Discovery of antimalarial drugs from secondary metabolites in actinomycetes culture library.

BACKGROUND: Natural products play a key role as potential sources of biologically active substances for the discovery of new drugs. This study aimed to identify secondary metabolites from actinomycete library extracts that are potent against the asexual stages of Plasmodium falciparum (P. falciparum). METHODS: Secondary metabolites from actinomycete library extracts were isolated from culture supernatants by ethyl acetate extraction. Comprehensive screening was performed to identify novel antimalarial compounds from the actinomycete library extracts (n = 28). The antimalarial activity was initially evaluated in vitro against chloroquine/mefloquine-sensitive (3D7) and-resistant (Dd2) lines of P. falciparum. The cytotoxicity was then evaluated in primary adult mouse brain (AMB) cells. RESULTS: Out of the 28 actinomycete extracts, 17 showed parasite growth inhibition > 50% at a concentration of 50 µg/mL, nine were identified with an IC50 value < 10 µg/mL, and seven suppressed the parasite significantly with an IC50 value < 5 µg/mL. The extracts from Streptomyces aureus strains HUT6003 (Extract ID number: 2), S. antibioticus HUT6035 (8), and Streptomyces sp. strains GK3 (26) and GK7 (27), were found to have the most potent antimalarial activity with IC50 values of 0.39, 0.09, 0.97, and 0.36 µg/mL (against 3D7), and 0.26, 0.22, 0.72, and 0.21 µg/mL (against Dd2), respectively. Among them, Streptomyces antibioticus strain HUT6035 (8) showed the highest antimalarial activity with an IC50 value of 0.09 µg/mL against 3D7 and 0.22 µg/mL against Dd2, and a selective index (SI) of 188 and 73.7, respectively. CONCLUSION: Secondary metabolites obtained from the actinomycete extracts showed promising antimalarial activity in vitro against 3D7 and Dd2 cell lines of P. falciparum with minimal toxicity. Therefore, secondary metabolites obtained from actinomycete extracts represent an excellent starting point for the development of antimalarial drug leads.

Effectiveness of a Lifestyle Improvement Support App in Combination with a Wearable Device in Japanese People with Type 2 Diabetes Mellitus: STEP-DM Study.

INTRODUCTION: Although the use of application (app)s and wearable devices supporting diabetes treatment has spread rapidly in recent years, evidence of their impact, especially in combination of them, is limited. TOMOCO™ is a lifestyle improvement support app that features interactive virtual conversations according to the programmed algorithm guiding users toward their goals of lifestyle improvement. We hypothesized that TOMOCO™ in combination with Fitbit, which accurately tracks users' activity level, would encourage people with type 2 diabetes mellitus (T2DM) to change their lifestyles and improve their glycated hemoglobin (HbA1c) levels without changes in conventional therapy. Thus, we performed the present study to explore the effectiveness of this combination in Japanese participants with T2DM who had not achieved their glycemic targets. METHODS: In this single-arm exploratory study, participants with T2DM used the TOMOCO™ and Fitbit in addition to the conventional diet/exercise therapy and anti-diabetic drug for 12 weeks. They were provided with feedback/advice by health care providers based on the TOMOCO™ and Fitbit records. The primary endpoint was the change in HbA1c from baseline to the end of the observation period. Data were expressed as mean ± standard deviation. RESULTS: Fifty-nine (96.7%) of the 61 participants (male, 42 [71.2%]; age, 60.1 ± 8.7 years; HbA1c level, 7.48 ± 0.37% at screening) completed the study. At the end of the observation period, the HbA1c was significantly reduced (- 0.41 ± 0.41%, p < 0.001). This trend was consistent across the preselected patient characteristics, including sex, age, and body mass index. However, it was more pronounced in the participants with earlier stages of behavioral changes defined by the transtheoretical model at baseline. CONCLUSIONS: The unique features of TOMOCO™ in combination with Fitbit, together with conventional therapy, may promote a healthy lifestyle and thus contribute to improving HbA1c in people with T2DM. CLINICAL TRIAL REGISTRATION: jRCT1070220007.

