Clinically compliant cryopreservation of differentiated retinal pigment epithelial cells.

BACKGROUND AIMS: Age-related macular degeneration (AMD) is the most common cause of blindness in elderly patients within developed countries, affecting more than 190 million worldwide. In AMD, the retinal pigment epithelial (RPE) cell layer progressively degenerates, resulting in subsequent loss of photoreceptors and ultimately vision. There is currently no cure for AMD, but therapeutic strategies targeting the complement system are being developed to slow the progression of the disease. METHODS: Replacement therapy with pluripotent stem cell-derived (hPSC) RPEs is an alternative treatment strategy. A cell therapy product must be produced in accordance with Good Manufacturing Practices at a sufficient scale to facilitate extensive pre-clinical and clinical testing. Cryopreservation of the final cell product is therefore highly beneficial, as the manufacturing, pre-clinical and clinical testing can be separated in time and location. RESULTS: We found that mature hPSC-RPE cells do not survive conventional cryopreservation techniques. However, replating the cells 2-5 days before cryopreservation facilitates freezing. The replated and cryopreserved hPSC-RPE cells maintained their identity, purity and functionality as characteristic RPEs, shown by cobblestone morphology, pigmentation, transcriptional profile, RPE markers, transepithelial resistance and pigment epithelium-derived factor secretion. Finally, we showed that the optimal replating time window can be tracked noninvasively by following the change in cobblestone morphology. CONCLUSIONS: The possibility of cryopreserving the hPSC-RPE product has been instrumental in our efforts in manufacturing and performing pre-clinical testing with the aim for clinical translation.

Unlicensed/Off-Label Drug Prescriptions at Hospital Discharge in Children: An Observational Study Using Routinely Collected Health Data.

BACKGROUND: Unlicensed and off-label (UL/OL) prescriptions have been associated with an increased risk of drug-related problems. Data of their prevalence at hospital discharge remain insufficient. We aimed to describe the prevalence of UL/OL drugs in outpatient prescriptions at discharge in children. METHODS: We conducted a retrospective study using the routinely collected health data of children at discharge from 2014 to 2016. The primary reference source for determining licensed labelling was the summaries of product characteristics (SPCs) in a French industry-independent formulary named Thériaque. We described the characteristics of UL/OL prescriptions at discharge and looked for predictors of UL/OL prescriptions. RESULTS: We included 2536 prescriptions of 479 children. Licensed, OL, and UL prescriptions accounted for 58.6% (95% CI: 56.7-60.5), 39.2% (95% CI: 37.3-41.1), and 2.3% (95% CI: 1.7-2.9), respectively. A total of 323 (74%) children received at least one UL/OL drug. Among the licensed drugs, bronchodilators (8.8%) and analgesics (8.6%), and among the OL drugs, antibiotics (2.8%), were the most prescribed. The younger age of the children and higher number of drugs they received increased the probability of UL/OL prescriptions (unadjusted p-value of ≤0.05). CONCLUSION: The prevalence of UL/OL prescriptions is about 40% at discharge from a pediatric university hospital in France.

Mitochondrial DNA damage triggers spread of Parkinson's disease-like pathology.

