective functions, and many studies have tried to use PEDF to treat CNV animal models. Mori et al. found that intravitreous injection of an adenoviral vector encoding PEDF (AdPEDF) could promote the expression of PEDF mRNA in the eye, which could be measured by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and immunohistochemical staining for PEDF protein throughout the retina. Then, they tried the intravitreous or subretinal injection of AdPEDF.10 in mice with laser-induced CNV, mice with VEGF-induced neovascularization, and mice with ischemia-induced retinal neovascularization. They suggested that PEDF gene transfer by intraocular injection of PEDF-containing vectors might provide a promising approach for treatment of ocular neovascularization . Anotherβ-actin promoter. They found that overexpression of PEDF inhibits laser-induced CNV in mouse models [An adeno-associated viral (AAV) vector containing expression construct coding for PEDF could inhibit CNV . Intraoce models .The sclera and choroid-RPE are permeable to PEDF and ovalbumin proteins, and these proteins can traverse the subconjunctiva to reach the retina . SubconjLentivirus-mediated transfer of PEDF was shown to be effective for treating laser-induced CNV. Yu et al. evaluated the effects of lentivirus-mediated PEDF gene transfer in the treatment of laser-induced rat CNV. They found that lentiviral vectors appeared to be safe and effective for gene expression, with little immune response by the recipient . A lentiSemkova et al. generated a high-capacity adenovirus (HC-Ad) vector expressing human PEDF, transduced cultured iris pigment epithelial (IPE) cells. They transplanted these modified cells to the subretinal space of laser-induced CNV rats and found that they inhibited neovascularization and improved the survival of photoreceptors .The functional response of RPE cells to PEDF is polarized to the apical side. The PEDF secretion pattern from the RPE cells is predominantly apical . VEGF re2O2-induced RPE monolayer permeability was examined. The results showed that PEDF could prevent H2O2-induced RPE permeability changes and preserve the barrier function of RPE cells against oxidative stress [2O2-induced redistribution of the junctional proteins and actin reorganization were decreased by using PEDF. He et al. [2O2-induced RPE cell death and oxidative stress disruption of mitochondrial reticular networks. Also, they found that an increasing PEDF dose had an overall slightly better effect on mitochondrial function. So, a possible mechanism for PEDF's neuroprotective actions might be through restoring mitochondrial dynamics perturbed by both aging and oxidative stress conditions. Subramanian et al. evidenced that PEDF-R protects RPE cells from oxidative stress-induced cell death via inhibiting 5-lipoxygenase (5-LOX) [The protective effect of PEDF on the barrier function of the RPE and the He stress . H2O2-ine et al.  investig (5-LOX) . PEDF-R 3 and established an in vitro model system to study RPE degeneration and protection. They found that the cytotoxicity could be decreased by using PEDF. PEDF decreased ARPE-19 cytotoxicity with 6 mM and 7 mM NaIO3 but did not increase the cell viability in response to 6-8 mM NaIO3 [It was reported that PEDF protects RPE cells from toxic damage. Nadal-Nicolas and Becerra induced cytotoxicity injury to ARPE-19 cells with NaIOmM NaIO3 .in vitro and in vivo the role of PEDF on the extrusion of calcium using specific Ca2+ pump inhibitors in models of rd1 mice. They found that PEDF acted on PMCA to reduce intracellular calcium at early stages of the rod photoreceptor cell death cascade. A decrease in the number of photoreceptors with high intracellular Ca2+ was achieved after treatment with PEDF by restraining calpain activation. PEDF protects the degenerating retina by decreasing intracellular calcium [The mechanism by which PEDF protects photoreceptor cells is not clear. Wang et al. evaluated the neuroprotective and anti-inflammatory effects of PEDF on the retinal lesions in a DKO rd8 mouse model of progressive, focal retinal degeneration, mimicking certain features of human atrophic AMD. They found that PEDF potently stabilizes photoreceptor degeneration via suppression of both the apoptotic and inflammatory pathways . One stu calcium . The resin vivo by using the retinal degeneration (rd) and retinal degeneration slow (rds) mutant mouse models. They found that a significant decrease in the death of photoreceptors was caused by injecting the purified protein into the vitreous body of the eye in the two mutants [Cao et al. demonstrated that the intravitreal injection of PEDF at one day or two days before exposure to constant light could protect photoreceptor cells from damage. When PEDF was used in combination with the basic fibroblast growth factor (bFGF), there was an enhancement of the functional status of the photoreceptor cells beyond the protection provided by the factor alone whereas PEDF given at, or after, the onset of constant light provided little or no protection . They su mutants . Their r−/− retina was reconstituted with WT RPE, rhodopsin expression and outer nuclear layer (ONL) thickness were partially restored [in vivo by using eye drops containing a small peptide fragment, PEDF 17-mer. The results showed that application of the PEDF 17-mer peptide enhanced photoreceptor survival and partially prevented retinal degeneration in Mitf mutant mice.PEDF may offer new therapeutic strategies in the treatment of retinal detachment and other degenerations that are induced by dystrophies of the RPE . The exprestored . Also, tin vitro and in vivo caused significant and specific reduction in caspase 2 (CASP2) mRNA and protein in retinal ganglion cells (RGC), and RGC neuroprotection is proportional to suppressed CASP2 levels [PEDF protects nerves by different mechanisms. One study reported that the neuroprotective effect of PEDF is associated with both antiapoptotic and anti-inflammatory activities on retinal neuronal cells . The pre2 levels .in vitro and in vivo. They confirmed previous reports that PEDF promoted RGC survival in vitro and in vivo. Also, they demonstrated for the first time that PEDF promoted RGC neurite outgrowth/axon regeneration. Combining PEDF with cAMP did not enhance PEDF-induced RGC neuroprotection but did enhance RGC axon regeneration [Müller cell-derived PEDF could protect RGC from hypoxia and growth factor withdrawal-induced cell death. The number of viable retinal ganglion cells (RGC) increases when cocultured with Müller glial cells, which could release PEDF . Vigneswneration .Microvessels are composed of two types of cells, endothelial cells (ECs) and pericytes. Pericytes control the overgrowth of neighboring ECs under physiological conditions and play an important role in the maintenance of microvascular homeostasis. Pericytes tighten the tight junction of ECs to reduce endothelial permeability. They play an important role in maintaining the integrity of the inner blood-retinal barrier (BRB) by surrounding ECs . One stu2O2-induced apoptosis and dysfunction through its antioxidative properties via glutathione peroxidase (GPx) [Oxidative stress-induced apoptosis and increased ratio of angiopoietin-2/angiopoietin-1 in pericytes could disrupt the pericyte-endothelial cell interactions, thus promoting angiogenesis by inducing VEGF mRNA levels in retinal pericytes. Amano et al. found that PEDF could protect retinal pericytes against high-glucose or Hse (GPx) . OxidatiYamagishi et al. investigated whether PEDF proteins could protect against advanced glycation end product- (AGE-) induced injury in retinal pericytes. They found that a specific PEDF binding protein existed in the plasma membrane of cultured pericytes, suggesting a possible cell surface receptor for PEDF in its inhibition of AGE-induced reactive oxygen species (ROS) generation and the subsequent decrease in DNA synthesis and apoptotic cell death in pericytes .PEDF could bind to extracellular matrix (ECM) components such as collagens and glycosaminoglycans (GAGs). The different biological activities of PEDF could be based on the interaction with collagens and GAGs.Collagen is the predominant extracellular matrix protein which plays an important role in cell adhesion and migration. The PEDF recognizes collagen in a sequence- and conformation-specific manner . Yasui eAt the molecular level, PEDF has binding sites for collagens and glycosaminoglycans. The collagen-binding property of PEDF may play a role in modifying its antiangiogenic effects in the eye . The assβ (TGF-β) has been linked to the development of fibrosis in a number of diseases, and tissue fibrosis is primarily attributable to the TGF-β1 isoform [β1. Macrophage-derived TGF-β1 is thought to promote fibrosis by directly activating resident mesenchymal cells including epithelial cells.Vascular changes often accompany the development of fibrosis. Transforming growth factor- isoform . Circulaβ1 and connective tissue growth factor (CTGF). Also, it reduced the production of extracellular matrix (ECM) protein production in a diabetic kidney [β1 and CTGF expression. They suggested that the reduction of ECM accumulation induced by PEDF might be mediated in part by the downregulation of TGF-β1.Wang et al. injected streptozotocin- (STZ-) induced diabetic rats with an adenovirus expressing PEDF (Ad-PEDF) to evaluate its effects in diabetes. Administration of Ad-PEDF could prevent the overexpression of fibrogenic factors, TGF-c kidney . This meβ has been recognized as a modulator of ECM formation, such as fibronectin and collagen. TGF-β is a major pathogenic factor in diabetic nephropathy, and it is increased under high-glucose conditions. One study found that PEDF blocked high glucose-induced TGF-β overexpression in primary human glomerular mesangial cells (HMCs) [TGF-s (HMCs) .