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Oxaliplatin-induced peripheral neurotoxicity (OIPN) is a debilitating side effect that afflicts approximately 90% of patients that is initiated by OCT2-dependent uptake of oxaliplatin in dorsal root ganglion (DRG) neurons. The antidepressant drug duloxetine has been used to treat OIPN, although its usefulness in preventing this side effect remains unclear. We hypothesized that duloxetine has OCT2-inhibitory properties and can be used as an adjunct to oxaliplatin-based regimens to prevent OIPN. Transport studies were performed in cells stably transfected with mouse or human OCT2 and in isolated mouse DRG neurons ex vivo. Wild-type and OCT2-deficient mice were used to assess effects of duloxetine on hallmarks of OIPN, endogenous OCT2 biomarkers, and the pharmacokinetics of oxaliplatin, and the translational feasibility of a duloxetine-oxaliplatin combination was evaluated in various models of colorectal cancer. We found that duloxetine potently inhibited the OCT2-mediated transport of several xenobiotic substrates, including oxaliplatin, in a reversible, concentration-dependent manner, and independent of species and cell context. Furthermore, duloxetine restricted access of these substrates to DRG neurons ex vivo and prevented OIPN in wild-type mice to a degree similar to the complete protection observed in OCT2-deficient mice, without affecting the plasma levels of oxaliplatin. Importantly, the uptake and cytotoxicity of oxaliplatin in tumor cell lines in vitro and in vivo were not negatively influenced by duloxetine. The observed OCT2-targeting properties of duloxetine, combined with the potential for clinical translation, provide support for its further exploration as a therapeutic candidate for studies aimed at preventing OIPN in patients with cancer requiring treatment with oxaliplatin. Significance: We found that duloxetine has potent OCT2-inhibitory properties and can diminish excessive accumulation of oxaliplatin into DRG neurons. In addition, pretreatment of mice with duloxetine prevented OIPN without significantly altering the plasma pharmacokinetics and antitumor properties of oxaliplatin. These results suggest that intentional inhibition of OCT2-mediated transport by duloxetine can be employed as a prevention strategy to ameliorate OIPN without compromising the effectiveness of oxaliplatin-based treatment.

BackgroundAmyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. The approximately 50 known ALS-associated genes do not fully explain its heritability, which suggests the existence of yet unidentified causative genes. We report results of studies aimed at identification of the genetic cause of ALS in a pedigree (three patients) without mutations in the common ALS-causative genes.MethodsClinical investigations included thorough neurological and non-neurological examinations and testings. Genetic analysis was performed by exome sequencing. Functional studies included identification of altered splicing by PCR and sequencing, and mutated proteins by western blot analysis. Apoptosis in the presence and absence of tunicamycin was assessed in transfected HEK293T cells using an Annexin-PE-7AAD kit in conjunction with flow cytometry.ResultsClinical features are described in detail. Disease progression in the patients of the pedigree was relatively slow and survival was relatively long. AnRNF13mutation was identified as the cause of the recessively inherited ALS in the pedigree. The gene is highly conserved, and its encoded protein (RING finger protein 13) can potentially affect various neurodegenerative-relevant functions, including protein homeostasis. TheRNF13splice site mutation caused expression of two mis-spliced forms ofRNF13mRNA and an aberrant RNF13 protein, and affected apoptosis.ConclusionRNF13was identified as a novel causative gene of recessively inherited ALS. The gene affects protein homeostasis, which is one of most important components of the pathology of neurodegeneration. The contribution ofRNF13to the aetiology of another neurodegenerative disease is discussed.

