A Phase 1 Study of Intravenous Busulfan as a Conditioning Regimen for Multiple Myeloma

The efficacy of melphalan (MEL) 140 mg/m 2 pre-transplant conditioning versus MEL 200 mg/m 2 for the elderly is still debated. We hypothesized that single-agent intravenous busulfan (BU) would show significant anti-myeloma efficacy and be better tolerated by elderly patients. A prospective 3+3 dose escalation study enrolled symptomatic multiple myeloma (MM) patients 65 years or older with SWOG performance 0–2 for treatment with intravenous BU pre-transplant at different administration levels. The primary objective was to determine the maximum tolerated dose (MTD) of BU that could be safely given over the least number of days. All patients, except one, received maintenance treatment post-transplant, mostly for 2 years. We enrolled 13 patients, mean age of 73 years (range 68–80). Pharmacokinetic analysis showed no greater than 2% accumulation in the 13 patients, confirming a lack of accumulation in the multi-dose regimen. No deaths occurred in the peri-transplant period. Grade 3/4 adverse effects were hematological, no dose-limiting toxicity was observed and MTD was not reached. Three patients developed grade 3 mucositis but none developed veno-occlusive disease. Ten (77%) patients achieved a complete remission (CR) post-transplant with a remarkably long average time to best response of 6.7 months (range: 6–14 m), and two attained a partial response. Median overall survival was 84 months (95% CI, 21–104) and the median progression-free survival was 60 months (95% CI, 9–93). Our results suggest that IV BU could be an alternative conditioning regimen to MEL 140 in elderly patients with MM, and supports future randomized trials

Crystal structure of Bacillus subtilis TrmB, the tRNA (m(7)G46) methyltransferase

The structure of Bacillus subtilis TrmB (BsTrmB), the tRNA (m(7)G46) methyltransferase, was determined at a resolution of 2.1 Å. This is the first structure of a member of the TrmB family to be determined by X-ray crystallography. It reveals a unique variant of the Rossmann-fold methyltransferase (RFM) structure, with the N-terminal helix folded on the opposite site of the catalytic domain. The architecture of the active site and a computational docking model of BsTrmB in complex with the methyl group donor S-adenosyl-l-methionine and the tRNA substrate provide an explanation for results from mutagenesis studies of an orthologous enzyme from Escherichia coli (EcTrmB). However, unlike EcTrmB, BsTrmB is shown here to be dimeric both in the crystal and in solution. The dimer interface has a hydrophobic core and buries a potassium ion and five water molecules. The evolutionary analysis of the putative interface residues in the TrmB family suggests that homodimerization may be a specific feature of TrmBs from Bacilli, which may represent an early stage of evolution to an obligatory dimer

Antifungal lipopeptides from Bacillus strains isolated from rhizosphere of Citrus trees

peer reviewedThe microbial ecology within the rhizosphere of Tylenchulus semipenetrans-infected Citrus L. trees was examined. Sixty bacterial strains were obtained from the roots of infected trees and from the eggs of T. semipenetrans. Among these strains some were obtained from the agar plates of two nematophagous fungi, Dactylellina gephyropaga (Drechsler) Ying Yang & Xing Z. Liu and Arthrobotrys conoides Drechsler. Bacterial strains were identified using 16S rDNA, gyrA and rpoB genes sequence analysis. Bacterial strains identified as Bacillus spp. were examined for their ability to synthesize surfactin, iturin, fengycin, kurstakin and bacillomycin using PCR amplification and sequencing of the encoding genes. Additionally, Bacillus strains were screened for their antifungal activity against F. solani, D. gephyropaga and A. conoides using the dual culture technique. Lipopeptide from whole cells and from supernatants of Bacillus spp. were screened using MALDI-TOF-MS analysis. The majority of the identified bacterial strains belong to the genus Bacillus with the predominance of B. cereus, B. thuringiensis, B. pumilus and B. subtilis. A total of fifteen Bacillus strains demonstrated an antifungal activity against F. solani, D. gephyropaga and A. conoides with the strongest effect found in B. amyloliquefaciens. The analysis of lipopeptides showed a high diversity of molecules, including majorly iturin C, bacillomycin D, fengycin A/B and Kurstakin found especially in B. subtilis strains. Moreover, MALDI-TOF-MS analysis showed that the responsible antibiotics for the antifungal Bacillus strains were associated with the presence of Surfactins/Pumilacidin and Fengycin A/B. Our results demonstrated the wide diversity of lipopeptides among Bacillus strains associated with citrus rhizosphere and demonstrated their antifungal ability. Our results extend the importance of Bacillus strains as potential candidates for antimicrobial activities due to their ability to synthesize and secrete cyclic lipopeptides