β-catenin in an ischemic heart [Endothelial mesenchymal transition (EndMT) plays a critical role in the pathogenesis and progression of interstitial and perivascular fibrosis after acute myocardial infarction (AMI). Zhang et al. reported that endogenous PEDF expression could inhibit cardiac fibrosis especially perivascular cardiac fibrosis by regulating EndMT via downregulating the transcriptional activation of ic heart .Wnt signaling promotes inflammation and oxidative stress. He et al. found that PEDF knockout enhanced unilateral ureteral obstruction- (UUO-) induced Wnt signaling activation, and PEDF inhibited the Wnt pathway-mediated fibrosis in renal proximal tubule epithelial cells . They suMany experiments have tried to examine the antifibrotic effect of PEDF. However, no application of PEDF antifibrosis in ophthalmology has been found.in vivo mouse model of carbon tetrachloride- (CCl4-) induced hepatic fibrosis [Tsai et al. found that the amount of PEDF was reduced dramatically in the fibrotic liver. The antifibrotic effect of PEDF is preserved in its 34-mer motif, and its antifibrotic activity was confirmed in an fibrosis . So, theβ1 (TGF-β1) expression. PEDF treatment could significantly attenuate VEGF expression and might lead to the inhibition of airway inflammation and airway remodeling [γ) [Inflammatory response can lead to increased collagen deposition and fibrosis . Principmodeling . Ho et aling [γ) . They obFamily history has a significant association with the incidence of AMD , which sThe relationship between PEDF polymorphisms and the risk of AMD in northern Chinese populations has been studied. The genotype and allele distribution of rs1136287 polymorphism were different between AMD patients and healthy people . The rs1Mattes et al. investigated a hypothesized association between these PEDF polymorphisms and the presence of exudative AMD in a white European population. They found that none of the investigated PEDF polymorphisms (rs12150053 and rs12948386) were likely a major risk factor for exudative AMD. Two polymorphisms did not confer a significantly increased risk for exudative AMD . TaqMan The etiology of AMD is multifactorial, with genetic risk factors contributing to the disease development and progression. Further studies on the role of PEDF gene variations in the pathogenesis of wet AMD are required.Myopia, especially high myopia, can lead to permanent loss of vision. CNV incidence increases in patients with high myopia. PEDF levels in the aqueous in high myopia patients have been investigated in many studies. But different studies come to different conclusions. Costagliola et al. investigated the VEGF and PEDF levels in patients with CNV secondary to pathological myopia (mCNV). Comparing with control samples, significantly lower levels of VEGF and PEDF in the aqueous humor were found in patients with mCNV. After intravitreal ranibizumab injection (IVR), the aqueous VEGF level was significantly reduced, but the PEDF level was significantly increased. An increase in PEDF levels may counteract the neovascularization process in mCNV . HoweverThe PEDF levels in Tenon's capsule of myopes were investigated. The level of “soluble” 45 kDa PEDF was reduced by twofold in Tenon's capsules of high myopia patients while the content of the “insoluble” 50 kDa form of PEDF was increased by fourfold . This reγ-secretase. So, a better understanding of the action of PEDF may provide a more effective therapeutic approach to the treatment of VEGF-induced pathogenesis in the eye. Furthermore, combined anti-VEGF and PEDF may play a synergistic role in the treatment of CNV. However, the potential toxicities or side effects caused by PEDF monotherapy or combination therapy with anti-VEGF need further investigation. The antioxidative effect of PEDF is promising as a way to treat retinal degenerative diseases. PEDF has an anti-inflammatory action, and whether it can suppress inflammation occurring in the retina and reduce macrophage infiltration and fibrosis needs to be investigated in the future. The role of PEDF in the eye is not clear, and further research is needed.PEDF is naturally present in the eye, and it not only functions as an antiangiogenic and neurotrophic/neuroprotective agent but also inhibits pathologically increased vascular permeability. The role of PEDF in antiangiogenesis and protection of pericytes can be applied to the treatment of diabetic retinopathy and CNV in the future. Also, the neurotrophic/neuroprotective effect of PEDF is more advantageous than single anti-VEGF drugs. PEDF inhibits the VEGF-induced barrier breakdown of RPE cell monolayers through the activation of


Growth hormone (GH) signaling plays a key role in mediating growth, development, metabolism, and lifespan regulation. However, the mechanisms of longevity regulation at the cellular and molecular level are still not well-understood. An important area in the field of GH research is in the development of advanced transgenic systems for conditional expression of GH signaling in a cell type- or tissue-specific manner. There have been many recent studies conducted to examine the effects of tissue-specific GHR disruption. This review updates our previous discussions on this topic and summarizes recent data on the newly-made tissue-specific GHR-KO mice including intestinal epithelial cells, bone, hematopoietic stem cells, cardiac myocytes, and specific brain regions. The data from these new genetically-engineered mice have a significant impact on our understanding of the local GH signaling function. Growth hormone (GH) is a major regulator of growth and development of an organism and the disruption of GH signaling (and subsequent IGF-1 disruption) has shown delay the aging process and associated diseases in mice. Disruption of GH-IGF-1 axis is the one of most successful and effective intervention which leads to increased lifespan in mice. In human patients with mutations in GHR gene, there is evidence of protection against cancer and diabetes . This hiGHR signaling in the intestines have been suggested to play an important role in the absorption of nutrients from food and in glucose homeostasis and maintenance of the intestinal epithelial barrier . RecombiIn our previous review, the characteristics of adipocyte-specific GHRKO mice (FaGHRKO) under the control of aP2-cre promoter/enhancer was discussed . Here, tIn a second study, Ran et al.  subjecteIt is well-known that GH signaling plays an important role in regulating lipid metabolism in the liver. Non-alcoholic fatty liver disease (NAFLD) is a widespread liver disorder and its risk increases with GH deficiency . PreviouLiver has an important part in how GH exerts its effects on the body by being the major producer of circulating IGF-1. By disrupting GHR in liver, we study the causal effects of direct GH action on the body independent of IGF-1. Many different lines of mice have been produced with GHR ablation in liver. Here we discuss the newest studies published after our last review.Pgc1α, Ampk, Sirt1, Nrf2, and Mfn2) in LiGHRKO mice and found that gene expression was altered with a sex differences being seen (de novo lipogenesis (DNL) in both males and females, but with hepatosteatosis developing only in males.One study sought to study the gene expression of mitochondrial biogenesis genes  and then fasted for 23 h. There were many interesting findings in these KO mice such as hypoglycemia, a fall in liver triglycerides and a reduction in liver autophagic vacuoles. All of these observations further underline the importance of GH action .GH signaling has shown to play an important role in proper cardiac functioning as evidenced from human patients with GH defects . On the inorganic) and Para Thyroid hormone levels were seen along with decreased bone formation indices when treated with PTH. GHR signaling was found to be required for proper bone growth, mineral acquisition and it functioned in conjunction with PTH.GH-IGF1 axis affects the risk of osteoporosis later in life by its signaling early in life . PTH (Parathyroid hormone) has been found to have both anabolic and catabolic effects on bone and it acts in conjunction with the GH/IGF-1 axis as IGF-1 is a mediator of PTH’s anabolic actions. The goal of the study conducted by Liu et al. was to see how osteocytes integrate signals from GH/IGF-1 pathway with PTH stimuli during bone acquisition. Liu et al. developed the DMP-GHRKO mouse line under the control of the murine dentin matrix protein 1 (Dmp1) promoter/enhancer elements . DMP-GHRHematopoietic stem cells (HSCs) express GHR and the role of GH signaling in its functioning combined with its relationship upon aging is unknown. The regenerative potential of HSC has found to be diminished during age. Stewart et al. generated and characterized a hematopoietic stem cell-specific GHRKO mice under the control of the mouse vav 1 oncogene (Vav1) promoter . It was List et al. generated and characterized the MuGHRKO mice using the muscle creatinine kinase (MCK) promoter to drive Cre expression . Data onGH and GHR are active in the central nervous system. GHR is known to be expressed