Here, we review the available tools to probe these questions in vivo within vertebrate model systems. We discuss how these tools have been used to date and consider future avenues of research.Autophagy is an intracellular clearance pathway that delivers cytoplasmic contents to the lysosome for degradation. It plays a critical role in maintaining protein homeostasis and providing nutrients under conditions where the cell is starved. It also helps to remove damaged organelles and misfolded or aggregated proteins. Thus, it is not surprising that defects in this pathway are associated with a variety of pathological conditions, such as neurodegeneration, cancer and infection. Pharmacological upregulation of autophagy is considered a promising therapeutic strategy for the treatment of neurodegenerative and infectious diseases. Studies in knockout mice have demonstrated that autophagy is essential for nervous system function, and data from invertebrate and vertebrate models suggest that the efficiency of autophagic processes generally declines with age. However, much of our understanding of the intracellular regulation of autophagy comes from  Most of these genes have vertebrate homologues that are named after their yeast counterparts. Interestingly, many of the yeast genes have more than one vertebrate homologue [In the initial steps of autophagy, a double-membraned, cup-shaped precursor  forms within the cytoplasm. The phagophore expands, engulfing substrates as it does so, and eventually the edges fuse to form a double-membraned vesicle, the autophagosome. This traffics along microtubules to the lysosome, with which it fuses resulting in the degradation of the autophagic contents . Autophae family . The atgomologue ,4, whichin vivo, it is necessary to label and visualize the phagophores and autophagosomes. However, few proteins are uniquely associated with autophagic vesicles and their precursors, with only one protein family (including LC3-II) known to label autophagic structures both prior to and after fusion with the lysosome. LC3 is one of several vertebrate homologues of ATG8. Mammalian cells have six ATG8 orthologues; the MAP1-LC3 (LC3) and GABARAP subfamilies (microtubule-associated protein 1 light chain 3 and GABA(A) receptor-associated protein families respectively), while zebrafish have eight (To follow this process ve eight . During ve eight . ATG4 isSince lipidated ATG8 proteins (such as LC3-II) are the only proteins which associate with pre-autophagosomal structures, autophagosomes and autolysosomes, they are widely accepted as being the best marker to distinguish autophagic vesicles from other cellular membranes ,6. Measuin vivo analysis. Similarly, to understand the role of autophagy in the pathogenesis of disease, it is important to study this process in the whole animal to investigate tissue-specific changes in flux, the difference in flux between young and old animals, and cell-autonomous versus non-cell-autonomous effects. In recent years, various transgenic reporters have been developed which may be useful to improve our understanding of autophagy in vivo. Together with advances in imaging such as CLEM (correlated light and electron microscopy) and lightsheet microscopy, we now have the tools to interrogate this process in living vertebrate animals. Although such imaging is in its infancy, here we review the available tools and highlight the future possibilities for studying autophagy in vivo.The majority of studies using these biochemical or fluorescent detection methods have only provided a snapshot of autophagic activity within a single tissue at a single time. Many studies have reported that basal levels of autophagy differ between different tissues, and we do not fully understand how these different rates are affected by pharmacological upregulation or disease pathology. Since upregulation of autophagy is considered to be a promising therapeutic strategy for the treatment of a range of disorders, including neurodegeneration, infectious disease and cancer ,12, it i2.in vivo in vertebrates and provided novel insights about its regulation in both physiological and pathological conditions. The overexpression of Atg8 homologues fused with GFP had been previously described in other species, such as yeast, Caenorhabditis elegans, Dictyostelium discoideum, Drosophila melanogaster and Arabidopsis thaliana [The use of a fusion construct comprising green fluorescent protein (GFP) tagged to LC3 was the first approach to examine autophagy thaliana –16.GFP-LC3, like endogenous LC3, becomes conjugated to the phagophore and remains on the membrane after the complete closure of the autophagosome. Autophagosomes labelled with GFP-LC3 are evident as puncta or ring-like structures by fluorescence microscopy –19. GFP-ex vivo real-time observations of GFP-LC3 positive autophagic structures [The generation of transgenic mice expressing GFP-LC3 under the control of a ubiquitous promoter has allowed the post-mortem examination of GFP-LC3 localization by high-resolution microscopy and in almost all tissues . The oveructures ,25.An important consideration in the analysis of such reporter lines is to determine whether the fluorescent protein is a faithful reporter of the endogenous protein. Kuma and colleagues  demonstrin vivo since they are amenable to most forms of fluorescent imaging due to their size and transparency. Furthermore, analysis is not restricted to embryonic stages, as their rapid development permits the analysis of functioning organs in larvae at free-swimming stages. Zebrafish have eight homologues of Atg8  promoter [et al. detected autophagy at approximately 15 h.p.f., evidenced by the presence of autophagosomes visualized as GFP-LC3 puncta in the CMV:GFP-LC3 transgenic reporter line [in vivo by adding LysoTracker to the embryo medium [Zebrafish are potentially a more tractable model to study autophagy  of Atg8  with higpromoter . Both trpromoter . Howeverter line . The benter line . The fuso medium . A dramao medium .in vivo imaging in this GFP-LC3 zebrafish line, for example, to study the role of autophagy in blastema formation and regeneration following fin amputation [Hs1bp3, a phosphoinositide-binding PX domain-containing protein, increased the number of GFP-LC3 puncta visualized directly along the trunk of morphants compared with control embryos, and this increase was greater after chloroquine treatment, suggesting increased autophagic flux in vivo [spns1, a putative lysosomal H+-carbohydrate transporter involved in senescence and in the late stages of the autophagy/lysosome pathway. Morpholino knockdown of spns1 resulted in an accumulation of GFP puncta visualized by confocal microscopy in live embryos and was also observed in spns1 mutants [in vivo in these examples, these studies relied on analysis of single-time-point images to assess autophagosome number and did not exploit the full potential of studying these events in the living organism.Several studies have exploited the ability to perform putation , or in tputation . The abiputation  in zebra in vivo . A simil mutants . Careful mutants . Althougin vivo observations has been in the study of the innate immune response [in vivo light microscopy and ex vivo electron microscopy imaging opens new directions for studying the role of autophagy in infectious diseases.One example of the power of using zebrafish for response . Transgeresponse . The comShigella have been used to study the process of bacterial clearance in vivo. Engulfed bacteria were observed to be sequestered in GFP-positive autophagosomes [Mycobacterium marinum infection in zebrafish, the bacteria were frequently found associated with GFP-LC3-positive vesicles, and these associations were particularly abundant in leucocytes. By correlative light and electron microscopy, the precise location of intracellular bacteria could be elucidated  by determining the ultrastructure of GFP-LC3-positive structures [Transgenic GFP-LC3 zebrafish infected with agosomes , a findiructures .These studies highlight the importance of verifying the properties of the LC3-labelled structures. Although LC3 is the best-established marker to identify autophagosomes, it can also be associated with single membranes on phagosomes within macrophages and other phagocytic cell types where it functions in a process called LC3-associated phagocytosis (LAP) . In thisin vivo or in primary cultures, there are important caveats to consider. GFP-LC3 was initially described to localize exclusively on autophagic membranes. However, GFP-LC3 protein can aggregate in an autophagy-independent manner without being conjugated to PE leading to misinterpretation of the results, especially during transient expression of the transgene [Although these transgenic reporters are powerful tools for studying autophagic processes ransgene . For exaransgene , and as ransgene .In vivo, chloroquine or ammonium chloride treatments may be employed to reduce vacuolar acidification, although these treatments are likely to be toxic at saturating concentrations and therefore, at best, can only be considered to be a partial lysosomal block. Such an approach has been used to measure cardiac autophagic flux in vivo in mCherry-LC3 transgenic mice [Since fluorescently tagged-LC3 labels the surface of all autophagic structures, from the formation of the phagophore to the autolysosome, no conclusions can be made about autophagy flux or dynamics by simply measuring the number of puncta. An increase in GFP-LC3 puncta may occur as a result of an increase in autophagosome formation but also could be the consequence of an impairment of autolysosome formation . In cellnic mice . A clearnic mice ,10 or winic mice . If the nic mice .A further important consideration is the degradation of GFP-LC3, which can generate free GFP fragments that may accumulate depending on the acidity of the lysosomes and degradative capacity of lysosomal compartments . In cell3.in vitro [in vitro to study the mechanisms regulating the maturation of autophagosomes and the fusion to lysosomes in the degradative process. Owing to this pH-dependent quenching of the GFP-LC3 fluorescence, only mRFP-LC3 can be detected in autolysosomes (i.e. these appear red only), whereas autophagosomes can be visualized by both fluorophores (i.e. these appear yellow)  .Figure in vivo mouse model expressing mRFP-GFP-LC3 was generated by Li and colleagues in 2014 [in vivo under stress conditions, such as starvation and disease. In these RFP-EGFP-LC3 mice, autophagic vacuoles were visualized as RFP- and EGFP-positive puncta, similar to in vitro observations in cells expressing the same construct. The model was first used to evaluate the role of autophagy in ischemia-reperfusion injury in the kidney using primary cell culture. In addition, primary cortical neurons from an independently generated mouse line have been used to investigate the interplay between chaperone proteins and autophagy [in vivo and increase autophagic flux in larvae zebrafish [synJ1-deficient zebrafish [in vivo in synJ1-deficient zebrafish, resulting in the accumulation of autophagosomes. Modulation of the PIP2 regulator, Arf6, by expressing a constitutively active mutant of Arf6, rescued the defects seen in cones of synJ1-deficient fish. These results suggest that Arf6a modulates positively the levels of PIP2, a substrate for SynJ1, and hence that both Arf6 and SynJ1 play a role in the same pathway to regulate autophagy in cone photoreceptors [in vivo.The first  in 2014 . Expressutophagy . Tandem ebrafish . Stable ebrafish –50. Liveeceptors . These sHowever, as with the analysis of GFP-tagged LC3, there are additional factors to be considered when using this tandem red-green fluorescent LC3 fusion protein. First, the red and green fluorescence from unconjugated LC3 exists in the cytosol of all cells. When autophagic flux is low, this background is higher. As the LC3 becomes conjugated and more puncta appear, the background fluorescence decreases . IdentifThe development of a new generation of fluorescent probes may help with some of these difficulties. A new dual-fluorescence probe was recently generated by the Mizushima group comprising GFP-LC3-RFP-LC3DG . The expMice and zebrafish expressing GFP-LC3-RFP-LC3DG were developed to evaluate autophagic flux in different tissues and validated to confirm that the reporter responds appropriately to drug-induced autophagy upregulation . Althougin vivo. Both