exploratory research and hypothesis in medicine

EXPLORATORY RESEARCH AND HYPOTHESIS IN MEDICINE

CONTENTS 2018 3(4):73–86

Editorial

CRISPR/Cas9 in Human Research—A Call for Unity in Medical EthicsChristopher Brooks 73

Review Article

Death-associated Protein Kinase 1 Impairs the Hippocampo-prefrontal Cortical Circuit and Medi-ates Post-stroke DepressionXiao Ke, Sehui Ma, Yufen Zhang, Yao Yi, Hongyan Yu, Dian Yu, Lei Pei 74

Case Report

Lambl’s Excrescences in Congenital Heart Disease and Other Clinical Situations: A Series of 17 CasesJohn Jairo Araujo, Tareq Rahimy 79

Letter to the Editor

Autophagy Beyond Homeostasis: Does Autophagy Dysregulation Drive Colorectal Cancer? Com-ment on Shawn Gurwara et al; Altered Expression of Autophagy-related Genes in Human Colon CancerAnand Singh 84

Reviewer Acknowledgement

2018 Reviewer Acknowledgement 86

Exploratory Research and Hypothesis in Medicine 2018 vol. 3 | 73

Copyright: © 2018 Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 4.0 International License (CC BY-NC 4.0), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Editorial

CRISPR/Cas9 in Human Research—A Call for Unity in Medical Ethics

Christopher Brooks*

Stemline Therapeutics, Inc., New York, USA

The CRISPR-associated RNA-guided endonuclease Cas9 system has revolutionized the field of genetic editing, having almost un-limited potential in bioengineering and medicine. The ability to remove a pathophysiological abnormality through therapeutic in-tervention using the CRISPR/Cas9 system represents a true possi-bility of treating rare and complex genetic disorders. Since first be-ing described in 2012, more than 1,500 publications have reported the use of this technology in various scientific areas. At least 10 companies are currently exploring the utility of this type of gene editing in human diseases.

Recently, an investigator at the Southern University of Sci-ence and Technology in Shenzhen, China, Dr. Jiankui He, reported the first use of gene editing in humans, whereby he described the birth of twin girls lacking the CCR5 gene.1 As first reported at the Human Genome Editing Conference held in Hong Kong in late November of 2018, Dr. He described the as-yet-unsubstantiated results of using the CRISPR/Cas9 technology to genetically re-move the CCR5 gene from human embryos, resulting in live birth. Since then, the scientific and medical communities have largely denounced this work, notably including the Committee of Genome Editing of the Genetics Society of China and of the Chinese Soci-ety for Stem Cell Research.2

Although Dr. He’s work has not been confirmed either infor-mally or through peer-reviewed publication, it has raised serious concerns and important discussions on the ethics of human re-search with regard to gene editing and the urgent need for inter-national agreement on the ethical responsibilities that will follow in this field. The medical ethics of human embryo research have been debated for many years, but the report of a scientist pursuing genetic manipulation through human birth has escalated the need for establishing a firm commitment to strong ethical standards in medical research.

There have been great advances in the understanding of CRIS-

PR/Cas9 over a relatively short period of time; however, some studies, including one in 2016 describing genome damage, large deletions and genetic crossover events as a result of the gene edit-ing process, have served to underscore the reality that we do not yet fully understand the changes occurring and the potential patho-genic consequences of such events.3 Without complete knowledge of the process itself nor of the potential serious consequences of the end result, it is imperative that we, as a scientific community, pause the further use of gene editing of human embryos, at least until the knowledge base has progressed. The rogue use of CRIS-PR/Cas9 and failure of peer-reviewed oversight threatens the fun-damental basis of scientific research as a whole.

Research that pushes established boundaries in an ethical manner, that questions previously held dogma, and that constantly strives to improve and refine scientific knowledge represents the core aim of Exploratory Research and Hypotheses in Medicine. Yet, these types of explorations must be conducted in a methodical manner and not applied to human research until the consequences are fully under-stood and a consensus is established on acceptable standards. We are not there yet with the CRISPR/Cas9 technology, and until we are rogue human research must not happen: primum non nocere.

References

[1] MarchioneM.Chinese researcher claimsbirthof first gene-editedbabies—twingirls.AssociatedPress.November25,2018.

[2] WangH, Li J, LiW, Gao C,WeiW. CRISPR twins: a condemnationfrom Chinese academic societies. Nature 2018;564(7736):345.doi:10.1038/d41586-018-07777-0.

[3] Kosicki M, Tomberg K, Bradley A. Repair of double-strand breaksinduced by CRISPR-Cas9 leads to large deletions and complex re-arrangements. Nat Biotechnol 2018;36(8):765–771. doi:10.1038/nbt.4192.

Received: December 24, 2018; Accepted: December 24, 2018*Correspondence to: Christopher Brooks, Stemline Therapeutics, Inc., New York, USA. E-mail: [email protected] to cite this article: Brooks C. CRISPR/Cas9 in Human Research—A Call for Unity in Medical Ethics. Exploratory Research and Hypothesis in Medicine 2018;3(4):73. doi: 10.14218/ERHM.2018.00027.

http://creativecommons.org/licenses/by-nc/4.0/

Exploratory Research and Hypothesis in Medicine 2018 vol. 3 | 74–78


Review Article

Introduction

In the past three decades, stroke has become a major disease that seriously jeopardizes the health and life of China’s residents, and it has placed a heavy burden on patients, their families, and soci-ety. According to the newly released 2016 Stroke Epidemiology Report, there are 70 million stroke patients in China, including 2 million new stroke-suffering people per year; moreover, 67.3% to 80.5% of the stroke patients represent cases of ischemic stroke.1 A recent meta-analysis of 61 cohort studies showed that 31% of stroke survivors experienced post-stroke depression (PSD) with-

in 5 years.2 PSD is one of the common complications associated with stroke events, and exhibits a series of depressive symptoms and corresponding physical signs. In addition to the neurologi-cal deficits, PSD patients also have emotional disorders, such as self-blame and decreased interest. Severe cases may have suicidal thoughts. Delayed, intervention may lead to suicide.

