Deneddylating enzyme SENP8 regulates neuronal development.

Neddylation is a cellular process in which the neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) is conjugated to the lysine residue of target proteins via serial enzymatic cascades. Recently, it has been demonstrated that neddylation is required for synaptic clustering of metabotropic glutamate receptor 7 (mGlu7) and postsynaptic density protein 95 (PSD-95), and the inhibition of neddylation impairs neurite outgrowth and excitatory synaptic maturation. Similar to the balanced role of deubiquitylating enzymes (DUBs) in the ubiquitination process, we hypothesized that deneddylating enzymes can regulate neuronal development by counteracting the process of neddylation. We find that the SUMO peptidase family member, NEDD8 specific (SENP8) acts as a key neuronal deneddylase targeting the global neuronal substrates in primary rat cultured neurons. We demonstrate that SENP8 expression levels are developmentally regulated, peaking around the first postnatal week and gradually diminishing in mature brain and neurons. We find that SENP8 negatively regulates neurite outgrowth through multiple pathways, including actin dynamics, Wnt/ß-catenin signaling, and autophagic processes. Alterations in neurite outgrowth by SENP8 subsequently result in the impairment of excitatory synapse maturation. Our data indicate that SENP8 plays an essential role in neuronal development and is a promising therapeutic target for neurodevelopmental disorders.

Pathogenic GRM7 Mutations Associated with Neurodevelopmental Disorders Impair Axon Outgrowth and Presynaptic Terminal Development.

Metabotropic glutamate receptor 7 (mGlu7) is an inhibitory heterotrimeric G-protein-coupled receptor that modulates neurotransmitter release and synaptic plasticity at presynaptic terminals in the mammalian central nervous system. Recent studies have shown that rare mutations in glutamate receptors and synaptic scaffold proteins are associated with neurodevelopmental disorders (NDDs). However, the role of presynaptic mGlu7 in the pathogenesis of NDDs remains largely unknown. Recent whole-exome sequencing (WES) studies in families with NDDs have revealed that several missense mutations (c.1865G>A:p.R622Q; c.461T>C:p.I154T; c.1972C>T:p.R658W and c.2024C>A:p.T675K) or a nonsense mutation (c.1757G>A:p.W586X) in the GRM7 gene may be linked to NDDs. In the present study, we investigated the mechanistic links between GRM7 point mutations and NDD pathology. We find that the pathogenic GRM7 I154T and R658W/T675K mutations lead to the degradation of the mGlu7 protein. In particular, the GRM7 R658W/T675K mutation results in a lack of surface mGlu7 expression in heterologous cells and cultured neurons isolated from male and female rat embryos. We demonstrate that the expression of mGlu7 variants or exposure to mGlu7 antagonists impairs axon outgrowth through the mitogen-activated protein kinase (MAPK)-cAMP-protein kinase A (PKA) signaling pathway during early neuronal development, which subsequently leads to a decrease in the number of presynaptic terminals in mature neurons. Treatment with an mGlu7 agonist restores the pathologic phenotypes caused by mGlu7 I154T but not by mGlu7 R658W/T675K because of its lack of neuronal surface expression. These findings provide evidence that stable neuronal surface expression of mGlu7 is essential for neural development and that mGlu7 is a promising therapeutic target for NDDs.SIGNIFICANCE STATEMENT Neurodevelopmental disorders (NDDs) affect brain development and function by multiple etiologies. Metabotropic glutamate receptor 7 (mGlu7) is a receptor that controls excitatory neurotransmission and synaptic plasticity. Since accumulating evidence indicates that the GRM7 gene locus is associated with NDD risk, we analyzed the functional effects of human GRM7 variants identified in patients with NDDs. We demonstrate that stable neuronal surface expression of mGlu7 is essential for axon outgrowth and presynaptic terminal development in neurons. We found that mitogen-activated protein kinase (MAPK)-cAMP-protein kinase A (PKA) signaling and subsequent cytoskeletal dynamics are defective because of the degradation of mGlu7 variants. Finally, we show that the defects caused by mGlu7 I154T can be reversed by agonists, providing the rationale for proposing mGlu7 as a potential therapeutic target for NDDs.

