A case of hyperphosphatemic familial tumoral calcinosis due to maternal uniparental disomy of a GALNT3 variant.

Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare, inherited autosomal recessive disorder caused by fibroblast growth factor-23 (FGF23), N-acetylgalactosaminyltransferase 3 (GALNT3), or Klotho (KL) gene variants. Here, we report the case of a Japanese boy who presented with a mass in his left elbow at the age of three. Laboratory test results of the patient revealed normocalcemia (10.3 mg/dL) and hyperphosphatemia (8.7 mg/dL); however, despite hyperphosphatemia, serum intact FGF23 level was low, renal tubular reabsorption of phosphate (TRP) level was inappropriately increased, and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) level was inappropriately normal. Genetic analysis revealed maternal uniparental disomy (UPD) of chromosome 2, which included a novel GALNT3 variant (c.1780-1G>C). Reverse transcription-polymerase chain reaction (RT-PCR) analysis of GALNT3 mRNA confirmed that this variant resulted in the destruction of exon 11. We resected the mass when the patient was five years old, owing to its gradual enlargement. No relapse or new pathological lesions were observed four years after tumor resection. This is the first case report of a Japanese patient with HFTC associated with a novel GALNT3 variant, as well as the first case of HFTC caused by maternal UPD of chromosome 2 that includes the GALNT3 variant.

An efficient CRISPR interference-based prediction method for synergistic/additive effects of novel combinations of anti-tuberculosis drugs.

Tuberculosis (TB) is treated by chemotherapy with multiple anti-TB drugs for a long period, spanning 6 months even in a standard course. In perspective, to prevent the emergence of antimicrobial resistance, novel drugs that act synergistically or additively in combination with major anti-TB drugs and, if possible, shorten the duration of TB therapy are needed. However, their combinatorial effect cannot be predicted until the lead identification phase of the drug development. Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a powerful genetic tool that enables high-throughput screening of novel drug targets. The development of anti-TB drugs promises to be accelerated by CRISPRi. This study determined whether CRISPRi could be applicable for predictive screening of the combinatorial effect between major anti-TB drugs and an inhibitor of a novel target. In the checkerboard assay, isoniazid killed Mycobacterium smegmatis synergistically or additively in combinations with rifampicin or ethambutol, respectively. The susceptibility to rifampicin and ethambutol was increased by knockdown of inhA, which encodes a target molecule of isoniazid. Additionally, knockdown of rpoB, which encodes a target molecule of rifampicin, increased the susceptibility to isoniazid and ethambutol, which act synergistically with rifampicin in the checkerboard assay. Moreover, CRISPRi could successfully predict the synergistic action of cyclomarin A, a novel TB drug candidate, with isoniazid or rifampicin. These results demonstrate that CRISPRi is a useful tool not only for drug target exploration but also for screening the combinatorial effects of novel combinations of anti-TB drugs. This study provides a rationale for anti-TB drug development using CRISPRi.

High contrast between lumbar nerve roots and surrounding structures using dual echo 3D turbo spin echo additional fusion images.

PURPOSE: We performed lumbar spinal magnetic resonance imaging of three-dimensional (3D) dual echo volumetric isotropic turbo spin echo acquisition (DE-VISTA) and constructed DE-VISTA additional fusion images (DE-VISTA-AFI), which is the addition of DE-VISTA proton density-weighted images (DE-VISTA-PDWI) to DE-VISTA T2-weighted images (DE-VISTA-T2WI). The aim of this study was to clarify whether DE-VISTA-AFI was able to clearly delineate spinal nerve roots. METHODS: A total of 677 patients underwent lumbar MR imaging, and the signal ratio (SR) between cerebrospinal fluid and nerve roots inside the dural sac and the SR between fat and nerve roots outside the dural sac were estimated using DE-VISTA-AFI, DE-VISTA-PDWI, DE-VISTA-T2WI, and 2D-T2WI. RESULTS: The SR between cerebrospinal fluid and nerve roots inside the dural sac on DE-VISTA-AFI was higher than that on DE-VISTA-PDWI (p < 0.0001) and on 2D T2WI (p < 0.0001). The SR between the fat tissue and nerve roots outside the dural sac on DE-VISTA-AFI was higher than that on DE-VISTA-PDWI (p < 0.0001) and 2D T2WI (p < 0.0001). CONCLUSION: DE-VISTA-AFI could clearly delineate the entire length of the lumbar nerve roots that run from the cauda equina in the spinal fluid through to the fat in the lateral recess, intervertebral foramen, and outside the intervertebral foramen.

