Exploring the substrate stereoselectivity and catalytic mechanism of nonribosomal peptide macrocyclization in surugamides biosynthesis.

SurE, the first reported penicillin-binding protein-like thioesterase (PBP-like TE), is known as a new off-loading cyclase, which catalyzes heterochiral coupling in nonribosomal peptides (NRPs). However, the structural rationale for substrate stereoselectivity and enzymatic mechanism remains mysterious. Here, computational models, integrating MD simulations and QM/MM methods, unveiled SurE's substrate recognition and catalytic process. An oxyanion hole stabilized the C-terminal D-residue during recognition. Residue R446 anchored the substrate for macrocyclization. A vital hydrogen-bonding network (Y154, K66, N156), verified by mutation results, was responsible for the recognition of N-terminal L-residue and involvement in catalytic process with a calculated 19.4 kcal/mol energy barrier. Four novel-designed peptide precursors were effectively cyclized into cyclopeptides by SurE based on computational analysis. Our results provide a comprehensive understanding of SurE's catalytic mechanism and guiding design of versatile PBP-like TEs for novel macrocyclic NRPs.

Bilateral thalamic and brainstem anaplastic astrocytoma: A case report.

RATIONALE: Bilateral thalamic glioma is extremely rare and characterized by strictly limited involvement of bilateral thalami. To investigate its clinical and neuroimaging features, we herein reported a rare case of anaplastic astrocytoma (AA) involving both thalami and the brainstem and reviewed the literature. PATIENT CONCERNS: A-33-year-old Chinese woman was referred to our department owing to persistent headache and nausea and vomiting. Neurological examination showed mild cognitive impairment and positive Kernig sign. DIAGNOSIS: Brain magnetic resonance imaging (MRI) demonstrated asymmetrical and swollen lesions involving both thalami, midbrain and pontine tegmentum, without restricted diffusion or enhancement. On day 7 after admission, she was transferred to the department of neurosurgery and underwent a stereotactic brain biopsy of the right thalamic lesion. Histopathological features and immunohistochemistry were consistent with AA, IDH wild-type, World Health Organization grade III. INTERVENTIONS: She was administrated with mannitol and glycerin fructose for decreasing intracranial pressure. OUTCOMES: In spite of receiving chemotherapy, she died on 2-month after her initial diagnosis. LESSONS: AA involving in both thalami and brainstem is a rare entity with poor prognosis. The clinicians and radiologists should deepen their awareness of the specific MRI feature of bilateral thalamic involvement. When MRI alone is insufficient, the utility of stereotactic biopsy is essential for making a definitive diagnosis.

A novel TNKS/USP25 inhibitor blocks the Wnt pathway to overcome multi-drug resistance in TNKS-overexpressing colorectal cancer.

Modulating Tankyrases (TNKS), interactions with USP25 to promote TNKS degradation, rather than inhibiting their enzymatic activities, is emerging as an alternative/specific approach to inhibit the Wnt/ß-catenin pathway. Here, we identified UAT-B, a novel neoantimycin analog isolated from Streptomyces conglobatus, as a small-molecule inhibitor of TNKS-USP25 protein-protein interaction (PPI) to overcome multi-drug resistance in colorectal cancer (CRC). The disruption of TNKS-USP25 complex formation by UAT-B led to a significant decrease in TNKS levels, triggering cell apoptosis through modulation of the Wnt/ß-catenin pathway. Importantly, UAT-B successfully inhibited the CRC cells growth that harbored high TNKS levels, as demonstrated in various in vitro and in vivo studies utilizing cell line-based and patient-derived xenografts, as well as APCmin/+ spontaneous CRC models. Collectively, these findings suggest that targeting the TNKS-USP25 PPI using a small-molecule inhibitor represents a compelling therapeutic strategy for CRC treatment, and UAT-B emerges as a promising candidate for further preclinical and clinical investigations.