N-linked glycosylation of the mGlu7 receptor regulates the forward trafficking and transsynaptic interaction with Elfn1.

Metabotropic glutamate receptor 7 (mGlu7) regulates neurotransmitter release at the presynaptic active zone in the mammalian brain. The regulation of mGlu7 trafficking into and out of the plasma membrane by binding proteins within the C-terminal region of mGlu7 governs the bidirectional synaptic plasticity. However, the functional importance of the extracellular domain of mGlu7 has not yet been characterized. N-glycosylation is an abundant posttranslational modification that plays crucial roles in protein folding and forward trafficking, but the role of N-glycosylation in mGlu7 function remains unknown. In this study, we find that mGlu7 is N-glycosylated at four asparagine residues in heterologous cells and rat cultured neurons. We demonstrate that N-glycosylation is essential for forward transport and surface expression of mGlu7. Deglycosylated mGlu7 is retained in the ER, obstructing expression on the cell surface, and is degraded through the autophagolysosomal degradation pathway. In addition, we identify the binding domain of mGlu7 to Elfn1, a transsynaptic adhesion protein. We find that N-glycosylation of mGlu7 promotes its interaction with Elfn1, thereby enabling proper localization and stable surface expression of mGlu7 at the presynaptic active zone. These findings provide evidence that N-glycans act to modulate the surface expression, stability, and function of mGlu7.

Crystal structure of a transcription factor, GerE (PaGerE), from spore-forming bacterium Paenisporosarcina sp. TG-14.

In cold and harsh environments such as glaciers and sediments in ice cores, microbes can survive by forming spores. Spores are composed of a thick coat protein, which protects against external factors such as heat-shock, high salinity, and nutrient deficiency. GerE is a key transcription factor involved in spore coat protein expression in the mother cell during sporulation. GerE regulates transcription during the late sporulation stage by directly binding to the promoter of cotB gene. Here, we report the crystal structure of PaGerE at 2.09 Šresolution from Paenisporosarcina sp. TG-14, which was isolated from the Taylor glacier. The PaGerE structure is composed of four α-helices and adopts a helix-turn-helix architecture with 68 amino acid residues. Based on our DNA binding analysis, the PaGerE binds to the promoter region of CotB to affect protein expression. Additionally, our structural comparison studies suggest that DNA binding by PaGerE causes a conformational change in the α4-helix region, which may strongly induce dimerization of PaGerE.

Structural and functional analysis of a dimeric fumarylacetoacetate hydrolase (EaFAH) from psychrophilic Exiguobacterium antarcticum.

Fumarylacetoacetate hydrolase (FAH) is essential for the degradation of aromatic amino acids as well as for the cleavage of carbon-carbon bonds in metabolites or small organic compounds. Here, the X-ray crystal structure of EaFAH, a dimeric fumarylacetoacetate hydrolase from Exiguobacterium antarcticum, was determined, and its functional properties were investigated using biochemical methods. EaFAH adopts a mixed ß-sandwich roll fold with a highly flexible lid region (Val73-Leu94), and an Mg2+ ion is bound at the active site by coordinating to the three carboxylate oxygen atoms of Glu124, Glu126, and Asp155. The hydrolytic activity of EaFAH toward various substrates, including linalyl acetate was investigated using native polyacrylamide gel electrophoresis, activity staining, gel filtration, circular dichroism spectroscopy, fluorescence, and enzyme assays.

Structural and functional characterization of a novel cold-active S-formylglutathione hydrolase (SfSFGH) homolog from Shewanella frigidimarina, a psychrophilic bacterium.