Actinocrispum wychmicini gen. nov., sp. nov., a novel member of the family Pseudonocardiaceae, isolated from soil.

A novel actinomycete, designated MI503-A4T, was isolated from soil. Comparative analysis of 16S rRNA gene sequences indicated that MI503-A4T was phylogenetically related to members of the family Pseudonocardiaceae. The most closely related genus was Kibdelosporangium (95.7-96.2 % sequence similarity). Substrate mycelia were branched and pale yellow to pale yellowish-brown. Straight- to zigzag-shaped aerial mycelia were observed, but Sporangium-like structures were absent. The whole-cell hydrolysate contained meso-diaminopimelic acid. The muramic acid residues in the peptidoglycan were N-acetylated. Whole-cell sugars were rhamnose, ribose, arabinose and galactose (cell wall chemotype IV). The predominant menaquinone was MK-9(H4). A small amount of MK-8(H4) was also detected. The DNA G+C content was 70.3-71.1 mol%. Polar lipids contained diphosphatidylglycerol, phosphatidylethanolamine and hydroxyl-phosphatidylethanolamine. Cellular fatty acid analysis of MI503-A4T detected predominantly iso-C14 : 0 (11.5 %), iso-C15 : 0 (13.3 %) and iso-C16 : 0 (35.7 %). Phenotypic and phylogenetic characteristics differentiated MI503-A4T from members of all genera within the family Pseudonocardiaceae with validly published names. Therefore, MI503-A4T is proposed to be a representative of a novel species in a novel genus, Actinocrispum wychmicini gen. nov., sp. nov. The type strain of the type species is MI503-A4T (=NBRC 109632T=DSM 45934T).

A New Screen for Tuberculosis Drug Candidates Utilizing a Luciferase-Expressing Recombinant Mycobacterium bovis Bacillus Calmette-Guéren.

Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is needed to not only to shorten the medication period but also to treat multi-drug resistant and extensively drug-resistant TB. Mycobacterium tuberculosis (Mtb) grows slowly and only multiplies once or twice per day. Therefore, conventional drug screening takes more than 3 weeks. Additionally, a biosafety level-3 (BSL-3) facility is required. Thus, we developed a new screening method to identify TB drug candidates by utilizing luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren (rBCG). Using this method, we identified several candidates in 4 days in a non-BSL-3 facility. We screened 10,080 individual crude extracts derived from Actinomyces and Streptomyces and identified 137 extracts which possessed suppressive activity to the luciferase of rBCG. Among them, 41 compounds inhibited the growth of both Mtb H37Rv and the extensively drug-resistant Mtb (XDR-Mtb) strains. We purified the active substance of the 1904-1 extract, which possessed strong activity toward rBCG, Mtb H37Rv, and XDR-Mtb but was harmless to the host eukaryotic cells. The MIC of this substance was 0.13 µg/ml, 0.5 µg/ml, and 2.0-7.5 µg/ml against rBCG, H37Rv, and 2 XDR-strains, respectively. Its efficacy was specific to acid-fast bacterium except for the Mycobacterium avium intracellular complex. Mass spectrometry and nuclear magnetic resonance analyses revealed that the active substance of 1904-1 was cyclomarin A. To confirm the mode of action of the 1904-1-derived compound, resistant BCG clones were used. Whole genome DNA sequence analysis showed that these clones contained a mutation in the clpc gene which encodes caseinolytic protein, an essential component of an ATP-dependent proteinase, and the likely target of the active substance of 1904-1. Our method provides a rapid and convenient screen to identify an anti-mycobacterial drug.

Androprostamines A and B, the new anti-prostate cancer agents produced by Streptomyces sp. MK932-CF8.