In the field of neurodegenerative diseases, especially sporadic Parkinson's disease (sPD) with dementia (sPDD), the question of how the disease starts and spreads in the brain remains central. While prion-like proteins have been designated as a culprit, recent studies suggest the involvement of additional factors. We found that oxidative stress, damaged DNA binding, cytosolic DNA sensing, and Toll-Like Receptor (TLR)4/9 activation pathways are strongly associated with the sPDD transcriptome, which has dysregulated type I Interferon (IFN) signaling. In sPD patients, we confirmed deletions of mitochondrial (mt)DNA in the medial frontal gyrus, suggesting a potential role of damaged mtDNA in the disease pathophysiology. To explore its contribution to pathology, we used spontaneous models of sPDD caused by deletion of type I IFN signaling (Ifnb-/-/Ifnar-/- mice). We found that the lack of neuronal IFNß/IFNAR leads to oxidization, mutation, and deletion in mtDNA, which is subsequently released outside the neurons. Injecting damaged mtDNA into mouse brain induced PDD-like behavioral symptoms, including neuropsychiatric, motor, and cognitive impairments. Furthermore, it caused neurodegeneration in brain regions distant from the injection site, suggesting that damaged mtDNA triggers spread of PDD characteristics in an "infectious-like" manner. We also discovered that the mechanism through which damaged mtDNA causes pathology in healthy neurons is independent of Cyclic GMP-AMP synthase and IFNß/IFNAR, but rather involves the dual activation of TLR9/4 pathways, resulting in increased oxidative stress and neuronal cell death, respectively. Our proteomic analysis of extracellular vesicles containing damaged mtDNA identified the TLR4 activator, Ribosomal Protein S3 as a key protein involved in recognizing and extruding damaged mtDNA. These findings might shed light on new molecular pathways through which damaged mtDNA initiates and spreads PD-like disease, potentially opening new avenues for therapeutic interventions or disease monitoring.

Drug-related risk of hospital readmission in children with chronic diseases, a systematic review.

BACKGROUND: Drug-related problems (DRPs) are one of the leading causes of hospital readmissions. Children with chronic diseases are more likely to experience DRPs than adults. The burden and characteristics of drug-related readmissions at and after hospital discharge in children remain unclear. OBJECTIVE: We aimed to summarize the impact of DRPs at and after hospital discharge on the risk of readmissions in children with chronic diseases. METHODS: We conducted a systematic review searching PubMed from inception until January 2022. Study selection criteria were studies assessing the impact of different factors at discharge and after discharge on the risk of hospital readmissions in children with chronic diseases, reporting an assessment of DRPs. DRP could be the only risk factor assessed or one among others. Included studies were assessed with the Risk of Bias in Non-Randomized Studies - of Exposure (ROBINS-E) tool. We summarized the qualitative impact of the reported DRPs on hospital readmission as conclusive (significant association) or inconclusive. RESULTS: Of the 4734 studies initially identified, 13 met inclusion criteria. Eleven studies were retrospective, using electronic health records. The studies assessed the impact of DRPs at or after discharge according to the type of medication (in 6 studies), number of medication (in 5 studies) and medication nonadherence (in 2 studies). From the 44 reported associations between DRPs and the risk of readmission 26 (59% [95% CI, 43%-73%]) were conclusive, of which 81% increased the risk and 19% decreased the risk, and 17 (39% [95% CI, 24%-55%]) were inconclusive. CONCLUSION: The impact of DRPs on hospital readmissions in children with chronic diseases displayed conflicting results, estimated associations having potentially a serious risk of bias. We need more evidence with a lower risk of bias.

Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation.

Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) are a promising cell source to treat age-related macular degeneration (AMD). Despite several ongoing clinical studies, a detailed mapping of transient cellular states during in vitro differentiation has not been performed. Here, we conduct single-cell transcriptomic profiling of an hESC-RPE differentiation protocol that has been developed for clinical use. Differentiation progressed through a culture diversification recapitulating early embryonic development, whereby cells rapidly acquired a rostral embryo patterning signature before converging toward the RPE lineage. At intermediate steps, we identified and examined the potency of an NCAM1+ retinal progenitor population and showed the ability of the protocol to suppress non-RPE fates. We demonstrated that the method produces a pure RPE pool capable of maturing further after subretinal transplantation in a large-eyed animal model. Our evaluation of hESC-RPE differentiation supports the development of safe and efficient pluripotent stem cell-based therapies for AMD.

IFN-ß rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1.