A cohort study of 51,119 PSD cases found that the PSD patients had a significantly higher risk of mortality than healthy people. PSD seriously affects patients’ lives and places heavy burdens on their families and society.3 Regarding the pathogenesis of PSD, it is considered to be a direct result of specific injury of neuro-anatomical structure after stroke or an indirect consequence of negative psychological reactions in patients, and is believed to be the result of a combination of psychological, social and biological factors, such as the severity of stroke, brain damage, sensorimo-tor dysfunction, and cognitive impairment.4 Although the patho-genesis of PSD may be related to impairment of the monoamine system, dysfunction of hypothalamic pituitary adrenal (HPA) axis, destruction of prefrontal cortical circuit, imbalance of glutamate neurotransmitters and proinflammatory factors, the specific neural circuit and molecular mechanisms have not yet been elucidated.5

For clinical treatment of PSD, a meta-analysis of a total of 1,655 patients in 16 studies showed that antidepressants produced only a slight benefit, with statistically insignificant difference in

Death-associated Protein Kinase 1 Impairs the Hippocampo-prefrontal Cortical Circuit and Mediates Post-stroke Depression

Xiao Ke1,2, Sehui Ma1,2, Yufen Zhang1,2, Yao Yi1,2, Hongyan Yu1,2, Dian Yu1,2 and Lei Pei1,2*

1Department of Neurobiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China; 2The Institute for Brain Research (IBR), Collaborative Innovation Center for Brain Science,

Huazhong University of Science and Technology, Wuhan 430030, China

Abstract

Post-stroke depression (PSD) is a common and complex post-stroke neuropsychiatric disorder, which not only delays functional recovery but also increases mortality, and which currently lacks effective drug therapy. The pathogenesis of PSD is associated with impairment of the subcortical neural circuits and alterations of synaptic plasticity and neurotransmitters, but the exact mechanisms of PSD remain unknown. Our previous work indicates that the death-associated protein kinase 1 (DAPK1) mediates neuronal death after stroke. Genetic deletion of DAPK1 gene or blocking DAPK1 signal in the PSD mouse model can not only alleviate cerebral ischemic injury but also relieve PSD-like behaviors. Our previous work has also demonstrated the following results. First, the neural circuit of dorsal CA1 (dCA1) to medial prefrontal cortex (mPFC) (dCA1-mPFC) is selectively impaired after stroke. Second, the DAPK1 signal is involved in the impairment of dCA1-mPFC neural circuit after stroke. Third, genetic deletion of the DAPK1 gene or blocking of the DAPK1 signal alleviates the injury of dCA1-mPFC neural circuit after stroke and improves PSD-like behaviors. In conclusion, we hypothesize that activated DAPK1 signal after stroke induces apoptosis in the hippocampal dCA1 neurons, leading to loss of the dCA1-mPFC glutamatergic projections, synaptic injury, decrease of glutamate release, inhibition of mPFC neurons, and finally onset of PSD. We hope to further replenish the mechanisms of PSD and provide new insights for PSD treatment.

Keywords: DAPK1; Neural circuit; Synaptic injury; Post-stroke depression.Abbreviations: CaM, calmodulin; DAPK1, death-associated protein kinase 1; HPA, hypothalamic pituitary adrenal; mPFC, medial prefrontal cortex; PSD, post-stroke depression.Received: October 23, 2018; Revised: December 21, 2018; Accepted: December 21, 2018*Correspondence to: Lei Pei, Department of Neurobiology, School of Basic Medi-cine, Tongji Medical College, Huazhong University of Science and Technology, Wu-han 430030, China. E-mail: [email protected] to cite this article: Ke X, Ma S, Zhang Y, Yi Y, Yu H, Yu D, Pei L. Death-associated Protein Kinase 1 Impairs the Hippocampo-prefrontal Cortical Circuit and Mediates Post-stroke Depression. Exploratory Research and Hypothesis in Medicine 2018;3(4):74–78. doi: 10.14218/ERHM.2018.00018.


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Ke X. et al: DAPK1 and post-stroke depression Explor Res Hypothesis Med

efficacy. Even worse, antidepressants are liable to increase side effects on the gastrointestinal and central nervous systems, such as confusion, excessive sedation, and tremors. In addition, psy-chological intervention for PSD is also ineffective.6 PSD is closely related to the patients’ poor prognosis, in that it can delay hospital stay, affect recovery of neurological function, and cause loss of independent living ability and even death. However, specific and effective treatments for PSD are unavailable. Therefore, clarifying the pathogenesis of PSD and developing more effective preven-tion and treatment strategies are of great clinical significance for promoting functional rehabilitation and improving quality of life in PSD patients.

Ischemic stroke causes depression

In the 1980s, Robinson et al.7 found that reducing the cerebral blood supply in rats could decrease the concentration of catechola-mines and hormones, and lead to acute stress response. Due to the decrease of these neurotransmitters and hormones, the rats became slower on the wheel and the animals’ willingness to exercise was also reduced.7 In 2012, El Husseini et al.8 found that most patients with transient ischemic attack developed depression finally, as did stroke patients with functional impairment. A prospective study in China showed that about 30% of patients had depression at 2 weeks after onset of stroke, and the prevalence of PSD was 31% at 1 year.

The incidence of PSD is high, and the risk exists for a long time. Fifteen years after stroke, the prevalence of depression is still as high as 31.2%.9 A recent study funded by the Beijing Mu-nicipal Science and Technology Commission was conducted with 520 outpatients and hospitalized stroke patients. The incidence of PSD in this group was 34.2%, with mild cases accounting for 20.2% and moderate cases accounting for 10.4%. The prevalence of PSD was 39% at 1 month, 53% at 3–6 months, and 24% at 1 year after stroke, respectively. If the severity of depression is not distinguished and is collectively referred to as PSD, the incidence is 20–70%.10

The above studies show that ischemic stroke can cause depres-sion.

Hippocampal CA1 Injury in PSD

In ischemic stroke, hippocampal CA1 neurons are more sensitive to ischemic injury than other regions. By using a rodent model of global cerebral ischemia to simulate transient ischemic attacks, a previous study found selective and persistent death of pyrami-dal neurons in the hippocampal CA1 region, while hippocampal CA3, dentate gyrus, and most cortical neurons were unaffected. Transient cerebral ischemia caused by cardiac arrest or thora-cotomy can also cause selective and longer-lasting cell death in the hippocampal CA1 region.11 Recent studies have also found that some proteins are abnormally expressed in the hippocampal CA1 region, such as the hamartoma protein Tsc1.12 Mitochondrial dysfunction may be another reason for hippocampal CA1 region resistance to ischemia in stroke.13 In addition, a mouse model of forebrain ischemia also demonstrated persistent damage to neu-rons in the hippocampal CA1 area.14 Recently, we successfully established a PSD mouse model using light-induced forebrain ischemia,15 and we similarly observed degeneration of hippocam-pal CA1 neurons. The above studies collectively suggest that is-chemic stroke can cause selective neuronal damage in the hip-

