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The neuronal ceroid lipofuscinoses (NCL) are genetic degenerative disorders of brain and retina. NCL with juvenile onset (JNCL) is genetically heterogeneous but most frequently caused by mutations of CLN3. Classical juvenile CLN3 includes a rare protracted form, which has previously been linked to autophagic vacuolar myopathy (AVM). Our study investigates the association of AVM with classic, non-protracted CLN3. Evaluation of skeletal muscle biopsies from three, non-related patients with classic, non-protracted and one patient with protracted CLN3 disease by histology, immunohistochemistry, electron microscopy, and Sanger sequencing of the coding region of the CLN3 gene. We identified a novel heterozygous CLN3 mutation (c.1056+34C>A) in one of our patients with classic, non-protracted CLN3 disease. The skeletal muscle of all CLN3 patients was homogeneously affected by an AVM characterized by autophagic vacuoles with sarcolemmal features and characteristic lysosomal pathology. Our observations show that AVM is not an exceptional phenomenon restricted to protracted CLN3 but rather a common feature in CLN3 myopathology. Therefore, CLN3 myopathology should be included in the diagnostic spectrum of autophagic vacuolar myopathies. Show less

Mutations in the CLN3 gene lead to so far an incurable juvenile-onset neuronal ceroid lipofuscinosis (JNCL) or Batten disease that starts at the age of 4-6 years with a progressive retinopathy leading to blindness. Motor disturbances, epilepsy and dementia manifest during several following years. Most JNCL patients carry the same 1.02-kb deletion in the CLN3 gene, encoding an unusual transmembrane protein, CLN3 or battenin. Based on data of genome-wide expression profiling in CLN3 patients with different rate of the disease progression [Mol. Med., 2011, 17: 1253-1261] and our bioinformatic analysis of battenin protein-protein interactions in neurons we propose that CLN3 can function as a molecular chaperone for some plasma membrane proteins, being crucially important for their correct folding in endoplasmic reticulum. Changes in spatial structure of these membrane proteins lead to transactivation of the located nearby receptors. Particularly, CLN3 interacts with a subunit of Na/K ATPase ATP1A1 which changes its conformation and activates the adjacent epidermal growth factor receptor (EGFR). As a result, a large amount of erroneously activated EGFR generates MAPK signal cascades (ERK1/ERK2, JNKs and p38) from cell surface eventually causing neurons' death. Molecular mechanism of the juvenile form of Batten disease (JNCL), which is based on the excessive activation of signaling cascades in a time of the radical increase of neuronal membranes' area in the growing brain, have been proposed and substantiated. The primary cause of this phenomenon is the defective function of the CLN3 protein that could not act properly as molecular chaperone for some plasma membrane proteins in the endoplasmic reticulum. The incorrect three-dimensional structure of at least one such protein, ATP1A1, leads to unregulated spontaneous and repetitive activation of the SRC kinase that transactivates EGFR with the subsequent uncontrolled launch of various MAPK cascades. Possible ways of treatment of patients with JNCL have been suggested. This article was reviewed by Konstantinos Lefkimmiatis, Eugene Koonin and Vladimir Poroikov. Show less

Juvenile Batten disease (JBD) is an inherited disorder that is characterized by the development of blindness, seizures, and progressive motor, psychiatric, and cognitive impairment. A model of JBD expressing the predominant human mutation (Cln3 Show less