in neurons that express leptin receptor, agouti-related protein, kisspeptin receptor, proopiomelanocortin (POMC) prohormone and steroidogenic factor-1 (SF1). Agouti-related protein neurons in the ARH (arcuate nucleus) are known to be major regulators of energy homeostasis. One of the major known functions of GH in the brain is that it promotes neuroendocrine adaptations during food deprivation. GH has also been found to signal the AgRP neurons under the same food deprived conditions. Leptin receptor expressing neurons of hypothalamus which also express GHR are known to be involved in metabolic adaptations that conserve energy during food restriction (FR).Cady et al. generated and characterized a mutant mouse line LepRb GHRKO with gene ablation in cells expressing leptin receptor . Furigo Bohlen et al.  developePregnancy is a time where a mammal experiences widespread metabolic changes and adaptations for the development of the offspring and the later birth. GH also plays some role in this process . A studyIn order to study the effects of GH action in anorexigenic neurons which express the proopiomelanocortin (POMC) prohormone, POMC GHR KO mice  model wavia specific neuronal populations, SF1 GHRKO mice were generated with GHR ablation in the ventromedial nucleus (VMH) neurons  . In orde neurons . In LepRThere have been numerous interesting studies on tissue-specific GH effects published in last few years and have been briefly summarized here. Of these some were tissues for which mouse models were already developed  while others were new such as heart, bone, intestine, HSCs, and different brain regions (AN took the lead in writing the manuscript. All authors contributed to the article and approved the submitted version.Our studies and preparation of this article were supported by NIH grants K01 AG048264 and AG057734 (LS).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Small form-factor sensors are widely used in minimally invasive intravascular diagnostic procedures. Manufacturing complexities associated with miniaturizing current fiber-optic probes, particularly for multi-parameter sensing, severely constrain their adoption outside of niche fields. It is especially challenging to rapidly prototype and iterate upon sensor designs to optimize performance for medical devices. In this work, a novel technique to construct a microscale extrinsic fiber-optic sensor with a confined air cavity and sub-micron geometric resolution is presented. The confined air cavity is enclosed between a 3 μm thick pressure-sensitive distal diaphragm and a proximal temperature-sensitive plano-convex microlens segment unresponsive to changes in external pressure. Simultaneous pressure and temperature measurements are possible through optical interrogation via phase-resolved low-coherence interferometry (LCI). Upon characterization in a simulated intravascular environment, we find these sensors capable of detecting pressure changes down to 0.11 mmHg (in the range of 760 to 1060 mmHg) and temperature changes of 0.036 °C (in the range 34 to 50 °C). By virtue of these sensitivity values suited to intravascular physiological monitoring, and the scope of design flexibility enabled by the precision-fabricated photoresist microstructure, it is envisaged that this technique will enable construction of a wide range of fiber-optic sensors for guiding minimally invasive medical procedures. I.in vivo use Miniature minimally-invasive sensors allow for fine spatial resolution and targeted point measurements at critical sites, particularly for biomedical diagnostics Fiber-optic sensors present a favorable alternative for use in minimally invasive medical applications. Of particular interest is their immunity to EMI, magnetic resonance imaging (MRI) compatibility, and high mechanical flexibility 2, metal films, nano-diamond, ceramics, and organic polymers have been investigated in this regard Fiber-optic pressure and temperature sensors comprise one or more responsive optical cavities that are interferometrically interrogated to measure corresponding deflections. Such probes can be formed by affixing an optical cavity structure consisting of a deformable diaphragm extrinsic to a waveguiding optical fiber. Materials such as Si, SiOet al. to form an SU-8 based cylindrical extrinsic structure with a single air-filled optical cavity 3D laser lithography is of interest for precision microfabrication of fiber-optic sensors. Early work on this paradigm was done by Hill In this work, we demonstrate the precision microfabrication of such a fiber-optic sensor that is simultaneously responsive to changes in both pressure and temperature within the intravascular physiological range TPP microfabrication of structures directly onto the cleaved end-faces of optical fibers have been reported to form lenses, gratings and micro-spring elements We have overcome these challenges through a combination of freeform 3D multi-cavity sensor design, optical path raytracing, TPP and single-step microstructure bonding to an SM optical fiber. Optical path length changes of the low-finesse cavities in these sensors were interrogated by phase-resolved low-coherence interferometry (LCI). We then characterized these devices to evaluate their structural design fidelity and application potential for sensing pressure and temperature changes in the range of minimally invasive interventions in a simulated intravascular environment.II.The extrinsic sensing element of our fiber-optic probe comprised a cylindrical cap structure with three optically reflecting interfaces along the beam path forming two distinct low-finesse optical cavities . Here, tA.Materials and Methods section). Using low SLD illumination power is desirable as it reduces the likelihood of beam-induced heating effects in the extrinsic sensor element, and/or the surrounding media beyond the partially transmissive distal deformable diaphragm (for instance blood when considering the intravascular space). To enable LCI signal detection at lower illumination power, the sensor element design was modified to enhance coupling of the back-reflected beam from each cavity surface into the core of the SM optical fiber. In contrast, within the case of parallel reflecting surfaces as is the design norm for FP cavities in fiber-optic pressure sensors This multi-cavity design was further modified with the aim of improving detection sensitivity and reducing the super-luminescent diode (SLD) illumination power required for LCI interrogation  was evaluated against a comparable reference sensor design (Sref) where the polymer block segment had a flat surface instead of a curved lensing surface, shown in Materials and Methods section) that the beam spot at the outer surface of the deformable diaphragm (indicated as PD) was found to diverge significantly more in Sref compared to Slensed., yielding an almost 4-fold larger beam spot irradiance area with Sref.Furthermore, the lensing capability of this integrated sensor . The presence of low-finesse optical cavities causes the interference pattern observed at the spectrometer in the LCI interrogation system, and the changes in optical path length of each cavity can be monitored as variations in this interference pattern Δz1 and Δz2, considering the optical cavities highlighted in in z2, while z2 would only respond to changes in temperature due to thermal expansion of the TPP-printed extrinsic sensor element material.These fiber-optic sensors were designed for interrogation by phase-resolved low-coherence interferometry (LCI). The Michelson interferometer sensor interrogation setup used in this study is based on the miniaturized setup described by Coote III.A.Using the results of the raytracing optimization, a freeform 3D computer-aided design (CAD) of the sensor element was formulated for TPP microfabrication. To simplify maneuvering the extrinsic microscale sensor element for fiber integration . Thereafter, these structures were safely detached from the substrate for integration to the 125 μm diameter SM optical fiber. A summary of these fabrication steps is shown in This CAD design formed the basis of defining the femtosecond laser beam trajectory for TPP microfabrication. TPP processing parameters were modified in order to achieve smooth surface topography, in addition to reducing the X-Y and Z scanning tolerances to below 0.3 μm each. Microstructure fabrication involved laser illumination to induce TPP within a proprietary IP-S photoresist droplet (Nanoscribe) on an ITO-coated glass substrate. Photoresist development, i.e., washout of unilluminated resist, was performed by immersing in propylene-glycol-monomethyl-ether-acetate (PGMEA)  with bean-shaped cross-section were incorporated along the periphery of the lensing segment , complete removal of all unpolymerized photoresist material through the designated washout holes was verified , while surface discontinuities on the curved lens surface are estimated to be <350 nm by virtue of the TPP lithography system resolution Materials and Methods).Further, through TPP fabrication of the transverse cross-section, and photoresist development using our modified protocol and microstructure detachment . Following SM fiber positioning as per design  under an optical microscope, as illustrated in lensed) was evaluated against a comparable reference sensor design (Sref) where the polymer block segment had a flat surface instead of a curved lensing surface , indicated in Furthermore, the lensing capability of