mTagRFP and mWasabi-LC3 are much brighter than mRFP/mCherry and EGFP fluorescence. mWasabi is also more acid-sensitive than EGFP and hence more easily quenched in the acidic environment of autolysosomes [in vivo models have been created using this construct. Similarly Rosella, a tandem reporter of the fast maturing red fluorescent protein dsRed.T3 with GFP, has been successfully used to track labelled cytosolic proteins, mitochondria or the nucleus to the autophagic vacuole in yeast [in vivo, although no further data have been published to date.The use of other fluorescent tandem reporters with different pH-sensitivities, such as mWasabi (pKa at 6.5 versus pKa 5.9 for GFP) leads to a faster loss of fluorescence in the autolysosomes  and may ysosomes . In addiysosomes . These cysosomes . Howeverin yeast ,55. Rosein yeast . These a4.In contrast to the use of dual fluorophores to label LC3, new approaches have been developed in recent years which allow one to measure autophagic flux using a single fluorophore. dKeima, a coral-derived fluorophore, has a bimodal excitation spectrum (438 and 550 nm) with an emission spectrum peak at 620 nm . The difIn addition, Keima can be targeted to either proteins or organelles. For example, Keima targeted to mitochondria (Mito-Keima) has been used to evaluate mitochondrial autophagy (mitophagy) in cell culture . Mito-Ke5.per se but to measure the clearance of autophagic substrates. Tau is a microtubule-associated protein which is known to be an autophagy substrate [in vivo (An alternative approach is not to measure autophagosomes ubstrate . Zebrafiubstrate  and alsoubstrate . Such st[in vivo . This ap6.in vivo. Indeed, such approaches have been applied to the in vivo trafficking of mitochondria [in vivo in such disease states will provide valuable information on which steps of the pathway can be manipulated for therapeutic benefit.To date, much of our understanding of autophagosome formation, trafficking and degradation have come from work in cell lines or in primary cell culture. The elegant work of the Holzbaur group in studying trafficking in primary neurons has revealed important aspects of autophagosome trafficking and biogenesis –64. Givechondria –67. Cavechondria . In addichondria ,70 sugge


Moreover, it is estimated that about one-third of the cases of ERinvolve young male afro-descendants with low physical conditioning and extremedehydration, occurring during summer military training courses.5Additionally, the cutaneous microcirculation is now considered as an accessible andrepresentative vascular bed for the assessment of systemic microcirculatoryreactivity and density.5Considering that ER has already been shown to be related to decreased systemicendothelium-dependent vasodilation in the systemic circulation in the experimentalsetting,6 it is reasonableto speculate that ER is also associated with significant systemic microcirculatorydysfunction. Moreover, there is no description in the specialized literature of theassociation of ER with microvascular endothelial function is humans. To the best ofour knowledge, this is the first report on the detrimental outcomes of ER onendothelium-dependent systemic microvascular reactivity in human beings.The assessment of systemic endothelial microvascular reactivity has already beenproven to be essential in the investigation of the pathophysiology of cardiovascularand metabolic diseases.This case report is part of an observational research study without anyintervention investigating the impact of special military training courses oncytokine profile and microvascular reactivity and the risk of developing ER inBrazilian Air Force military personnel who fully completed a five-week trainingperiod. This study was performed in accordance with the Declaration of Helsinkiof 1975 (revised in 2013). The case report was approved by the InstitutionalReview Board (IRB) of the National Institute of Cardiology of the Ministry ofHealth, Rio de Janeiro, Brazil under protocol number # CAAE49792515.6.0000.5272. The subject read and signed the informed consent formapproved by the IRB. The patient was encouraged to share his perception of theclinical event that occurred during special military training with hiscolleagues.2maxof 54.66 ml/kg/min). The patient presented no significant information on pastpersonal or familial past medical history, including ER, and did not use anymedication nor oral supplements during the period of military training. Wetested the patient’s blood using hemoglobin electrophoresis, which showed theabsence of hemoglobin S. Thus, we can consider that the patient did not presentsickle-cell trait. He was diagnosed with ER on the second day of militarytraining. He had performed running exercise in a combat uniform and transportinga 15 Kg kit including shield and gun, with a limited intake of water, and afterbeing exposed to tear and pepper gases for 45 minutes. On the day before, he hadrun 2,400 meters in 12 minutes, and on both occasions, the running exerciseswere performed in warm (32ºC) and humid (86% relative humidity) conditionstypical of the summer season in Rio de Janeiro, Brazil.The patient was a 21-year-old and physically fit afro-descendant Brazilian AirForce military trainee, who spontaneously applied for riot control militarytraining. The military was considered to have excellent aerobic endurance forhis age range (20-29 years of age), using the Cooper run test , dark-coloredurine, lower limb edema and gait difficulty.7 During the post-occlusivereactive hyperemia (PORH) test, arterial occlusion was performed withsupra-systolic pressure using a sphygmomanometer for 3 min. Following therelease of pressure, the maximum flux was measured. Measurements of skin bloodflow were divided by the mean arterial pressure to yield the cutaneous vascularconductance (CVC) in APU/mmHg. The capillary density, defined as the number ofperfused capillaries per mm2 of skin area, was assessed byhigh-resolution intra-vital color microscopy . Thedorsum of the non-dominant middle phalanx was used for image acquisition. Imageswere acquired and saved for posterior off-line analysis using a semi-automaticintegrated system . The mean capillarydensity was calculated as the arithmetic mean of the number of visible  capillaries in three contiguous microscopic fields of 1mm2 each, as described previously.8The evaluations of microvascular reactivity were performed one day before thebeginning of military training and one day after hospital discharge, both in themorning between 8 and 12 AM and after a 12-hour fast. Microcirculatory testswere performed after a 20-minute rest in the supine position in atemperature-controlled room (23 ± 1°C). Microvascular reactivity wasevaluated using a laser speckle contrast imaging system  in combination with skin iontophoresisof acetylcholine (ACh) for noninvasive and continuous measurement of cutaneousmicrovascular perfusion changes .9 and to maintain adequate urine output. The plasmacytokine analysis is presented in Laboratory testing of the subject is shown in Of note, one day after hospital discharge, systemic endothelium-dependentmicrovascular reactivity was severely impaired. These results can be observedboth in the pharmacological (acetylcholine-induced) and physiological(PORH-induced) microvascular vasodilator responses . Finally9After six days of hospitalization and two additional weeks of rest at home, thesubject had fully recovered and was able to return to his normal activities.Prognosis of ER is generally good if full recovery occurs.This case report demonstrates that ER after strenuous physical exercise, performed inadverse environmental conditions and with limited water access, can be paralleled bypersistent systemic microvascular dysfunction, detectable up to 1 week after thebeginning of symptoms and even after normalization of muscle enzymes and completeresolution of renal dysfunction. In fact, a marked reduction of theendothelium-dependent systemic microvascular reactivity induced by bothpharmacological (acetylcholine) and physiological  stimuli was observed one week after the diagnosis of ER. Moreover,skin capillary function, measured as post-ischemic capillary recruitment, was alsosignificantly impaired, suggesting a loss of vasodilatory reserve and autoregulatorycapacity and the existence of severe microvascular endothelial dysfunction.10 On theother hand, strenuous exercise increases oxidative metabolism and produces apro-oxidant environment, and consequent endothelial dysfunction,11 while regular and moderatephysical activity promotes an antioxidant state and preserves endothelialfunction.10 Thus,high-intensity exercise training in previously untrained individuals, such as thatof special military training, could be detrimental to the promotion of vascularhealth.Exercise training of moderate intensity is well-known to induce beneficial effects onthe occurrence of cardiovascular diseases through the preservation of vascularendothelial function.12 Eventhough we did not obtain cytokine plasma levels on the day of hospital admission, weobserved an increase both in pro-inflammatory  and anti-inflammatory cytokines after hospital discharge, compared to values obtainedimmediately before military training. The plasma levels of muscle-derived IL-6,which is considered to be a key mediator released during exhaustiveexercise,13 usually startsto increase within the first hour of prolonged exercise and continues to risedepending on the duration of the exercise.13 In fact, it is well established that the elevation ofpro-inflammatory cytokines at the time of muscle injury influences the synthesis ofacute phase proteins and the expression of anti-inflammatory cytokines, as aphysiological response to offset the inflammatory response.13 Moreover, it has been consistentlyshown that there is a rise in anti-inflammatory cytokines, IL-1ra and IL-10,following endurance exercise lasting longer than 2 h.13 Yet, the plasma levels of TNF-a were not increasedone week after ER, suggesting that this cytokine has a different kinetics profile,compared with the aforementioned pro-inflammatory cytokines. Plasma levels of INF-gdid not show important variations in the present case. Actually, most studies in theliterature failed to demonstrate a significant rise in plasma IFN-g afterexercise.13The microvascular alterations described above were simultaneous with alterations ofthe profile of plasma cytokines. Nevertheless, it is clearly not possible toestablish a link between both phenomena in the present case report, since othermetabolic changes could also be involved in the initiation of microvasculardysfunction. It is well known that ER is acutely associated with the production ofpro-inflammatory cytokines.Strengths and limitations of our experimental approach should be considered. The useof laser-based skin microvascular flowmetry, as well as the evaluation of the levelsof plasma cytokines, is not yet possible in clinical practice. One major strength ofthe present case report is the demonstration of persistent systemic endothelialmicrovascular dysfunction and systemic inflammatory reaction after clinical andlaboratory regression of ER. The long-lasting vascular inflammatory process observedin the present clinical case could have implications in the prognosis of patientspresenting with ER. Nevertheless, it was impossible to retest these parameters inlonger time intervals in the present case.ER may be accompanied by systemic microvascular dysfunction even after the resolutionof symptoms and normalization of conventional laboratory tests. The microcirculatorydisturbance is concurrent with alterations of plasma levels of both pro- andanti-inflammatory cytokines. Accordingly, ER should always be considered in theclinical scenario of muscle pain and disability, fever and dark urine after heavyexercise, including that performed for professional reasons. Besides that, the casereport shows that ER may be associated with other complex and potentially severeconditions, which are microvascular dysfunction and systemic inflammation. These arenovel findings which we would like to add to the clinicians´ reasoning. If theevaluation of microvascular function is made available clinically, it may be anotherpotentially interesting evaluation to be performed in patients with ER.Nevertheless, more studies are needed to clarify the association betweenmicrovascular dysfunction and ER, as well as its clinical implications.