Isolation of Hydrazide-alkenes with Different Amino Acid Origins from an Azoxy-alkene-Producing Mutant of Streptomyces rochei 7434AN4.

A triple mutant (strain KA57) of Streptomyces rochei 7434AN4 produces an azoxy-alkene compound, KA57A, which was not detected in a parent strain or other single and double mutants. This strain accumulated several additional minor components, whose structures were elucidated. HPLC analysis of strain KA57 indicated the presence of two UV active components (KA57D1 and KA57D2) as minor components. They exhibited a maximum UV absorbance at 218 nm, whereas a UV absorbance of azoxy-alkene KA57A was detected at 236 nm, suggesting that both KA57D1 and KA57D2 contain a different chromophore from KA57A. KA57D1 has a molecular formula of C12H22N2O2, and NMR analysis revealed KA57D1 is a novel hydrazide-alkene compound, (Z)-N-acetyl-N'-(hex-1-en-1-yl)isobutylhydrazide. Labeling studies indicated that nitrogen Nß of KA57D1 is derived from l-glutamic acid, and the isobutylamide unit (C-1 to C-3, 2-Me, and Nα) originates from valine. KA57D2 has a molecular formula of C13H24N2O2, and its structure was determined to be (Z)-N-acetyl-N'-(hex-1-en-1-yl)-2-methylbutanehydrazide, in which a 2-methylbutanamide unit was shown to originate from isoleucine. Different biogenesis of the Nα atom (l-serine for KA57A, l-valine for KA57D1, and l-isoleucine for KA57D2) indicates the relaxed substrate recognition for nitrogen-nitrogen bond formation in the biosyntheses of KA57A, KA57D1, and KA57D2.

Isolation and characterization of blackish-brown BY2-melanin accumulated in cultured tobacco BY-2 cells.

The tobacco BY-2 cell line is one of the most utilized plant cell lines. After long-term culture, the cells turn brown to black, but the causal pigment is unknown. We successfully isolated a blackish-brown pigment from BY-2 cells cultured for 3 weeks. Morphological and spectroscopic analyses indicated that the pigment had similar features to a melanin-like substance reported previously. Furthermore, physicochemical analyses revealed that this pigment possessed most of the properties of melanin-like pigments. In addition, the high nitrogen content suggested that it differed from common plant melanins classified as allomelanins, suggesting a novel eumelanin-like pigment: "BY2-melanin". This is the first example showing that eumelanin-like pigments are produced in the cultures of plant cells for which the accumulation of melanin has not been reported. This tobacco BY-2 cell culture technique may represent a customizable and sustainable alternative to conventional melanin production platforms, with significant potential for industrial and pharmacological applications.

Accumulation of lankamycin derivative with a branched-chain sugar from a blocked mutant of chalcose biosynthesis in Streptomyces rochei 7434AN4.

Lankamycin, a macrolide antibiotic produced by Streptomyces rochei 7434AN4, exhibits a moderate antimicrobial activity and acts as a synergistic pair with carbocyclic antibiotic lankacidin C by binding to the ribosome exit tunnel. Its biosynthetic gene (lkm) cluster (orf24-orf53) is located on the largest plasmid pSLA2-L (210,614 bp). Our group possesses a variety of lankamycin derivatives and macrolide-modification enzymes including P450 enzymes and glycosyltransferases, which may lead to expand the chemical library of bioactive macrolides. Here we constructed a mutant of a 3-ketoreductase gene lkmCVI (orf42) involved in d-chalcose biosynthesis, and its metabolite was isolated and structure-elucidated. Accumulation of novel lankamycin derivative harboring a branched-chain deoxysugar, 5-O-(4',6'-dideoxy-3'-C-acetyl-d-ribo-hexopyranosyl)-3-O-(4″-O-acetyl-l-arcanosyl)-lankanolide, indicated that LkmCVI acts as a gate keeper enzyme for d-chalcose synthesis in lankamycin biosynthesis.