Biosynthesis of trialkyl-substituted aromatic polyketide NFAT-133 involves unusual P450 monooxygenase-mediating aromatization and a putative metallo-beta-lactamase fold hydrolase.

The bacterial trialkyl-substituted aromatic polyketides are structurally featured with the unusual aromatic core in the middle of polyketide chain such as TM-123 (1), veramycin A (2), NFAT-133 (3) and benwamycin I (4), which were discovered from Streptomyces species and demonstrated with antidiabetic and immunosuppressant activities. Though the biosynthetic pathway of 1-3 was reported as a type I polyketide synthase (PKS), the PKS assembly line was interpreted inconsistently, and it remains a mystery how the compound 3 was generated. Herein, the PKS assembly logic of 1-4 was revised by site-mutagenetic analysis of the PKS dehydratase domains. Based on gene deletion and complementation, the putative P450 monooxygenase nftE1 and metallo-beta-lactamase (MBL) fold hydrolase nftF1 were verified as essential genes for the biosynthesis of 1-4. The absence of nftE1 led to abolishment of 1-4 and accumulation of new products (5-8). Structural elucidation reveals 5-8 as the non-aromatic analogs of 1, suggesting the NftE1-catalyzed aromatic core formation. Deletion of nftF1 resulted in disappearance of 3 and 4 with the compounds 1 and 2 unaffected. As a rare MBL-fold hydrolase from type I PKSs, NftF1 potentially generates the compound 3 through two strategies: catalyze premature chain-offloading as a trans-acting thioesterase or hydrolyze the lactone-bond of compound 1 as an esterase.

The theoretical model and sinusoidal optimization strategy for oscillating shear valve type continuous waves generator.

Continuous-wave mud generator is widely adopted for real-time downhole data transmission of deep wells in the oil industry. There have been many studies on rotor structure optimization of continuous rotary valve tools but few studies on the different optimization mechanisms of oscillating shear valve tools. In this paper, a theoretical model of pressure wave generation for oscillating shear valve tools was presented, and the reason for the non-sinusoidal waveform was analyzed theoretically for the first time. A series of rotor rotation strategies were proposed to improve the sinusoidal properties of pressure waves without changing the mechanical structure of the rotor. Finally, CFD with different parameters was conducted to verify the feasibility of the optimization scheme. The numerical results give the recommended range of parameters under different control schemes. In addition, the relationship between throttle pressure drop and pressure wave waveform is found through comparison with previous research of the team, which provides a new research idea for the optimization of the pressure wave in the future.

Iterative-Acting Thioesterase from Polyketide Biosynthesis Accepts Diverse Nucleophilic Alcohols to Yield Oxazole-Containing Esters.

The biosynthesis of antitumor oxazole-containing conglobatin is directed by a multienzyme assembly line of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), in which an uncanonical iterative-acting C-terminal thioesterase domain, Cong-TE, ligated two fully elongated chains/conglobatin monomers on the terminal acylcarrier protein and subsequently cyclized the resulting dimer to a C2-symmetric macrodiolide. Screening of the conglobatin producer for secondary metabolites led to the discovery of two new compounds conglactones A (1) and B (2), possessing inhibitory activities to phytopathogenic microorganisms and cancer cells, respectively. The compounds 1 and 2 feature the ester bond-linked hybrid structures consisting of an aromatic polyketide benwamycin I (3) and one (for 1)/two (for 2) molecules of the conglobatin monomer (5). Genetic mutational analysis revealed that the production of 1 and 2 was correlated with the biosynthetic pathways of 3 and 5. Biochemical analysis indicated that 1 and 2 were produced by Cong-TE from 3 and an N-acetylcysteamine thioester form of 5 (7). Furthermore, the substrate compatibility of Cong-TE was demonstrated by enzymatically generating a bunch of ester products from 7 and 43 exotic alcohols. This property of Cong-TE was further validated by producing 36 hybrid esters in the fermentation of conglobatin producer fed with nonindigenous alcohols. This work shows a prospect of developing Cong-TE for green synthesis of valuable oxazole-containing esters, thus complementing the environmentally unfriendly chemosynthesis strategies.