Mutations in genes traditionally associated with syndromic retinal disease are increasingly found to cause nonsyndromic inherited retinal degenerations. Mutations in CLN3 are classically associated with juvenile neuronal ceroid lipofuscinosis, a rare neurodegenerative disease with early retinal degeneration and progressive neurologic deterioration, but have recently also been identified in patients with nonsyndromic inherited retinal degenerations. To our knowledge, detailed clinical characterization of such cases has yet to be reported. To provide detailed clinical, electrophysiologic, structural, and molecular genetic findings in nonsyndromic inherited retinal degenerations associated with CLN3 mutations. A multi-institutional case series of 10 patients who presented with isolated nonsyndromic retinal disease and mutations in CLN3. Patient ages ranged from 16 to 70 years; duration of follow-up ranged from 3 to 29 years. Longitudinal clinical evaluation, including full ophthalmic examination, multimodal retinal imaging, perimetry, and electrophysiology. Molecular analyses were performed using whole-genome sequencing or whole-exome sequencing. Electron microscopy studies of peripheral lymphocytes and CLN3 transcript analysis with polymerase chain reaction amplification were performed in a subset of patients. There were 7 females and 3 males in this case series, with a mean (range) age at last review of 37.1 (16-70) years. Of the 10 patients, 4 had a progressive late-onset rod-cone dystrophy, with a mean (range) age at onset of 29.7 (20-40) years, and 6 had an earlier onset rod-cone dystrophy, with a mean (range) age at onset of 12.1 (7-17) years. Ophthalmoscopic examination features included macular edema, mild intraretinal pigment migration, and widespread atrophy in advanced disease. Optical coherence tomography imaging demonstrated significant photoreceptor loss except in patients with late-onset disease who had a focal preservation of the ellipsoid zone and outer nuclear layer in the fovea. Electroretinography revealed a rod-cone pattern of dysfunction in 6 patients and were completely undetectable in 2 patients. Six novel CLN3 variants were identified in molecular analyses. This report describes detailed clinical, imaging, and genetic features of CLN3-associated nonsyndromic retinal degeneration. The age at onset and natural progression of retinal disease differs greatly between syndromic and nonsyndromic CLN3 disease, which may be associated with genotypic differences. Show less

Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is a fatal lysosomal storage disease caused by autosomal recessive mutations in CLN3. JNCL is typified by progressive neurodegeneration that has been suggested to occur from excessive excitatory and impaired inhibitory synaptic input; however, no studies to date have directly evaluated neuronal function. To examine changes in neuronal activity with advancing disease, electrophysiological recordings were performed in the CA1 hippocampus (HPC) and visual cortex (VC) of acute brain slices from Cln3 Show less

Clinical trials have been conducted for the neuronal ceroid lipofuscinoses (NCLs), a group of neurodegenerative lysosomal diseases that primarily affect children. Whereas clinical rating systems will evaluate long-term efficacy, biomarkers to measure short-term response to treatment would be extremely valuable. To identify candidate biomarkers, we analyzed autopsy brain and matching CSF samples from controls and three genetically distinct NCLs due to deficiencies in palmitoyl protein thioesterase 1 (CLN1 disease), tripeptidyl peptidase 1 (CLN2 disease), and CLN3 protein (CLN3 disease). Proteomic and biochemical methods were used to analyze lysosomal proteins, and, in general, we find that changes in protein expression compared with control were most similar between CLN2 disease and CLN3 disease. This is consistent with previous observations of biochemical similarities between these diseases. We also conducted unbiased proteomic analyses of CSF and brain using isobaric labeling/quantitative mass spectrometry. Significant alterations in protein expression were identified in each NCL, including reduced STXBP1 in CLN1 disease brain. Given the confounding variable of post-mortem changes, additional validation is required, but this study provides a useful starting set of candidate NCL biomarkers for further evaluation. Show less

CLN diseases are rare lysosomal storage diseases characterized by progressive axonal degeneration and neuron loss in the CNS, manifesting in disability, blindness, and premature death. We have previously demonstrated that, in animal models of infantile and juvenile forms of CLN disease (CLN1 and CLN3, respectively), secondary neuroinflammation in the CNS substantially amplifies neural damage, opening the possibility that immunomodulatory treatment might improve disease outcome. First, we recapitulated the inflammatory phenotype, originally seen in mice in autopsies of CLN patients. We then treated mouse models of CLN1 and CLN3 disease with the clinically approved immunomodulatory compounds fingolimod (0.5 mg/kg/day) and teriflunomide (10 mg/kg/day) by consistent supply in the drinking water for 5 months. The treatment was well tolerated and reduced T cell numbers and microgliosis in the CNS of both models. Moreover, axonal damage, neuron loss, retinal thinning, and brain atrophy were substantially attenuated in both models, along with reduced frequency of myoclonic jerks in Ppt1 Show less