this integrated sensor  agrees well with the reference pressure sensor. Over longer duration measurements exceeding 30 minutes however, signal drift was found to be present (similar to other polymer-based interferometric sensors in literature) The pressure response of this sensor was tested and found to approximate linear behavior in comparison to the bulk pressure reference sensor in the characterization setup. Evaluating the response of these microfabricated sensors in the range of 760 mmHg to 1060 mmHg , the pressure sensitivity of cavity 1 and φ2 are plotted in i), was found to approximate linear behavior with an R2 value of 0.981. In this temperature range, the sensitivity of φ1 (corresponding to cavity z1) was found to be 0.052 rad/ °C, with a noise floor value of 18.9 mrad . The temperature resolution was found to be 0.036 °C in this case.Response of the sensor to temperature changes was evaluated by applying controlled thermal gradients to the sensor by implementing a programmable temperature feedback in the outer water bath . Care waThe temperature response in these optical probes depend upon the thermal expansion and thermo-optic coefficient (change in refractive index with response to temperature) of the extrinsic sensing element material, in this case the proprietary IP-S photoresist. Non-linear temperature response beyond 50 °C could be attributed to the thermal expansion of the polymeric sensor element in 3D not being completely anisotropic along the optical axis in our sensor design. These dynamics could be varied and studied in detail by implementing modified TPP-printed multi-cavity sensor designs ensuring anisotropic thermal expansion in a large temperature range, or by using alternate materials, such as glass or metallic composites. High resolution TPP fabrication of these materials was recently reported and could be modified for implementation to fiber-optic probes Materials and Methods section). The bonding of the extrinsic sensing element to the optical fiber  and the sensor response (interrogated using the LCI system) were found to remain undamaged even after immersion in a water bath for a duration of 14 days. Additionally, the sensor signals were observed to be intact following mechanical impacts against the inner walls of the sealed water-filled polycarbonate tubes in the characterization setup  cavity was constructed using a novel method that involved high resolution TPP 3D printing, with a freeform design suited to simple and irreversible bonding to an SM optical fiber for measurements in a remote fluidic environment. The optical design of the monolithic multi-cavity extrinsic sensing structure was optimized through raytracing simulations to minimize beam divergence within its optical cavities to improve sensor SNR for efficient performance. Importantly, the fiber-optic integration was performed using non-hygroscopic transparent cyanoacrylate adhesive to enable irreversible bonding of the sensing element to the optical fiber to enable prolonged operation within an aqueous environment, for instance the intravascular space. The technique described here lends well to high resolution micro-sensor prototyping with superior structural fidelity and design flexibility. Moreover, interrogating these low-finesse cavity fiber-optic sensors with phase-resolved LCI in a simulated intravascular environment yielded pressure and temperature sensitivities well within the range required for intravascular sensing catheters and intracranial pressure measurements as per medical device standardization requirements (FDA title-21B §870.1200 and §882.1620 respectively) While the proof-of-concept design implemented in this paper comprised within a single microstructure - a microlens, air-cavity and a robust microscale polymeric diaphragm supported only at its edges, there is scope for inclusion of additional cavity elements with tunable mechanical and optical functionalities. Instead of forming an air cavity, additional materials  could be introduced into the TPP structure by micro-pipetting into the air cavity spaces of such fiber-optic probes for requisite optical, bio-chemical and mechanical sensing operations. Moreover, beyond the use of photoresists, this technique could be extended to multiphoton 3D printed fiber-optic extrinsic probes where the microscale extrinsic sensing structures are formed using alternate materials such glass or metals to lower sensor drift, and/or for operation in other measurement ranges.In addition to extending this sensor construction paradigm to myriad materials for precision TPP-based microfabrication, design modifications for alternative sensing applications could be explored. For instance, a single air-filled optical cavity design could be adapted to form miniature high-finesse cavities for fiber-optic ultrasound sensing by metallizing the distal end of the optical fiber and proximal end of the deformable diaphragm to enhance cavity finesse. Along with the interferometric measurement of physiological pressure and temperature, such sensors could be modified for multiplexed sensing and biomolecule detection by including biochemically functionalized optically responsive external surfaces of the distal deformable diaphragms. The optical design may also be modified to deliver focused optical energy for tissue ablation, light-guides for mm-scale endoscopic cameras and/or diffractive optical elements integrated to optical fibers for measurements in opto-genetics. Moreover, use of recently developed photoresist material with long-term biocompatibility (for instance IP-Visio) to form the probe microstructure could enable development of minimally invasive implantable devices.Combined with the ongoing innovation in parallelized femtosecond laser-based material processing and adaptive optics, these developments could enable large-scale production of precision microfabricated fiber-optic probes for use in a variety of areas within and beyond biomedical sensing. Therefore, this work presents significant inroads in the field of fiber-optic sensors in general, and MIS probes in particular.VI.A.et al.Sensor interrogation was performed through phase-resolved LCI with the sensing fiber connected in a Michelson interferometer configuration , based oB.z1 and z2 in z2 by Δz2. A temperature increase causes the photoresist material to expand, modifying the cavity length z1 by Δz1. The distal deformable diaphragm also undergoes thermal expansion contributing to Δz2, depending on the design of the sensor element. These changes in optical path length can be monitored as variations in the complex argument of the inverse Fourier-transformed spectrum at its maxima, as detailed in our previous work The LCI sensing mechanism can be described by reference to the two-cavity design of the extrinsic sensing element, with optical cavity lengths indicated as 1z and 2z, and small temperature and pressure-induced variations in these optical cavity lengths, Δz1 and Δz2, can be obtained from the complex argument of the IFT at these maxima, according to Choma et al., I(k) is the intensity spectrum as a function of wavenumber k, zj is the baseline optical cavity length, which includes the geometric length and refractive index, j is an integer equal to 1 or 2 signifying each of the two optical cavities, λc is the central wavelength of the SLD, and t is time, referenced to a start time t0. The complex argument signal, φj forms the output of the sensor. In this work, two such complex argument signals φ1 and φ2, corresponding to the optical cavity lengths z1 and z2 were analyzed. These signals were calibrated against reference pressure and temperature sensors, to allow measurement of absolute changes in pressure and temperature using this fiber optic probe.Briefly, an inverse Fourier transform (IFT) is applied to the intensity spectrum obtained from the spectrometer. The maxima of the complex magnitude of the IFT can be shown to be proportional to the baseline optical cavity lengths, C.et al., Ray tracing for the optical system representing the sensor design was performed on OpticStudio 15.6 software  using the Physical Optics propagation method in the Sequential ray tracing mode. The basic design paradigm used for these structures is indicated in The surface curvatures and dimensions of the various cavity elements were optimized, while also considering possible positioning errors during sensor integration by relative light source decentering  by ±1 μm in the optical system Two cases of decentered source are depicted as the red and green paths and the case of centered source as the blue path in D.The complete sensor element, including the lateral holding element, was designed using computer aided design (CAD) software . This 3D CAD design for the sensor element was converted from Standard Tessellation Language (STL) format to the native General Writing Language (GWL) data format of the TPP system using DeScribe software (Nanoscribe GmbH). Subsequently, the GWL file was loaded into the system's control software NanoWrite (Nanoscribe GmbH) for print execution.The extrinsic sensor element was fabricated on a glass substrate coated with Indium Tin Oxide (ITO), using a commercial TPP system  and its 25x objective. During TPP printing, polymerization inside a droplet of liquid UV curable resin  was induced at the focus of a 780 nm femtosecond laser beam by two-photon absorption. The beam could be rapidly steered laterally (X-Y) within a hexagonal block (350 × 350 × 200 μm). For