Supplementary Table S1. Percentage of OCT2 inhibition by compounds reported to reduce platinum-induced toxicities. Supplementary Table S2. Validation of human, rat, and murine overexpressed cells by evaluating their ability to accumulate known prototypical transport substrates. Supplementary Figure S1. Chemical structure of duloxetine. Supplementary Figure S2. IVIS imaging of tumor bearing mice. Supplementary Figure S3. Metabolites of duloxetine do not inhibit OCT2 function. Supplementary Figure S4. Duloxetine is an OCT2 inhibitor that extensively binds to extracellular membrane. Supplementary Figure S5. Duloxetine does not prevent peripheral neurotoxicity associated with vincristine and paclitaxel. Supplementary Figure S6. Paw withdrawal force measured by VFH instrument before the start of the treatment in wild-type (WT) and OCT1/2(-/-) mice (n=5-10 per group). Supplementary Figure S7. Activity of oxaliplatin in various colorectal cancer cell lines. Supplementary Figure S8. Effect of duloxetine on sciatic and caudal nerve velocity and amplitude.

Oxaliplatin-induced peripheral neurotoxicity (OIPN) is a debilitating side effect that afflicts approximately 90% of patients that is initiated by OCT2-dependent uptake of oxaliplatin in dorsal root ganglion (DRG) neurons. The antidepressant drug duloxetine has been used to treat OIPN, although its usefulness in preventing this side effect remains unclear. We hypothesized that duloxetine has OCT2-inhibitory properties and can be used as an adjunct to oxaliplatin-based regimens to prevent OIPN. Transport studies were performed in cells stably transfected with mouse or human OCT2 and in isolated mouse DRG neurons ex vivo. Wild-type and OCT2-deficient mice were used to assess effects of duloxetine on hallmarks of OIPN, endogenous OCT2 biomarkers, and the pharmacokinetics of oxaliplatin, and the translational feasibility of a duloxetine-oxaliplatin combination was evaluated in various models of colorectal cancer. We found that duloxetine potently inhibited the OCT2-mediated transport of several xenobiotic substrates, including oxaliplatin, in a reversible, concentration-dependent manner, and independent of species and cell context. Furthermore, duloxetine restricted access of these substrates to DRG neurons ex vivo and prevented OIPN in wild-type mice to a degree similar to the complete protection observed in OCT2-deficient mice, without affecting the plasma levels of oxaliplatin. Importantly, the uptake and cytotoxicity of oxaliplatin in tumor cell lines in vitro and in vivo were not negatively influenced by duloxetine. The observed OCT2-targeting properties of duloxetine, combined with the potential for clinical translation, provide support for its further exploration as a therapeutic candidate for studies aimed at preventing OIPN in patients with cancer requiring treatment with oxaliplatin.Significance:We found that duloxetine has potent OCT2-inhibitory properties and can diminish excessive accumulation of oxaliplatin into DRG neurons. In addition, pretreatment of mice with duloxetine prevented OIPN without significantly altering the plasma pharmacokinetics and antitumor properties of oxaliplatin. These results suggest that intentional inhibition of OCT2-mediated transport by duloxetine can be employed as a prevention strategy to ameliorate OIPN without compromising the effectiveness of oxaliplatin-based treatment.

Sandhoff disease is a rare fatal infantile neurologic disorder. Adult onset Sandhoff is even rarer. Variability of clinical features in adult onset Sandhoff patients and overlaps between these and features of other neurologic diseases have sometimes led to mis-diagnosis. We describe an adult onset Sandhoff disease affected individual whose clinical presentation were also consistent with the Brown-Vialetto-Van Laere syndrome (BVVL) diagnosis. Screening of BVVL-causing genes, SLC52A3 and SLC52A2, did not identify candidate disease-causing mutations, but exome sequencing revealed compound heterozygous mutations in the known Sandhoff disease-causing gene, HEXB. Decreased blood hexosaminidase activity and evidence of cerebellar atrophy confirmed Sandhoff disease diagnosis. To the best of our knowledge, this is the first report of a Sandhoff disease case that mimics BVVL and that presents with prominent cranial nerve involvement. For differential diagnosis, measurement of hexosaminidase activity and MRI should quickly be performed. Genetic analysis can be done for confirmation of diagnosis.