Osteoid osteoma (OO) apparent in the intra-articular region of the elbow is very rare. Although computed tomography-guided excision and radiofrequency ablation have been recognized as useful treatment options, arthroscopic excision has recently received focus as an alternative strategy for lesions close to neurovascular structures or intra- and juxta-articular lesions. We herein report a 17-year-old female who underwent arthroscopic treatment for intra-articular OO located at the olecranon/coronoid fossa. Her symptoms included elbow pain that was exacerbated at night and contracture of elbow flexion-extension, and she was diagnosed with intra-articular OO after 12 months of symptomatic history. Arthroscopically, thorough synovectomy for both the anterior and posterior aspects of the joint enabled definition of the tumor margin with hyperemic alteration and excision of the lesion as an en bloc specimen. At the 12-month follow-up, the patient had no recurrence of elbow limitation or pain. This case report describes the advantages of arthroscopic treatment, including a low-invasive approach and easy accessibility to the whole intra-articular space, which can provide clear visualization of the tumorous lesion. Osteoid osteoma (OO) is a benign bone tumor generally apparent in the first to third decades. It has been reported that approximately 50% of OOs occur in the femur and tibia with the diaphyseal shaft or toward the metaphysis, with the elbow joint very rarely affected . AlthougIn addition to tumor excision through the open approach, computed tomography- (CT-) guided excision and radiofrequency ablation have been recognized as viable treatment options , 5. ArthA 17-year-old right-handed female presented in our outpatient clinic with a 12-month history of pain and limited motion of her left elbow. The symptoms included mild pain during the day time that became increasingly severe at night. A physical examination revealed elbow contracture with a maximal extension of -20° and flexion of 125° Figures . The patUnder a diagnosis of intra-articular OO at the elbow, the patient underwent arthroscopic treatment to excise the lesion. A routine anteromedial portal was created 1 cm anterior and 2 cm proximal to the medial epicondyle, and the scope was introduced. Severe proliferation of synovial tissues was observed throughout the joint space . A 4 mm We herein reported a case of arthroscopic excision for intra-articular OO of the elbow joint. The advantages of this technique include a low-invasive approach and easy accessibility to both the anterior and posterior aspects of the tumorous lesion. Under the classical open approach, in contrast, a relatively long incision is required, especially for determining the accurate degree of excision; consequently, this approach carries a risk of scar formation, residual pain, and contracture. Arthroscopically, thorough synovectomy enabled us to identify the tumorous lesion with hyperemic alteration. Arthroscopic maneuvers such as capsular release and debridement can be added to improve the joint motion. This patient did not undergo such release since the loss of the range of motion was considered mild. Bhatia  suggesteTo our knowledge, 5 case reports and 1 case series have described a total of 17 patients who underwent arthroscopic treatment for elbow OOs , 6–10; aThe difficulty in diagnosing OO based on imaging findings has been mentioned in cases with severe inflammatory changes, such as a prominent periosteal reaction, exaggerated synovial hypertrophy, and joint effusion with extensive bone marrow and soft tissue edema . In addiWe herein report a 17-year-old female who underwent arthroscopic treatment for intra-articular OO located at the elbow. This case report describes the advantages of arthroscopic treatment, including a low-invasive approach and easy accessibility to the whole intra-articular space, which can provide clear visualization of the tumorous lesion.


Statin-induced necrotizing autoimmune myopathy (SINAM) is a rare side effect of statin use which manifests as progressive muscle weakness. Because statins are a widely prescribed medication for coronary artery disease, hyperlipidemia, and many other diseases, many patients are at risk of developing SINAM or one of the many other statin-induced myopathies. Due to identification of an antibody specific to this disease, we were able to diagnose SINAM in a patient whose symptoms had progressed to the extent that they were debilitating. Our case describes SINAM in a patient undergoing treatment with a statin for an extended period of time, diagnosis of the disease process, treatment, and resolution of symptoms. Statins are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors and are some of the most widely prescribed medications for treating atherosclerotic disease in order to reduce the morbidity and mortality for both coronary and cerebral vascular diseases. Most statin-induced myotoxicity is self-limiting with myalgia and transient elevation of muscle enzymes followed by complete recovery within weeks to months upon the discontinuation of the statin. Statin-induced necrotizing autoimmune myopathy (SINAM) is a rare side effect reported in about 2-3 out of 100,000 people who use statins. We present a case of SINAM manifested in a patient with over ten years of simvastatin use who was recently switched to atorvastatin.A 66-year-old female with a medical history of hypertension, hyperlipidemia, diabetes mellitus type 2, and obesity presented with hip pain after a fall. Radiograph of the hip was negative for dislocation or fracture. On physical exam, she had proximal muscle weakness greater in the lower extremities, and the rest of her examination was negative for skin rashes, lymphadenopathy, joints inflammation, and pedal edema. Her EKG showed normal sinus rhythm with no ST-T changes. Blood work showed elevated CPK (9767), CKMB 101.50), and aldolase (60) levels and LFT derangement (AST-302 and ALT404)  have been one of the most widely prescribed medications, and treatment with statins has effectively reduced the cardiovascular morbidity and mortality. Evidence presented by the American College of Cardiology/American Heart Association Blood Cholesterol Treatment Task Force solidified statins as the only cholesterol-lowering agent that showed mortality benefit. The new guidelines have expanded the statin-eligible population in the United States from 43 to 56 million, a total increase of 13 million. This number further approaches 1 billion worldwide , 8. A stAs statins are an extensively used class of drugs, clinicians should be aware of SINAM and patients should be promptly evaluated at the earliest signs of inflammatory muscle disease which mimics polymyositis. It is important to differentiate statin-associated inflammatory myopathies from other self-limited myopathies, because the disease will not subside following discontinuation of the statin . Muscle


Development of eco-friendly polymer foams is an urgent research topic because of the serious environmental pollution caused by trash heaps and the time-release of harmful gases. Polymer PVC foam using azodicarbonamide as a chemical foaming agent has been highly requested for further improvement due to the residual ammonia gas that continuously leaks out. Here, we demonstrate an effective and costless process for the reduction of releasing ammonia from PVC foams using the overcoat technology of deodorants. We have selected four candidate materials, gelite, zeolite, terra alba, and fumed silica as original materials for the deodorant of ammonia, and they showed an ammonia deodorization rate (ADR) of over 80% without any treatment except the fumed silica. When we over-coated the UV-curing agent mixed deodorants on the PVC foams (thickness ~300 µm), the ADR of the terra alba and zeolite complex foams was remarkably higher than 90%, however, the specific gravity and chromaticity were not changed within 20%. This indicates that our developed process using deodorant layer for ammonia reduction has a high potential for the production of eco-friendly polymer foams. Polymer foams have many useful attributes such as flexibility, low density, impact resistance, and heat transfer capacity compared with conventional rigid polymers . These fSo far, various attempts have been made to remove unregulated ammonia, formamide, and formaldehyde leaking from the polymer foams ,22,23. FIn this study, we have probed organic-inorganic hybrid deodorants, which reduce the releasing ammonia gas captured inside the foam. The hybrid deodorants are composed of UV-curable resin and inorganic porous materials such as gelite, zeolite, terra alba, and fumed silica. The porous material’s deodorization rate is preliminarily tested according to the temperature and deodorizing time. The hybrid deodorants are over-coated on the PVC foam body and sequentially UV-cured. The specific gravity and chromaticity of the final foams have been controlled to be changed within 20% compared to the reference. The hybrid deodorants tested the deodorization of ammonia. PVC sol , ADCA chemical foaming agents (Dongin Semichem Co. Ltd.), UV-curing agents , gelite , zeolite , terra alba , hydrophilic fumed silica, hydrophobic fumed silica hydrochloric acid (HCl) . Gelite and HCl were mixed by a mechanical stirrer at 100 °C for 1.5 h. Then the HCl-treated gelite was thoroughly washed with water and vacuum dried for 24 h.First, PVC sol and 3.4 parts ADCA foaming agent were mixed by a mechanical stirrer. Then the PVC mixture was bar-coated on the release paper (the wet thickness ≈ 1 mm). The bar-coated PVC mixture was foamed in a convection oven at 230 °C for 2 min 10 s [The surface and cross section morphology of the deodorants and the foams over-coated by the deodorants were measured by scanning electron microscope (SEM) (JEOL/JSM-6701f). The ammonia deodorization rate (ADR) of deodorants was tested by FITI Testing & Research Institute. The specific gravity of the foams over-coated by the deodorants was measured by a hydrometer according to the ASTM-D792 standard test method. The specific gravity of deodorants was calculated from the weight and the volume obtained by using a measuring cylinder. Chromaticity of the foams over-coated by the deodorants was measured by a spectroeye according to the ASTM-E1164 standard test method. Ammonia emission concentration of the foams over-coated by the deodorants was tested by Korea Polymer Testing & Research Institute. The brief test process is: The foams were cut into 2 pieces of 10 cm × 10 cm, respectively. Then each 2 pieces of the cut foams were put in a 5 L Tedlar bag filled with nitrogen gas and the Tedlar bags were sealed. The sealed Tedlar bags were placed in a convection oven for 1 h at 25 °C or 80 °C. After 1 h, ammonia emission concentration of the foams over-coated by the deodorants was measured by an ammonia detector tube.In this work, we used six different deodorants, gelite, HCl-gelite, zeolite, terra alba, and hydrophobic/hydrophilic fumed silica, which have been recognized by a good deodorizing ability ,28,30. FFor the stable and durable over-coating layer, we mixed the deodorants with a UV-curing agent. The use of a UV-curing agent is helpful to avoid the sintering process that induces the decrease of specific surface area and porosity. After over-coating the deodorants, the change of specific gravity and chromaticity of the PVC foams was measured and the results are shown in According to the process as schematically shown in In this work, we have developed deodorants of ammonia gas applying for the production of eco-friendly foams using nanoporous materials such as gelite, zeolite and terra alba. The deodorant layers over-coated on PVC foams worked well for the reduction of time-releasing ammonia. Based on the consideration of all results, although the deodorant layers using terra alba and zeolite showed a high ARD value over than 90%, the terra alba showed the best deodorizing performance of ammonia on the PVC foam. We believe that this work provides a promising solution to reduce the time-released hazardous gases from the synthetic polymer foams used in everyday life.