Mitochondrial homeostasis is essential for providing cellular energy, particularly in resource-demanding neurons, defects in which cause neurodegeneration, but the function of interferons (IFNs) in regulating neuronal mitochondrial homeostasis is unknown. We found that neuronal IFN-ß is indispensable for mitochondrial homeostasis and metabolism, sustaining ATP levels and preventing excessive ROS by controlling mitochondrial fission. IFN-ß induces events that are required for mitochondrial fission, phosphorylating STAT5 and upregulating PGAM5, which phosphorylates serine 622 of Drp1. IFN-ß signaling then recruits Drp1 to mitochondria, oligomerizes it, and engages INF2 to stabilize mitochondria-endoplasmic reticulum (ER) platforms. This process tethers damaged mitochondria to the ER to separate them via fission. Lack of neuronal IFN-ß in the Ifnb-/- model of Parkinson disease (PD) disrupts STAT5-PGAM5-Drp1 signaling, impairing fission and causing large multibranched, damaged mitochondria with insufficient ATP production and excessive oxidative stress to accumulate. In other PD models, IFN-ß rescues dopaminergic neuronal cell death and pathology, associated with preserved mitochondrial homeostasis. Thus, IFN-ß activates mitochondrial fission in neurons through the pSTAT5/PGAM5/S622 Drp1 pathway to stabilize mitochondria/ER platforms, constituting an essential neuroprotective mechanism.

Relationship between adverse drug reactions and unlicensed/off-label drug use in hospitalized children (EREMI): A study protocol.

INTRODUCTION: To date, few studies have shown a significant association between off-label drug use and adverse drug reactions (ADRs). The main aims of this study is to evaluate the relationship between adverse drug reactions and unlicensed or off-label drugs in hospitalized children and to provide more information on prescribing practice, the amplitude, consequences of unlicensed or off-label drug use in pediatric inpatients. METHODS: In this multicenter prospective study started from 2013, we use the French summaries of product characteristics in Theriaque (a prescription products guide) as a primary reference source for determining pediatric drug labeling. The detection of ADRs is carried out spontaneously by health professionals and actively by research groups using a trigger tool and patients' electronic health records. The causality between suspected ADRs and medication is evaluated using the Naranjo and the French methods of imputability independently by pharmacovigilance center. All suspected ADRs are submitted for a second evaluation by an independent pharmacovigilance experts. STRENGTH AND LIMITATIONS OF THIS STUDY: For our best knowledge, EREMI is the first large multicenter prospective and objective study in France with an active ADRs monitoring and independent ADRs validation. This study identifies the risk factors that could be used to adjust preventive actions in children's care, guides future research in the field and increases the awareness of physicians in off-label drug use and in detecting and declaring ADRs. As data are obtained through extraction of information from hospital database and medical records, there is likely to be some under-reporting of items or missing data. In this study the field specialists detect all adverse events, experts in pharmacovigilance centers assess them and finally only the ADRs assessed by the independent committee are confirmed. Although we recruit a high number of patients, this observational study is subject to different confounders.

Effects of a single dose of psilocybin on behaviour, brain 5-HT2A receptor occupancy and gene expression in the pig.

Psilocybin has in some studies shown promise as treatment of major depressive disorder and psilocybin therapy was in 2019 twice designated as breakthrough therapy by the U.S. Food and Drug Administration (FDA). A very particular feature is that ingestion of just a single dose of psilocybin is associated with lasting changes in personality and mood. The underlying molecular mechanism behind its effect is, however, unknown. In a translational pig model, we here present the effects of a single dose of psilocybin on pig behaviour, receptor occupancy and gene expression in the brain. An acute i.v. injection of 0.08 mg/kg psilocybin to awake female pigs induced characteristic behavioural changes in terms of headshakes, scratching and rubbing, lasting around 20 min. A similar dose was associated with a cerebral 5-HT2A receptor occupancy of 67%, as determined by positron emission tomography, and plasma psilocin levels were comparable to what in humans is associated with an intense psychedelic experience. We found that 19 genes were differentially expressed in prefrontal cortex one day after psilocybin injection, and 3 genes after 1 week. Gene Set Enrichment Analysis demonstrated that multiple immunological pathways were regulated 1 week after psilocybin exposure. This provides a framework for future investigations of the lasting molecular mechanisms induced by a single dose of psilocybin. In the light of an ongoing debate as to whether psilocybin is a safe treatment for depression and other mental illnesses, it is reassuring that our data suggest that any effects on gene expression are very modest.