Androgen receptor (AR) is a validated target in all clinical states of prostate cancer. Androprostamines A and B, the new inhibitors of androgen receptor, were isolated from Streptomyces sp. MK932-CF8. Their structures were determined by the spectroscopic analysis, degradation studies and synthesis. Androprostamines showed potent inhibitory effect against androgen-dependent growth of human prostate cancer cells without cytotoxicity and repressed the androgen-induced expression of AR-regulated genes.

Waldiomycin, a novel WalK-histidine kinase inhibitor from Streptomyces sp. MK844-mF10.

WalK, a histidine kinase, and WalR, a response regulator, make up a two-component signal transduction system that is indispensable for the cell-wall metabolism of low GC Gram-positive bacteria. WalK inhibitors are likely to show bactericidal effects against methicillin-resistant Staphylococcus aureus . We discovered a new WalK inhibitor, designated waldiomycin, by screening metabolites from actinomycetes. Waldiomycin belongs to the family of angucycline antibiotics and is structurally related to dioxamycin. Waldiomycin inhibits WalK from S. aureus and Bacillus subtilis at IC50s 8.8 and 10.2 µM, respectively, and shows antibacterial activity with MICs ranging from 4 to 8 µg ml(-1) against methicillin-resistant S. aureus and B. subtilis.

Intervenolin, a new antitumor compound with anti-Helicobacter pylori activity, from Nocardia sp. ML96-86F2.

Because stromal cells can regulate the growth and metastasis of tumor cells, a compound that modulates the interaction between the stromal cells and the tumor cells can control the tumor progression. In the course of our screening for such a compound, we have isolated a new compound, intervenolin, from the culture broth of Nocardia sp. ML96-86F2. Intervenolin inhibits the growth of human gastric and colorectal cancer cell lines in the coculture with the respective organ-derived stromal cells more strongly than that of the cancer cells cultured alone. Intervenolin shows antitumor effect against a xenograft model of human colorectal cancer cells in vivo. Furthermore, intervenolin exerts selective anti-Helicobacter pylori effect.

Vegfrecine, an inhibitor of VEGF receptor tyrosine kinases isolated from the culture broth of Streptomyces sp.

A new inhibitor of VEGF receptor tyrosine kinases, vegfrecine (1), was isolated from the culture broth of Streptomyces sp. MK931-CF8. The molecular structure of 1 was determined by NMR and MS analysis combined with synthesis. Compound 1 showed potent inhibitory activity against vascular endothelial growth factor receptor (VEGFR) tyrosine kinases in in vitro enzyme assays, but platelet-derived growth factor receptors (PDGFRs), fibroblast growth factor receptor (FGFR), and epidermal growth factor receptor (EGFR) responded only weakly. Compound 1 is a promising new selective VEGFR inhibitor for investigating new treatments of cancer and inflammatory diseases.

Migracins A and B, new inhibitors of cancer cell migration, produced by Streptomyces sp.

In the course of screening for breast cancer cell migration inhibitors, we isolated two novel compounds, migracins A and B from the culture broth of Streptomyces sp. MI264-NF2. Their structures are related to those of luminacins previously isolated from Streptomyces. Migracins A and B inhibited breast cancer cell migration, monitored by wound healing assay with IC50 values of 1.31 and 1.99 µg ml(-1), respectively, in human breast carcinoma MDA-MB-231 cells without showing any cytotoxicity. Migracins also inhibited the migration of human lung adenocarcinoma A549 cells and human fibrosarcoma HT-1080 cells. Therefore, migracins may become new cancer metastasis inhibitors.

Amycolamicin: a novel broad-spectrum antibiotic inhibiting bacterial topoisomerase.

The abuse of antibacterial drugs imposes a selection pressure on bacteria that has driven the evolution of multidrug resistance in many pathogens. Our efforts to discover novel classes of antibiotics to combat these pathogens resulted in the discovery of amycolamicin (AMM). The absolute structure of AMM was determined by NMR spectroscopy, X-ray analysis, chemical degradation, and modification of its functional groups. AMM consists of trans-decalin, tetramic acid, two unusual sugars (amycolose and amykitanose), and dichloropyrrole carboxylic acid. The pyranose ring named as amykitanose undergoes anomerization in methanol. AMM is a potent and broad-spectrum antibiotic against Gram-positive pathogenic bacteria by inhibiting DNA gyrase and bacterial topoisomerase IV. The target of AMM has been proved to be the DNA gyrase B subunit and its binding mode to DNA gyrase is different from those of novobiocin and coumermycin, the known DNA gyrase inhibitors.