Characterization of the surugamide biosynthetic gene cluster of TUA-NKU25, a Streptomyces diastaticus strain isolated from Kusaya, and its effects on salt-dependent growth.

Kusaya, a traditional Japanese fermented fish product, is known for its high preservability, as it contains natural antibiotics derived from microorganisms, and therefore molds and yeasts do not colonize it easily. In this study, the Streptomyces diastaticus strain TUA-NKU25 was isolated from Kusaya, and its growth as well as the production of antibiotics were investigated. Strain TUA-NKU25 showed advantageous growth characteristics in the presence, but not in the absence, of sodium chloride (NaCl). Antimicrobial assay, high-performance liquid chromatography, and electrospray ionization-mass spectrometry analysis showed that this strain produced surugamide A and uncharacterized antimicrobial compound(s) during growth in the presence of NaCl, suggesting that the biosynthesis of these compounds was upregulated by NaCl. Draft genomic analysis revealed that strain TUA-NKU25 possesses a surugamide biosynthetic gene cluster (sur BGC), although it is incomplete, lacking surB/surC. Phylogenetic analysis of strain TUA-NKU25 and surugamide-producing Streptomyces showed that sur BGC formed a clade distinct from other known groups.

Bioinspired computational design of lankacidin derivatives for improvement in antitumor activity.

Background: The 17-membered polyketide, lankacidin C, exhibits considerable antitumor activity as a microtubule stabilizer by binding to the paclitaxel binding site. Method: Esterification of the C-7/C-13 hydroxyl in lankacidin C was performed with acetyl, cinnamoyl and hydrocinnamoyl groups and their antitumor activity was assessed to improve the cytotoxicity of lankacidins through bioinspired computational design. Results: Compared with the cytotoxicity of parent lankacidin C against the HeLa cell line, 13-O-cinnamoyl-lankacidin C demonstrated sevenfold higher cytotoxicity. Furthermore, 7,13-di-O-cinnamoyl-lankacidin C exhibited considerable antitumor activity against three tested cell lines. Conclusion: C13-esterification by a cinnamoyl group dramatically improved antitumor activity, in agreement with computational predictions. This finding provides a potential substrate for next-generation lankacidin derivatives with significant antitumor activity.

Effect of sodium-glucose cotransporter 2 inhibitor medication on new prescriptions of antihypertensives, antigout/antihyperuricemics and antidyslipidemics in Japan: Analysis using the JMDC Claims Database.

AIMS/INTRODUCTION: This study aimed to investigate the effects of sodium-glucose cotransporter 2 inhibitors (SGLT2i) on new prescriptions of drugs, including antihypertensives, antigout/antihyperuricemics and antidyslipidemics, for the treatment of lifestyle-related diseases in Japanese patients with diabetes mellitus using the JMDC Claims Database. MATERIALS AND METHODS: Patients with type 2 diabetes mellitus who were newly treated with SGLT2i or other oral antidiabetic drugs and had not been prescribed any antihypertensives, antigout/antihyperuricemics or antidyslipidemics for at least 1 year were extracted from the database. Using propensity score calibration matching (1:1), we assessed the proportion of patients who started the aforementioned concomitant medications within 2 years, and the risk ratio of SGLT2i to other antidiabetic medication groups was calculated. RESULTS: In 856,796 patients with diabetes mellitus, 734, 1,197 and 703 propensity score calibration-matched patients in each group were analyzed for the prescription of antihypertensives, antigout/antihyperuricemics and antidyslipidemics, respectively. The new prescriptions of antihypertensives and antigout/antihyperuricemics were lower in the SGLT2i group than those in the other oral antidiabetic drug group (risk ratio 0.66 and 0.37, respectively), whereas those of antidyslipidemics were more common in the SGLT2i group (risk ratio 1.43). CONCLUSIONS: New prescriptions of antihypertensives or antigout/antihyperuricemics were lower for patients taking SGLT2i than those taking other oral antidiabetic drugs, probably due to a reduction of blood pressure and uric acid levels by SGLT2i. The more frequent prescriptions of antidyslipidemics might partially reflect a moderate increase in low-density lipoprotein cholesterol levels as a result of sodium-glucose cotransporter 2 inhibition.