Identification of unique and shared mitochondrial DNA mutations in neurodegeneration and cancer by single-cell mitochondrial DNA structural variation sequencing (MitoSV-seq).

BACKGROUND: Point mutations and structural variations (SVs) in mitochondrial DNA (mtDNA) contribute to many neurodegenerative diseases. Technical limitations and heteroplasmy, however, have impeded their identification, preventing these changes from being examined in neurons in healthy and disease states. METHODS: We have developed a high-resolution technique-Mitochondrial DNA Structural Variation Sequencing (MitoSV-seq)-that identifies all types of mtDNA SVs and single-nucleotide variations (SNVs) in single neurons and novel variations that have been undetectable with conventional techniques. FINDINGS: Using MitoSV-seq, we discovered SVs/SNVs in dopaminergic neurons in the Ifnar1-/- murine model of Parkinson disease. Further, MitoSV-seq was found to have broad applicability, delivering high-quality, full-length mtDNA sequences in a species-independent manner from human PBMCs, haematological cancers, and tumour cell lines, regardless of heteroplasmy. We characterised several common SVs in haematological cancers (AML and MDS) that were linked to the same mtDNA region, MT-ND5, using only 10 cells, indicating the power of MitoSV-seq in determining single-cancer-cell ontologies. Notably, the MT-ND5 hotspot, shared between all examined cancers and Ifnar1-/- dopaminergic neurons, suggests that its mutations have clinical value as disease biomarkers. INTERPRETATION: MitoSV-seq identifies disease-relevant mtDNA mutations in single cells with high resolution, rendering it a potential drug screening platform in neurodegenerative diseases and cancers. FUNDING: The Lundbeck Foundation, Danish Council for Independent Research-Medicine, and European Union Horizon 2020 Research and Innovation Programme.

A study on preterm births during 2013-2015, Shiraz, Iran.

Preterm birth is the leading cause of neonatal and infant mortality and a substantial portion of neonatal morbidities. The perinatal mortality and morbidity statistics in developing countries are inadequate. In this study, we assessed prevalence and health outcomes of preterm deliveries in tertiary care university hospitals. A retrospective study of hospital records of premature babies born in all the five governmental tertiary care settings during the time interval of 2013-2015 in Shiraz was conducted. Result of this study showed that there was an overall 127.6 premature births per 1000 live births in the study duration. 23.8% of premature newborn had RDS and Incidence of prematurity with RDS was 82.4 per thousand live births. 52.6% of premature newborns were hospitalised in NICU and 8.5% had ROP. Five percent suffered from sepsis and 1% suffered from NEC. Overall mortality was nearly 10% of all the premature newborn. In conclusion, this study showed that premature births and its complications for newborn need to be addressed more in Iran. Impact statement Preterm birth is the leading cause of neonatal mortality and morbidities. Mortality and morbidity statistics related to preterm infants are important healthcare indicators implying the quality of the perinatal health care system and are prerequisite for the identification of problems and implementation of preventive measures. However, the perinatal mortality and morbidity statistics in developing countries are inadequate. The aim of this study was to assess prevalence and health outcome of preterm deliveries in tertiary care university hospitals in Shiraz city, Iran. This study showed that prematurity rate was 12.7%. Among preterm cases, 52.6% were admitted to NICU. RDS occurred among 23.8% of the total premature neonates and 8.2% of the total live births. The incidence of NEC was 1/0% of the total premature neonates and 0.4% of the total live births. The incidence of sepsis was 5% of the premature neonates and 1.9% of the total live births and the incidence of ROP was 8.5% among the premature neonate. The overall mortality of premature neonates was 9.9% of the total premature neonates and 1.2% of the total live births. Rigorous measures for prevention of premature births and its complications for newborns are required in Iran.