fabricating structures larger than the predefined microscale volume, printing was performed across multiple parallelepiped blocks angled along the z-axis at 10°. The complete structure was realized by stitching together individual blocks such that no slice lines fell along the path of the sensor interrogation beam path designated in the ray trace, with some additional allowance along the periphery . The average fabrication time for each sensor element was 54 minutes  using the above mentioned commercial TPP system and print settings.Following TPP printing, the microstructures were developed by immersing in propylene-glycol monomethyl-ether-acetate (PGMEA) for 180 minutes to ensure complete washout of uncured resist from the complete structure, and subsequent rinsing in isopropyl-alcohol (IPA) for 15 minutes was performed to ensure removal of residual developer. The 3D printed structures were lifted-off the ITO-glass substrate with extra-fine tip, acid- resistant tweezers. These steps are represented schematically in E.The surface roughness of the proximal surface of the diaphragm was characterized using an AFM (Veeco Dimension 3100) in a transverse cross-section of a test sample consisting of only 45 μm of the distal-most section of the sensing element, to allow the AFM tip to access the measurement surface. The average surface roughness values were obtained by evaluating the AFM data on NanoScope Analysis software package in the tapping mode over an area of 5 μm square.F.The pressure and temperature responses of the sensor were measured with a pressure-sealed water chamber. Pressure within this chamber was varied with an electro-pneumatic regulator  and monitored with a custom configured pressure transducer . Pulsed pressure variations with different step sizes and duty cycles were induced within this chamber across the absolute range of 760 mmHg (atmospheric) to 1060 mmHg.For temperature sensitivity measurements, the chamber was placed in a programmable water bath with its temperature varied from 21 to 55 °C. The pressure within the sealed water chamber was kept constant during temperature sensitivity measurements. The reference temperature sensing thermocouple , was positioned adjacent to the precision 3D printed fiber-optic sensor. Interferometric phase changes were calibrated to values from the corresponding reference sensor with a linear transformation


Teaching Point: Uterine involvement of B-cell acute lymphoblastic leukaemia is exceedingly rare. Globular enlargement of the uterus with diffuse homogeneous signal intensity is suggestive for the diagnosis on magnetic resonance imaging (MRI) in clinically suspected cases. A 41-year-old woman presented at the emergency department with severe metrorrhagia. It had started a few weeks ago and was gradually increasing in severity. A blood transfusion was even needed to compensate for the anaemia. A hemorrhagic myoma uteri was initially suspected on clinical examination but upon further inquiries, the patient stated she was recently diagnosed with relapsed B-cell acute lymphoblastic leukaemia (B-ALL).As a first imaging evaluation ultrasound was performed, showing a non-specific uterine enlargement. Magnetic resonance imaging (MRI) of the pelvis was added for further exploration. MRI confirmed the diffuse uterine enlargement and demonstrated a thickened, poorly defined (nearly ‘washed out’) junctional zone. The uterine signal was homogeneously slightly hyperintense on T2-weighted images Figure  and homoB-ALL is an acute malignancy of immature lymphocytes in the bone marrow and blood. The most commonly affected extramedullary sites are the testes and the central nervous system. Infiltration of the female reproductive system is often found microscopically in relapsed B-ALL, but it is rarely clinically obvious. Furthermore, the uterus is rarely the dominant site of involvement . The cliUltrasound is the first choice for imaging investigation, and although it can provide an initial evaluation of uterine mass, MRI is recommended for further assessment. Global enlargement of the uterus due to diffuse infiltration of the B-ALL with a homogeneous hypo- to isointense signal on T1-weighted images and slight hyperintense signal on T2-weighted images are most commonly seen. The junctional zone and uterine border are usually spared and pelvic lymphadenopathy is common. This imaging appearance is, however, not specific and the disease can also manifest as multiple nodules or a large lobulated uterine mass. In the latter case, the absence of necrosis despite the large size of the tumour and the intact junctional zone are diagnostic clues for haematological invasion. In rare cases, the architecture may be disturbed and invasion into nearby organs can occur [


Drosophila melanogaster and classified genes by the expression level in S2 cells  as well as the number of tissues in which a gene was expressed . Chromatin in 5′-regions of genes we classified on four states according to the hidden Markov model (4HMM). Only the Aquamarine chromatin state we considered as Active, while the rest three states we defined as Non-Active. Surprisingly, about 20/40% of genes with 5′-regions mapped to Active/Non-Active chromatin possessed the minimal/at least modest RPKM and BoE. We found that regardless of RPKM/BoE the genes of Active chromatin possessed the regular nucleosome arrangement in 5′-regions, while genes of Non-Active chromatin did not show respective specificity. Only for genes of Active chromatin the RPKM/BoE positively correlates with the number of nucleosome sites upstream/around TSS and negatively with that downstream TSS. We propose that for genes of Active chromatin, regardless of RPKM value and BoE the nucleosome arrangement in 5′-regions potentiates transcription, while for genes of Non-Active chromatin, the transcription machinery does not require the substantial support from nucleosome arrangement to influence gene expression.We analyzed the whole-genome experimental maps of nucleosomes in  Nucleosomes are the basic packaging units of chromatin. Their positioning has a critical impact on the regulation of transcription. A wide variety of chromatin proteins can affect nucleosome positions ,2. BesidIntrinsic DNA sequence preferences due to the thermodynamic costs associated with wrapping stiff DNA around the histone octamer influence on nucleosome positions in vivo ,9. A widExperimental data on nucleosome positioning in genomes come from a number of related techniques ,10,11. FMaps of chromatin states were a consequence of recent advances in experimental and bioinformatics approaches for genome analysis. First, we earlier developed an approach to simultaneously map the interband material cytologically and in the genome using transposon insertion tags . This alThese advances in cytology and bioinformatics allowed partitioning the genome into four chromatin types referred to as Aquamarine, Lazurite, Malachite, and Ruby ,30. We hThe canonical nucleosome arrangement Nuc–1/NDR/Nuc+1 is typical for housekeeping genes; stress-responsive genes have noncanonical nucleosome arrangement due to delocalized, i.e., uniformly distributed nucleosomes . PartialWe proceeded from the earlier analysis of one cell line  to the aSilent ;Modest ;High .We classified all genes according to RPKM value (reads per kilobase million) in S2 cells  on threeTissue-specific ;Modest ;Constitutive .We computed the breadth of expression (BoE) as the number of tissues in which a gene was expressed. We considered genes satisfied the criterion RPKM > 3 to compute BoE. We classified genes by BoE on three classes:To incorporate the chromatin landscape in the analysis we considered the whole-genome 4-state fragmentation model 4HMM ,30. We cThe distribution of Active genes into classes of RPKM value and BoE demonstrated that the majority of genes had at least modest BoE or RPKM 81.5%, B. Hence,We appealed to experimental data on the mapping of resistant/sensitive nucleosomes see . We compthe strong nucleosome formation sites downstream TSS;the depletion of resistant nucleosome sites upstream TSS;the regular ladder of sensitive nucleosomes upstream TSS.Note that peaks at positions Nuc−2, Nuc−1, and Nuc+1 are completely absent in genes with modest/high RPKM or in “modest BoE”/constitutive genes.Thus, for Non-Active genes respecting modest/high RPKM or “modest BoE”/constitutive classes B, we didstrongly significant negative CC for resistant nucleosomal sites around TSS  for tissue-specific and modest; moderate negative CC for sensitive nucleosomal sites for tissue-specific  and modest ;strongly significant positive CC downstream TSS  for tissue-specific and modest;moderately significant positive CC for sensitive nucleosomal sites upstream TSS in tissue-specific ) and modest .In this section, we study dependencies between the number of mapped nucleosome sites around TSS and expression measures RPKM/BoE. This analysis was performed separately for Active and Non-Active genes. We applied Kendall’s rank correlation coefficients (CC) see  to reveaIn Non-Active genes the 1st and 3rd trends are not observed; the 2nd trend is even opposite for tissue-specific and is absent for the rest genes.Constitutive Active and Non-Active genes did not show significant correlation . Howeverabout 20% of Active genes were either silent or tissue-specific;about 40% of Non-Active genes had at least modest RPKM or BoE.The development of whole-genome maps of chromatin states ,33,34,35Then, along the