Spinal-bulbar muscular atrophy (SBMA) (Kennedy's disease) is a motor neuron disease. Kennedy's disease is nearly exclusively caused by mutations in the androgen receptor encoding gene (AR). The results of studies aimed at identification of the genetic cause of a disease that best approximates SBMA in a pedigree (four patients) without mutations in AR are reported.Clinical investigations included thorough neurological and non-neurological examinations and testing. Genetic analysis was performed by exome sequencing using standard protocols. UBA1 mutations were modeled on the crystal structure of UBA1.The clinical features of the patients are described in detail. A missense mutation in UBA1 (c.T1499C; p.Ile500Thr) was identified as the probable cause of the non-Kennedy SBMA in the pedigree. Like AR, UBA1 is positioned on chromosome X. UBA1 is a highly conserved gene. It encodes ubiquitin-like modifier activating enzyme 1 (UBA1) which is the major E1 enzyme of the ubiquitin-proteasome system. Interestingly, UBA1 mutations can also cause infantile-onset X-linked spinal muscular atrophy (XL-SMA). The mutation identified here and the XL-SMA causative mutations were shown to affect amino acids positioned in the vicinity of UBA1's ATP binding site and to cause structural changes.UBA1 was identified as a novel SBMA causative gene. The gene affects protein homeostasis which is one of most important components of the pathology of neurodegeneration. The contribution of this same gene to the etiology of XL-SMA is discussed.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. The approximately 50 known ALS-associated genes do not fully explain its heritability, which suggests the existence of yet unidentified causative genes. We report results of studies aimed at identification of the genetic cause of ALS in a pedigree (three patients) without mutations in the common ALS-causative genes.Clinical investigations included thorough neurological and non-neurological examinations and testings. Genetic analysis was performed by exome sequencing. Functional studies included identification of altered splicing by PCR and sequencing, and mutated proteins by western blot analysis. Apoptosis in the presence and absence of tunicamycin was assessed in transfected HEK293T cells using an Annexin-PE-7AAD kit in conjunction with flow cytometry.Clinical features are described in detail. Disease progression in the patients of the pedigree was relatively slow and survival was relatively long. An

Introduction: Acinetobacter baumannii known as a main agent in nosocomial infections and its drug resistance is a medical problem in hospitals. In the present study, growth control of Acinetobacter by Oregano essential oil was investigated in-vitro and in experimental wounds on rats. Materials and Methods: Oregano essential oil (OES) was prepared by a Clevenger apparatus in a hydrodistillation process. Susceptibility of clinical isolate of Acinetobacterto essential oil was evaluated by disk diffusion and microbroth dilution methods. Ketamine and xylazine were used for surgical anesthesia induction in rats. The backs of 30 rats were shaved and an experimental wound in one square inch was prepared between two scapulae. All the ulcers were infected with fresh culture of Acinetobacter. The rats were divided into three equal groups for treatment by OES twice a day, treatment with silversulfuridine ointment (SSO) and control group without any treatment. Infection symptoms were assessed until the wound was fully granulated. Results: MIC and MBC were as 4 mg/ml and 6 mg/ml, respectively. Blood culture of control group and SSO contained A. baumannii but was negative in treatment group. The cultivation and examination of the wounds and its secretions revealed the absence of A. baumannii in treatment group. Wound healing was faster in the treatment group than in the control groups. Conclusion: Infection of the wounds infected with A. baumannii was interestingly controlled by OES and subsequently accelerated the wound healing process in Wistar rats. Therefore, OES recommends for evaluation in a clinical trial.