TiO2-NPs are able to carry antibiotics to target cells enhancing the antimicrobial efficiency; so that these nanoparticles are generally used in antibiotic capsules, like lincomycin, added as a dye. Lincomycin is usually used to treat pregnancy bacterial vaginosis and its combination with TiO2-NPs arises questions on the potential effects on fetus health. This study investigated the potential impact of TiO2-NPs and lincomycin co-exposure on human amniocytes in vitro. Cytotoxicity was evaluated with trypan blue vitality test, while genotoxic damage was performed by Comet Test, Diffusion Assay and RAPD-PCR for 48 and 72 exposure hours. Lincomycin exposure produced no genotoxic effects on amniotic cells, instead, the TiO2-NPs exposure induced genotoxicity. TiO2-NPs and lincomycin co-exposure caused significant increase of DNA fragmentation, apoptosis and DNA damage in amniocytes starting from 48 exposure hours. These results contribute to monitor the use of TiO2-NPs combined with drugs in medical application. The potential impact of antibiotics with TiO2-NPs during pregnancy could be associated with adverse effects on embryo DNA. The use of nanomaterials in drugs formulation should be strictly controlled in order to minimize risks.Titanium dioxide nanoparticles (NPs-TiO TiO2-NPs are applied in medicine as photosensitizer for photodynamic therapy -3′) . The amp to 100% .The data were expressed as mean and standard deviation (SD). Differences in the percentage of cell viability, DNA damage and apoptosis genomic stability among the experimental groups were analyzed with the unpaired Student’s t-test using GraphPad Prism 6. Only results with p-value ≤ 0.05 were considered statistically significant.2-NPs and TiO2-NPs combined with lincomycin underwent chromatographic separation  was at 196 nm, whereas the second (Band II) at 240 nm  with respect to the negative control . Each type of cell has its own vulnerability to NPs; in fact, the metabolic rate, the antioxidant enzyme machinery and DNA repair capabilities of each cell type responded differently based on the concentration of TiO2-NPs, which induced different toxicity levels. Moreover, several crystalline forms of NP-TiO2 and the aggregation of NPs determined a change in their effects due to the increase in their size. Sadhukha and collaborators [The nanoparticles large-scale application has raised the attention on the possible adverse effects on the exposed organism’s health. Titanium dioxide is among the most used nanoparticles in different industrial sectors. NPs-TiOembranes ,56,57,58borators  demonstrelopment . In the elopment , the DLSborators ,62. In oborators  showed h2 nanoparticles. This response could be different related to the exposure time and dosage. The results of this study are in accordance with Boland and collaborators [2-NPs and demonstrated how titanium dioxide nanoparticles are harmful to cell survival by activating an exuberant apoptotic response. The latter, to be activated, it requires the internalization of the TiO2 nanoparticles since the accumulation in lysosomes leads to their rupture and to the release of hydrolases such as cathepsins and intracellular ROS production. The primary mechanism of NPs induced toxicity is due to oxidative stress, resulting in damage to cellular membranes and biological macromolecules [2-NPs induced DNA damage in amniocytes at a concentration of 10 μg/L. Thus, the dose comparison of our study with earlier reports [2-NPs. Furthermore, the nanomaterials released in the cytoplasm allow their access to essential biomolecules causing damage. In addition, the nanoparticles can reach the nucleus, in fact, a high fragmentation was also found by Patel and collaborators in 2017 [2 could induce ·OH radicals which are probably responsible for the DNA damage in the exposed cells [2-NPs and lincomycin induced genotoxic effects without any cytotoxic one; in fact the co-exposure times affected the cell survival and induced changes in genetic material which do not cause cytotoxic effects. However, this mechanism has not been clarified and needs further investigations. Human amniocyte co-treated with TiO2-NPs and lincomycin showed the increase of DNA fragmentation, induction of apoptotic process and DNA damage. The results of Comet Assay demonstrate that the co-exposure induced a statistically significant increase of DNA strand breaks starting from 48 exposure hour. The data obtained by Diffusion assay highlighted the increase of amniotic cell apoptosis from 48 to 72 exposure hours after TiO2-NPs and lincomycin co-exposure. Similarly, the DNA genomic stability was statistically reduced after the co-exposure.In order to protect the cell, we suggest that DNA repair activity would be activated as a consequence of DNA damage in cells exposed to TiOborators  who onlyolecules ,66,67,68 reports ,70 sugge in 2017 . The auted cells . There aed cells ,74,75. Ted cells . This st2-NPs alone. The results of this work suggested that the combination of TiO2-NPs and lincomycin cause an intermediate level of genotoxicity to amniocytes compared to the exposure of each single compound which by themselves are quite toxic to these cells. Nevertheless, the combination tested produce less toxicity that the same quantity of NPs probably because the co-exposure could affect the bioavailability of the NPs or their biophysical characteristics . In fact, extracted UV spectra of lincomycin and TiO2-NPs showed that when media with TiO2-NPs combined with lincomycin was injected, in each chromatogram, a single peak was detected and its retention time seemed almost superimposable to that of TiO2-NPs. The observation of this hyperchromic effect, together with the disappearance in the HPLC chromatogram of the peak related to pure lincomycin, allowes us to hypothesize the interactions have been established. Therefore, the results demonstrate that the combination of TiO2-NPs and lincomycin leads to the aggregate formation reducing the genotoxcity compared with the dispersed nanoparticles.However, we stress that although co-exposure induces genotoxic effects, the latter are still lower than the exposure to TiO2-NPs with organic pollutants (CdCl2 and dioxin) in Dicentrarchus labrax in sea water [2-NPs reduced CdCl2-induced effects and DNA damage in Mytilus galloprovincialis, whereas, additive effects were no observed [2-NPs and lincomycin on human amniotic cells, rather co-exposure reduces the TiO2-NPs genotoxicity. Considering that Trojan horse effect is controversial, it could be hypothesized that these nanoparticles could influence the activity of the lincomycin in such a way that could improve its efficacy and thus lead to dosage reduction.The loss or gain of genotoxicity drugs and/or pollutants carried by titanium dioxide nanoparticles could depend on the molecule carried and media, determining or not the “Trojan horse effect”. However, the experimental evidence on “Trojan horse effect” is conflicting, in fact, it has been demonstrated no interaction of the TiOea water ,39. Instobserved . Accordi2-NPs during pregnancy should be thoroughly investigated and exposure to NPs must be prevented or minimized. Nevertheless, it must be considered that the behavior of the substances tested is relative to an in vitro system that use a single concentration substances, so, it is necessary to complement the in vitro testing by performing a complete genotoxicity assay on a large range of concentrations and by accurately assessing the level of cytotoxicity; then, by performing methodologies able to display relevant genetic events . In this way, a complete in vitro genotoxicity profile will be available. As a result, further studies on other in vivo models could be considered to clarify this aspect, so as to carry out prevention interventions on maternal exposure. The data assume that the genotoxicity studies of NPs must be recommended during a comprehensive assessment of the safety of TiO2-NPs and novel types of NPs and nanomaterials.The exposure to TiO


In the epigenetics field, large-scale functional genomics datasets of ever-increasing size and complexity have been produced using experimental techniques based on high-throughput sequencing. In particular, the study of the 3D organization of chromatin has raised increasing interest, thanks to the development of advanced experimental techniques. In this context, Hi-C has been widely adopted as a high-throughput method to measure pairwise contacts between virtually any pair of genomic loci, thus yielding unprecedented challenges for analyzing and handling the resulting complex datasets. In this review, we focus on the increasing complexity of available Hi-C datasets, which parallels the adoption of novel protocol variants. We also review the complexity of the multiple data analysis steps required to preprocess Hi-C sequencing reads and extract biologically meaningful information. Finally, we discuss solutions for handling and visualizing such large genomics datasets. The total length of DNA contained in a human cell would be 2 m long if completely stretched, i.e., considering the cumulative size of 6 billion nucleotides composing a diploid genome. However, such a long polymer must fit into a nucleus with an average diameter of 10 μm, i.e., five orders of magnitude shorter than the genome  contacts  the restriction enzymes used in the experimental procedure and by (2) the sequencing depth. Over the years, we have witnessed an attempt to increase the resolution of Hi-C data by working on these parameters, resulting in available datasets characterized by increasing size and resolution, reaching very high numbers of sequenced reads, especially for mammalian genomes. In addition, specific protocol variations have been proposed with the aim of improving the resolution.The classical Hi-C technique involves restriction digestion of a formaldehyde cross-linked genome with sequence specific restriction enzymes, followed by fill in and repair of digested ends with the incorporation of biotin-linked nucleotides. The repaired ends are then re-ligated. Finally, the cross-linking is reversed and associated proteins are degraded. This produces the ligation products which are then nonspecifically sheared, generally by sonication, and enriched for sheared fragments containing the ligation junction, using a biotin pull-down strategy, and finally sequenced using paired-end sequencing  first of all aligned to the reference genome, (2) then filtered to remove spurious signal, and then read counts are (3) binned and (4) normalized. The last two steps are often performed simultaneously but involve distinct choices that affect the characteristics of the normalized contact matrix obtained as final output  normalization methods. The explicit normalization methods are based on the explicit definition of a set of biases known to be associated to Hi-C reads or high-throughput sequencing in general, thus affecting the resulting read counts per restriction fragment. These include the fragment length, its GC content and mappability. Correction factors are computed for each of the considered biases and their combination, then applied to the read counts per genomic bin  the identification of compartments, 2) calling TADs, and (3) point of interactions, also termed loop calling when referred to  calling https://github.com/dekkerlab/cworld-dekker) and in the HiTC R package . Other methods are not based on a one-dimensional score, but aim to identify the best partitioning of the contact matrix in TADs based on clustering algorithms, such as HiCseg or ClusterTAD  calculates for each bin the degree of upstream and downstream interaction biases and is segmented with a hidden Markov model (HMM) to derive TADs in DomainCaller  approaches. Fit-Hi-C , Capture-HiC (cHi-C) is a more advisable technique .FASTQ and BAM are the current standard for sequenced and aligned reads, respectively. When it comes to the creation of the read pairs (pairs file) and the interaction map files, these are saved in formats that vary greatly among the tools Fig. . The basThe most intuitive way to save the Hi-C map is as a plain text symmetric matrix, where the first column and first row contain the bin identifiers. HOMER  used for visualization by the 3D Genome Browser . ThanksSeveral tools address these issues and display Hi-C contact maps as heatmaps, supporting matrices saved in binary formats to allow fast retrieval of the data. Juicebox (Durand et al. Other tools such as WashU Epigenome Browser (Zhou et al. Finally, HiCExplorer instead is a more complex framework available as command line tools or Galaxy module for a web interface (Wolff et al. Overall, the rapid widespread adoption of Hi-C and its variants have spurred an explosive growth of complexity and size in available chromatin 3D architecture datasets. This, coupled with the rapid flourishing of many data analysis approaches, has already raised substantial concerns in the field about the need for common standards and guidelines (Marti-Renom et al.