BACKGROUND: S-Formylglutathione is hydrolyzed to glutathione and formate by an S-formylglutathione hydrolase (SFGH) (3.1.2.12). This thiol esterase belongs to the esterase family and is also known as esterase D. SFGHs contain highly conserved active residues of Ser-Asp-His as a catalytic triad at the active site. Characterization and investigation of SFGH from Antarctic organisms at the molecular level is needed for industrial use through protein engineering. RESULTS: A novel cold-active S-formylglutathione hydrolase (SfSFGH) from Shewanella frigidimarina, composed of 279 amino acids with a molecular mass of ~ 31.0 kDa, was characterized. Sequence analysis of SfSFGH revealed a conserved pentapeptide of G-X-S-X-G found in various lipolytic enzymes along with a putative catalytic triad of Ser148-Asp224-His257. Activity analysis showed that SfSFGH was active towards short-chain esters, such as p-nitrophenyl acetate, butyrate, hexanoate, and octanoate. The optimum pH for enzymatic activity was slightly alkaline (pH 8.0). To investigate the active site configuration of SfSFGH, we determined the crystal structure of SfSFGH at 2.32 Å resolution. Structural analysis shows that a Trp182 residue is located at the active site entrance, allowing it to act as a gatekeeper residue to control substrate binding to SfSFGH. Moreover, SfSFGH displayed more than 50% of its initial activity in the presence of various chemicals, including 30% EtOH, 1% Triton X-100, 1% SDS, and 5 M urea. CONCLUSIONS: Mutation of Trp182 to Ala allowed SfSFGH to accommodate a longer chain of substrates. It is thought that the W182A mutation increases the substrate-binding pocket and decreases the steric effect for larger substrates in SfSFGH. Consequently, the W182A mutant has a broader substrate specificity compared to wild-type SfSFGH. Taken together, this study provides useful structure-function data of a SFGH family member and may inform protein engineering strategies for industrial applications of SfSFGH.

Peroxide-dependent oxidation reactions catalyzed by CYP191A1 from Mycobacterium smegmatis.

OBJECTIVES: To find the catalytic activities of CYP191A1 from Mycobacterium smegmatis, in which functions of most P450s are unknown, by using a set of reductase systems, peroxides, and various substrates including fatty acids and human drugs. RESULTS: CYP191A1 was functionally expressed in Escherichia coli and purified. Its catalytic activities were examined with fatty acids, chromogenic and fluorogenic substrates, and several human P450 substrates, in the presence of six different types of electron transfer systems, such as rat NADPH-P450 reductase, Candida NADPH-P450 reductase, ferredoxin/ferredoxin reductase, putidaredoxin/putidaredoxin reductase, and peroxides (H2O2 and t-butyl hydroperoxide). The reactions catalyzed by CYP191A1 included the hydroxylation and O-dealkylation of several substrates. CONCLUSIONS: CYP191A1 preferentially catalyzes the peroxide-dependent oxidation of various substrates over the reductase-dependent reaction. Its peroxygenase activity may be used an effective biocatalytic tool to synthesize the metabolites of drugs.

Marine Antifreeze Proteins: Structure, Function, and Application to Cryopreservation as a Potential Cryoprotectant.

Antifreeze proteins (AFPs) are biological antifreezes with unique properties, including thermal hysteresis(TH),ice recrystallization inhibition(IRI),and interaction with membranes and/or membrane proteins. These properties have been utilized in the preservation of biological samples at low temperatures. Here, we review the structure and function of marine-derived AFPs, including moderately active fish AFPs and hyperactive polar AFPs. We also survey previous and current reports of cryopreservation using AFPs. Cryopreserved biological samples are relatively diverse ranging from diatoms and reproductive cells to embryos and organs. Cryopreserved biological samples mainly originate from mammals. Most cryopreservation trials using marine-derived AFPs have demonstrated that addition of AFPs can improve post-thaw viability regardless of freezing method (slow-freezing or vitrification), storage temperature, and types of biological sample type.

Engineering Synthetic Multistress Tolerance in Escherichia coli by Using a Deinococcal Response Regulator, DR1558.