Brown-Vialetto-Van Laere (BVVL) and Fazio-Londe are disorders with amyotrophic lateral sclerosis-like features, usually with recessive inheritance. We aimed to identify causative mutations in 10 probands. Neurological examinations, genetic analysis, audiometry, magnetic resonance imaging, biochemical and immunological testings, and/or muscle histopathology were performed. Mutations in known causative gene SLC52A3 were found in 7 probands. More importantly, only 1 mutated allele was observed in several patients, and variable expressivity and incomplete penetrance were clearly noted. Environmental insults may contribute to variable presentations. Putative causative mutations in other genes were identified in 3 probands. Two of the genes, WDFY4 and TNFSF13B, have immune-related functions. Inflammatory responses were implicated in the patient with the WDFY4 mutation. Malfunction of the immune system and mitochondrial anomalies were shown in the patient with the TNFSF13B mutation. Prevalence of heterozygous SLC52A3 BVVL causative mutations and notable variability in expressivity of homozygous and heterozygous genotypes are being reported for the first time. Identification of WDFY4 and TNFSF13B as candidate causative genes supports conjectures on involvement of the immune system in BVVL and amyotrophic lateral sclerosis.

Background SPG11 mutations can cause autosomal recessive hereditary spastic paraplegia (ARHSP) and juvenile amyotrophic lateral sclerosis (JALS). Because these diseases share some clinical presentations and both can be caused by SPG11 mutations, it was considered that definitive diagnosis may not be straight forward. Methods The DNAs of referred ARHSP and JALS patients were exome sequenced. Clinical data of patients with SPG11 mutations were gathered by interviews and neurological examinations including electrodiagnosis (EDX) and magnetic resonance imaging (MRI). Results Eight probands with SPG11 mutations were identified. Two mutations are novel. Among seven Iranian probands, six carried the p.Glu1026Argfs*4‐causing mutation. All eight patients had features known to be present in both ARHSP and JALS. Additionally and surprisingly, presence of both thin corpus callosum (TCC) on MRI and motor neuronopathy were also observed in seven patients. These presentations are, respectively, key suggestive features of ARHSP and JALS. Conclusion We suggest that rather than ARHSP or JALS, combined ARHSP/JALS is the appropriate description of seven patients studied. Criteria for ARHSP, JALS, and combined ARHSP/JALS designations among patients with SPG11 mutations are suggested. The importance of performing both EDX and MRI is emphasized. Initial screening for p.Glu1026Argfs*4 may facilitate SPG11 screenings in Iranian patients., Nineteen patients of eight families with SPG11 mutations are described. Two previously unreported SPG11 mutations are reported. Definitive diagnosis of ARHSP or JALS is sometimes very difficult. Rather than ARHSP or JALS, combined ARHSP/JALS is the appropriate description for some patients with SPG11 mutation. Criteria for ARHSP, JALS, and combined ARHSP/JALS designations among patients with SPG11 mutations are suggested.

Charcot-Marie-Tooth (CMT) is a common neuropathy, and hereditary motor and sensory neuropathy with proximal predominance (HMSN-P) is a recently described rare neuromuscular disease. Although many genes have been implicated for CMT, TFG is the only known HMSN-P-causing gene. Within the framework of diagnostic criteria, clinical variation is evident among CMT-diagnosed and also HMSN-P-diagnosed individuals. Mutations that cause p.(Pro285Leu) and p.(Gly269Val) in TFG were earlier reported as cause of HMSN-P in two Iranian pedigrees. Here, we report the identification of p.(Gly269Val) in TFG as cause of CMT in a large Iranian pedigree. The clinical features of patients of the three pedigrees are presented and critically compared. Similarities between the two HMSN-P-diagnosed pedigrees with different TFG mutations, and differences between the two differentially diagnosed pedigrees with the same p.(Gly269Val) mutation were evident. The clinical features of the HMSN-P pedigree with the p.(Pro285Leu) and the CMT pedigree with the p.(Gly269Val) mutation were clearly congruent with the respective diagnoses, whereas the features of the HMSN-P-diagnosed pedigree with the p.(Gly269Val) were intermediate between the other two pedigrees. It is therefore suggested that the clinical features of the three Iranian pedigrees with TFG mutations and diagnosed with HMSN-P or CMT represent a continuum.