pocampal CA1 area.Because the hippocampus has wide projections to other brain

regions that regulate mood and stress, it is considered to be a criti-cal regulatory site for depression,16 and the “hippocampal theory” has been proposed and has supplemented the “monoamine neuro-transmitter hypothesis”, which is a widely accepted theory for the pathogenesis of depression. Meanwhile, a large number of mag-netic resonance imaging studies have found reduced volume of hippocampus in PSD patients.17 Animal studies have also shown the reduction of apical dendrites in the hippocampal CA1 neu-rons. The decreased number of new neurons may be associated with hippocampal volume reduction and depression.18 Protecting hippocampal neurons or promoting neurogenesis could relieve symptoms of depression.19 Because the pyramidal neurons in the hippocampal CA1 area are mainly glutamatergic neurons, the de-crease of glutamate neurotransmitter caused by the loss of pyrami-dal neurons in the hippocampal CA1 area may lead to the occur-rence of PSD. In addition, the hippocampus has a certain inhibitory effect on the activity of the hypothalamic-pituitary-adrenal gland axis. Therefore, inhibition of the HPA axis is weakened after hip-pocampal injury,20 which leads to dysfunction of the HPA axis and induces depression finally.21 Thus, damage to the hippocampal CA1 area may be involved in the development of PSD.

Abnormal hippocampo-prefrontal cortical circuit causes de-pression

Imaging of the patients’ brain with depression and histological examination of post-mortem brain have revealed abnormalities in the prefrontal cortex, cingulate gyrus, hippocampus, striatum, and almonds.22 At present, many brain regions have been report-ed to be involved in the pathophysiology of depression. First, deep brain electrical stimulation at the anterior cingulate cortex and nucleus accumbens exhibits an antidepressant-like effect on individuals with refractory depression. This effect is thought to inhibit the activity of the brain region through blockade of the depolarization of axonal fibers.23 Second, dopaminergic neural projections in the midbrain (ventral tegmental area to nucleus accumbens) increased activity-dependent release of brain de-rived neurotrophic factor, which mediates susceptibility to social stress. This effect may be partially produced by phosphorylation of the transcription factor cAMP response element binding pro-tein.24 Third, the decreased concentration of neurotrophic factors (such as brain derived neurotrophic factor) reduces the degree of hippocampal nerve regeneration and neuronal processing and complexity. These effects are partly produced by increasing cor-tisol concentration and decreasing the activity of the cAMP re-sponse element binding protein.25 Fourth, peripherally released metabolic hormones, such as ghrelin and leptin, can produce mood-related alterations by acting on the hypothalamus and sev-eral regions of the limbic system (such as hippocampus, ventral tegmental area, and nucleus accumbens).26

In addition, a large number of autopsy and neuro-imaging stud-ies have found that patients with depression have reduced gray matter volume and decreased nerve fiber density in the prefrontal cortex and hippocampus, while the intermediate prefrontal cortex (mPFC) and hippocampus are critical brain regions thought to be involved in depression,27 and mPFC receives abundant synaptic projections from the hippocampal CA1 region. Therefore, it is speculated that the CA1-mPFC circuit participates in the cognitive function of depression. The study also found that mPFC mainly receives glutamatergic projection fibers from the CA1 area. In the

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Ke X. et al: DAPK1 and post-stroke depressionExplor Res Hypothesis Med

rat model of depression, the synaptic plasticity of the CA1-mPFC circuit was also found to be impaired.14,28 Our preliminary results also found the loss of dCA1-mPFC projection fibers in the is-chemic stroke mouse model. In summary, impaired hippocampal-prefrontal circuit caused by ischemic stroke can cause depression.

Ischemic stroke activates the death-associated protein kinase 1 (DAPK1) death signal in the hippocampal CA1 area and in-duces PSD

Our previous study found that DAPK1 interacts with the excitatory glutamate receptor (GluN2B subunit) and induces neuronal cell death in a mouse model of ischemic stroke, which demonstrates that DAPK1 is a pivotal molecule in neuronal death after ischemic stroke.29,30 DAPK1 is a Ca2+/calmodulin (CaM)-dependent serine/threonine protein kinase, consisting of a kinase domain, a CaM-binding motif, eight repeats of anchored proteins, and a death do-main. The CaM-binding motif contains binding sites and a self-in-hibition area.31 Binding of DAPK1 to the CaM-binding site results in DAPK1 conformational changes, reactivation of CaM blocked active sites, and DAPK1 activation.32 Previous studies have shown that activated DAPK1 is involved in tumor cell death in lymphoma. DAPK1-induced apoptosis has been reported as mainly modulated by Fas, tumor necrosis factor,33 apoptotic protease,34 and p53.35

DAPK1 is associated with nerve injury and aging-related neu-rological diseases, such as stroke, epilepsy, and Alzheimer’s dis-ease. Recently, we reported that DAPK1 activation is involved in the impairments of synaptic transmission, spatial learning and memory.36 Notably, DAPK1 also mediates depression-like symp-toms in chronic stress. Inhibiting DAPK1 signal by genetic de-letion of the DAPK1 gene or pharmacological interruption with an interfering peptide that specifically blocks DAPK1 binding to the GluN2B subunit, both reverse molecular changes and synap-tic protein loss caused by chronic stress in the prefrontal cortex, and finally exert rapid and long-lasting antidepressant effects.37 In addition, DAPK1 mediates synaptic long-term depression by preventing the binding of Ca2+/CaM-dependent protein kinase II with GluN2B, which in turn affects learning, and cognitive and emotional functions.38 These studies have collectively suggested that the DAPK1 death signal may mediate the development of depression-like symptoms after ischemic stroke.