Cellular robustness is an important trait for industrial microbes, because the microbial strains are exposed to a multitude of different stresses during industrial processes, such as fermentation. Thus, engineering robustness in an organism in order to push the strains toward maximizing yield has become a significant topic of research. We introduced the deinococcal response regulator DR1558 into Escherichia coli (strain Ec-1558), thereby conferring tolerance to hydrogen peroxide (H2O2). The reactive oxygen species (ROS) level in strain Ec-1558 was reduced due to the increased KatE catalase activity. Among four regulators of the oxidative-stress response, OxyR, RpoS, SoxS, and Fur, we found that the expression of rpoS increased in Ec-1558, and we confirmed this increase by Western blot analysis. Electrophoretic mobility shift assays showed that DR1558 bound to the rpoS promoter. Because the alternative sigma factor RpoS regulates various stress resistance-related genes, we performed stress survival analysis using an rpoS mutant strain. Ec-1558 was able to tolerate a low pH, a high temperature, and high NaCl concentrations in addition to H2O2, and the multistress tolerance phenotype disappeared in the absence of rpoS. Microarray analysis clearly showed that a variety of stress-responsive genes that are directly or indirectly controlled by RpoS were upregulated in strain Ec-1558. These findings, taken together, indicate that the multistress tolerance conferred by DR1558 is likely routed through RpoS. In the present study, we propose a novel strategy of employing an exogenous response regulator from polyextremophiles for strain improvement.

Suppression of colitis-associated carcinogenesis through modulation of IL-6/STAT3 pathway by balsalazide and VSL#3.

BACKGROUND AND AIM: Recent studies suggest that the anti-inflammatory agent balsalazide (BSZ) and probiotic agent VSL#3 have potential therapeutic benefits for the treatment of patients with inflammatory bowel disease. However, their effectiveness in preventing colitis-associated carcinogenesis (CAC) remains uncertain. The aim of the present study was to determine the chemopreventive effects of BSZ and VSL#3 in the murine azoxymethane (AOM)/dextran sodium sulfate (DSS) model. METHODS: C57B/L6J mice were randomly divided into four groups: CAC group, BSZ group, VSL#3 group, and BSZ + VSL#3 group. After 2 weeks, the AOM/DSS model was induced by AOM injection followed by two cycles of 2% DSS. RESULTS: During first and second cycles of DSS, the number of F4/80-positive macrophages was significantly lower in the drug-treated groups compared with the CAC group (P < 0.05). At the endpoint, the total numbers of tumors in the drug-treated groups were significantly low compared with the CAC group (P < 0.05), and the drug-treated groups had significantly lower F4/80-positive macrophages in the tumor stroma (P < 0.01). The protein production of macrophage inflammatory protein 1 beta, monocyte chemoattractant protein-1, interleukin (IL)-6, and IL-10 in the colon tissues decreased in concordance with the plasma concentrations of the cytokines (P < 0.05). The drug-treated groups revealed lower expression of p-STAT3 compared with the CAC group. In addition, BCL2 decreased, and BAX increased markedly in the BSZ + VSL#3 group. CONCLUSIONS: These results revealed that BSZ and VSL#3 have chemopreventive effects against CAC through IL-6/STAT3 suppression. BSZ and VSL#3 could be suitable options for chemoprevention of colorectal cancer.

Crystal Structure of a Putative Cytochrome P450 Alkane Hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and Its Conformational Substrate Binding.