The MC1R gene plays a crucial role in pigmentation synthesis. Loss-of-function MC1R variants, which impair protein function, are associated with red hair color (RHC) phenotype and increased skin cancer risk. Cultured cutaneous cells bearing loss-of-function MC1R variants show a distinct gene expression profile compared to wild-type MC1R cultured cutaneous cells. We analysed the gene signature associated with RHC co-cultured melanocytes and keratinocytes by Protein-Protein interaction (PPI) network analysis to identify genes related with non-functional MC1R variants. From two detected networks, we selected 23 nodes as hub genes based on topological parameters. Differential expression of hub genes was then evaluated in healthy skin biopsies from RHC and black hair color (BHC) individuals. We also compared gene expression in melanoma tumors from individuals with RHC versus BHC. Gene expression in normal skin from RHC cutaneous cells showed dysregulation in 8 out of 23 hub genes (CLN3, ATG10, WIPI2, SNX2, GABARAPL2, YWHA, PCNA and GBAS). Hub genes did not differ between melanoma tumors in RHC versus BHC individuals. The study suggests that healthy skin cells from RHC individuals present a constitutive genomic deregulation associated with the red hair phenotype and identify novel genes involved in melanocyte biology. Show less

Upon environmental changes, proliferating cells delay cell cycle to prevent further damage accumulation. Yeast Cip1 is a Cdk1 and Cln2-associated protein. However, the function and regulation of Cip1 are still poorly understood. Here we report that Cip1 expression is co-regulated by the cell-cycle-mediated factor Mcm1 and the stress-mediated factors Msn2/4. Overexpression of Cip1 arrests cell cycle through inhibition of Cdk1-G1 cyclin complexes at G1 stage and the stress-activated protein kinase-dependent Cip1 T65, T69, and T73 phosphorylation may strengthen the Cip1and Cdk1-G1 cyclin interaction. Cip1 accumulation mainly targets Cdk1-Cln3 complex to prevent Whi5 phosphorylation and inhibit early G1 progression. Under osmotic stress, Cip1 expression triggers transient G1 delay which plays a functionally redundant role with another hyperosmolar activated CKI, Sic1. These findings indicate that Cip1 functions similarly to mammalian p21 as a stress-induced CDK inhibitor to decelerate cell cycle through G1 cyclins to cope with environmental stresses.A G1 cell cycle regulatory kinase Cip1 has been identified in budding yeast but how this is regulated is unclear. Here the authors identify cell cycle (Mcm1) and stress-mediated (Msn 2/4) transcription factors as regulating Cip1, causing stress induced CDK inhibition and delay in cell cycle progression. Show less

How cells coordinate growth and division is key for size homeostasis. Phosphorylation by G1-CDK of Whi5/Rb inhibitors of SBF/E2F transcription factors triggers irreversible S-phase entry in yeast and metazoans, but why this occurs at a given cell size is not fully understood. We show that the yeast Rim15-Igo1,2 pathway, orthologous to Gwl-Arpp19/ENSA, is up-regulated in early G1 and helps promoting START by preventing PP2A Show less

Neuronal ceroid lipofuscinosis (NCL), also known as Batten disease, refers to a group of severe neurodegenerative disorders that primarily affect children. The most common subtype of the disease is caused by loss-of-function mutations in CLN3, which is conserved across model species from yeast to human. The precise function of the CLN3 protein is not known, which has made targeted therapy development challenging. In the social amoeba Dictyostelium discoideum, loss of Cln3 causes aberrant mid-to-late stage multicellular development. In this study, we show that Cln3-deficiency causes aberrant adhesion and aggregation during the early stages of Dictyostelium development. cln3 Show less

CLN3 is a rare lysosomal storage disorder. The majority of the patients suffer from neurological degeneration in the first decade of life leading to death in the second or third decade. One of the first symptoms is a rapid visual decline from retinal degeneration. The aim of this study was to correlate the retinal changes in CLN3 as seen with spectral domain optical coherence tomography (SD-OCT) with functional data in patients in the first years after the subjective onset of ocular symptoms. Three unrelated children aged from 5.6 to 8.8 years, and with molecularly confirmed CLN3, underwent a comprehensive ophthalmological examination including visual acuity, fundus photography, fundus autofluorescence (FAF), electrophysiology (multifocal ERG), Goldmann visual fields, and SD-OCT. A predominant loss of the first and second neuron retinal layers progressing from the macula to the periphery was identifed. The retinal nerve fibre layer (RNFL) displayed gliosis and an irregular lining of the inner limiting membrane. Compared to the preferential reduction of photoreceptor layer thickness in other maculopathies with pan-retinal involvement, the thickness of the first and second neuron layers was reduced simultaneously in CLN3. Functional testing by multifocal ERG reflected the degenerative progress. Semiquantitative evaluation revealed a generally reduced FAF. This is the first detailed morphological evaluation of CLN3 patients in the first years after the subjective onset of ocular symptoms. CLN3 is characterized by an early degeneration predominant of the first and second neuron compared to other macular and generalized retinal dystrophies. Imaging is instrumental for early diagnosis and gene-directed molecular analysis of this fatal disorder. Show less