Recently, we successfully established a PSD mouse model by using light-induced forebrain ischemia,15 and further detected pro-tein-protein interactions in the hippocampal CA1 region via mass spectrometry. We found that DAPK1 interacted with caytaxin, which was firstly reported in 2003 by Bomar and his colleagues.39 This finding revealed that mutations in the ATCAY gene encoding caytaxin caused Cayman ataxia, an autosomal recessive disorder characterized by poor muscle coordination, mental retardation, loss of head control and eye movements, and difficulties in speak-ing and walking. In addition, caytaxin is abundantly expressed in neurons of the cortex, cerebellum, hippocampus, olfactory bulb, and basal ganglia.33

Akamatsu and colleagues used full-length caytaxin as a bait to perform yeast two-hybridization with mouse adult brain cDNA,40 and found that caytaxin can drive the protein light chain of ki-nesin-1 through its N-terminal ELEWED sequence (amino acids 115–120) and participate in axonal transportation. Kinesin, a key molecule for axonal transportation, can be combined with a vari-ety of linker molecules, such as TRAK1/2 and JIP1, to regulate mitochondrial axonal transportation of amyloid precursor protein. Studies have also shown that caytaxin can act as a linker molecule

for kinesin, and transport mitochondria along the axons to neu-rites, which provide the required energy for various biological processes, such as presynaptic vesicle fusion and neurotransmit-ter release.34 Further, it has been indicated that caytaxin plays an important role in mitochondrial axonal transportation and the syn-aptic energy supply.

Our recent study demonstrated that DAPK1 interacts with cay-taxin, causing apoptosis of hippocampal CA1 neurons. In cultured primary neurons, we employed virus co-transfected DAPK1-re-lated plasmids (activated DAPK1 (ΔCaM) and inactive DAPK1 (K42A)) with caytaxin. Mitochondria staining showed that ΔCaM decreased the number of mitochondria in the neuronal processes, as compared with K42A. However, co-transfected ΔCaM with mu-tant caytaxin (S46A) showed alleviation of mitochondrial reduc-tion. In addition, co-transfection of ΔCaM and wild-type caytaxin caused a relative decrease both in the frequency and amplitude of miniature excitatory post-synaptic currents, suggesting that the serine 46 of caytaxin is essential for DAPK1-induced presynaptic energy deficiency and synaptic dysfunction.

Based on the above studies, we hypothesized that after ischemic stroke, activated DAPK1 causes phosphorylation of the caytaxin protein at serine 46 (pS46), which interrupts the formation of mi-tochondrial axonal transport complexes, leading to presynaptic mitochondrial reduction, following energy deprivation, then caus-ing impairment of synaptic transmission, and ultimately induc-ing neuronal apoptosis. Using the conditional DAPK1 knockout (DAPK1−/−) mice, we found that the apoptosis of hippocampal dCA1 neurons was decreased and that the depression-like symp-toms were effectively alleviated in PSD mice. In addition, the membrane-permeable small molecule polypeptide Tat-CTX com-posed of caytaxin (43-EDSSSPPST-51) was synthesized to block the binding of the DAPK1 to caytaxin, and to reduce apoptosis of hippocampal dCA1 neurons and alleviate PSD-like symptoms in mice.

Conclusions

In conclusion, based on previous studies, we hypothesize that acti-vated DAPK1 signal after stroke induces apoptosis in hippocampal dCA1 neurons, which leads to loss of dCA1-mPFC glutamatergic projections, synaptic injury, decrease of glutamate release, inhibi-tion of mPFC neurons, and causes onset of PSD finally. By ge-netically knocking out the DAPK1 gene (DAPK1−/−) or blocking DAPK1 death signal with exogenous peptide may be possible to inhibit the apoptosis of dCA1 neurons and reduce the impairment of dCA1-mPFC neural circuit, thereby alleviating PSD (Fig. 1).

Future research directions

The proposed hypothesis has important theoretical significance for elucidating the molecular and neural circuit mechanisms of PSD, and will provide new insights for the treatment of PSD. In the fu-ture, the following issues need to be further verified. The first is clarifying the molecular mechanism of DAPK1 death signal medi-ating the decrease of dCA1-mPFC glutamatergic projection after ischemic stroke. The second is revealing the reasons behind the differences between PSD animals and non-PSD animals. And, the last but not least involves information that will benefit the treat-ment of PSD, such as determining the efficacy and safety of the exogenous peptide Tat-CTX that interferes with the DAPK1 death signal.


Ke X. et al: DAPK1 and post-stroke depression Explor Res Hypothesis Med

Acknowledgments

This work was supported by the National Natural Science Foun-dation of China (Grant Nos. 81571078 and 81870932) and the Medjaden Academy & Research Foundation for Young Scientists (Grant No. MJR20160057).

Conflict of interest

The authors declare they have no conflict of interests related to this publication.

Author contributions

Designed the study (LP), drafted the manuscript (XK), contributed to the critical revision of the manuscript for important intellectual content (SM, YZ, YY, HY and DY). All authors approved the ver-sion to be published.

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Fig. 1. Schematic diagram of the hypothesis. The neural circuit and molecular mechanism of post-stroke depression (PSD). (a) Normal conditions; (b) Stroke conditions.


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[33] Cohen O, Inbal B, Kissil JL, Raveh T, Berissi H, Spivak-Kroizaman T, et al. DAP-kinase participates in TNF-alpha- and Fas-induced apoptosis and its function requires the death domain. J Cell Biol 1999;146(1):141–148. doi:10.1083/jcb.146.1.141.

[34] Jin Y, Gallagher PJ. Antisense depletion of death-associated protein kinase promotes apoptosis. J Biol Chem 2003;278(51):51587–5193. doi:10.1074/jbc.m309165200.

[35] Raveh T, Droguett G, Horwitz MS, DePinho RA, Kimchi A. DAP ki-nase activates a p19ARF/p53-mediated apoptotic checkpoint to suppress oncogenic transformation. Nat Cell Biol 2001;3(1):1–7. doi:10.1038/35050500.

[36] Zhu H, Yan H, Tang N, Li X, Pang P, Li H, et al. Impairments of spa-tial memory in an Alzheimer’s disease model via degeneration of hippocampal cholinergic synapses. Nat Commun 2017;8(1):1676. doi:10.1038/s41467-017-01943-0.

[37] Li SX, Han Y, Xu LZ, Yuan K, Zhang RX, Sun CY, et al. Uncoupling DAPK1 from NMDA receptor GluN2B subunit exerts rapid antidepressant-like effects. Mol Psychiatry 2018;23(3):597–608. doi:10.1038/mp.2017.85.

[38] Goodell DJ, Zaegel V, Coultrap SJ, Hell JW, Bayer KU. DAPK1 medi-ates LTD by making CaMKII/GluN2B binding LTP specific. Cell Rep 2017;19(11):2231–2243. doi:10.1016/j.celrep.2017.05.068.

[39] Bomar JM, Benke PJ, Slattery EL, Puttagunta R, Taylor LP, Seong E, et al. Mutations in a novel gene encoding a CRAL-TRIO domain cause human Cayman ataxia and ataxia/dystonia in the jittery mouse. Nat Genet 2003;35(3):264–269. doi:10.1038/ng1255.