Isolation, Biosynthetic Investigation, and Biological Evaluation of Maniwamycin G, an Azoxyalkene Compound from Streptomyces sp. TOHO-M025.

A new maniwamycin analogue, maniwamycin G, was isolated from Streptomyces sp. TOHO-M025 as a major product. Maniwamycin G has a molecular formula of C12H22N2O4, and its extensive NMR analysis revealed that maniwamycin G contains a methoxycarbonyl group instead of an amide as found in maniwamycin F. Its C-2 and C-3 configurations were determined to be (2R, 3R) by circular dichroism spectrum and a modified Mosher method, respectively. The biosynthetic origin of maniwamycin G was investigated using isotope-labeled compounds. The carbon source of maniwamycin G is four acetate units (C-1', C-2'; C-3', C-4'; C-5', C-6'; and C-4, C-5) and l-serine (C-1 to C-3). The nitrogen atom attached at C-2 (Nα) originates from serine, whereas the nitrogen atom of a hexen-1-yl amine unit (Nß) is derived from glutamic acid. The quorum-sensing inhibitory activity of maniwamycin G was 2-fold lower than that of maniwamycin F.

Amelioration of Murine Diabetic Nephropathy with a SGLT2 Inhibitor Is Associated with Suppressing Abnormal Expression of Hypoxia-Inducible Factors.

Diabetic nephropathy (DN), once manifested, is unlikely to completely recover. Factors that influence DN progression were explored by investigating the process of glomerulosclerosis and interstitial fibrosis and chronological changes in glucose, albuminuria, hyperfiltration, and expressions of sodium-glucose cotransporter 2 (SGLT2) and hypoxia-inducible factors (HIFs) up to 50 weeks in inducible cAMP early repressor transgenic mice, a model of severe DN. Long-term intervention with the SGLT2 inhibitor canagliflozin or islet transplantation or heminephrectomy was used. Inducible cAMP early repressor transgenic mice exhibited progressive diabetic glomerulosclerosis and mild interstitial fibrosis, and expressed extensive HIF-1α and HIF-2α in glomerulus and tubules, with sustained hyperfiltration up to 50 weeks. Canagliflozin ameliorated glomerulosclerosis/interstitial fibrosis gradually and reduced HIF overexpression. Islet-transplanted mice exhibited no amelioration. None of the heminephrectomized diabetic mice survived the hyperfiltration overload, but all of the canagliflozin-treated mice survived with re-expressions of HIF-1α and HIF-2α. These results suggest that persistent glomerular hyperfiltration might initiate glomerular injury, and persistent overexpression of HIFs could promote the development of glomerulosclerosis and interstitial fibrosis. Canagliflozin attenuated both changes. Oxidative stress or hypoxia was undetectable in this model. The abnormal expression of HIF-1α and HIF-2α may be a potential therapeutic target for preventing glomerulosclerosis and interstitial fibrosis.

Three 4-monosubstituted butyrolactones from a regulatory gene mutant of Streptomyces rochei 7434AN4.

Extensive metabolite analysis of Streptomyces rochei 7434AN4 was performed to discover uncharacterized secondary metabolites. A mutant strain of S. rochei, in which two regulatory genes srrC (a tetR-type repressor) and srrY (SARP-type activator) were inactivated, accumulated three 4-monosubstituted γ-butyrolactones YT02-A, YT02-B, and KH01-A, which were not detected in the parent strain. Their structures were identified as 4,10-dihydroxy-10-methyldodecan-4-olide, 4,10-dihydroxy-10-methylundecan-4-olide, and 4-hydroxy-11-oxo-10-methyldodecan-4-olide. A structural comparison indicated that the three butanolides and the signaling molecules, termed S. rochei butenolides (SRBs), could share common C12 or C13 fatty acids for their biosynthesis intermediates, however, these three butanolides did not induce antibiotic production even at 50 µM concentration (1000-folds of the minimum antibiotic-inducing concentration of SRBs) in S. rochei.