Area postrema syndrome with linear enhancement along the surface of the brainstem and fourth ventricle in autoimmune GFAP astrocytopathy.

BACKGROUND: Glial fibrillary acidic protein (GFAP) astrocytopathy, a novel autoimmune disease of the nervous system, was first defined in 2016. To our knowledge, area postrema syndrome (APS) with linear enhancement along the surface of the brainstem and fourth ventricle is extremely rare in this disorder. CASE PRESENTATION: A Chinese woman presented with intractable nausea and vomiting after onset of flu-like symptoms. Brain magnetic resonance imaging (MRI) disclosed abnormal signal intensities in the dorsal medulla oblongata including area postrema. Besides, linear enhancement surrounding the surface of the brainstem and fourth ventricle was visualized after gadolinium injection. Cerebrospinal fluid (CSF) analysis showed increased cell count and protein. A cell-based assay was positive for anti-GFAP IgG in CSF. She was diagnosed with autoimmune GFAP astrocytopathy and treated with high-dose glucocorticoid. The patient received a quick recovery with entire resolution of the initial abnormalities. CONCLUSIONS: Isolated APS can be the initial manifestation of autoimmune GFAP astrocytopathy. Linear enhancement surrounding the surface of the brainstem and fourth ventricle is another neuroradiological hallmark.

Switching Prenyl Donor Specificities in Squalene Synthase-Like Aromatic Prenyltransferases from Bacterial Carbazole Alkaloid Biosynthesis.

Lavanduquinocin (LDQ) is a potent neuroprotective carbazole alkaloid from Streptomyces species that features a rare cyclic monoterpene/cyclolavandulyl moiety attached to the tricyclic carbazole nucleus. We elucidated the biosynthetic logic of LDQ by enzymatically reconstituting the total biosynthetic pathway and identified the genes required for generating the cyclolavandulyl moiety in LDQ based on mutagenetic analysis, including a cyclolavandulyl diphosphate synthase gene ldqA and a squalene synthase-like aromatic prenyltransferase gene ldqG. LdqG is homologous to carbazole prenyltransferases, NzsG and CqsB4, discovered from the biosynthetic pathways of two bacterial carbazoles, neocarazostatin and carquinostatin. Based on analysis of the sequences and modeled protein structures, further in vitro and in vivo site-directed mutagenetic analyses led to identification of two residue sites, F53 and C57 in NzsG vs I54 and A58 in LdqG, which play crucial roles in governing the prenyl donor specificities toward cyclolavandulyl, dimethylallyl, and geranyl diphosphates. By applying this knowledge in strain engineering, prenyl donor delivery was rationally switched to produce the desired prenylated carbazoles. The study provides an opportunity to rationally manipulate the prenylation modification to carbazole alkaloids, which could influence the biological activities by increasing the affinity for membranes as well as the interactions with cellular targets.

The stroke mechanism, clinical presentation, and radiological feature of bilateral medial medullary infarction.

BACKGROUND: Bilateral medial medullary infarction (BMMI) is a rare type of posterior circulation stroke. The aim of this study is to characterize its stroke mechanisms, clinical manifestations, neuroradiological features, and prognosis. METHODS: From January 2015 to June 2021, a retrospective review of 15 patients diagnosed with BMMI was conducted. The clinical and neuroradiological features were summarized by our experienced neurologists. RESULTS: Fifteen patients (12 male, 3 female), ranging in age from 48 to 72 years, satisfied the inclusion criteria. The common clinical presentations included motor weakness (100%), deep sensory disturbance (93.3%), vertigo/dizziness (80%), dysarthria (93.3%), and dysphagia (66.7%). Vertically, infarct lesions in the rostral medulla were observed in all included patients. Horizontally, "heart appearance," "Y appearance," and "fan appearance" infarcts occurred in 9 cases (60%), 5 cases (33.3%), and 1 (6.7%) case, respectively. Patients (53.3%) had severe stenosis or occlusion in unilateral vertebral artery (VA), and 33.3% had normal findings in the vertebrobasilar artery. Patients (93.3%) achieved poor prognosis. CONCLUSION: BMMI is more frequently located in the rostral medulla and comprises three forms of infarction. The two main stroke etiologies of BMMI are large-artery atherosclerosis (LAA) and small vessel disease (SVD). BMMI is always associated with bad clinical outcome.