This study aimed to estimate overreporting  and underreporting  rates in self-reported IGD assessment compared with clinical diagnosed IGD. The study population consisted of 45 with IGD and 228 without IGD based on clinical diagnosis from the Internet User Cohort for Unbiased Recognition of Gaming Disorder in Early Adolescence (iCURE) study. All participants completed self-reported IGD assessments. Clinical interviews were conducted blindly by trained mental health professionals based on DSM-5 IGD criteria. Self-assessed average daily amount of gaming time and game genre were measured. Psychological characteristics, including anxiety, suicidality, aggression, self-control, self-esteem, and family support, were obtained from the baseline survey. The false-negative rate for self-reported IGD assessment was 44%. The false-negative group reported less time playing online games than the IGD group, though their psychological characteristics were similar to those of the IGD group. The false-positive rate was 9.6%. They reported more time playing online games than non-IGD group, though their psychological characteristics were similar to those of non-IGD group except self-control. The discrepancy of IGD diagnoses between self-reports and clinical diagnosis revealed limitations of self-measurements. Various strategies are required to overcome the methodological shortfalls of self-reports for the assessment of IGD. However, these measures may lack credibility due to biased responding. In general, research participants may respond in a way that makes them look as good as possible. In particular, self-report biases may be introduced when responding to sensitive questions, such as those related with addictive behaviours, sexual experiences, and delinquency. Self- measurements may also be prone to exaggeration. Some evidence suggests that individuals tend to over-report their pro-environmental behaviour7. Several IGD assessments have been examined to test the diagnostic validity of DSM-5 IGD criteria including the 10-item Internet Gaming Disorder Test (IGDT-10)8, IGD-209, IGDS-SF910, Video Game Dependency Scale (CASA)6, and IGD scales with both polychotomous and dichotomous methods to determine the most practical scale for IGD diagnosis11. Population based surveys did not include clinician diagnosis but used Latent Class Analysis in the group with the highest likelihood of meeting the nine criteria as the gold standard to investigate the cut-off threshold proposed in the DSM-59.In 2013, the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) proposed diagnostic criteria for Internet gaming disorder (IGD) as a condition warranting further empirical and clinical research. The suggested IGD criteria paved the way for measuring IGD in a consistent manner. Many studies have used the DSM-5 diagnostic criteria to estimate the prevalence of IGD, and most have confirmed IGD using self-administered questionnaires12. However, false positive and false negative results were still present in the self-report assessment. To the best of our knowledge, no studies have been conducted comparing self-reported IGD with clinical interviews based on DSM-5 criteria in a population-based study. Therefore, this study aimed to estimate the false-positive and false-negative rates in an IGD self-report assessment compared with a clinical assessment of IGD made by mental health professionals based on DSM-5 IGD diagnostic criteria. The false-negative (underreporting) group was defined as adolescents who did not have IGD on self-report, but were diagnosed with IGD in the clinical diagnostic interview. The false-positive (overreporting) group was defined as adolescents who had IGD on self-report, but who were assessed as not having IGD in the clinical diagnostic interview.Although self-reports may have limitations in psychiatric epidemiological studies, few studies have considered the accuracy of self-reported assessment in making a diagnosis of IGD. A previous study reported that the sensitivity and specificity of self-reported Internet addiction measurement were around 85%, indicating good diagnostic accuracy13. This study investigated whether psychosocial characteristics which are well known risk and protective factors related to the development of IGD such as anxiety14, suicidality15, aggression16, self-control17, self-esteem18, and family support20 differed between adolescents in the false-positive and false-negative groups. We hypothesized that the false positive group was related to the non-IGD group, and the false negative group was related to the IGD group with regard to psychological characteristics related to development of IGD.Because prevalence estimates using self-reported assessment can be biased because of false-negative and false positive rates, it is important to estimate the magnitudes and direction of bias by measuring false-negative and false positive rates. Longitudinal follow-up studies will reveal whether the discrepancies between self-reported assessments and clinical interview were genuine IGD or not. However, since the current study was based on the baseline results, we performed an exploratory analysis to evaluate whether the false-negative and the false positive groups were related to the non-IGD or IGD group. IGD risk factors can predict who is more likely to become addictedP = 0.050).Table P < 0.001).In the IGD group, 18 (40.0%) had started playing Internet games before entering elementary school, 15 (33.3%) started between the first and third grades, and 11 (24.4%) started in or after the fourth grade. In the non-IGD group, 28 (12.3%) had started playing Internet games before entering elementary school, 96 (42.3%) started between the first and third grades, and 67 (29.5%) started in or after the fourth grade. The IGD group began playing Internet games earlier than the non-IGD group (The self-reported IGD assessment had a sensitivity of 55.6% (95% CI: 45.2–67.1%) and specificity of 90.4% (95% CI: 83.0–97.2%) (Table P < 0.001)  Table .Table 3GAll psychological characteristics were significantly different among the four groups in ANOVA and Chi-square test. Anxiety, aggression, self-esteem, familial support, and experience of suicidality during the past year were not different between non-IGD and false positive groups in Duncan and Dunn post-hoc analyses. The false negative group was similar to the IGD group with regard to the five psychosocial characteristics. Compared to non-IGD, the mean scores of self-control were high in the false positive and false negative groups Table .Table 4IThis study identified some discrepancies between self-measurements and clinically verified IGD diagnoses among adolescents. The false-negative rate was 44% (95% CI: 31.9–58.8%), and the false-positive rate was 9.6% (95% CI: 6.4–14.2%). Several factors may explain the false-negative findings, including social desirability effects, a dislike for participating in the study, and limitations of self-assessment tools.21. Adolescents at risk for various disorders may not wish to disclose certain facts about themselves, and they may purposefully under-report socially undesirable risk behaviours. Social desirability effects can threaten validity of results because responses are, by definition, systematically biased. Respondents, wishing to present themselves in a favorable light, may consciously or unconsciously tailor their answers to make their behaviours appear less deviant and more socially desirable23. Williams et al.23 postulated that social-desirability bias leads players to underreport their playing time23. Individuals playing online games may also under-report gaming time for other reasons . The mode of data collection may influence the degree of social-desirability bias24. Research on this topic has found that the further a mode is from face-to-face interaction, the less is the extent of social-desirability bias. It should also be noted that some respondents may over-rate participation in socially deviant behaviours, particularly during developmental stages like adolescence that are characterized by an increase in seemingly rebellious behaviours.It can be difficult to accurately assess risk behaviours in adolescents with self-reported questionnairesSome adolescents simply might not have been inclined to participate in the survey itself. Perhaps they considered the survey tedious and uninteresting; as a result, it is not possible to rule out reluctant answering. Unwillingness to participate could have affected the false negative rate when considering different situations between self-report and clinical interview. Self-report assessment administered by unit of class, 30 adolescents was connected the website in the computer room at the same time without any enforcement, but clinical interview was conducted through 1:1 interview by psychiatrists and certified clinical psychologists. In the context of 1:1 interviews with a probing process to identify IGD symptoms, students would have been less affected by their willingness to participate than in a self-administered survey.The discrepancy between self-reported and clinically diagnosed IGD might also be due to the limitations of the “yes” or “no” response method as a self-reported assessment tool. When IGD was assessed using self-report among adolescents with the dichotomous response method of “yes” or “no,” the respondents might have faced difficulties accurately describing their responses in nuanced situations. This may have introduced error because the response type forced the responder to make a choice between the two options. The inherent limitations of a “yes” or “no” response-based questionnaire make it challenging to obtain precise responses from subjects in assessing IGD based on DSM-5. To overcome the limitations of self-report assessment tools, clinicians or mental health specialists may need to evaluate IGD symptoms through a probing process.The false-negative adolescents reported less time playing online games than IGD adolescents. However, the assessments of psychological characteristics showed similar results between the false-negative and IGD groups of adolescents in all assessments. The false-negative group might have under-reported their game-use behaviour or IGD symptoms in self-report assessments. Twenty of 45 IGD case produced a false negative response. Among these 20, four adolescents responded insincerely by marking all of the same numbers on the first stage self-report IGD assessment (IGUESS). They even responded with the same number on the reverse questions, contradictory to logic, which were included to assess insincere responses in the self-report assessment. Another four adolescents appeared to carefully review the entire questionnaire and reported no IGD symptoms. Twelve of 20 false negative adolescents might have underreported their IGD symptoms due to social desirability or the results of the limitations of self-assessment tools.st-, 4th-, 7th-, and 9th-grade students. Once students are classified into the high-risk group, they will be guided to receive an intervention. This process may make the false-negative rate of Korea higher than other countries.In Korea, the government-led youth game use factual survey is evaluated every year for 1The false-positive group reported more time playing online games than non-IGD adolescents. However, they showed a similar pattern to the non-IGD group of adolescents in most psychological characteristics, with the exception of self-control. Lack of self-control might be increased impulsiveness. Externalizing characteristics may be related to the behaviours of exaggeration or boasting about game skills or about the perception of power or influence in a peer group of adolescents.The prevalence of IGD was 1.94% , which may be an underestimate. To determine the overall prevalence of IGD, we randomly sampled 232 adolescents out of the 2,319 (10%) enrolled in the iCURE study. Among 232, 73 were met the inclusion criteria for the second-stage clinical diagnostic interview, and 153 did not meet the criteria. Among 153, two were diagnosed with IGD in the clinical interview, although they did not meet the inclusion criteria for the second-stage clinical diagnostic interview. Based on these results, 20 adolescents were likely to be diagnosed with IGD among the adolescents who did not meet the criteria for the second-stage clinical diagnostic interview. However, we identified only 8 IGD cases through the second-stage diagnostic interview criteria, so we estimate that approximately 12 IGD cases may have been missed.This study had several limitations. First, we selected a representative sample of 10% from the total population due to practical concerns, but only four of these individuals were diagnosed with IGD . Because the representative sample size was relatively small, our results may have been affected by random