Overexpression of SRO_3163, a homolog of Streptomyces antibiotic regulatory protein, induces the production of novel cyclohexene-containing enamide in Streptomyces rochei.

Streptomyces antibiotic regulatory proteins (SARPs) are well characterized as transcriptional activators for secondary metabolites in Streptomyces species. Streptomyces rochei 7434AN4 harbors 15 SARP genes, among which 3 were located on a giant linear plasmid pSLA2-L and others were on the chromosome. Some SARP genes were cloned into an integrative thiostrepton-inducible vector pIJ8600, and their recombinants were cultivated. The recombinant of SARP gene, SRO_3163, accumulated a UV-active compound YM3163-A, which was not detected in the parent strain and other SARP recombinants. Its molecular formula was established to be C8H11NO. Extensive NMR analysis revealed that YM3163-A is a novel enamide, 2-(cyclohex-2-en-1-ylidene)acetamide, and its structure was confirmed by chemical synthesis including Horner-Wadsworth-Emmons reaction and ammonolysis.

Production of Agrocinopine A by Ipomoea batatas Agrocinopine Synthase in Transgenic Tobacco and Its Effect on the Rhizosphere Microbial Community.

Agrobacterium tumefaciens is a bacterial pathogen that causes crown gall disease on a wide range of eudicot plants by genetic transformation. Besides T-DNA integrated by natural transformation of plant vegetative tissues by pathogenic Agrobacterium spp., previous reports have indicated that T-DNA sequences originating from an ancestral Agrobacterium sp. are present in the genomes of all cultivated sweet potato (Ipomoea batatas) varieties analyzed. Expression of an Agrobacterium-derived agrocinopine synthase (ACS) gene was detected in leaf and root tissues of sweet potato, suggesting that the plant can produce agrocinopine, a sugar-phosphodiester opine considered to be utilized by some strains of Agrobacterium spp. in crown gall. To validate the product synthesized by Ipomoea batatas ACS (IbACS), we introduced IbACS into tobacco under a constitutive promoter. High-voltage paper electrophoresis followed by alkaline silver nitrate staining detected the production of an agrocinopine-like substance in IbACS1-expressing tobacco, and further mass spectrometry and nuclear magnetic resonance analyses of the product confirmed that IbACS can produce agrocinopine A from natural plant substrates. The partially purified compound was biologically active in an agrocinopine A bioassay. A 16S ribosomal RNA amplicon sequencing and meta-transcriptome analysis revealed that the rhizosphere microbial community of tobacco was affected by the expression of IbACS. A new species of Leifsonia (actinobacteria) was isolated as an enriched bacterium in the rhizosphere of IbACS1-expressing tobacco. This Leifsonia sp. can catabolize agrocinopine A produced in tobacco, indicating that the production of agrocinopine A attracts rhizosphere bacteria that can utilize this sugar-phosphodiester. These results suggest a potential role of IbACS conserved among sweet potato cultivars in manipulating their microbial community.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Renal, cardiovascular and safety outcomes of canagliflozin in patients with type 2 diabetes and nephropathy in East and South-East Asian countries: Results from the Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation Trial.