Investigation of carbonyl amidation and O-methylation during biosynthesis of the pharmacophore pyridyl of antitumor piericidins.

Piericidins are a large family of bacterial α-pyridone antibiotics with antitumor activities such as their anti-renal carcinoma activity exhibited recently in nude mice. The backbones of piericidins are derived from ß, δ-diketo carboxylic acids, which are offloaded from a modular polyketide synthase (PKS) and putatively undergo a carbonyl amidation before α-pyridone ring formation. The tailoring modifications to the α-pyridone structure mainly include the verified hydroxylation and O-methylation of the C-4' position and an unidentified C-5' O-methylation. Here, we describe a piericidin producer, terrestrial Streptomyces conglobatus, which contains a piericidin biosynthetic gene cluster in two different loci. Deletion of the amidotransferase gene pieD resulted in the accumulation of two fatty acids that should be degraded from the nascent carboxylic acid released by the PKS, supporting the carbonyl amidation function of PieD during α-pyridone ring formation. Deletion of the O-methyltransferase gene pieB1 led to the production of three piericidin analogues lacking C-5' O-methylation, therefore confirming that PieB1 specifically catalyses the tailoring modification. Moreover, bioactivity analysis of the mutant-derived products provided clues regarding the structure-function relationship for antitumor activity. The work addresses two previously unidentified steps involved in pyridyl pharmacophore formation during piericidin biosynthesis, facilitating the rational bioengineering of the biosynthetic pathway towards valuable antitumor agents.

Unilateral cerebral cortical encephalitis (CCE) with positive anti-MOG antibodies: A case report.

RATIONALE: Nowadays, myelin oligodendrocyte glycoprotein (MOG)-antibody-associated disease (MOGAD) is regarded as an independent inflammatory demyelinating disease. Here, we report a rare case of unilateral cerebral cortical encephalitis (CCE) with positive anti-MOG antibodies. PATIENT CONCERNS: A 19-year-old woman was admitted to our hospital owing to acute onset fever and headache. Four days later, she experienced a focal seizure that progressed to generalized tonic-clonic seizures. DIAGNOSIS: Brain magnetic resonance imaging (MRI) demonstrated cortical lesions in the left cerebral hemisphere on T2-weighted fluid-attenuated inversion recovery imaging. The patient was positive for anti-MOG antibodies in serum and diagnosed with anti-MOG antibody-associated unilateral CCE. INTERVENTIONS: She was administrated with intravenous methylprednisolone followed by oral corticosteroids. OUTCOMES: On day 14 after admission, a repeat MRI revealed partial resolution of the initial abnormalities. The patient received a quick recovery without residual symptoms. CONCLUSIONS: Unilateral CCE with positive anti-MOG antibodies has emerged as a special clinical phenotype of MOGAD. It should be emphasized that the characteristic neuroradiological features of CCE would be an important clue to the correct diagnosis of MOGAD.

Defense responses of sulfur dioxygenase to sulfide stress in the razor clam Sinonovacula constricta.