Rapidly progressive glomerulonephritis associated with PR3-ANCA positive subacute bacterial endocarditis.

Patients with bacterial endocarditis often have renal complications. This report presents the case of an elderly man with rapidly progressive glomerulonephritis (RPGN) associated with subacute bacterial endocarditis (SBE) due to Enterococcus faecalis infection. The patient was positive for anti-proteinase 3-antineutrophil cytoplasmic antibody (PR3-ANCA) and rheumatoid factor (RF) with hypocomplementemia. Treatment for SBE with antibiotics and the surgical replacement of the affected valves resulted in an improvement of RPGN, the disappearance of PR3-ANCA and RF, and the normalization of hypocomplementemia. This rare case suggests the importance of recognizing the cause of positive PR3-ANCA, because SBE could be an occult cause of RPGN mimicking ANCA-associated vasculitis.

Hyponatremia in a patient with scleroderma renal crisis: a potential role of activated renin-angiotensin system.

BACKGROUND: Scleroderma renal crisis is an important complication of scleroderma (systemic sclerosis) that is associated with significant morbidity and mortality. On the other hand, hyponatremia has never been reported in patients with scleroderma renal crisis. CASE PRESENTATION: A 66-year-old man with scleroderma was admitted to our hospital for an evaluation of renal dysfunction and extreme hypertension. The laboratory evaluation revealed remarkably high plasma renin activity in association with microangiopathic hemolytic anemia, and the anti-RNA polymerase III antibody assessment was positive. The patient was diagnosed with scleroderma renal crisis and was started treatment with enalapril maleate, an angiotensin-converting enzyme inhibitor. During hospitalization, the patient developed symptomatic hyponatremia three times and each laboratory analysis revealed improperly high levels of antidiuretic hormone without signs of extracellular fluid volume depletion as well as remarkably high plasma renin activities and angiotensin levels. However, hyponatremia has not been demonstrated to occur as a result of combined therapy with candesartan cilexetil, an angiotensin II receptor blocker, and aliskiren fumarate, a direct renin inhibitor. The plasma renin activities and angiotensin levels were normalized and the renal function was maintained after treatment. CONCLUSIONS: To our best knowledge, this is the first documented case of scleroderma renal crisis complicated with hyponatremia. This report also suggests that the activated renin-angiotensin system may play a role in the development of hyponatremia and that hyponatremia should be taken into consideration as a rare but possible complication associated with screloderma renal crisis.

High plasma pentosidine level is accompanied with cardiovascular events in hemodialysis patients.

BACKGROUND: Cardiovascular disease is a major complication in patients with end-stage renal disease (ESRD). The accumulation of advanced glycation end products (AGEs) is facilitated in these patients. The aim of this study was to investigate the relationship between circulating AGEs and cardiovascular events in hemodialysis patients. METHODS: The plasma level of pentosidine, a well-defined AGEs, was measured in 110 hemodialysis patients who were prospectively followed for 90 months. The relationship between plasma pentosidine level and cardiovascular events was assessed using Kaplan-Meier and Cox regression analysis. RESULTS: Thirty-nine cardiovascular events (14 coronary heart disease and 25 strokes) occurred during the follow-up period. Multivariable Cox proportional hazard analysis showed that plasma pentosidine levels (HR 1.040, 95% CI 1.022-1.058, p < 0.01) were correlated to increased risk for cardiovascular events. When patients were divided into four groups according to plasma pentosidine levels, Kaplan-Meier analysis revealed that cardiovascular events in the highest pentosidine group were significantly greater than in the other groups (p < 0.01 in lower and low, and p < 0.05 in high pentosidine groups). CONCLUSION: The plasma pentosidine level predicts cardiovascular events in hemodialysis patients. The effects of lowering circulating AGE levels on cardiovascular events should be examined in ESRD patients.

Isolation and structure elucidation of a novel androgen antagonist, arabilin, produced by Streptomyces sp. MK756-CF1.