Enzymatic alkane hydroxylation reactions are useful for producing pharmaceutical and agricultural chemical intermediates from hydrocarbons. Several cytochrome P450 enzymes catalyze the regio- and stereo-specific hydroxylation of alkanes. We evaluated the substrate binding of a putative CYP alkane hydroxylase (CYP153D17) from the bacterium Sphingomonas sp. PAMC 26605. Substrate affinities to C10-C12 n-alkanes and C10-C14 fatty acids with Kd values varied from 0.42 to 0.59 µM. A longer alkane (C12) bound more strongly than a shorter alkane (C10), while shorter fatty acids (C10, capric acid; C12, lauric acid) bound more strongly than a longer fatty acid (C14, myristic acid). These data displayed a broad substrate specificity of CYP153D17, hence it was named as a putative CYP alkane hydroxylase. Moreover, the crystal structure of CYP153D17 was determined at 3.1 Å resolution. This is the first study to provide structural information for the CYP153D family. Structural analysis showed that a co-purified alkane-like compound bound near the active-site heme group. The alkane-like substrate is in the hydrophobic pocket containing Thr74, Met90, Ala175, Ile240, Leu241, Val244, Leu292, Met295, and Phe393. Comparison with other CYP structures suggested that conformational changes in the ß1-ß2, α3-α4, and α6-α7 connecting loop are important for incorporating the long hydrophobic alkane-like substrate. These results improve the understanding of the catalytic mechanism of CYP153D17 and provide valuable information for future protein engineering studies.

Regioselective hydroxylation of omeprazole enantiomers by bacterial CYP102A1 mutants.

A large set of Bacillus megaterium CYP102A1 mutants are known to metabolize various drugs to form human metabolites. Omeprazole (OMP), a proton pump inhibitor, has been widely used as an acid inhibitory agent for the treatment of gastric acid hypersecretion disorders. It is primarily metabolized by human CYP2C19 and CYP3A4 to 5'-OH OMP and a sulfone product, respectively. It was recently reported that several CYP102A1 mutants can oxidize racemic and S-OMP to 5'-OH OMP and that these mutants can further oxidize 5'-OH racemic OMP to 5'-COOH OMP. Here, we report that the S- and R-enantiomers of OMP are hydroxylated by 26 mutants of CYP102A1 to produce 1 major metabolite (5'-OH OMP) regardless of the chirality of the parent substrates. Although the binding of R-OMP to the CYP102A1 active site caused a more apparent change of heme environment compared with binding of S-OMP, there was no correlation between the spectral change upon substrate binding and catalytic activity of either enantiomer. The 5'-OH OMP produced from racemic, S-, and R-OMP could be obtained with a high conversion rate and high selectivity when the triple R47L/F87V/L188Q mutant was used. These results suggest that bacterial CYP102A1 mutants can be used to produce the human metabolite 5'-OH OMP from both the S- and R-enantiomers of OMP.

Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs.

Human drug metabolites produced by cytochrome P450 enzymes are critical for safety testing and may themselves act as drugs or leads in the drug discovery and development process. Here, highly active chimeric fusion proteins (chimeras) were obtained by reductase domain swapping of mutants at key catalytic residues of the heme domain with that of a natural variant (CYP102A1.2) of P450 BM3 (CYP102A1.1) from Bacillus megaterium. Random mutagenesis at the heme domain of the chimera was also used to generate chimeric mutants that were more active and diverse than the chimeras themselves. To determine whether the chimeras and several mutants of the highly active chimera displayed enhanced catalytic activity and, more importantly, whether they acquired activities of biotechnological importance, we measured the oxidation activities of the chimeras and chimeric mutants toward human P450 substrates, mainly drugs. Some of the chimeric mutants showed high activity toward typical human P450 substrates including drugs. Statin leads, especially chiral products, with inhibitory effects toward HMG-CoA reductase could be obtained from metabolites of statin drugs generated using these chimeric mutants. This study reveals the critical role of the reductase domain for the activity of P450 BM3 and shows that chimeras generated by domain swapping can be used to develop industrial enzymes for the synthesis of human metabolites from drugs and drug leads.

Predicting CYP2C19 catalytic parameters for enantioselective oxidations using artificial neural networks and a chirality code.