variability.Second, clinically diagnosed IGD was evaluated by only one mental health professional per each person rather than by multiple observers. However, the diagnostic evaluations occurred through four-day workshops to ensure the reliability of the diagnosis. Although the mental health specialists conducted clinical diagnostic interviews according to the DSM-5 diagnostic criteria, it may have been difficult to determine whether the self-report was false-positive or false-negative because the threshold of the DSM-5 IGD diagnostic criteria have not been clearly established through formal testing. Considering an IGUESS cutoff score for IGD risk of 10 or higher, and an IGUESS score of 6 points is expected to be associated with about 2 subclinical symptoms, almost all of the adolescents with problems in gaming behaviours from the entire cohort were likely to have undergone the second-stage clinical diagnostic interview, so we believe that most cases were confirmed. We also included in the second-stage diagnostic interview anyone who responded insincerely by marking all of the same numbers on the IGUESS or anyone who reported a suicidal experience during the past year. These efforts helped ensure that we could identify all participants with IGD.Despite these efforts, we cannot exclude the possibility that the prospective second-stage diagnostic interview missed participants with IGD because of the limitations of self-report. Two of four adolescents were diagnosed with IGD among the random sample; it is possible that the false-negative rate was an underestimate. Self-reported assessment is a convenient survey method, but it cannot always detect false reporting by respondents, so the results might be biased. The reason for the high false-negative rate may be due to repetitive exposure to similar questions among Korean students.25.Self-reported IGD assessments have the possibility of introducing bias which would affect the validity of the measurement. To overcome the methodological shortfalls of self-reports for the assessment of IGD, future research may incorporate other objective measurements, include reverse questionnaires with contradictory to logic, use Likert scale assessment, or apply randomized response techniques for gathering self-reported assessment in sensitive questions with protection of privacyAdolescents in the false positive or false negative group could be in a subclinical stage. Natural history of false positive and false negative groups should be examined to determine whether they belong to the IGD or non-IGD group through longitudinal follow-up. Since the iCURE study was designed as a prospective longitudinal study, the results will be revealed in future studies.Because the false-positive or false-negative rate may be influenced by various personal, environmental, or situational factors, it is difficult to generalize our results to other age groups, populations of different ethnic, racial or cultural compositions, or individuals living in rural areas or other countries. In addition, although the sub-cohort participants consisted of a representative sample from 21 schools, most schools included in the iCURE study were registered in Seoul, so the results may be most relevant to a metropolitan environment.Internet User Cohort for Unbiased Recognition of Gaming Disorder in Early Adolescence (iCURE) study was conducted in Korea. The study was fully reviewed and approved by the Institutional Review Board of The Catholic University of Korea (MC140NM10085) and conducted in accordance with the Declaration of Helsinki in 201326. Informed consent was acquired from all participants and their parents or legal guardians following explanation of the nature of the principles of research, including confidentiality and the freedom of choice to participate. In addition, the current study received approval from the Institutional Review Board of The Catholic University of Korea for data analysis (MC17ENSI0108). The iCURE data management board released de-identified iCURE data.In 2015 through 2017, baseline assessment for the 27. The iCURE study has enrolled 2,319 adolescents from the 3rd, 4th and 7th grades. All participants completed self-administered IGD assessments using both the Internet Game Use-Elicited Symptom Screen (IGUESS)28 and the Internet Gaming Disorder Questionnaire (IGDQ) as a self-report version of the highly Structured Clinical Interview for DSM-5 IGD (SCI-IGD)29. A second-stage assessment for making a clinical diagnosis was made for those participants who had one of the following indications: a score of six or higher on the IGUESS; anyone who responded insincerely by marking all of the same numbers on the IGUESS including the reverse questions in contradictory to logic; or anyone who reported a suicidal experience during the past year. These participants were given diagnostic interviews. Participants who scored 6 points in the IGUESS were expected to have about two subclinical IGD symptoms. A total of 834 among 2,319 adolescents participated in the second-stage clinical diagnostic interview. Forty-three adolescents were verified as having IGD through the two-phase assessment.The protocol for the iCURE study has been published previouslyTwo hundred thirty-two adolescents were selected as a representative random sample of 10% of the iCURE study participants, stratified by school. All of these adolescents completed the clinical diagnostic interviews. Some of these adolescents were part of both the representative sample and the second-stage diagnostic interviews. Of the 232 adolescents, 4 were diagnosed with IGD in the clinical interviews, including 2 who were included in the second-stage clinical diagnostic process and 2 who did not meet the second-stage clinical-interview indications but were diagnosed with IGD through the clinical diagnostic interview. This case sub-cohort consisted of 45 participants with IGD (case) and 228 participants without IGD (non-case), as diagnosed clinically  and structural validity . Moderate to strong correlations with depressive symptoms, anxiety, hyperactivity, and gaming hours supported the criterion validity of this scale (in submission). Meeting five or more of the DSM-5 criteria was revealed to be the best cut-off to distinguish the normal group from the clinically impaired group29.The IGDQThe clinical diagnostic interviews for IGD were conducted face-to-face by five psychiatrists and four experienced clinical psychologists. To ensure reliability, all of the mental health specialists who participated in the clinical interviews completed a four-day workshop. They were kept blind to the results of the self-reported assessments. At the end of each day of diagnostic interviews, they discussed any ambiguous diagnoses and to make final determinations. The interviews assessed participants based on the nine DSM-5 IGD criteria, in accordance with the international consensus for IGD represented in the DSM-5. Individuals with positive responses to five or more of the IGD criteria and functional impairments were classified into the IGD group. The interviews were confidentially conducted in an independent space within the participants’ schools. The overall IGD diagnostic percent agreement was 91%, and overall percent agreement in each of the nine IGD criteria ranged from 79% to 90%.Socio-demographic characteristics, including gender and family structure , were obtained from the iCURE study baseline assessment data.Self-reported assessments of participants’ gaming usage were obtained from the baseline survey, including questions regarding first exposure, average daily amount of time spent playing online games during weekdays and weekend days, and the titles of the games that they had played during the past 12 months. Online games were classified into 5 categories: role-playing games, shooter games, simulation games, arcade games, and unknown.Psychological characteristics of anxiety, suicidality, aggression, self-control, self-esteem, and family support were obtained to evaluate the psychological profiles of the self-assessed IGD and clinically diagnosed IGD groups compared with those of adolescents without positive assessments of IGD.30. This scale is reliable and showed a Cronbach’s alpha of 0.91 in the current study.To examine trait anxiety, we used the Korean translation of the Trait Anxiety Inventory for Children (TAIC) developed by Spielberger and colleagues. The Korean TAIC is a 3-point 20-item inventory that asks respondents to indicate how frequently they feel worried, bothered, or nervous. The total score ranges from 20 to 6031. Participants were asked whether they had seriously considered committing suicide, made a plan to commit suicide, or had attempted suicide during the past year. The presence or absence of suicide ideation, plans, and attempts was based on the participant response (yes or no).The presence of suicidal ideation, suicide plans, and suicide attempts was determined through the following direct questions derived from the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I)32. This measure detects feelings of self-acceptance, self-respect, and generally positive self-evaluation. Each item is rated on a 5-point Likert scale, ranging from 1 (strongly disagree) to 5 (strongly agree). The total score ranges from 10 to 50. This measure detects feelings of self-acceptance, self-respect, and generally positive self-evaluation. In the current study, this scale was reliable, with a Cronbach’s alpha of 0.83.Self-esteem was measured using 10 items from the Self-Esteem Scale33. Each item is rated on a 4-point scale: (4) strongly agree, (3) somewhat agree, (2) somewhat disagree, and (1) strongly disagree. Higher scores indicate greater problem with self-control. This scale was reliable, with a Cronbach’s alpha of 0.86 in the current study.Self-control was assessed with the 10-item Self-Control Scale of Gottfredson and Hirschi (1990)34. This scale was reliable, with a Cronbach’s alpha of 0.88 in the current study.Aggression was evaluated by the Buss-Perry Aggression Questionnaire with a 29-item, 5-point Likert scale measurement. Higher scores indicate greater aggressionet al.35. Items are rated on a 5-point Likert scale, from 1 (never) to 5 . Peer support was used in the analysis. Higher scores indicated that the participant perceived greater social support from peers35. This scale was reliable, with a Cronbach’s alpha of 0.94.Social support was evaluated with the Social Support Appraisals Scale (SSAS). This scale evaluated the child’s subjective appraisal of family, peer, and teacher support to address perceived support. It is a 41-item self-report instrument developed by Dubow Socio-demographic characteristics were summarized with numbers and percentages for categorical variables or means and standard deviations (SDs) for continuous variables. The study population was divided into four groups according to either concordance or discordance of the self-reported and clinically diagnosed IGD. The non-IGD comparison group included participants who were evaluated as not having IGD in both the self-report and clinical interview. The IGD group included anyone who had IGD on both the self-report and clinical interview. The false-negative group included adolescents who did not have IGD on self-report, but were diagnosed with IGD in the clinical diagnostic interview. The false-positive group included adolescents who had IGD on self-report, but who were assessed as not having IGD in the clinical diagnostic interview. False positive rate is calculated as the fraction of the number of classified as non-IGD in clinical diagnosis as the denominator and the number evaluated an IGD in self-reported assessment as the numerator. False negative rate is calculated as the fraction of the number of patients classified as IGD in clinical diagnosis as the denominator and the number of patients evaluated as non-IGD in self-reported assessment as the numerator. Sensitivity and specificity were calculated, considering the mental health professionals’ clinical interviews as the gold standard.The t-test, chi-square test, and Fisher exact test were used to determine differences between the four groups.Differences in psychological characteristics among the four groups were analysed using ANOVA for continuous variables and Chi-square tests for categorical variables. If there was a significant group difference in ANOVA, Duncan multiple comparison test was used for post-hoc comparison. If there was a significant group difference in Chi-square test, Dunn multiple comparison test was used for post-hoc comparison. All tests are two-tailed, and p-values less than 0.05 were considered statistically significant. All analyses were performed with SAS software package version 9.4 .The datasets generated during and/or analysed during the current study are available from the corresponding author.