BACKGROUND: Sulfide is a well-known toxicant widely distributed in the culture environment. As a representative burrowing benthic bivalve, the razor clam Sinonovacula constricta is highly sulfide tolerant. Mitochondrial sulfide oxidation is an important way for sulfide detoxification, where sulfur dioxygenase (SDO) is the second key enzyme. OBJECTIVE: To investigate the mechanism of sulfide tolerance in S. constricta, the molecular characterization of its SDO (designated as ScSDO) was studied. METHODS: The cDNA sequence of ScSDO was cloned by RACE technique. The response of ScSDO in gills and livers of S. constricta was investigated during sulfide exposure (50, 150, and 300 µM sulfide) for 0, 3, 6, 12, 24, 48, 72, and 96 h by qRT-PCR. Moreover, the temporal expression of ScSDO protein in S. constricta gills after exposure to 150 µM sulfide was detected by Western blot. The subcellular location of ScSDO was identified by TargetP 1.1 prediction and Western Blot analysis. RESULTS: The full-length cDNA of ScSDO was 2914 bp, encoding a protein of 304 amino acids. The deduced ScSDO protein was highly conserved, containing the signature HXHXDH motif of the metallo-ß-lactamase superfamily and two metal-binding sites, of which metal-binding site I is known to be the catalytically active center. Subcellular localization confirmed that ScSDO was located only in the mitochondria. Responding to the sulfide exposure, distinct time-dependent increases in ScSDO expression were detected at both mRNA and protein levels. Moreover, the gills exhibited a higher ScSDO expression level than the livers. CONCLUSIONS: All of our results suggest that ScSDO plays an important role in mitochondrial sulfide oxidation during sulfide stress, making S. constricta highly sulfide tolerant. In addition, as a respiratory tissue, the gills play a more critical role in sulfide detoxification.

Bilateral symmetrical deep gray matter involvement and leptomeningeal enhancement in a child with MOG-IgG-associated encephalomyelitis.

BACKGROUND: Currently, myelin oligodendrocyte glycoprotein (MOG)-IgG-associated encephalomyelitis (MOG-EM) is regarded as an independent inflammatory demyelinating disease. Magnetic resonance imaging (MRI) abnormalities occur in 44.4% of patients with MOG-EM. However, symmetrical deep gray matter involvement with leptomeningeal enhancement is rarely described in the literature. CASE PRESENTATION: A 3-year-old boy was admitted to our hospital because of acute onset fever, headache, vomiting and disturbance of consciousness. Neurological examination showed somnolence, neck stiffness and positive Kernig's sign. Brain MRI demonstrated bilateral symmetrical lesions in the basal ganglia and thalamus as well as diffuse leptomeningeal enhancement along the sulci of bilateral hemisphere. Cerebrospinal fluid analysis demonstrated increased cell count (7 cells/mm3, mononuclear cells dominant) and protein (1.17 g/L) without glucose and chloride abnormality. Work-up for infectious and autoimmune causes, serum MOG IgG was positive by cell based assay. Therefore, a diagnosis of MOG-EM was established according to the international recommendatory criteria in 2018. He was administrated with intravenous methylprednisolone followed by oral corticosteroids and had recovered completely within 1 week. CONCLUSIONS: In the setting of meningoencephalitis-like clinical presentation with bilateral symmetrical deep gray matter involvement, MOG-EM should be distinguished from other infectious and autoimmune disorders, such as Epstein-Barr virus (EBV) encephalitis, Japanese encephalitis and Anti-NMDA receptor (NMDAR) encephalitis. Besides, aseptic meningitis associated with leptomeningeal enhancement may be an atypical phenotype of MOG-EM.

Applying molecular networking for targeted isolation of depsipeptides.

LC-HRMS/MS molecular networking enabled the targeted isolation of three new neoantimycin analogs (1, 3, 5) and two known ones (2, 4) from the culture broth of Streptomyces conglobatus RJ8. After derivatization into C1-hydroxyl form compounds (6-10) respectively, the absolute structures of 1-5 were clearly determined by analyzing the hydrolyzed components from 6-10. Compounds 2 and 3 were confirmed to be a pair of epimers with different stereochemistry at C-2, and so were 4 and 5. This is the first report of the isolation and characterization of epimers of NATs. The most abundant eight compounds we obtained were subjected to a cytotoxicity assay, 1 and 6 exhibited excellent cytotoxicity with the lowest IC50 value in the picomolar range against six human carcinoma cell lines while 7 and 8 showed potent cytotoxicity against PC-9 and PC-9/GR cell lines.