AIMS/INTRODUCTION: The sodium-glucose cotransporter 2 inhibitor, canagliflozin, reduced kidney failure and cardiovascular events in the Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation (CREDENCE) trial. We carried out a post-hoc analysis to evaluate the efficacy and safety of canagliflozin in a subgroup of participants in East and South-East Asian (EA) countries who are at high risk of renal complications. MATERIALS AND METHODS: Participants with an estimated glomerular filtration rate of 30 to <90 mL/min/1.73 m2 and urinary albumin-to-creatinine ratio of >300-5,000 mg/g were randomized to 100 mg of canagliflozin or a placebo. The effects of canagliflozin treatment on pre-specified efficacy and safety outcomes were examined using Cox proportional hazards regression between participants from EA countries (China, Japan, Malaysia, the Philippines, South Korea and Taiwan) and the remaining participants. RESULTS: Of 4,401 participants, 604 (13.7%) were from EA countries; 301 and 303 were assigned to the canagliflozin and placebo groups, respectively. Canagliflozin lowered the risk of primary outcome (composite of end-stage kidney disease, doubling of serum creatinine level, or renal or cardiovascular death) in EA participants (hazard ratio 0.54, 95% confidence interval 0.35-0.84). The effects of canagliflozin on renal and cardiovascular outcomes in EA participants were generally similar to those of the remaining participants. Safety outcomes were similar between the EA and non-EA participants. CONCLUSIONS: In the CREDENCE trial, the risk of renal and cardiovascular events was safely reduced in participants from EA countries at high risk of renal events.

Chemoenzymatic synthesis, computational investigation, and antitumor activity of monocyclic lankacidin derivatives.

We investigated the importance of the δ-lactone ring (C1-C5) in lankacidin C using chemoenzymatic synthesis and computational prediction and assessing biological activity, including antitumor activity. Pyrroloquinoline quinone-dependent dehydrogenase (Orf23) in Streptomyces rochei was used in the chemoenzymatic synthesis of lankacyclinone C, a novel lankacidin C congener lacking the δ-lactone moiety. Orf23 could convert the monocyclic lankacidinol derivatives, lankacyclinol and 2-epi-lankacyclinol, to the C-24 keto compounds, lankacyclinone C and 2-epi-lankacyclinone C, respectively, elucidating the relaxed substrate specificity of Orf23. Computational prediction using molecular dynamics simulations and the molecular mechanics/generalized Born-surface area protocol indicated that binding energy values of all the monocyclic derivatives are very close to those of lankacidin C, which may reflect a comparable affinity to tubulin. Monocyclic lankacidin derivatives showed moderate antitumor activity when compared with bicyclic lankacidins, suggesting that the δ-lactone moiety is less important for antitumor activity in lankacidin-group antibiotics.

Discovery of a Polyamino Acid Antibiotic Solely Comprising l-ß-Lysine by Potential Producer Prioritization-Guided Genome Mining.

While the genome mining approach has enabled the rational exploration of untapped bioactive natural products, in silico identifications of their biosynthetic genes are often unconnected to the actual production of the corresponding molecules in native strains due to the genetic dormancy. We report here the rational discovery of an unexplored cationic homo polyamino acid (CHPA) antibiotic by potential producer prioritization-guided genome mining. Mining the genome of γ-poly-d-diaminobutyric acid (poly-d-Dab)-producing Streptoalloteichus hindustanus NBRC 15115, which was selected based on the finding that the known CHPAs are universally co-produced in pairs, identified a putative CHPA synthetase, PblA, as a potential candidate being expressed actively. Bioinformatic and biochemical analyses of PblA provided the critical clue that its polymer product could be an unusual CHPA consisting of l-ß-lysine. Instrumental analyses of the metabolites from S. hindastanus indeed revealed the production of an unprecedented linear CHPA, ε-poly-l-ß-lysine, concomitantly with poly-d-Dab. The CHPA we discovered exerted excellent antimicrobial activity against a broad spectrum of microorganisms, including bacteria and fungi, and was revealed to show resistance against nonspecific proteolytic enzymes. This study marks the first report of the efficacy of the strain prioritization-guided genome mining strategy for the discovery of bioactive CHPAs.

Lung cancer screening by single-shot dual-energy subtraction using flat-panel detector.