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Case Report

Introduction

Lambl’s excrescences (LEx), also referred to as valvular strands, are thin, filiform and hypermobile processes first described on the aortic valve (AV) by Bohemian physician Vilem Dusan Lambl in 1856.1 They begin as small thrombi on the coaptation points of the endocardial surfaces and have the potential to embolize to distant organs and cause stroke or myocardial infarction. These strands are almost exclusively seen on the left side of the heart at the valve closure site, where there is endothelial damage due to wear and tear. However, they can occur anywhere on the semilunar valves and they are found in natural or prosthetic valves.2,3

LEx are most frequent in adults over 60 years-old, but pediatric cases have also been described. Phillips et al.4 reported a low prev-alence of only 1.7% in 700 pediatric patients assessed with tran-

sthoracic echocardiography (TTE). In general, they are benign and do not require intervention or more aggressive invasive imaging. LEx can occur as singular or multiple strands. They are referred to as ‘giant’ LEx if they form a complex that is greater than or equal to 2 cm in diameter; the literature associates these giant LEx with ischemic stroke, angina, and even myocardial infarction.5,6 They are also described in association with rheumatic valvulitis, aortic stenosis, and bicuspid AV.7 Differential diagnoses for LEx include myxomas, thrombi, cardiac neoplasms, vegetations and metasta-ses. From a histologic standpoint, LEx are similar to cardiac papil-lary fibroelastomas. Specifically, they are composed of fibroelastic and hyalinized stroma covered by a layer of endothelial cell lin-ing.8

Materials and methods

We present here 17 consecutive cases (8 females), with a mean age of 23.7 years (0–76 years), who were examined by the cardiology department of a Level III hospital, and who, for various reasons (Table 1), underwent echocardiography [14 transthoracic echocar-diograms (TTEs) and 3 transesophageal echocardiograms (TEEs), all performed by the same expert examiner]. A multifrequency transducer (3–7 MHz) was used in a parasternal long axis echo-cardiographic view of the left ventricle, focusing the study on the outflow tract and AV, with zoom images and second harmonic im-aging for the adults. In the TEE cases, a 14-mm multiplane probe (4–7 MHz) was used in the medial transesophageal plane with a 135° angulation over the left ventricular outflow tract and AV. Both studies used the same equipment, namely the Vivid S5 ultrasound

Lambl’s Excrescences in Congenital Heart Disease and Other Clinical Situations: A Series of 17 Cases

John Jairo Araujo1* and Tareq Rahimy2

1Vall d’Hebron University Hospital, Barcelona, Catalonia, Spain; Department of Clinic and Research Cardiology, Meintegral Clinic, Colombia; 2University of zagreb school of medicine; Pilgrim Hospital, UK

Abstract

Lambl’s excrescences have a low prevalence in the general population. Although they occur most frequently in adults over 60 years-old, pediatric cases have been described. The cases in adulthood are associated with ischem-ic stroke, but in childhood they are asymptomatic. The aim of reporting on this case series is to show the associa-tion or coexistence of Lambl’s excrescences with some congenital heart diseases (CHDs), of which there are no known descriptive case series in adults or children. We present 17 patients (8 females), with a mean age of 23.7 years; among these cases, 64.7% were under 18 years-old. We found that 94% of the Lambl’s excrescences were located on the aortic valve. In 47% (8 cases), they coincided with a CHD (with 6 of those individuals being under 18 years-old). We propose the hypothesis that Lambl’s excrescences could have a congenital origin or coexist with CHD. No complications were found throughout the follow-up. Lambl’s excrescences could be more frequent than currently reported in the literature, and more research should be done on their significance in CHD-associated stroke.

Keywords: Lambl’s excrescences; Congenital heart disease; Stroke.Abbreviations: CHD, congenital heart disease; CHDs, congenital heart diseases; LEx, Lambl’s excrescences; AV, aortic valve; AVs, aortic valves; TTE, transthoracic echocardiography; TTEs, transthoracic echocardiograms; TEE, transesophageal echocardiogram; TEEs, transesophageal echocardiograms; MV, mitral valve; VSD, ventricular septal defect.Received: October 07, 2018; Revised: December 12, 2018; Accepted: December 13, 2018*Correspondence to: John Jairo Araujo, Department of Clinic and Research Cardiol-ogy. Meintegral Clinic, Colombia, Postal code: 0956. Tel: +5768740201; E-mail: [email protected] to cite this article: Araujo JJ, Rahimy T. Lambl’s Excrescences in Congeni-tal Heart Disease and Other Clinical Situations: A Series of 17 Cases. Exploratory Research and Hypothesis in Medicine 2018;3(4):79–83. doi: 10.14218/ERHM.2018. 00016.



Araujo JJ. et al: Lambl’s excrescences in congenital heart diseaseExplor Res Hypothesis Med

Table 1. Demographic, anatomical and echocardiographic variables

Case Sex Age Reason Echo Location Main diagnoses Management Follow-up outcome

1 M 14 murmur TTE aortic valve minimal aortic valve regurgitation

echocardiographic surveillance

asymptomatic at 12 month follow-up

2 F 12 syncope TTE aortic valve none echocardiographic surveillance


3 F 26 pulmonary hypertension

TTE aortic valve DORV + subaortic VSD + pulmonary hypertension

specific management for pulmonary hypertension sildenafil + bosentan, echocardiographic surveillance

Eisenmenger syndrome, no neurological event at 18 month follow-up

4 M 32 septic shock TEE aortic valve Non-stenotic bicuspid valve

aspirin 100 mg, echocardiographic surveillance

asymptomatic, no neurological event at 14 month follow-up

5 F 40 syncope TTE aortic valve minimal aortic valve regurgitation

aspirin 100 mg, atorvastatin 20 mg


6 F 4 ventricular septal defect

TTE aortic valve restrictive perimembranous septal defect

aortic regurgitation, surgical repair VSD echocardiographic surveillance

minimal aortic valve regurgitation at 8 month follow-up

7 F 74 mitral valve regurgitation

TEE aortic valve moderate functional mitral regurgitation, heart failure

surgical valve replacement, anticoagulation warfarin therapy, echocardiographic surveillance