Cytochromes P450 (CYP for isoforms) play a central role in biological processes especially metabolism of chiral molecules; thus, development of computational methods to predict parameters for chiral reactions is important for advancing this field. In this study, we identified the most optimal artificial neural networks using conformation-independent chirality codes to predict CYP2C19 catalytic parameters for enantioselective reactions. Optimization of the neural networks required identifying the most suitable representation of structure among a diverse array of training substrates, normalizing distribution of the corresponding catalytic parameters (k(cat), K(m), and k(cat)/K(m)), and determining the best topology for networks to make predictions. Among different structural descriptors, the use of partial atomic charges according to the CHelpG scheme and inclusion of hydrogens yielded the most optimal artificial neural networks. Their training also required resolution of poorly distributed output catalytic parameters using a Box-Cox transformation. End point leave-one-out cross correlations of the best neural networks revealed that predictions for individual catalytic parameters (k(cat) and K(m)) were more consistent with experimental values than those for catalytic efficiency (k(cat)/K(m)). Lastly, neural networks predicted correctly enantioselectivity and comparable catalytic parameters measured in this study for previously uncharacterized CYP2C19 substrates, R- and S-propranolol. Taken together, these seminal computational studies for CYP2C19 are the first to predict all catalytic parameters for enantioselective reactions using artificial neural networks and thus provide a foundation for expanding the prediction of cytochrome P450 reactions to chiral drugs, pollutants, and other biologically active compounds.

Self-Aggregating Tau Fragments Recapitulate Pathologic Phenotypes and Neurotoxicity of Alzheimer's Disease in Mice.

In tauopathy conditions, such as Alzheimer's disease (AD), highly soluble and natively unfolded tau polymerizes into an insoluble filament; however, the mechanistic details of this process remain unclear. In the brains of AD patients, only a minor segment of tau forms ß-helix-stacked protofilaments, while its flanking regions form disordered fuzzy coats. Here, it is demonstrated that the tau AD nucleation core (tau-AC) sufficiently induced self-aggregation and recruited full-length tau to filaments. Unexpectedly, phospho-mimetic forms of tau-AC (at Ser324 or Ser356) show markedly reduced oligomerization and seeding propensities. Biophysical analysis reveal that the N-terminus of tau-AC facilitates the fibrillization kinetics as a nucleation motif, which becomes sterically shielded through phosphorylation-induced conformational changes in tau-AC. Tau-AC oligomers are efficiently internalized into cells via endocytosis and induced endogenous tau aggregation. In primary hippocampal neurons, tau-AC impaired axon initial segment plasticity upon chronic depolarization and is mislocalized to the somatodendritic compartments. Furthermore, it is observed significantly impaired memory retrieval in mice intrahippocampally injected with tau-AC fibrils, which corresponds to the neuropathological staining and neuronal loss in the brain. These findings identify tau-AC species as a key neuropathological driver in AD, suggesting novel strategies for therapeutic intervention.

Correlation of Body Composition via Bioelectrical Impedance Analysis and Motor Function and Recovery of Upper Extremity in Patients Undergoing Stroke Rehabilitation.

Bioelectrical impedance analysis (BIA) has been used to investigate the body compositions and predict functional outcomes in patients with stroke, while the role of BIA to predict motor function or recovery in stroke has not been clarified. This study aimed to investigate relationship between body composition measured by BIA and upper limb motor function and recovery. Body compositions (soft tissue lean mass, phase angle, body fat mass and body water) of fifty patients who are admitted to the stroke rehabilitation unit were segmentally analyzed via BIA. The motor recovery of upper extremity (UE) was evaluated via Fugl-Meyer Assessment (UE-FMA) at the time of transfer and discharge. Correlations between body composition and UE-FMA at discharge were analyzed using Spearman correlation coefficient. Multiple regression analysis was used to determine the regression between body composition and motor function and recovery. The Δ Phase angle, the difference of both sides was significantly linearly inversely correlated with UE-FMA at discharge. However, in multiple regression analysis, body compositions including phase angle did not significantly predict motor function at discharge or motor recovery. The Δ Phase angle is related to the severity of upper limb motor function at discharge in subacute stroke patients, and further studies are needed to determine its value as a predictor for motor recovery.

Case Report: The success of face analysis technology in extremely rare genetic diseases in Korea: Tatton-Brown-Rahman syndrome and Say-Barber -Biesecker-Young-Simpson variant of ohdo syndrome.