PURPOSE: The purpose of this study was to evaluate the usefulness of single-shot dual-energy subtraction (DES) method using a flat-panel detector for lung cancer screening MATERIALS AND METHODS: The subjects were 13,315 residents (5801 males and 7514 females) aged 50 years or older (50-97 years, with an intermediate value of 68 years) who underwent lung cancer screening for a period of 1 year and 6 months from January 2019 to June 2020. We investigated whether the number of lung cancers detected, the detection rate, and the rate of required scrutiny changed, when DES images were added to the judgment based on conventional chest radiography. RESULTS: When DES images were added, the number and percentage of cancer detection increased from 16 (0.12%) to 23 (0.17%) (P < 0.05). Five of the newly detected 7 lung cancers were in the early stages of resectable cancer. The rate of participants requiring scrutiny increased slightly from 1.1 to 1.3%. CONCLUSION: DES method improved the detection of lung cancer in screening. The increase in the percentage of participants requiring scrutiny was negligible.

Substrate specificity of two cytochrome P450 monooxygenases involved in lankamycin biosynthesis.

To elucidate the gross lankamycin biosynthetic pathway including two cytochrome P450 monooxygenases, LkmK and LkmF, we constructed two double mutants of P450 genes in combination with glycosyltransferase genes, lkmL and lkmI. An aglycon 8,15-dideoxylankanolide, a possible substrate for LkmK, was prepared from an lkmK-lkmL double mutant, while a monoglycoside 3-O-l-arcanosyl-8-deoxylankanolide, a substrate for LkmF, was from an lkmF-lkmI double mutant. Bioconversion of lankamycin derivatives was performed in the Escherichia coli recombinant for LkmK and the Streptomyces lividans recombinant for LkmF, respectively. LkmK catalyzes the C-15 hydroxylation on all 15-deoxy derivatives, including 8,15-dideoxylankanolide (a possible substrate), 8,15-dideoxylankamycin, and 15-deoxylankamycin, suggesting the relaxed substrate specificity of LkmK. On the other hand, LkmF hydroxylates the C-8 methine of 3-O-l-anosyl-8-deoxylankanolide. Other 8-deoxy lankamycin/lankanolide derivatives were not oxidized, suggesting the importance of a C-3 l-arcanosyl moiety for substrate recognition by LkmF in lankamycin biosynthesis. Thus, LkmF has a strict substrate specificity in lankamycin biosynthesis.

mRNAs in urinary nano-extracellular vesicles as potential biomarkers for non-invasive kidney biopsy.

Urinary nano-extracellular vesicles (NVs), including exosomes and microvesicles, are considered potential biomarkers for kidney diseases using liquid biopsies. However, clinical application of urinary NVs has not yet been validated. In the present study, the levels of mRNAs in urinary NVs in animal models of kidney disease were assessed. Urine samples were collected from the animal models and urinary NVs were isolated by ultracentrifugation. Gene expression levels of kidney injury markers in urinary NVs and renal tissue were quantified by reverse transcription-quantitative PCR. The mRNA levels of desmin, a podocyte injury marker, in urinary NVs was markedly increased in the puromycin aminonucleoside (PAN) nephritis model, in parallel with enhanced desmin expression in kidney tissues. The expression of regulator of calcineurin 1 and the podocin to nephrin ratio (PNR) were also increased in the PAN nephritis model. Treatment with prednisolone mitigated these changes in gene expression as well as proteinuria. PNR, which is considered a predictive marker of glomerular dysfunction, in urinary NVs was highly correlated with urinary protein excretion (P<0.01). Furthermore, PNR in urinary NVs of Zucker diabetic fatty rats, a diabetic kidney disease model, was correlated with urinary albumin excretion (P<0.01). These results suggest that changes in mRNA levels of urinary NVs reflect the disease status of kidney tissues and their functional alterations. Collectively, mRNA analysis of urinary NVs may be used as a liquid biopsy tool for improved classification and performance of risk prediction to determine the severity of kidney diseases.