length of promoter, we computed the profiles of the average numbers of resistant/sensitive nucleosome sites for Active and Non-Active genes and categorized them by RPKM value and BoE. Notably, these subsets revealed clear diversity in the nucleosome arrangement around TSS . We concNext, we tested the significance of correlations between the number of resistant/sensitive nucleosomal sites  and RPKMIt was estimated  that almThe genome-wide location of nucleosomes during zebrafish embryogenesis indicated that well-positioned nucleosome arrays appeared on thousands of promoters during genome activation . It is iThe recent analysis of chromatin folding for various epigenetic states of chromatin  supporteOur conclusions are in agreement with a number of recent reports:for Active state potentiates transcription, i.e., a more regular nucleosome arrangement in promoter points to higher expression level and higher number of expressed tissues;for Non-Active state does not influence gene expression, i.e., the expression level/breadth and the nucleosome positioning are independent.Earlier , genes oWe took the nucleosome mapping data   ,30. We cD:We computed Kendall’s rank correlation coefficients (CC) to estimate the significance of correlations between the expression measure (BoE or RPKM) and the number of nucleosomes mapped in a specific position. To estimate the significance of Kendall’s rank CC we applied the normal approximation  with theN denotes the sample size.Active genes demonstrated clear pattern of nucleosome arrangement around TSS; the regular nucleosome positioning correlates with the expression level/breadth;Non-Active genes did not show such clear specificity in nucleosome positioning; the correlation between the regular nucleosome positioning and expression level or breadth either is absent, not conservative, or shows even the opposite significant trend compared to that of Active genes.We took the whole-genome map of chromatin states and referred “Active” and “Non-Active” genes according to the overlap of their 5′-regulatory regions with domains of Active and Non-Active chromatin states. Our analysis confirmed that classification of all genes according chromatin states allowed deducing the better explanation for the influence of nucleosome positioning on gene transcription activity. We found that irrespective to the expression level or the number of expressed tissues:The analysis of 5-state model of Filion et al.  supporte


The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that has been shown to be an essential regulator of a broad spectrum of biological activities required for maintaining the body’s vital functions. AhR also plays a critical role in tumorigenesis. Its role in cancer is complex, encompassing both pro- and anti-tumorigenic activities. Its level of expression and activity are specific to each tumor and patient, increasing the difficulty of understanding the activating or inhibiting roles of AhR ligands. We explored the role of AhR in tumor cell lines and patients using genomic data sets and discuss the extent to which AhR can be considered as a therapeutic target. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that has multiple critical cellular functions . It beloSince the early 90s, AhR has been defined as an essential environmental sensor that enables the activation or inhibition of cellular pathways in response to a broad spectrum of ligands in a cell-type- and context-specific manner ,10. MoreHere, we summarize the role of AhR in cancer mechanisms, based on previous studies and the analysis of a set of genetic and genomic databases. Then, we discuss the conditions required to consider AhR as a therapeutic target.AhR alterations in cancer by interrogating available genomic data  searchable on the cBioPortal for cancer genomics online platform  ..AhR mRNAAhR expression, AhR-correlated signatures can also be considered to evaluate the potential effectiveness of a treatment. Indeed, we found that these gene signatures correlate with the efficacy of molecules previously highlighted in AhR expression and the correlated transcriptional signature to define specific anti-tumor strategies.In addition to the level of −/− MDA-MB 231 TNBC cells  [https://www.ncbi.nlm.nih.gov/gds/?term=GSE129221] [The raw data count matrix from the RNA seq data was obtained from the GEO database for the previous experiments on the lung-cancer cell lines (sensitive or resistant to an EGFR inhibitor: gefitinib) GSE79688 [SE79688]  and GSE1E129221] .The expression heatmap of differentially-expressed genes between samples was obtained for a log2-fold change using the ComplexHeatmap 2.0.0  package


Immunization programmes against hepatitis-B are being carried out since more than three decades but still HBV is a major public health problem. Hepatitis B virus (HBV) genome consists of circular and partial double stranded DNA. Due to partial double stranded DNA, it uses an RNA intermediate during replication. This replicative strategy of HBV and lack of polymerase proofreading activity give rise to error occurrences comparable to retroviruses. The low fidelity of polymerase, overlapping reading frames and high replication rate produces many non-identical variants at every cycle of replication. Therefore, HBV spreads with mutations and variations. The mutations have been reported both in non-structural as well as structural genes of HBV genome. Recent advances in molecular biology have made easier to analyse these mutations. Hepatitis B antiviral therapy and immunization are all influenced by genetic variability. The analysis and understanding of these mutations are important for therapy against hepatitis B and updating of diagnostic tools. The present review discusses about mutations occurring in whole HBV genome. The mutation occurring both in structural and non-structural genes and non-coding regions have been described in details. It is much more informative because most of literature available, covers only individual gene or DNA regions of HBV. The virus attacks the liver and can cause both acute and chronic disease 2.The best method for analysis of mutation is Nucleotide sequence analysis. Mutations can be analysed region wise. It can be done for single gene region by determining and comparing nucleotide sequence of the specific gene 3.Sequence variations in the core promoter are limited because of its pivotal role in viral replication. The double mutation, A to T transversion at 1762 and G to A transition at 1764, is often present in patients with chronic hepatitis, hepatocellular carcinoma, fulminant hepatitis, and less often in asymptomatic carriers, and in carriers without HBV markers 4.The core promoter overlaps with X gene, therefore core promoter mutations often affect the structure and thus presumably the function of X protein. Nearly all deletions/insertions in the BCP  are responsible for frame shift in X gene and lead to the production of truncated X proteins. These X proteins lack a domain in the C terminus (around aa position 130–140), which is highly conserved and is essential for trans activation activity. It was reported that these mutations affecting the crucial domain for transactivation, suppress the transcription of both precore and core mRNA in vitroX-ORF overlaps the C-terminus of the P-gene and the N-terminus of the C-gene. Therefore, mutations within the X-region can affect three regions at a time. X deletions created fusion-protein between the polymerase and the core-protein (PC-proteins) or between the polymerase and a 3′truncated HBx (PX-proteins) in vitroX mutants are suspected to lead to a transmissible HBV infection, which cannot be detected by conventional test system because the classical serological markers are missing. The probable explaination being, HBx seems to be non-essential to viral life cycle 5.HBV variants have been revealed with blocked HBeAg synthesis due to mutations in the precore region. Hepatitis B flares occur during the HBsAg immune clearance phase in HBsAg-positive individuals. Hepatitis B flares also occur in the HBeAg-negative individuals, but less frequently, highlighting the role of HBeAg in liver damage in vivo. Less common precore mutations resulting in HBeAg negativity include iniation codon mutations (at position 1814 or 1815), a nonsense mutation at 1874 and frameshift mutations. Mutations in the pre core codon 17 (G1862T) which occurs in the bulge of ε seems to alter the processing of the HBeAg precursor protein, leading to an HBeAg negative phenotype Three types of mutations leading to pre-C defective variants are known: first type of mutations inactivate the pre-C ATG; second type are insertion or deletion in the pre-C region that lead to a frame shift; and third type mutations lead to a stop codon in pre-C region. All three types of mutations prevent translation of the precore protein from the pre-C mRNA, whereas the translation of the core protein from the pregenomic-C mRNA remains unaffected If HBeAg-minus mutants are selected by immune mechanisms, then appearance of mutants is an immune escape phenomenon. The report that precore-mutants were associated with non-responsiveness to interferon therapy or with only a transient therapeutic effect supports this hypothesis Studies on epidemiology of precore mutants reveled that HBeAg mutant were associated with hepatocellular carcinoma to mild or asymptomatic liver disease. HBV DNA from liver tissues especially from hepatocellular carcinoma cells was found