The Hospital Value Based Purchasing Program (HVBP) in the United States, announced in 2010 and implemented since 2013 by the Centers for Medicare and Medicaid Services (CMS), introduced payment penalties and bonuses based on hospital performance on patient 30-day mortality and other indicators. Evidence on the impact of this program is limited and reliant on the choice of program-exempt hospitals as controls. As program-exempt hospitals may have systematic differences with program-participating hospitals, in this study we used an alternative approach wherein program-participating hospitals are stratified by their financial exposure to penalty, and examined changes in hospital performance on 30-day mortality between hospitals with high vs. low financial exposure to penalty.Our study examined all hospitals reimbursed through the Medicare Inpatient Prospective Payment System (IPPS) – which include most community and tertiary acute care hospitals – from 2009 to 2016. A hospital’s financial exposure to HVBP penalties was measured by the share of its annual aggregate inpatient days provided to Medicare patients (“Medicare bed share”). The main outcome measures were annual hospital-level 30-day risk-adjusted mortality rates for acute myocardial infarction (AMI), heart failure (HF) and pneumonia patients. Using difference-in-differences models we estimated the change in the outcomes in high vs. low Medicare bed share hospitals following HVBP.n = 540) and low (n = 1362) Medicare bed share hospitals, respectively. High Medicare bed share hospitals were more likely to have smaller bed size and less likely to be teaching hospitals, but ownership type was similar among both Medicare bed share groups.. Among low Medicare bed share (control) hospitals, baseline (pre-HVBP) 30-day mortality was 16.0% (AMI), 10.9% (HF) and 11.4% (pneumonia). In both high and low Medicare bed share hospitals 30-day mortality experienced a secular decrease for AMI, increase for HF and pneumonia; differences in the pre-post change between the two hospital groups were small (< 0.12%) and not significant across all three conditions.In the study cohort of 1902 US hospitals, average Medicare bed share was 61 and 41% in high (HVBP was not associated with a meaningful change in 30-day mortality across hospitals with differential exposure to the program penalty. The three aims of better health, better care, and lower costs of the U.S. Department of Health and Human Services’ National Quality Strategy are at the focal-point of the Centers for Medicare and Medicaid Services’ (CMS’) notion of value . The AffEvidence on the impact of HVBP on the target outcomes is limited. Two prior studies, which found that HVBP was not associated with any change in 30-day mortality, compared mortality changes in IPPS hospitals with those in non-IPPS hospitals , 6. ThesUsing publicly reported data on hospital performance from 2009 to 2016, we examined the association between HVBP incentive size and changes in 30-day mortality by comparing pre- to post-HVBP changes in hospital 30-day mortality among high Medicare share hospitals with those of low Medicare share hospitals. We examined 30-day mortality changes for the three admission cohorts  that were all introduced into the HVBP in 2014.The data for this study comes from two publicly available sources: CMS Hospital Compare  . Specifically, we defined a dichotomous indicator of high Medicare share hospitals that groups hospitals with Medicare share inpatient days greater than or equal to 55% as high Medicare share hospitals and the other hospitals as low Medicare share hospitals. Due to a dearth of prior work or theoretical guidelines in grouping hospitals by Medicare share, we also examined different categorizations including with different cut-offs and examined the sensitivity of study findings to these alternative cut-offs.. , 19. DueThe hospital characteristics included in our analysis as regression covariates were: bed size ; teaching hospital status - a binary variable indicating membership in the Council of Teaching Hospitals; ownership ; and region . In addition, we also included time-varying characteristics: hospital average daily patient census; proportion of low income patients at hospital level ; state-level annual poverty rate; state-level annual unemployment rate.We compared the hospital characteristics of the high vs. low Medicare share hospitals in the baseline year 2009. We plotted annual average mortality rate for each condition from 2009 to 2016 for high and low Medicare share hospitals; compared the baseline difference in mortality rate between high and low Medicare share hospitals for each condition; and estimated linear time series models to capture average change in annual mortality rates over the study period for the targeted conditions among the two groups of hospitals. We evaluated the association of the HVBP incentive size with the mortality outcomes using a difference-in-differences type approach, whereby pre- vs. post-HVBP changes in the outcome in high Medicare share hospitals were contrasted with corresponding changes in low Medicare share hospitals. In identifying pre- and post-HVBP periods, we note that while the HVBP was announced in March 2010, with passage of the ACA, the 30-day mortality measures for AMI, HF, and pneumonia were introduced in FY 2014. However, hospital performance on 30-day mortality was based on all eligible hospitalizations during the prior 3 year period, and for the first program year (FY 2014) hospital performance was based on discharges during July 1, 2011 – June 30, 2013 . Since tFor our main analysis, to estimate the association between hospital mortality performance and the degree of exposure to the HVBP, we used linear regression models with difference-in-differences specifications that included indicators for high Medicare share hospitals and post-HVBP time period, and for the interaction (product) of high Medicare share hospitals and post-HVBP. We also included as covariates, indicators of the aforementioned hospital characteristics measured in the baseline year 2009). The coefficient estimate of the interaction term gives the excess pre- to post-HVBP change in mortality rate for high Medicare share hospitals compared to that for low Medicare share hospitals 09. The c. As the Given potential changes in 30-day readmission rate for AMI, HF, and pneumonia associated with the Hospital Readmission Reduction Program (HRRP), another CMS incentive program that was introduced alongside the HVBP in 2010, there may be unintended spillover effects (of the HRRP) on 30-day mortality rates for the aforementioned conditions . As a seAll statistical analyses in this study were performed using Stata version 14.1 . The InsThe final study sample comprised 1902 hospitals in each year between 2009 and 2016. We compared the characteristics of the high Medicare share hospitals vs. the low Medicare share hospitals in 2009  and for pneumonia was − 0.2% vs. 0.2% [p-value = 0.003].Figure The results of the association between a hospital’s degree of reliance on Medicare patients (and thus the effective incentive size under the HVBP) and 30-day risk adjusted mortality rate for AMI, HF, and pneumonia using the difference-in-differences analysis are presented in Table The validity of our difference-in-differences results rests on the assumption that there were no pre-HVBP trend differences between high and low Medicare share hospitals. Therefore, we compared the pre-HVBP mortality trends between high and low Medicare share hospitals . For HF and pneumonia admission cohorts the pre-HVBP trends were similar among the two hospital groups , instead of the 6 years in the main analysis, showed no significant change in mortality – and in the case of pneumonia cohort using 2014–2016 data, a significant not meaningful change – associated with HVBP . These results are broadly consistent with findings from other studies that used critical access hospitals or hospitals in the state of Maryland as the comparison group . AdditioThere are a number of possible explanations for the lack of evidence of improvement in patient outcomes under the HVBP. First, the financial incentives under the HVBP may have been too small – the maximum penalty was 1% (2013), 1.25% (2014), 1.5% (2015), and 1.75% (2016) – to elicit performance changes from hospitals , 29. SecOur study has several limitations. First, although reliance on Medicare revenues may be smaller for the low Medicare share hospitals, these hospitals still have an incentive to improve patient outcomes since part of their revenue comes from Medicare payments. However, the magnitude of this incentive is likely to be substantially smaller in comparison with that for high Medicare share hospitals. Thus, to the extent that incentives influence hospital behavior, efforts to reduce mortality under the HVBP should have been substantially greater in high-Medicare share hospitals. Second, the HVBP program was launched during the time that quality of care was improving . While tIn conclusion, our results indicate that HVBP was not associated with differential improvement in mortality outcomes across hospitals that differed in the extent of risk of penalty over the 4 years since the implementation of the program. Policy makers should re-evaluate whether providing monetary incentives to hospitals is an effective mechanism to motivate hospitals to perform better. There is considerable concern that this program may have unintended adverse consequences, particularly for financially vulnerable hospitals, by exacerbating the resource constraints in facilitating interventions to improve patient care; also, they may lead to undesirable gaming responses, such as patient cherry-picking or use of alternative diagnostic codes for non-targeted conditions . Alterna


Aspergillus species known to cause disease in humans, is related to increased virulence of the fungus.Mycoviruses that infect fungi generally do not have a significant effect on the host and, instead, reduce the toxicity of the fungi. However, recent studies have shown that polymycovirus-1, a mycovirus that infects Aspergillus fumigatus polymycovirus-1 (AfuPmV-1) and other mycoviruses known to infect Aspergillus spp. to examine the genetic characteristics of AfuPmV-1. In addition, codon usage analysis was performed to determine whether the nucleotide composition and codon usage characteristics of AfuPmV-1 were also present in other polymycoviruses and hypervirulence-related mycoviruses. Phylogenetic analysis was also performed to investigate their evolutionary relationship.Comparative analysis was performed of RdRP gene codon usage patterns of Analysis of nucleotide composition indicated that AfuPmV-1 had the highest GC content among analyzed mycoviruses and relative synonymous codon usage analysis indicated that all of the codons preferred by AfuPmV-1 ended with C or G, while codons ending with A or U were not observed. Moreover, the effective number of codons, the codon adaptation index, and correspondence analysis showed that AfuPmV-1 had greater codon preference compared with other mycoviruses and that AfuPmV-1 had relatively high adaptability to humans and fungi. These results were generally similar among polymycoviruses.Aspergillus spp. This difference may be related to the hypervirulence effect of AfuPmV-1. Analysis of AfuPmV-1 codon usage patterns could contribute to the identification and prediction of virulence effects of mycoviruses with similar genetic characteristics.The codon usage pattern of AfuPmV-1 differs from other mycoviruses that infect  Aspergillus fumigatus, was reported to have significant mild hypervirulence