The neuronal ceroid lipofuscinoses are a group of recessively inherited, childhood-onset neurodegenerative conditions. Several forms are caused by mutations in genes encoding putative lysosomal membrane proteins. Studies of the cell biology underpinning these disorders are hampered by the poor antigenicity of the membrane proteins, which makes visualization of the endogenous proteins difficult. We have used Drosophila to generate knock-in YFP-fusions for two of the NCL membrane proteins: CLN7 and CLN3. The YFP-fusions are expressed at endogenous levels and the proteins can be visualized live without the need for overexpression. Unexpectedly, both CLN7 and CLN3 have restricted expression in the CNS of Drosophila larva and are predominantly expressed in the glia that form the insect blood-brain-barrier. CLN7 is also expressed in neurons in the developing visual system. Analogous with murine CLN3, Drosophila CLN3 is strongly expressed in the excretory and osmoregulatory Malpighian tubules, but the knock-in also reveals unexpected localization of the protein to the apical domain adjacent to the lumen. In addition, some CLN3 protein in the tubules is localized within mitochondria. Our in vivo imaging of CLN7 and CLN3 suggests new possibilities for function and promotes new ideas about the cell biology of the NCLs. Show less

Neurodegenerative diseases characterized by aberrant accumulation of undigested cellular components represent unmet medical conditions for which the identification of actionable targets is urgently needed. Here we identify a pharmacologically actionable pathway that controls cellular clearance via Akt modulation of transcription factor EB (TFEB), a master regulator of lysosomal pathways. We show that Akt phosphorylates TFEB at Ser467 and represses TFEB nuclear translocation independently of mechanistic target of rapamycin complex 1 (mTORC1), a known TFEB inhibitor. The autophagy enhancer trehalose activates TFEB by diminishing Akt activity. Administration of trehalose to a mouse model of Batten disease, a prototypical neurodegenerative disease presenting with intralysosomal storage, enhances clearance of proteolipid aggregates, reduces neuropathology and prolongs survival of diseased mice. Pharmacological inhibition of Akt promotes cellular clearance in cells from patients with a variety of lysosomal diseases, thus suggesting broad applicability of this approach. These findings open new perspectives for the clinical translation of TFEB-mediated enhancement of cellular clearance in neurodegenerative storage diseases. Show less

The Neuronal Ceroid Lipofuscinoses (NCLs, Batten disease) are a group of inherited neurodegenerative disorders with variable age of onset, characterized by the lysosomal accumulation of autofluorescent ceroid lipopigments. The endoplasmic reticulum (ER) is a critical organelle for normal cell function. Alteration of ER homeostasis leads to accumulation of misfolded protein in the ER and to activation of the unfolded protein response. ER stress and the UPR have recently been linked to the NCLs. In this review, we will discuss the evidence for UPR activation in the NCLs, and address its connection to disease pathogenesis. Further understanding of ER-stress response involvement in the NCLs may encourage development of novel therapeutical agents targeting these pathogenic pathways. Show less

Start is the main decision point in eukaryotic cell cycle in which cells commit to a new round of cell division. It involves the irreversible activation of a transcriptional program by G1 CDK-cyclin complexes through the inactivation of Start transcriptional repressors, Whi5 in yeast or Rb in mammals. Here we provide novel keys of how Whi7, a protein related at sequence level to Whi5, represses Start. Whi7 is an unstable protein, degraded by the SCF Show less

Synapses are an early pathological target in many neurodegenerative diseases ranging from well-known adult onset conditions such as Alzheimer and Parkinson disease to neurodegenerative conditions of childhood such as spinal muscular atrophy (SMA) and neuronal ceroid lipofuscinosis (NCLs). However, the reasons why synapses are particularly vulnerable to such a broad range of neurodegeneration inducing stimuli remains unknown. To identify molecular modulators of synaptic stability and degeneration, we have used the Cln3 Show less