Mutation in ADORA1 identified as likely cause of early-onset parkinsonism and cognitive dysfunction.

BACKGROUND: We aimed to identify the genetic cause of neurological disease in an Iranian family whose manifestations include symptoms of parkinsonism and cognitive dysfunction. METHODS: Clinical data on the patients were gathered by interviews with parents, neurological examinations, and laboratory tests. Genetic analysis was performed by genome-wide single-nucleotide polymorphism homozygosity mapping and exome sequencing. The effect of putative disease-causing mutation was assessed by immunocytochemistry on HEK293 cells and Western blotting on proteins extracted from HEK293 cells transfected with wild-type and mutated genes. RESULTS: Homozygosity mapping and exome sequencing led to identification of a mutation in ADORA1 that causes p.Gly279Ser in the encoded protein, adenosine A1 receptor (A1 R), as the probable cause of disease. The mutation segregated with disease status in the family, affects a highly conserved amino acid, and was absent in 700 controls. CONCLUSIONS: The known biological activities of A1 R in brain functions including its physical interaction with and inhibitory effect on dopamine receptor D1 provide supportive evidence that disruptions of A1 R may result in neurological dysfunction. Also, recent evidence on the related adenosine A2B receptor marks the domain in which the mutation is positioned as important for function. Finally, ADORA1 is located within the Parkinson's disease locus PARK16, which has been identified in several populations. ADORA1 may be the PD susceptibility gene within this locus. The molecular mechanism by which p.Gly279Ser disrupts A1 R function remains unknown, but a quantitative effect on interaction with the dopamine receptor was not shown. © 2016 International Parkinson and Movement Disorder Society.

Identification of mutation in GTPBP2 in patients of a family with neurodegeneration accompanied by iron deposition in the brain.

We aimed to identify the genetic cause of a neurologic disorder accompanied with mental deficiency in a consanguineous family with 3 affected siblings by linkage analysis and exome sequencing. Iron accumulation in the brain of the patients was a notable phenotypic feature. A full-field electroretinography revealed generalized dysfunction of photoreceptors, bipolar cells, and amacrine cells. A splice site mutation in GTPBP2 that encodes GTP-binding protein 2 was identified in the patients and considered possible cause of their disease. The mutation was empirically shown to cause deletion of exon 9 of the gene and result in production of a truncated protein-lacking conserved C-terminus domains. GTPBP2 is a member of the GTPase superfamily of proteins. A recent report of identification of another splice site mutation in GTPBP2 in mice that causes neurodegeneration, and retinal damage provides supportive evidence for our finding. The conditions in the affected individuals of the family studied may define a novel form of neurodegeneration with brain iron accumulation, and GTPBP2 may be a novel neurodegeneration with brain iron accumulation gene.

Assessment of cytokine expression profile in acute myeloid leukemia patients before and after chemotherapy.

OBJECTIVE: One of the major goals of cancer treatment is the monitoring of chemotherapeutic protocols. Quantitative and comparative cytokine expression profiling could be reliable to be used for biomarkers in deadly and fast-growing cancers such as acute myeloid leukemia (AML). The present study aims to assess and further validate cytokines with probable effects on proliferation and maturation of blood cells in AML. MATERIALS AND METHODS: Gene expression levels of IL-1ß, IL-10, IL-8, TNF-α, and IFN-γ were analyzed before and after chemotherapy and after granulocyte colony-stimulating factor (G-CSF) therapy in 46 AML patients by an in-house quantitative comparative RT-PCR method. RESULTS: Our findings indicated that although the gene expression level of TNF-α was almost constant in all 3 samples, IL-1ß, IL-8, and IL-10 expression levels showed a decrease after chemotherapy and an increase after G-CSF therapy. On the other hand, the expression level of IFN-γ had a different pattern with an increase after chemotherapy and a decrease after G-CSF therapy. CONCLUSION: Taken together, the results of this study are in support of the idea that the analyzed cytokines could be useful biomarkers for AML treatment monitoring. However, further molecular epidemiological investigations are suggested to elaborate more cancer monitoring biomarkers.