to possess more mutants than HBV DNA from the serum. An increasing number of precore-mutants such as 28 stop codon mutations of the precore start codon  and/or frame shift mutations in serum and point mutations in the ‘a’ determinant of HBsAg from acute, chronic liver disease, HBV infected non-tumor tissues and finally hepatocellular carcinoma cells were discovered 6.HBcAg and HBeAg are highly cross-reactive at the T cell level. It therefore may be speculated that HBeAg presents immunogenic epitopes to the T cells thus protecting the HBcAg expressing hepatocytes against the immune system. After the selection of HBeAg-negative mutant the epitopes of HBcAg might come under the pressure of the immune system The core gene contains both humoral and cytotoxic T cell epitopes. Comparison of HBc/e (HBc + HBe) sequences with the corresponding wild-type sequence reveals that 75% of all amino acid sequences cluster at 36 hot-spot positions, which sum upto only 20% of the HBc/e sequence. Amino acid changes are more frequent in the central core protein domain Mutations inside the CTL-epitopes (aa18–27) of the core gene might create immune escape mutants leading to chronic viral persistence and severe liver disease. Naturally occurring changes inside CTL epitopes were found in South Eastern Asia 7.7.1In vivo models imply that dysregulation of surface protein expression/ or expression of mutant pre-S1 proteins can cause direct and immune-mediated liver cell injury. In specific S gene transgenic mice, relative over expression of preS1 to S protein was directly shown to be toxic to liver cells The large HBsAg (pre-S1) and medium HBsAg (pre-S2) are very important for virus clearance because they are more immunogenic and appear earlier in the course of infection than the small HBsAg 7.2Pre-S2 deletions cluster in a small segment at the pre-S2 amino terminus do not affect the pre-S2 protein sequence beyond amino acid position 23. All the deletions are in frame and corresponding variants express internally deleted pre-S1, pre-S2, and P protein. Variants with deletion in pre-S2 expressed HBsAg similar to wild type HBV in hepatoma cells, and were shown to be infectious for primary human hepatocytes 7.3in vitroin utero. The potential of the vaccine escape mutants to establish a long term carrier state and their transmissibility could be a potential problem for global vaccination in the future This region is believed to interact with protective or neutralizing anti-HBs antibodies elicited during natural infection as well as by vaccination with HBsAg Anti-HBs antibodies are also given as immunoprophylaxis to prevent reinfection of the liver graft after transplantation for HBV related fulminant or ESLD. Reinfection of the liver despite protective anti-HBs titres was found to be associated with mutations in the ‘a’ determinant clustered at aa 145 8.Deletions in the C gene may change the structure and expression of the P protein, deletions in the preS1 and preS2 regions remove sequence from the dispensable spacer region, and ‘a’ determinant mutations lead to changes or insertions in the RT domain. Mutations, which seriously alter the function of P protein, are seen in patients treated with nucleoside analogues lamivudine and famciclovir. Resistance to lamivudine therapy is associated with amino acid changes in the YMDD motif, namely methionine to valine or isoleucine 9.Variants containing large deletions or insertions, which affect several HBV genes and/or regulatory sequences, are defined as grossly altered. Three classes of such genomes are, with large deletions or insertions at termini with the direct repeat sequences; second genomes with an internal poly (dA) tail accompanied by an adjacent large deletion; and third, genomes with large deletions derived from spliced pregenomic RNA. Further, the first class can be divided into four types. The deletions either ends at DR1 and the adjacent sequences are collinear with the 3′end of the DNA minus strand (type A), or they end at DR2 and the adjacent sequences are collinear with the 5′end of the DNA plus strand (type B). In type C the genome is collinear with the 3′end of the DNA minus strand and deletions start at DR1. In type D the region between DR1 and DR2, the so called cohesive overlap region of the open circular HBV genome is partially or completely duplicated. Interestingly, virus-virus and virus-cellular junctions of chromosomally integrated HBV DNA also frequently map to DR1 and DR2 Genomes with an internal poly (dA) sequence have been identified as a very minor genome population in the serum and liver samples from the patients with different types of liver disease. These genomes are likely to be generated during genome replication in the core particle by a mechanism involving template switching and reverse transcription of the poly (A) tail of the RNA pregenome transA large fraction of the pregenomic RNA undergoes splicing in HBV infected liver and HBV DNA transfected hepatoma cells 10A number of variants of Hepatitis B virus are circulating among HBV infected patients around the world. These variants can be distinguished by using molecular biology techniques such as PCR and DNA sequencing. The mutations in HBV are not limited to particular gene or DNA fragment but have been detected throughout the genome. These mutations play significant roles in viral latency, immune escape, the pathogenesis of liver disease and resistance to antiviral therapies.


Deep learning algorithms are increasingly used for automatic medical imaging analysis and cardiac chamber segmentation. Especially in congenital heart disease, obtaining a sufficient number of training images and data anonymity issues remain of concern.Progressive generative adversarial networks (PG-GAN) were trained on cardiac magnetic resonance imaging (MRI) frames from a nationwide prospective study to generate synthetic MRI frames. These synthetic frames were subsequently used to train segmentation networks (U-Net) and the quality of the synthetic training images, as well as the performance of the segmentation network was compared to U-Net-based solutions trained entirely on patient data.Cardiac MRI data from 303 patients with Tetralogy of Fallot were used for PG-GAN training. Using this model, we generated 100,000 synthetic images with a resolution of 256 × 256 pixels in 4-chamber and 2-chamber views. All synthetic samples were classified as anatomically plausible by human observers. The segmentation performance of the U-Net trained on data from 42 separate patients was statistically significantly better compared to the PG-GAN based training in an external dataset of 50 patients, however, the actual difference in segmentation quality was negligible .We demonstrate the utility of PG-GANs for generating large amounts of realistically looking cardiac MRI images even in rare cardiac conditions. The generated images are not subject to data anonymity and privacy concerns and can be shared freely between institutions. Training supervised deep learning segmentation networks on this synthetic data yielded similar results compared to direct training on original patient data. Deep learning technology is currently in the process of revolutionizing medical diagnostic services . Convolun = 303 patients) was used to train progressive GAN networks, which in turn produced synthetic MRI frames that were utilized to manually produce segmentation maps for the training of downstream U-Net segmentation models. A second part (n = 42 patients) was utilized to train U-Nets directly on patient frames. The performance of these two U-Net models was subsequently compared with a third (independent) fraction of the data (n = 50 patients), not used for training either U-Net models. In addition, the quality of the synthetic PG-GAN images was assessed visually, and the degree of similarity to original MRI images was quantified using a statistical similarity index (for details see below).Fig. GANLib GitHub repository and implemented in TensorFlow [2, 162, 322, 642, 1282 and 2562 pixels, respectively. The number of filters and the batch size was adjusted accordingly (for details see below). A latent vector of dimension 64 was used as an input to the generator which consisted of blocks of 4 × 4 and 3 × 3 2-D convolution layers with leaky ReLU (leakiness 0.2) and a 2-D upscale layer. In analogy to the original model, new layer-blocks were added to both the generator and the discriminator incrementally, while existing layers remained trainable. Additional layers were faded in, doubling the resolution of the generator and the discriminator but allowing for a smooth transition in the process. The addition of minibatch standard deviation into the discriminator and pixel-wise feature vector normalization in the generator were also implemented as originally described [To generate realistic images of long and short axis cardiac MRI frames, two progressive GANs were built as described in detail by Karras et al. 2018 . The orinsorFlow . Adaptatwww.ClinicalTrials.gov; unique identifier, NCT00266188). Inclusion criteria were absence of an implantable cardioverter-defibrillator and a patient age at the time of MRI > 8 years. The MRIs were collected at 14 German centers using a pre-defined protocol. Further details on the MRI protocol as well as additional exclusion criteria have been reported by the study consortium previously [Overall, 6400 4-chamber long axis (LAX) MRI frames from 279 patients and 7015 2-chamber short axis (SAX) images from 303 patients  were used for training the PG-GANs. All patients had a diagnosis of congenital heart disease with a status post repair for tetralogy of Fallot - a form of cyanotic congenital heart disease which accounts for approximately 12% of adults with congenital heart disease under regular follow at specialized centers [eviously –12. All All study participants  gave written informed consent before the baseline MRI investigation and study inclusion, which were approved by the Ethics Committee . In addition, research within the framework of the National Register for Congenital Heart Defects is covered by Ethics Approval by the Charité Ethics Committee, Berlin, Germany.p-values are reported.To evaluate the quality of the synthetic PG-GAN network frames, a random selection of 200 PG-GAN derived, and 200 original MRI frames were presented to human investigators head to head. The operator was presented with two images in a random order arrangement  and was required to determine which image was of GAN origin. The number of correct answers is reported as a percentage of total pairs presented, representing a measure of the discriminatory ability of human operators. To test whether experienced cardiac MRI specialists may have a superior ability to recognize synthetic images compared to cardiologists not directly involved in cardiac MRI reporting the results were compared using the Fisher exact test and To identify similarities between the generated PG-GAN frames and natural MRI frames available in the dataset a multi-scale statistical similarity index (sliced Wasserstein) distance approach at various resolutions is adapted . To thisFor segmentation of cardiac chambers, a U-Net setup was employed . The netIn total, two pairs of U-Net models were produced. One pair (including a SAX and a LAX model) based on a training data set using original patient MRI frames and a second pair trained on a random sample of frames produced by the PG-GAN model.To assess performance differences between U-Nets trained on synthetic PG-GAN derived data from those trained directly on patient MRI frames, the Dice metric and percentage area variability  were assessed for both models compared to ground-truth masks produced manually on a set of frames from patients not used for model training. Details on the calculation of Dice metrics and percentage area variability have been reported in detail in the literature by us and others previously , 4. Brie2 to 1282 and 2562 pixels. All GANs trained as expected and yielded visually acceptable synthetic MRI frames. Based on the results of the multiscale statistical similarity between PG-GAN generated frames and actual patient MRI frames, Fig. The first aim of the study was to test the feasibility of training the PG-GANs on the data available. We found no evidence of training instability in our models. Figure p-value < 0.001 for both short and long axis frames). Overall, however, none of the PG-GAN derived frames was labelled as anatomically implausible by the reviewers.Presenting 200 pairs of randomly positioned images  to study subjects with various grades of experience showed that 68.7 and 85.3% of the short axis images generated by the PG GAN were recognized as such by experienced cardiologists  and CMR experts (RR and SO), respectively. For 4-chamber views the correct recognition rate was 72.2% for non-CMR specialists and 88.0% for the experienced CMR readers. The trained and experienced CMR-experts performed significantly better compared to the cardiologists not directly involved in cardiac MRI reporting (The performance of trained U-Net models was tested on a set of 100 MRI frames from patients not used for PG-GAN or U-Net training and the percentage variation as well as the Dice metric was quantified. Comparing segmentation networks (U-Net) trained on actual patient MRIs and those trained entirely on PG-GAN derived data showed only slight superiority in performance for the former. As shown in Table The current study demonstrates the use of GANs to generate synthetic cardiac MRI images of patients with congenital heart disease. As data quantity and quality are critical for training deep learning models, the proposed method should be useful to assist training downstream deep learning networks in the setting of rare medical conditions. The synthetic GAN images are not subject to data anonymity issues or privacy concerns and can be shared freely between medical institutions, allowing accelerated development of new diagnostic tools.Artificial intelligence and deep learning solutions are revolutionizing interpretation of medical images. It is hoped that these technologies will not only augment efficiency but also improve diagnostic quality. Most current implementations use image classifiers or segmentation networks to this end , 15. ThePrevious applications of GAN models to medical imaging include increasing the resolution of cardiac MRI images , de-aliaWe have not investigated whether dynamic series of images mimicking cardiac motion could be generated by adjusting the input vector. It has been reported that manipulating the latent vector can result is meaningful transitions between images. Due to the limited resolution and the fact that visually especially the blood pool is not perfectly modelled by the generator, the images created are partly distinguishable from actual patient frames. It is hoped that by optimizing the GAN network further, increasing computing power and potentially combining the PG-GAN setup with other downstream deep learning networks the image quality can be further improved. We can only speculate on the reasons why no evidence of training instability was evident for the PG-GAN in our study. This may be potentially related to the design of the PG-GAN making it less prone to such effects compared to conventional GAN setups .The current study illustrates the utility of PG-GANs for generating large amounts of realistically looking cardiac MRI images even in rare cardiac conditions. The generated images are not subject to data anonymity and privacy concerns and can be shared freely between institutions. As training supervised deep learning segmentation networks on this synthetic data yielded similar results compared to direct training on original patient data, we contend that this approach may find applications for training segmentation networks or improving accuracy of existing models by additional training on PG-GAN generated images.


To understand how Fear of falling (FoF) alters mobility, FoF was evaluated annually in 243 older adults (median age=70) with varying degrees of visual field loss from glaucoma, and Rasch-analyzed FoF scores associated with the likelihood of falling in the following year  and changes in physical activity . At lower FoF levels, each one-unit worsening in FoF was associated with a 2.73-fold higher odds of reporting a fall in the next year  but not with average daily steps taken (p = 0.44). At higher FoF levels, inter-year changes in FoF were not significantly associated with a fall in the next year (p = 0.78); but were associated with 407 fewer daily steps taken per one-unit change in FoF . FoF is an important driver of mobility; the specific aspects of mobility affected varies by the degree of FoF.


We propose the use of a machine learning algorithm to improve possible COVID-19 case identification more quickly using a mobile phone–based web survey. This method could reduce the spread of the virus in susceptible populations under quarantine. This is especially true for viral diseases that are easily and readily transmissible and have asymptomatic infectivity periods. The novel coronavirus (SARS-CoV-2) described in December 2019 (COVID-19) has resulted in major quarantines to prevent further spread, including major cities, villages, and public areas throughout China and across the globe.To reduce the time needed to identify a person under investigation (PUI) for COVID-19 and their rapid isolation, we propose to collect a basic travel history along with the more common signs and symptoms using a mobile phone–based online survey. Such data can be used in the preliminary screening and early identification of possible COVID-19 cases. Thousands of data points can be processed through an artificial intelligence (AI) framework that can evaluate individuals and stratify them into no risk, minimal risk, moderate risk, and high risk groups. The high-risk cases identified can then be quarantined earlier, thus decreasing the chance of spreading the virus  alerts or Health Check Recommended for Coronavirus (MHCRC) alerts (for possible identification and assessment) and No Health Check Recommended for Coronavirus (NCRC) alerts (for nonidentified respondents). These procedures are explained in steps 3 and 4 in Appendix 11 Several studies have promoted disease detection through AI models.15 The use of mobile phones19 and web-based portals21 have been tested successfully in health-related data collection. In addition, our proposed algorithm can be easily extended to identify individuals who might have any mild symptoms and signs. However, such techniques must be applied in a timely way for relevant and rapid results. Apart from cost-effectiveness, our proposed modeling method could greatly assist in identifying and controlling COVID-19 in populations under quarantine due to the spread of SARS-CoV-2.Applications of AI and deep learning can be useful tools in assisting diagnoses and decision making in treatment.