The neuronal ceroid lipofuscinoses (NCLs or Batten disease) are a group of inherited, fatal neurodegenerative disorders of childhood. In these disorders, glial (microglial and astrocyte) activation typically occurs early in disease progression and predicts where neuron loss subsequently occurs. We have found that in the most common juvenile form of NCL (CLN3 disease or JNCL) this glial response is less pronounced in both mouse models and human autopsy material, with the morphological transformation of both astrocytes and microglia severely attenuated or delayed. To investigate their properties, we isolated glia and neurons from Cln3-deficient mice and studied their basic biology in culture. Upon stimulation, both Cln3-deficient astrocytes and microglia also showed an attenuated ability to transform morphologically, and an altered protein secretion profile. These defects were more pronounced in astrocytes, including the reduced secretion of a range of neuroprotective factors, mitogens, chemokines and cytokines, in addition to impaired calcium signalling and glutamate clearance. Cln3-deficient neurons also displayed an abnormal organization of their neurites. Most importantly, using a co-culture system, Cln3-deficient astrocytes and microglia had a negative impact on the survival and morphology of both Cln3-deficient and wildtype neurons, but these effects were largely reversed by growing mutant neurons with healthy glia. These data provide evidence that CLN3 disease astrocytes are functionally compromised. Together with microglia, they may play an active role in neuron loss in this disorder and can be considered as potential targets for therapeutic interventions. Show less

Melanoma is an aggressive skin cancer that can rapidly metastasize to become fatal, if not diagnosed early. Despite recent therapeutic advances, management of melanoma remains difficult. Therefore, novel molecular targets and strategies are required to manage this neoplasm. This study was undertaken to determine the role of the sirtuin SIRT6 in melanoma. Employing a panel of human melanoma cells and normal human melanocytes, we found significant SIRT6 mRNA and protein upregulation in melanoma cells. Further, using a tissue microarray coupled with quantitative Vectra analysis, we demonstrated significant SIRT6 overexpression in human melanoma tissues. Lentiviral short hairpin RNA-mediated knockdown of SIRT6 in A375 and Hs 294T human melanoma cells significantly decreased cell growth, viability, and colony formation, induced G1-phase arrest and increased senescence-associated beta-galactosidase staining. As autophagy is important in melanoma and is associated with SIRT6, we used a qPCR array to study SIRT6 knockdown in A375 cells. We found significant modulation in several genes and/or proteins (decreases in AKT1, ATG12, ATG3, ATG7, BAK1, BCL2L1, CLN3, CTSB, CTSS, DRAM2, HSP90AA1, IRGM, NPC1, SQSTM1, TNF, and BECN1; increases in GAA, ATG10). Our data suggests that increased SIRT6 expression may contribute to melanoma development and/or progression, potentially via senescence-and autophagy-related pathways. Show less

Among Neuronal Ceroid Lipofuscinoses (NCLs), which are childhood fatal neurodegenerative disorders, the juvenile onset form (JNCL) is the most common. JNCL is caused by recessive mutations in the CLN3 gene. CLN3 encodes a lysosomal/endosomal transmembrane protein but its precise function is not completely known. We have previously reported that in baby hamster kidney (BHK) cells stably expressing myc-tagged human CLN3 (myc-CLN3), hyperosmotic conditions drastically increased myc-CLN3 mRNA and protein expression. In the present study, we analyzed the consequences of hyperosmolarity, and increased CLN3 expression on cathepsin D (CTSD) activity and prosaposin processing using BHK cells transiently or stably expressing myc-CLN3. We found that hyperosmolarity increased lysotracker staining of lysosomes, and elevated the levels of myc-CLN3 and lysosome-associated membrane protein-1 (LAMP1). Hyperosmolarity, independently of the expression level of myc-CLN3, decreased the levels of PSAP and saposin D, which are protein cofactors in sphingolipid metabolism. The lysosomal enzyme cathepsin D (CTSD) mediates the proteolytic cleavage of PSAP precursor into saposins A-D. Myc-CLN3 colocalized with CTSD and activity of CTSD decreased as myc-CLN3 expression increased, and clearly decreased under hyperosmotic conditions. Nevertheless, levels of CTSD measured by Western blotting were not altered under any studied condition. Our results suggest a direct involvement of CLN3 in the regulation of CTSD activity. J. Cell. Biochem. 118: 3883-3890, 2017. © 2017 Wiley Periodicals, Inc. Show less