The novel mutation p.Asp251Asn in the ß-subunit of succinate-CoA ligase causes encephalomyopathy and elevated succinylcarnitine.

SUCLA2 is one of several nuclear-encoded genes that can cause encephalomyopathy accompanied by mitochondrial DNA depletion. The disorder usually manifests in early childhood and leads to early death. The gene encodes one of the subunits of succinyl-CoA synthase, the enzyme that catalyzes the reversible conversion of substrates succinyl-CoA and ADP to products succinate and ATP in the tricarboxylic acid pathway. Thirty-two individuals harboring mutations in SUCLA2 have so far been reported, and five different mutations were observed among these individuals. Here we report identification of a novel mutation in SUCLA2 in two cousins affected with encephalomyopathy. The novel mutation causes p.Asp251Asn; the affected amino acid is likely positioned within the ATP-grasp domain of the encoded protein. As previously reported in other patients, we did not observe elevation of methylmalonic acid, the biochemical hallmark of patients with mutations in SUCLA2. We instead found elevated levels of succinylcarnitine.

FOXC1 in human trabecular meshwork cells is involved in regulatory pathway that includes miR-204, MEIS2, and ITGß1.

Forkhead box C1 (FOXC1) is a transcription factor that affects eye development. FOXC1 is implicated in the etiology of glaucoma because mutations in the gene are among the causes of Axenfeld-Rieger syndrome which is often accompanied by glaucoma. Glaucoma is the second leading cause of blindness. It is a complex disorder whose genetic basis in most patients remains unknown. Microarrays expression analysis was performed to identify genes in human trabecular meshwork (TM) primary cultures that are affected by FOXC1 and genes that may have roles in glaucoma. This represents the first genome wide analysis of FOXC1 target genes in any tissue. FOXC1 knock down by siRNAs affected the expression of 849 genes. Results on selected genes were confirmed by real time PCR, immunoblotting, and dual luciferase reporter assays. Observation of MEIS2 as a FOXC1 target and consideration of FOXC1 as a potential target of miR-204 prompted testing the effect of this micro RNA on expression of FOXC1 and several genes identified by array analysis as FOXC1 target genes. It was observed that miR-204 caused decreased expression of FOXC1 and the FOXC1 target genes CLOCK, PLEKHG5, ITGß1, and MEIS2 in the TM cultures. Expression of CLOCK, PLEKHG5, ITGß1 has not previously been reported to be affected by miR-204. The data suggest existence of a complex regulatory pathway in the TM part of which includes interactions between FOXC1, miR-204, MEIS2, and ITGß1. All these molecules are known to have TM relevant functions, and the TM is strongly implicated in the etiology of glaucoma.

Pantothenate kinase 2 mutation with eye-of-the-tiger sign on magnetic resonance imaging in three siblings.

BACKGROUND: Pantothenate kinase associated neurodegeneration (PKAN) is the most prevalent type of neurodegeneration with brain iron accumulation (NBIA) disorders characterized by extrapyramidal signs, and 'eye-of-the-tiger' on T2 brain magnetic resonance imaging (MRI) characterized by hypointensity in globus pallidus and a hyperintensity in its core. All PKAN patients have homozygous or compound heterozygous mutation in PANK2 gene. METHODS: Three sibling patients were diagnosed based on clinical presentations especially extrapyramidal signs and brain MRI. The exons and flanking intronic sequences of PANK2 were sequenced from DNA of leukocytes of the affected individuals. RESULTS: All patients were homozygous for c.C1069T, p.R357W in PANK2 gene. This mutation is well conserved in the homologous protein of distally related spices. CONCLUSION: In the current study we identified three siblings affected with PKAN, all of them have mutations in PANK2 gene. In MRI of all patients with PANK2 mutation eye-of-the-tiger sign was apparent.

Diabetic thermal hyperalgesia: role of TRPV1 and CB1 receptors of periaqueductal gray.