In the course of screening for a new type of androgen receptor (AR) antagonist, we isolated a novel compound, arabilin, with two structural isomers, spectinabilin and SNF4435C, produced by Streptomyces sp. MK756-CF1. Structure elucidation on the basis of the spectroscopic properties showed that arabilin is a novel polypropionate-derived metabolite with a p-nitrophenyl group and a substituted γ-pyrone ring. Arabilin competitively blocked the binding of androgen to the ligand-binding domain of AR in vitro. In addition, arabilin inhibited androgen-induced prostate-specific antigen mRNA expression in prostate cancer LNCaP cells.

Paleic acid, a fatty acid from Paenibacillus sp.: taxonomy, fermentation, isolation, structure determination, and anti-Mannheimia and -Pasteurella activity.

Paleic acid (1), an antibiotic, was obtained from a fermentation broth of Paenibacillus sp. BMK771-AF3. The compound is a fatty acid (9Z,16R)-16-hydroxy-9-octadecenoic acid ((R)-16-hydroxyoleic acid), whose isolation required protection of its polar functional groups. Mosher esters of paleic acid yielded information on the absolute configuration of secondary alcohol, and well-resolved (1)H NMR peaks around the double bond suggested that olefin adopted a Z geometry. Paleic acid showed potent antibacterial activity and narrow spectrum against Mannheimia haemolytica with MIC values ranging between 0.78 and 1.56 microg ml(-1).

Walkmycin B targets WalK (YycG), a histidine kinase essential for bacterial cell growth.

The WalK (a histidine kinase)/WalR (a response regulator, aka YycG/YycF) two-component system is indispensable in the signal transduction pathway for the cell-wall metabolism of Bacillus subtilis and Staphylococcus aureus. The inhibitors directed against WalK would be expected to have a bactericidal effect. After we screened 1368 culture broths of Streptomyces sp. by a differential growth assay, walkmycin A, B and C, which were produced by strain MK632-100F11, were purified using silica-gel column chromatography and HPLC. In this paper, the chemical structure of the major product (walkmycin B) was determined to be di-anthracenone (C(44)H(44)Cl(2)O(14)), which was very similar to BE40665A. MICs of walkmycin B against B. subtilis and S. aureus were 0.39 and 0.20 microg ml(-1), and IC(50) measurements against WalK were 1.6 and 5.7 microM, respectively. To clarify the affinity between WalK and walkmycin B, surface plasmon resonance was measured to obtain the equilibrium dissociation constant, K(D1), of 7.63 microM at the higher affinity site of B. subtilis WalK. These results suggest that walkmycin B inhibits WalK autophosphorylation by binding to the WalK cytoplasmic domain.

Pargamicin A, a novel cyclic peptide antibiotic from Amycolatopsis sp.

A novel cyclic peptide antibiotic, pargamicin A was isolated from the culture broth of an actinomycete strain. The producing organism, designated ML1-hF4, was identified as a member of the genus Amycolatopsis. Pargamicin A was identified as a novel cyclic hexapeptide antibiotic containing piperazic acid by various spectroscopic analyses. Pargamicin A showed potent antibacterial activity against Staphylococcus aureus strains including MRSA and Enterococcus faecalis/faecium strains including VRE.

Inhibitory activity of the hypoxia-inducible factor-1 pathway by tartrolone C.

Hypoxia-inducible factor-1 (HIF-1) is a central mediator of cellular responses to low oxygen and has recently become an important therapeutic target for solid tumor therapy. To identify small molecule inhibitors of the HIF-1 transcriptional activation, we have established a high through-put assay system using a stable transformant of mammalian cells that express a luciferase reporter gene construct containing a HIF-1 binding site. Using this system, we screened 5000 cultured broths of microorganisms, and we found that fermentation broth produced by Streptomyces strain 1759-27 showed significant inhibition of the reporter activity induced by hypoxic conditions. The active substance NBRI759-27 was purified and determined to be tartrolone C by several methods including X-ray crystallography. In the reporter gene assay, tartrolone C inhibited the HIF-1 transcriptional activity under hypoxic conditions with an IC50 value of 0.17 microg/ml.