Tatton-Brown-Rahman syndrome (TBRS) and Say-Barber-Biesecker- Young-Simpson variant of Ohdo syndrome (SBBYSS) are extremely rare genetic disorders with less than 100 reported cases. Patients with these disorders exhibit a characteristic facial dysmorphism: TBRS is characterized by a round face, a straight and thick eyebrow, and prominent maxillary incisors, whereas SBBYSS is characterized by mask-like facies, blepharophimosis, and ptosis. The usefulness of Face2Gene as a tool for the identification of dysmorphology syndromes is discussed, because, in these patients, it suggested TBRS and SBBYSS within the top five candidate disorders. Face2Gene is useful for the diagnosis of extremely rare diseases in Korean patients, suggesting the possibility of expanding its clinical applications.

Generation of human chiral metabolites of simvastatin and lovastatin by bacterial CYP102A1 mutants.

Recently, the wild-type and mutant forms of cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium were found to oxidize various xenobiotic substrates, including pharmaceuticals, of human P450 enzymes. Simvastatin and lovastatin, which are used to treat hyperlipidemia and hypercholesterolemia, are oxidized by human CYP3A4/5 to produce several metabolites, including 6'ß-hydroxy (OH), 3″-OH, and exomethylene products. In this report, we show that the oxidation of simvastatin and lovastatin was catalyzed by wild-type CYP102A1 and a set of its mutants, which were generated by site-directed and random mutagenesis. One major hydroxylated product (6'ß-OH) and one minor product (6'-exomethylene), but not other products, were produced by CYP102A1 mutants. Formation of the metabolites was confirmed by high-performance liquid chromatography, liquid chromatography-mass spectroscopy, and NMR. Chemical methods to synthesize the metabolites of simvastatin and lovastatin have not been reported. These results demonstrate that CYP102A1 mutants can be used to produce human metabolites, especially chiral metabolites, of simvastatin and lovastatin. Our computational findings suggest that a conformational change in the cavity of the mutant active sites is related to the activity change. The modeling results also suggest that the activity change results from the movement of several specific residues in the active sites of the mutants. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward simvastatin and lovastatin.

Bilateral Cerebral Ptosis in a Patient with Subdural Hemorrhage: a Case Report.

Although cerebral ptosis is rare, it is commonly associated with unilateral right cerebral hemisphere lesions. We report a case of a 79-year-old woman who presented with bilateral complete ptosis after a traumatic right fronto-temporo-parietal subdural hemorrhage (SDH). Bilateral ptosis was the primary manifestation of the acute right SDH, and the patient had no parenchymal lesion. Her prognosis was good, and she made a complete recovery. Right hemispheric hypoperfusion, as demonstrated on brain perfusion single-photon emission computed tomography, implied that the lateralization of eyelid control was in the right hemisphere, in line with previous reports.

Prominent Asymmetric Muscle Weakness and Atrophy in Seronegative Immune-Mediated Necrotizing Myopathy.

Immune-mediated necrotizing myopathy, a new subgroup of inflammatory myopathies, usually begins with subacute onset of symmetrical proximal muscle weakness. A 35-year-old male presented with severe asymmetric iliopsoas atrophy and low back pain with a previous history of left lower extremity weakness. Although his first left lower extremity weakness occurred 12 years ago, he did not receive a clear diagnosis. Magnetic resonance imaging of both thigh muscles showed muscle edema and contrast enhancement in patch patterns, and the left buttock and thigh muscles were more atrophied compared to the right side. Serum creatine kinase levels were elevated, and serologic testings were all negative. Genetic testing using a targeted gene-sequencing panel for neuromuscular disease including myopathy identified no pathogenic variants. Muscle biopsy on the right vastus lateralis showed scattered myofiber necrosis with phagocytosis and an absence of prominent inflammatory cells, consistent with seronegative necrotizing myopathy. Thus, unusual asymmetric muscle weakness and atrophy can be a manifestation of inflammatory myopathy.