8 M 9 history of Kawasaki disease

TTE aortic valve none aspirin 3 mg/kg/day, echocardiographic surveillance


9 M 11 murmur TTE aortic valve minimal aortic valve regurgitation



10 F 12 endocarditis TTE mitral biologic valve prosthesis

non-endocarditis mitral biologic valve prosthesis

aspirin 3 mg/kg/day, echocardiographic surveillance


11 M 46 leukemia TTE aortic valve none echocardiographic surveillance


12 M 0 cyanosis TTE aortic valve tetralogy of Fallot surgical repair at 6 months of age

no data yet

13 M 15 bicuspid aortic valve

TTE aortic valve non-stenotic bicuspid aortic valve

aspirin 100 mg, echocardiographic surveillance


14 M 16 syncope TTE aortic valve bicuspid valve, late repair of aortic coarctation



15 F 8 Fontan dysfunction evaluation

TTE aortic valve dilated left ventricle , sinus node dysfunction, minimal aortic valve regurgitation

aspirin 3 mg/kg/day, echocardiographic surveillance


16 M 8 aortic coarctation

TTE aortic valve aotic coarctation aortic angioplasty, echocardiographic surveillance


17 F 76 suspected endocarditis

TEE aortic valve minimal aortic valve regurgitation

aspirin 81 mg, atorvastain 20 mg, echocardiographic surveillance

asymptomatic, no neurological event at 15 month follow-up

Age: years; (case 12: 1 month old newborn); TTE: transthoracic echocardiogram; TEE: transesophageal echocardiogram; DORV: double outlet right ventricle; VSD: ventricular septal defect.


Araujo JJ. et al: Lambl’s excrescences in congenital heart disease Explor Res Hypothesis Med

machine (GE Healthcare, Chicago, IL, USA). Differential diagno-ses were ruled out, including laminar thrombi, endocarditis, and papillary fibroelastomas, among others.

Results

It is possible that the greater spatial and temporal resolution of the diagnostic equipment used, the transducer frequency, the use of windows with echocardiographic zoom, the experience of the ex-aminer, and having exams focused on the left ventricular outflow tract, allowed for the incidental diagnosis of LEx (Fig. 1). Of the 17 cases presented, 64.7% were under 18 years of age. In 94% of the cases, the LEx were located on the AV; only one was on the biological mitral valve (MV). Forty-seven percent (8 cases) had CHDs [2: ventricular septal defect (VSD); 1: tetralogy of Fallot; 2: bicuspid AV; 1: MV repair with biologic prosthesis; 1: tricuspid atresia and Fontan circulation; and 1: aortic coarctation).

There was no direct relationship between the reason for the study and the finding of LEx in any of the pediatric cases; it was a 100% incidental finding. In fact, in two cases of syncope evalua-tion, the finding of LEx was not expected, given its very low fre-quency in the pediatric population. In two of the pediatric cases, innocent murmurs were correlated with minimal AV regurgitation, and when the exam was focused on this structure, LEx was diag-nosed. In only one adult case was the exam directed towards find-ing the cause of syncope in the AV, but this diagnosis was not ex-pected and neither was it the direct cause of syncope. Cases of LEx associated with CHDs were found very close to the VSD and left ventricular outflow tract. Perhaps the reason for this is that with proximity to the VSD, hemodynamic shunt forces cause stress and damage to the surface of the aortic vellums and the development of LEx. Or, perhaps, LEx can be considered to also be a congenital

defect which accompanies these CHDs (Fig. 2).Treatment was conservative in most cases; seven individuals re-

ceived aspirin as preventive treatment (4: children; and 3: adults). The pediatric patients were medicated with aspirin due to their CHD. The adults were medicated with aspirin alone or in combi-nation with atorvastatin, according to their risk factors. There was no ictus in either group on follow-up. In patients who underwent corrective surgery (CHD, valvular disease), there were no surgical reports of LEx resection (Table 1).

Discussion

In 1856, Lambl first described small filiform processes on the ven-tricular surface of normal and abnormal AVs. In subsequent patho-logic series of normal valves, a 70% to 90% prevalence of LEx was reported, predominantly on the MV (70% to 85%), followed by the AV (62% to 90%) and the right-sided valves (8% to 20%).9 Recently, Osorio et al.10 analyzed 33 cases of LEx, and reported the affected valve was AV in 75% (25/33), followed by MV in 17% (6/33) and pulmonary valve in 2.8% (1/33). Salah et al.11 analyzed the cases published from 1981 to 2018; in that series, the affected valve was AV in 89% and MV in 7%, with one case in the pulmo-nary valve. Our series’ results are similar and congruent with those reported, with a high incidence on the AV.

The etiology of LEx is uncertain; the constant bending and buckling of leaflets can cause tearing of subendocardial collagen and elastic fibers, with subsequent formation of a small throm-bus, fibrin deposit, inflammation and fibrosis. Matsukuma et al.,12 reviewed autopsy files and retrieved a total of 126 cases of AVs without infectious endocarditis. Of these, 73 AVs were selected from patients without AV dysfunction and the remaining 53 were retrieved from patients with AV dysfunction, including aortic ste-

Fig. 1. Cases of LEx. (a–b) A 14 year-old male; long axis view of the aortic valve, with zoom (the LEx measured 5 mm) and short and long axis views of the aortic valve with minimal aortic valve regurgitation. (c) A 4 year-old female with VSD. Apical four chamber view (LEx in the aortic valve and comparative color) and showing minimal aortic valve regurgitation. (d) A 9 year-old male with a history of Kawasaki disease; LEx in the aortic valve. Abbreviations: LEx, Lambl’s excrescences; VSD, ventricular septal defect.


Araujo JJ. et al: Lambl’s excrescences in congenital heart diseaseExplor Res Hypothesis Med

nosis and aortic regurgitation. The results showed LEx [classical and/or non-ex LEx in 106 (84%)]. Out of these, 88 (70%) had fea-tures of classical LEx, 78 (62%) had features of non-ex LEx and 60 AVs (48%) had features consistent with both classical and non-ex LEx. In the present study, the incidence of LEx was calculated to be 84%, which is consistent with the previous studies. This study also further highlighted that non-ex LEx, which has previously re-ceived little attention, is more common than expected, especially in markedly deformed AVs. AV dysfunction does not seem to in-fluence the development of LEx but the morphological changes associated with AV dysfunction can foster non-ex LEx.12

In the adult, the embolic risk of valvular LEx was evaluated by Roldan et al.13 in a prospective study published in 2015; this study involved 90 healthy subjects and 88 patients with or without suspected cardioembolism and followed the cases clinically for ap-proximately 4 years. The prevalence of LEx in healthy subjects (38%) and in patients with (47%) or without (41%) suspected car-dioembolism was similar, irrespective of age or sex, and the pres-ence of LEx did not appear to be associated with future embolic events. These findings suggested that there may not be a direct causal link between ischemic stroke and the presence of the valve strands.13

A review of the current literature shows many different views on the clinical importance, pathophysiology, association with is-chemic events and management of LEx. However, there is a gen-eral consensus amongst most of the authors that LEx should be considered in the differential diagnosis of patients who present with signs of cardioembolism, until conclusive evidence suggests otherwise. The experts recommend performing a TEE on these pa-tients and treating them with dual antiplatelet therapy if LEx is found to be present. However, no definitive guidelines currently direct the management of LEx, and management is largely based upon case reports in the literature.