The clinical and neuroradiological features of patients of coexisting atraumatic convexity subarachnoid hemorrhage and large artery atherosclerosis stroke: A retrospective observational study.

ABSTRACT: Atraumatic convexity subarachnoid hemorrhage (c-SAH) concomitant with large artery atherosclerosis (LAA) stroke has been rarely discussed in the literature. Our aim in this study is to characterize the clinical and neuroradiological features of patients with LAA stroke and c-SAH.A retrospective study from a single institution was performed between January 2016 and June 2020. Only patients diagnosed with c-SAH and LAA stoke were included in this study. The clinical presentation and neuroimaging finding were summarized by our experienced neurologists.In total, 12 patients (8 men, 4 women), ranging in age from 45 to 75 years, were identified. All of them had cardiovascular risk factors and hypertension was the commonest (50%). Almost all patients presented hemiparesis (91.7%). Other clinical presentations included, dysarthria (41.7%), hemianesthesia (33.3%), facial palsy (33.3%), aphasia (16.7%), and cognitive impairment (8.3%). Internal border-zone (IBZ) infarction and cortical border-zone (CBZ) infarction occurred in 12 and 3 patients, respectively. c-SAH might occurred in different cortical sulcis. Percentages of frontal lobe, parietal lobe and fronto-parietal lobe were 41.7% (n = 5), 25% (n = 3) and 25% (n = 3), respectively. All ischemic lesions were ipsilateral to the sites of c-SAH. High-grade atherosclerotic stenosis of large artery was detected in all patients. The M1 segment of middle cerebral artery (MCA) is the second most common atherosclerotic artery after internal carotid artery (ICA).Our data suggest that LAA stroke is always ipsilateral to the site of c-SAH. Severe atherosclerotic changes can also been seen in the M1 segment of MCA apart from extracranial ICA. Moreover, border zone infarction may be a specific form of infarct when c-SAH is confronted with LAA stroke.

Targeted accumulation of selective anticancer depsipeptides by reconstructing the precursor supply in the neoantimycin biosynthetic pathway.

BACKGROUND: Neoantimycins are a group of 15-membered ring depsipeptides isolated from Streptomycetes with a broad-spectrum of anticancer activities. Neoantimycin biosynthesis is directed by the hybrid multimodular megaenzymes of non-ribosomal peptide synthetase and polyketide synthase. We previously discovered a new neoantimycin analogue unantimycin B, which was demonstrated to have selective anticancer activities and was produced from the neoantimycin biosynthetic pathway with a starter unit of 3-hydroxybenzoate, instead of the 3-formamidosalicylate unit that is common for neoantimycins. However, the low fermentation titre and tough isolation procedure have hindered in-depth pharmacological investigation of unantimycin B as an anticancer agent. RESULTS: In this work, we genetically constructed two unantimycin B producer strains and inhibited neoantimycins production by removing natO and natJ-L genes essential for 3-formamidosalicylate biosynthesis, therefore facilitating chromatographic separation of unantimycin B from the complex fermentation extract. Based on the ΔnatO mutant, we improved unantimycin B production twofold, reaching approximately 12.8 mg/L, by feeding 3-hydroxybenzoate during fermentation. Furthermore, the production was improved more than sixfold, reaching approximately 40.0 mg/L, in the ΔnatO strain introduced with a chorismatase gene highly expressed under a strong promoter for endogenously over-producing 3-hydroxybenzoate. CONCLUSION: This work provides a case of targeting accumulation and significant production improvement of medicinally interesting natural products via genetic manipulation of precursor biosynthesis in Streptomycetes, the talented producers of pharmaceutical molecules.