effects in the moth species, Galleria mellonella [Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1) found in the same fungus [Tetramycoviridae to Polymycoviridae as more viruses showing similar characteristics to AfuTmV-1 were discovered, including BbPmV-1, which infects the insect pathogenic fungus, Beauveria bassiana [Mycoviruses are viruses that infect fungi and are known to be infectious to most fungal species. While mycoviruses have little or no influence on the fungal host in most cases, some have been shown to control the pathogenicity of the host by increasing or decreasing the virulence , 2. To dllonella , and sime fungus . AfuTmV-bassiana   and the complete nucleotide sequences of the RdRP coding region of mycoviruses that infect Aspergillus species were used. Eight sets of data published between 2006 and 2015 were studied in the analysis. Other polymycoviruses and hypervirulence-related mycoviruses data were downloaded from NCBI for comparison (Table The AfuPmV-1 genome consists of four capped double-stranded RNAs (dsRNAs), of which the largest dsRNA1 segment encodes the RNA-dependent RNA polymerase (RdRP) , 6. The https://sourceforge.net/projects/codonw/) and CALcal  were used to conduct analyses of nucleotide composition, overall/local G + C content, relative synonymous codon usage (RSCU), effective number of codons (ENC), codon adaptation index (CAI), and correspondence analysis (COA) for each of the selected genetic data [Homo sapiens) and fungus (Aspergillus fumigatus) were acquired from the codon usage database (http://www.kazusa.or.jp/codon/) for comparison [Aspergillus (Group 1), polymycoviruses with a fungal host other than Aspergillus (Group 2), and newly reported mycoviruses that enhance host virulence (Group 3).The programs CodonW  to infer the influence of evolutionary processes on codon usage patterns. RdRP coding sequences were aligned with the MUSCLE algorithm, and the phylogenetic tree was constructed by applying the Maximum Likelihood method and the Kimura 2-parameter substitution model. The robustness of the tree was verified with the bootstrap value set to 1000 [The phylogenetic relationships among the mycoviruses used for the analysis were inspected and phylogenetic analysis was conducted using the MEGA7 program  and the other viruses showed ENC values > 50 /tryptophan (UGG) that do not have synonymous codons. Although there were exceptions in the COA results according to the third nucleotide in the codon, G- and C-ended codons formed one group and A- and U-ended codons formed another group  ended with either a C or G nucleotide. The distinctive codon usage pattern of AfuPmV-1 compared to other mycoviruses that infect Aspergillus spp. are pathogenic fungi that cause various symptoms in humans. The hypervirulence effect of mycoviruses can increase the toxicity of Aspergillus spp. in human hosts, and thus increase the severity of symptoms. Here, AfuPmV-1 was shown to have distinct patterns in some codon usage indexes compared to other mycoviruses that infect Aspergillus spp. The distinctive codon usage pattern of AfuPmV-1 demonstrated in the present study indicated the need for monitoring of mycoviruses with similar characteristics. Research on mycoviruses has generally focused on their hypovirulence effects on fungi that infect plants. With the discovery of polymycoviruses, further research on the hypervirulence effects of mycoviruses is needed, particularly with regard to mechanisms of virulence control in mycoviruses, such as AfuPmV-1, which infects human pathogenic fungi.


Immune checkpoint inhibitors have dramatically changed lung cancer treatment, demonstrating an overall survival benefit. There are limited data about re-challenge in patients with non-small cell lung cancer. We attempted to address this question for re-challenge of immune checkpoint inhibitor in patients with advanced non-small cell lung cancer.We retrospectively analyzed 11 patients with advanced non-small cell lung cancer treated with nivolumab and re-challenged with nivolumab/pemblorizumab at Kansai Medical University Hospital from December 2015 to December 2017.Three patients achieved PR and two patients were in SD. These patients were apt to be good responders to the initial treatment, to develop immune-related adverse events and to be immediately started on re-challenge with immune checkpoint inhibitor. The median PFS was 2.7  months. Five patients (45%) had mild to moderate immune-related adverse events.Our study shows the effectiveness of re-challenge of immune checkpoint inhibitors in a subset of non-small cell lung cancer patients. Re-challenge might become one of treatment option for advanced non-small cell lung cancer. The development of immune checkpoint inhibitors (ICPi) has dramatically changed lung cancer treatment, demonstrating an overall survival benefit. The PD-1 (programmed death-1) blocking antibodies nivolumab and pembrolizumab are currently approved for advanced non-small cell lung cancer (NSCLC). The PDL-1 (programmed death ligand-1) antibody atezolizumab was also recently approved for NSCLC. Nivolumab is the first ICPi to be approved for relapse and refractory NSCLC and has been the mainstay of treatment for NSCLC since 2015. Pembrolizumab has been approved for first-line treatment of NSCLC since 2016 in Japan. A PD-L1 expression level of >50% is essential for the use of pemblolizumab [A few reports of re-challenge of ICPi have been published in patients with melanoma and involve ipilimumab monotherapy or combination therapy of ipilimumab and nivolumab , 3, 4, 5The aims of this study were to evaluate the safety and efficacy of re-challenge of ICPi in patients with advanced NSCLC and to potentially elucidate factors that could identify patients that would gain the most benefit from re-challenge of ICPi.The clinical characteristics of the patients  included in this study are shown in Table All patients received nivolumab for the initial treatment of ICPi. In the initial treatment of ICPi, 5 patients (45%) achieved a partial response (PR) and 2 patients (18%) was in stable disease (SD) (Table Ten patients (91%) were received cytotoxic chemotherapies between the initial ICPi and re-challenge treatment. One patient was not received any treatment. Eight in ten patients were given docetaxel based regimens and two patients were received radiation. Median duration from initial ICPi to re-challenge of ICPi was 4.2 (1.0-12.7) months.In the re-challenge of ICPi, ten patients were administered nivolumab and one was administered pembrolizumab. Among the total 11 patients, 3 patients 27%) achieved a PR and 2 patients (18%) were in SD (Table 7% achievThe frequencies of adverse events(AEs) and irAEs are shown in Table The most common irAEs were rash (n=4) and diarrhea (n=4) and liver dysfunction (n=2).Severe irAEs (grade > 3) were not seen in both courses. There was no patient who discontinued treatment due to AE or irAEs.ICPi have dramatically changed lung cancer treatment over the past years, demonstrating an overall survival benefit. The question of re-challenge with the same therapeutic approach regularly occurs in daily practice. We attempted to address this question for ICPi in patients with advanced NSCLC.In our study, five in eleven patients (45%) achieved PR or SD, which suggests that a re-challenge of ICPi might be a promising treatment. However, the question occurs as to in which patients would gain best benefit from re-challenge.One of the parameter which reflects the efficacy of re-challenge could be the response to the initial treatment with ICPi. In the report of re-challenge of ICPi in patients with melanoma, six in eight patients who responded well in the initial treatment revealed good response to the re-challenge . In our In the duration between the two ICPi treatments, eight of the ten patients received docetaxel-based regimens. An important factor in the use of cytotoxic anticancer drugs is immunogenic cell death (ICD), in which the cytotoxic anticancer drug or radiotherapy elicits an immune response leading to cell death of the tumor cells , 16, 17.Although the length of time required to build the immune response to ICPi remains unclear, we speculate that the establishment of acquired immunity could require more time than building of spontaneous immunity. Thus, the duration from the initial treatment to re-challenge might be important. In the responders to the re-challenge, the median duration from the initial treatment to the re-challenge was 1.6 months, whereas, non-responder was 4.7 months. This result reveals that the duration between the initial treatment to the re-challenge might be concerned to the effectiveness of re-challenge. We assumed that the building of immune response might have started by the initial treatment and still kept building even after the initial treatment failure. Therefore, when the re-challenge starts, the building of the immune response might have been completed and immune response might occur promptly. However, after some interval without ICPi, the immune response might become reset, thus the response might be worse after long interval drug withdrawal. Our data suggests that it may be feasible to restart the re-challenge within three months from the last of the initial treatment.The expression of PD-L1 influence the therapeutic effect of ICPi . PositivThis study has some limitations, including its retrospective design, a single facility, and small sample. Statistical analysis could not be done due to the small number of cases. Since the basic experiment has not been carried out, the detailed mechanism is unknown. Furthermore, we did not use immune-related response criteria because this was not a prospective trial and most physicians were not yet familiar with such criteria. We were also unable to examine the expression of PD-L1 before treatment for all patients because of the limitation described above.In conclusion, our findings suggest that re-challenge of ICPi is tolerable and safe. Further, re-challenge may be a promising treatment in some patients who responded well in the initial treatment or who developed irAEs in the initial treatment, and the shorter duration to re-challenge may bring better response to re-challenge. Thus, these patients should be considered for re-challenge. However, additional studies are needed to identify patient populations most likely to gain the best benefit from re-challenge of ICPi and to clarify the precise mechanisms of anticancer immune response.2) was administered every 2 weeks until the occurrence of disease progression, or unacceptable toxicity. After cessation of the initial treatment of nivolumab, patients were received nivolumab  or pembrolizumab  as re-challenge until the occurrence of disease progression, unacceptable toxicity, withdrawal, or death. This study was conducted in accordance with the Declaration of Helsinki and the requirements of the institution's review board.We conducted a retrospective study of patients with advanced NSCLC who were treated with single-agent nivolumab after platinum failure and were received re-challenge of nivolumab/pembrolozumab from December 2015 to December 2017 at Kansai Medical University Hospital, Osaka, Japan. The initial nivolumab (3 mg/mTreatment responses of the patients were evaluated according to the Response Evaluation Criteria in Solid Tumors using whole-body computed tomography performed every 8 to 12 weeks. However, because of the possibility of pseudo-progression in patients considered to have progressive disease, tumor size was carefully evaluated with reference to the Guidelines for the Evaluation of Immune Therapy Activity in Solid Tumors . The eva