Neuronal ceroid lipofuscinosis (NCL), also referred to as Batten disease, is the most common form of childhood neurodegeneration. Mutations in CLN3 cause the most prevalent subtype of the disease, which manifests during early childhood and is currently untreatable. The precise function of the CLN3 protein is still not known, which has inhibited the development of targeted therapies. In the social amoeba Dictyostelium discoideum, loss of the CLN3 homolog, Cln3, reduces adhesion during early development, which delays streaming and aggregation. The results of the present study indicate that this phenotype may be at least partly due to aberrant protein secretion in cln3 Show less

CLN3 disease (Spielmeyer-Vogt-Sjogren-Batten disease, previously known as classic juvenile neuronal ceroid lipofuscinosis, NCL) is a pediatric-onset progressive neurodegenerative disease characterized by progressive vision loss, seizures, loss of cognitive and motor function, and early death. While no precise biochemical mechanism or therapies are known, the pathogenesis of CLN3 disease involves intracellular calcium accumulation that may trigger apoptosis. Our prior work in in vitro cell models of CLN3 deficiency suggested that FDA-approved calcium channel antagonists may have therapeutic value. To further evaluate the potential efficacy of this approach in an otherwise untreatable disorder, we sought to compare the therapeutic effects and underlying mechanisms in an animal model of CLN3 disease. Here, we used the well-characterized XT7 complete cln-3 knockout strain of C. elegans to evaluate the therapeutic efficacy of calcium channel antagonist therapy in a living animal model of Batten disease. Therapeutic effects of five calcium channel antagonists were evaluated on XT7 animal lifespan and in vivo mitochondrial physiology. Remarkably, maximal therapeutic efficacy in this model animal was observed with 1 μM flunarizine, the identical concentration previously identified in cell-based neuronal models of CLN3 disease. Specifically, flunarizine rescued the short lifespan of XT7 worms and prevented their pathophysiologic mitochondrial accumulation. These results confirm the treatment efficacy and dosing of flunarizine in cln-3 disease in a translational model organism. Clinical treatment trials in CLN3 human patients are now needed to test the dosing regimen and efficacy of flunarizine in individuals suffering with this otherwise untreatable and ultimately lethal neurologic disease. Show less

Solute carriers (SLCs) are vital as they are responsible for a major part of the molecular transport over lipid bilayers. At present, there are 430 identified SLCs, of which 28 are called atypical SLCs of major facilitator superfamily (MFS) type. These are MFSD1, 2A, 2B, 3, 4A, 4B, 5, 6, 6 L, 7, 8, 9, 10, 11, 12, 13A, 14A and 14B; SV2A, SV2B and SV2C; SVOP and SVOPL; SPNS1, SPNS2 and SPNS3; and UNC93A and UNC93B1. We studied their fundamental properties, and we also included CLN3, an atypical SLC not yet belonging to any protein family (Pfam) clan, because its involvement in the same neuronal degenerative disorders as MFSD8. With phylogenetic analyses and bioinformatic sequence comparisons, the proteins were divided into 15 families, denoted atypical MFS transporter families (AMTF1-15). Hidden Markov models were used to identify orthologues from human to Show less

Phosphorus is a pivotal element in all biochemical systems: it serves to store metabolic energy as ATP, it forms the backbone of genetic material such as RNA and DNA, and it separates cells from the environment as phospholipids. In addition to this "big hits", phosphorus has recently been shown to play an important role in other important processes such as cell cycle regulation. In the present review, we briefly summarize the biological processes in which phosphorus is involved in the yeast Saccharomyces cerevisiae before discussing our latest findings on the role of this element in the regulation of DNA replication in this eukaryotic model organism. We describe both the role of phosphorus in the regulation of G1 progression by means of the Cyclin Dependent Kinase (CDK) Pho85 and the stabilization of the cyclin Cln3, as well as the role of other molecule composed of phosphorus-the polyphosphate-in cell cycle progression, dNTP synthesis, and genome stability. Given the eminent role played by phosphorus in life, we outline the future of phosphorus in the context of one of the main challenges in human health: cancer treatment. [Int Microbiol 19(3):133-141 (2016)]. Show less