Hyperalgesia is one of the debilitating complications of diabetes. This condition might be caused by defects in central or peripheral processing of pain signals. In the present study we aim to see if diabetic hyperalgesia is related to changes in Transient Receptor Potential Vanilloid 1 (TRPV1) or Cannabinoid CB1 receptors of periaqueductal gray (PAG). Activation of glutamatergic projecting neurons in midbrain ventrolateral periaqueductal gray (VL-PAG) induces antinociception. Agonists of TRPV1 in VL-PAG increase firing of these glutamatergic neurons. CB1 receptor agonists also cause antinociception by decreasing Gamma Aminobutyric Acid (GABA) release in PAG and disinhibiting these glutamatergic neurons. In the present study antinociceptive effect of intra VL-PAG microinjections of CB1 and TRPV1 agonists [WIN55,212-2 (WIN) and capsaicin respectively] were compared in diabetic vs. non-diabetic rats, meanwhile mRNA expression of these receptors in PAG of diabetic and non-diabetic rats were evaluated by real time polymerase chain reaction (real time PCR) assay. Our results showed an attenuation of capsaicin antinociceptive effect (P<0.05) and TRPV1 receptor expression (P=0.023) but an increase in WIN antinociceptive effect (P<0.05) and CB1 receptor expression (P<0.001) in PAG of diabetic vs. non-diabetic rats. It is concluded that down-regulation of TRPV1 receptors in PAG is responsible for reduced antinociceptive effect of TRPV1 agonist. This finding may be an underlying cause of diabetic hyperalgesia. Up-regulation of CB1 receptors might be a compensatory mechanism but the precise elucidation of the effects of CB1 changes on disinhibition needs further studies.

Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli.

The present work aims to study a new NADH-cytochrome b5 reductase (cb5r) from Mucor racemosus PTCC 5305. A cDNA coding for cb5r was isolated from a Mucor racemosus PTCC 5305 cDNA library. The nucleotide sequence of the cDNA including coding and sequences flanking regions was determined. The open reading frame starting from ATG and ending with TAG stop codon encoded 228 amino acids and displayed the closest similarity (73%) with Mortierella alpina cb5r. Lack of hydrophobic residues in the N-terminal sequence was apparent, suggesting that the enzyme is a soluble isoform. The coding sequence was then cloned in the pET16b transcription vector carrying an N-terminal-linked His-Tag sequence and expressed in Escherichia coli BL21 (DE3). The enzyme was then homogeneously purified by a metal affinity column. The recombinant Mucor enzyme was shown to have its optimal activity at pH and temperature of about 7.5 and 40 degrees C, respectively. The apparent K(m) value was calculated to be 13 microM for ferricyanide. To our knowledge, this is the first report on cloning and expression of a native fungal soluble isoform of NADH-cytochrome b5 reductase in E. coli.

Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli

The present work aims to study a new NADH-cytochrome b5 reductase (cb5r) from Mucor racemosus PTCC 5305. A cDNA coding for cb s r was isolated from a Mucor racemosus PTCC 5305 cDNA library. The nucleotide sequence of the cDNA including coding and sequences flanking regions was determined. The open reading frame starting from ATG and ending with TAG stop codon encoded 228 amino acids and displayed the closest similarity (73 percent) with Mortierella alpina cb s r. Lack of hydrophobic residues in the N-terminal sequence was apparent, suggesting that the enzyme is a soluble isoform. The coding sequence was then cloned in the pET16b transcription vector carrying an N-terminal-linked His-Tag® sequence and expressed in Escherichia coli BL21 (DE3). The enzyme was then homogeneously purified by a metal affinity column. The recombinant Mucor enzyme was shown to have its optimal activity at pH and temperature of about 7.5 and 40 °C, respectively. The apparent Km value was calculated to be 13 μM for ferricyanide. To our knowledge, this is the first report on cloning and expression of a native fungal soluble isoform of NADH-cytochrome b5 reductase in E. coli.