Most authors agree that the choice between surgical interven-

tion versus conservative observation, including anticoagulation or antiplatelet therapy, needs to be decided on a case-by-case basis and should be based on the patient’s pre-operative risk stratifica-tion.14,15 Some experts recommend close follow-up with serial echocardiograms every 6 to 12 months for asymptomatic patients with LEx; children can also be included in this group. For patients with LEx who have experienced a stroke with no alternative source of emboli, treatment with anticoagulation (warfarin therapy) or an-tiplatelet therapy (aspirin, 81 – 200 mg per day, clopidrogrel 75 mg per day) has been suggested and reported in several cases. In other reports, dabigatran or rivaroxaban has been administered.16,17 Fi-nally, in patients with LEx who experience recurrent strokes while on anticoagulation, a surgical debridement of the LEx can be con-sidered.

Chu et al.18 analyzed 20 LEx cases. Nine were referred for surgical resection, with the following indications: primary or re-current ischemic stroke, angina, weakness and fatigue, headache, mitral localization in papillary muscle and chordae tendineae, and incidental finding. In the cases analyzed by Osorio et al.,10 the 2 month to 3 year follow-up period did not show any cases of recur-rence of LEx or stroke (only one stroke case was reported, but it was not related to LEx). However, there is as yet no clear guide in CHD, because LEx are infrequent and there are not enough cases currently reported. Our impartial and critical opinion is to perform surgical resection at the same time as the CHD is repaired. Con-servative management was the treatment of choice in 14/17 of the cases in our series; surgery was performed in only three cases, but for other reasons. However, the following hypothesis arise: 1. Can LEx be an isolated congenital defect? 2. Do LEx occur in associa-tion with CHD?

The low prevalence of LEx in the general population, and even more so in the pediatric population, gives rise to the following questions: What is the importance of knowing the diagnosis in childhood, given that relationship with cryptogenic ictus in adult-

Fig. 2. Cases of LEx. (a–b) A 15 year-old male; short axis view of the bicuspid aortic valve (a) and long axis view focusing on the outflow tract and aortic valve, with zoom. The yellow arrow shows LEx. (c) A 1 month-old newborn with tetralogy of Fallot; long axis view.Green arrow shows LEx in aortic valve. (d) A 26 year-old female with double outlet right ventricle. The yellow arrow shows LEx in aortic valve. Abbreviations: LEx, Lambl’s excrescences; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle.


Araujo JJ. et al: Lambl’s excrescences in congenital heart disease Explor Res Hypothesis Med

hood is known? Is it important to routinely look for LEx if they are asymptomatic in pediatric patients and become less visible as the children grow (lower echocardiographic resolution due to a diffi-cult acoustic window)? The best recommendation is to report these findings in the echocardiographic studies performed and follow-up during childhood, adolescence and adulthood, evaluating the risk factors and following the recommendations of the experts to initi-ate anticoagulation or antiplatelet therapy.

The importance of this case series report is the coincidence with CHD; there are no known descriptive case series in adults or chil-dren. This report also raises questions such as: What would be the best course of action when LEx are found? Should they be resected during CHD surgical repair? The real dilemma lies in that, due to their low frequency, they are not routinely looked for, CHD repair focuses only on the cardiac defect, and LEx may remain as a per-sistent residual following CHD repair.

Conclusions

This case series leads us to think that LEx cases are really much more prevalent than currently reported in the literature. Further-more, they are associated with CHD and may be of some impor-tance in the cause of CHD-associated stroke. Many times, patients with unrepaired and repaired CHD have an embolic stroke as a result of CHD residuals (e.g., residual VSD following atrioven-tricular canal repair). The fact that LEx and CHD are an associa-tion that heightens the risk of stroke in this population arouses our research interest.

These case descriptions motivate the search for LEx related to CHD, and pose the question of whether their origin is also con-genital or rather a direct consequence of the classic description of natural valvular wear and tear, increased by the hemodynamic effects of CHD. At present, there are no large series or controlled studies which can answer these questions. However, this contribu-tion of cases will undoubtedly awaken the interest of more expert investigators.

Acknowledgments

There was no private or public sector funding for this research.


The authors have no conflict of interests related to this publication.


John Jairo Araujo: collected the data, performed echocardiograms, wrote the paper; Tareq Rahimy: collected the data, wrote the paper.

References

[1] Lambl VD. Papillare exkreszenzen an der semilunar-klappe der aorta. Wien Med Wochenschr 1856;6:244–247.

[2] Aziz F, Baciewicz FA Jr. Lambl’s excrescences: review and recommen-dations. Tex Heart Inst J 2007;34(3):366–368.

[3] Kamran H, Patel N, Singh G, Pasricha V, Salifu M, McFarlane SI. Lam-bl’s excrescences: A case report and review of the literature. Clin Case Rep Rev 2016;2(7):486–488. doi:10.15761/ccrr.1000254.

[4] Phillips A, Qureshi M, Eidem B, Cetta F. Lambl’s excrescences in chil-dren: Improved detection via transthoracic echocardiography. Con-genit Heart Dis 2018;13(2):251–253. doi:10.1111/chd.12560.

[5] Yacoub H, Walsh A, Pineda C. Cardioembolic stroke secondary to lambl’s excrescence on the aortic valve: a case report. J Vasc Interv Neurol 2014;7(3):23–25.

[6] Pizzuti A, Parisi F, Mosso L, Cali’ Quaglia F, Tomasello A. Acute myocardial infarction in a patient with two-vessel oclusion and a large lambl’s excrescence. Case Rep Cardiol 2016:8370212. doi:10.1155/2016/8370212.

[7] Ozturk C, Haqmal H, Balta S, Demirkol S, Unlu M, Koklu M, et al. The association of severe aortic stenosis and narrow aortic root in a young patient—What is the etiology: Rheumatic valvulitis or lambl’s excrescences? Int J Cardiol 2016;208:87–91. doi:10.1016/j.ijcard.2016.01.018.

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[13] Roldan C, Schevchuck O, Tolstrup K, Roldan P, Macias L, Qualls C, et al. Lambl’s excrescences: association with cerebrovascu-lar disease and pathogenesis. Cerebrovasc Dis 2015;40(0):18–27. doi:10.1159/000381906.

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Letter to the Editor

Autophagy Beyond Homeostasis: Does Autophagy Dysregulation Drive Colorectal Cancer? Comment on Shawn Gurwara et al;

Altered Expression of Autophagy-related Genes in Human Colon Cancer

Anand Singh*

Thoracic Surgery Branch, National Cancer Institute – NIH Center for Cancer Research, 10 Center Drive, Room 3-5888W, Bethesda, MD 20892 USA

Autophagy is an evolutionarily conserved and regulated catabolic process that ensures the degradation of damaged organelles and excessive or misfolded proteins in lysosomes for the recycling of essential amino acids and energy.1 Autophagy is constitutively ac-tive at basal level in a majority of cell types and plays an important role in cellular homeostasis via maintaining cell organelles’ and proteins’ turnover as well as their functions. In addition to the ho-meostatic function, autophagy also acts as a pro-survival pathway and can be activated in response to different stress conditions, such as nutrient starvation, organelle damage, metabolic stress, endo-plasmic reticulum stress, accumulation of non-functional proteins, and radiation or chemotherapy treatment. Recent studies have demonstrated that dysregulation of autophagy pathways may be involved in the pathogenesis of diverse diseases, such as myopa-thy, neurodegeneration, aging, microbial infection, inflammatory bowel disease, and cancer.2

In cancer, the role of autophagy is quite complicated and de-pends on tumor type and stage. During the early stage of tumori-genesis, autophagy usually acts as a tumor suppressor by clearing the damaged cellular content, reducing reactive oxygen species’ level and thereby DNA damage. Whereas in established tumors or an advanced stage of the tumor, it may act as a tumor promoter, as tumor cells utilize enhanced autophagy to survive under low oxy-gen and low nutrient conditions. The core machinery of autophagy is encoded by autophagy-related genes (ATGs). As of now, 36 ATG genes have been identified. Recent studies have revealed that modulation of such autophagy genes as BECN1 (ATG6), ATG5, ATG7, ATG16L1, etc. are associated with the pathogenesis of vari-ous cancers.1,3

In the current issue of Exploratory Research and Hypothesis in Medicine, Shawn Gurwara et al. has reported that dysregula-tion of autophagy pathways could be a leading mechanism in the carcinogenesis of human colorectal cancer. In their study, it was observed that ATGs were downregulated in both early- and late-stage colon cancer compared to normal colon mucosa. Although

the sample size of this study was small, 17 ATGs were found to be significantly downregulated in human colon cancer; these in-clude ATG4A, ATG4C, ATG4D, CTSD, CTSS, ESR1, GAA, and GABARAP. Further, the authors confirmed the similar mRNA ex-pression profile for ATG4A, ATG4C, ATG4D, GAA, GABARAP, CTSD, and CTSS in a large TCGA dataset of colon adenocarci-noma and rectum adenocarcinoma (known as COAD/READ) patients. Though this study claims the possible role of ATGs in colon cancer, the limitations of this study are its small cohort, which includes only six tumor specimens, three for each early and late colon cancers, and the lack of protein expression data for the dysregulated ATGs.

The autophagy pathway is mediated by many ATGs, among which the proteases of ATG4 family play an important role in the conjugation of ATG8 to lipid membranes and the deconjugation of ATG8 from the autophagosome. The activity of ATG4 family genes is critical and highly specific for targeting and autophagic vacuole formation. Recent studies by others have demonstrated the antitumor role of ATG4C and ATG4D in fibrosarcoma and breast cancer.4,5 In agreement with previous studies, the findings by Gurwara et al. provide new insights into the pathogenesis of colon cancer and suggest the tumor suppressor role of autophagy in colon cancer. In the future, it will be interesting to study the role of these dysregulated ATGs in in vitro systems as well as in vivo colon cancer models for the development of new therapeutic regimens.


The author has no conflict of interests related to this publication.


Manuscript writing (AS).

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Received: October 30, 2018; Revised: November 10, 2018; Accepted: December 10, 2018*Correspondence to: Anand Singh, Thoracic Surgery Branch, National Cancer Insti-tute - NIH Center for Cancer Research, 10 Center Drive, Room 3-5888W, Bethesda, MD 20892 USA. Tel: 240-8583-874 (desk); E-mail: [email protected] to cite this article: Singh A. Autophagy Beyond Homeostasis: Does Au-tophagy Dysregulation Drive Colorectal Cancer? Comment on Shawn Gurwara et al; Altered Expression of Autophagy-related Genes in Human Colon Cancer. Ex-ploratory Research and Hypothesis in Medicine 2018;3(4):84–85. doi: 10.14218/ERHM.2018.00020.



Singh A. Autophagy Beyond Homeostasis Explor Res Hypothesis Med

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Exploratory Research and Hypothesis in Medicine 2018 vol. 3 | 86


Reviewer Acknowledgement

☆DOI: 10.14218/ERHM.2018.000RA

2018 Reviewer Acknowledgement

Editorial Office of Exploratory Research and Hypothesis in Medicine

We thank the following reviewers for their contribution and support in 2018.

Sarah Farukhi Ahmed United StatesAli Akdogan TurkeySudheer Arava IndiaMiguel Borja MexicoChristopher Brooks United StatesChang Chen United StatesBohao Chen United StatesPatricia M Crittenden United StatesMonica De la Fuente SpainAhmed M Elsodany EgyptEngin Erturk TurkeyEzzatollah Fathi

IranYair Feld IsraelAlfredo Franco-Obregón SingaporeJamil Ghahhari ItalyNianqiao Gong ChinaSanna Isosävi FinlandHans O Kalkman SwitzerlandAbbas Khani SwitzerlandTae Ho Lee United StatesFernando Lopes CanadaSusumu Matsukuma JapanDavid Miller United Kingdom

Ahmed E. Abdel Moneim EgyptSabina Passamonti ItalySedat Saylan TurkeyYou Shang ChinaAnand Singh United StatesTomoyasu Sugiyama JapanHemant D. Une AustraliaYu N Utkin RussiaLanjing Zhang United StatesWeilin Zhang ChinaPeng Zhang China


exploratory research and hypothesis in medicine

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