Formation of the conserved pseudouridine at position 55 in archaeal tRNA

Pseudouridine (Ψ) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Ψ55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are (pfu)Cbf5, a protein known to play a role in RNA-guided modification of rRNA, and (pfu)PsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. (pfu)PsuX is hereafter renamed (pfu)Pus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, (pfu)Pus10 efficiently complements an Escherichia coli strain deficient in the bacterial Ψ55 synthase TruB. These results indicate that it is probable that (pfu)Cbf5 or (pfu)Pus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein

CRISPR/Cas9 screen in human iPSC‐derived cortical neurons identifies NEK6 as a novel disease modifier of C9orf72 poly(PR) toxicity

Introduction The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one. Methods We performed a kinome-wide CRISPR/Cas9 knock-out screen in human induced pluripotent stem cell (iPSC) -derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex-vivo studies. Results Knock-down of NIMA-related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock-down of nek6 also ameliorated the poly(PR)-induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53-related DNA damage. Discussion We identified NEK6, which regulates poly(PR)-mediated p53-related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS

Isolation and characterization of Pseudomonas putida mutants affected in arginine, ornithine and citrulline catabolism: Function of the arginine oxidase and arginine succinyltransferase pathways

Pseudomonas putida mutants impaired in the utilization of arginine are affected in either the arginine succinyltransferase pathway, the arginine oxidase route, or both. However, mutants affected in one of the pathways still grow on arginine as sole carbon source. Analysis of the products excreted by both wild-type and mutant strains suggests that arginine is mainly channelled by the oxidase route. Proline non-utilizing mutants are also affected in ornithine utilization, confirming the role of proline as an intermediate in ornithine catabolism. Mutants affected in ornithine cyclodeaminase activity still grow on proline and become unable to use ornithine. Both proline non-utilizing mutants and ornithine-cyclodeaminase-minus mutants are unable to use citrulline. These results, together with induction of ornithine cyclodeaminase when wild-type P. putida is grown on citrulline, indicate that utilization of citrulline as a carbon source proceeds via proline with ornithine as an intermediate. Thus in P. putida, the aerobic catabolism of arginine on the one hand and citrulline and ornithine on the other proceed by quite different metabolic segments.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Comparative studies on the degradation of guanidino and ureido compounds by Pseudomonas

The utilization of guanidino and ureido compounds was studied in several Pseudomonas species. Multiple routes of agmatine catabolism were found. All members of the homology group I of Pseudomonas use the initial deamination of agmatine to carbamoylputrescine which is subsequently converted to putrescine. In Pseudomonas indigofera, the catabolism of agmatine can also occur via an initial hydrolysis of the amidino group to putrescine catalyzed by an agmatine amidinohydrolase. A third pathway was found in Pseudomonas cepacia, namely oxidative deamination producing guanidinobutyraldehyde catalyzed by agmatine dehydrogenase, followed by formation of guanidinobutyrate and removal of urea by guanidinobutyrate amidinohydrolase to produce 4-aminobutyrate. Novel amidinohydrolases were characterized in P. putida for the utilization of arcaine and audouine, and in P. cepacia for arcaine, homoarginine and guanidinovalerate. Guanidinovalerate amidinohydrolase was also detected in P. doudoroffii. Some of these amidinohydrolases accept more than one substrate, e.g. guanidinobutyrate and guanidinovalerate utilization by P. doudoroffii and P. cepacia, the catabolism of arcaine and audouine by P. putida, and the degradation of arcaine and homoarginine by P. cepacia.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Alternative Cell Sources for Liver Parenchyma Repopulation: Where Do We Stand?

Acute and chronic liver failure is a highly prevalent medical condition with high morbidity and mortality. Currently, the therapy is orthotopic liver transplantation. However, in some instances, chiefly in the setting of metabolic diseases, transplantation of individual cells, specifically functional hepatocytes, can be an acceptable alternative. The gold standard for this therapy is the use of primary human hepatocytes, isolated from livers that are not suitable for whole organ transplantations. Unfortunately, primary human hepatocytes are scarcely available, which has led to the evaluation of alternative sources of functional hepatocytes. In this review, we will compare the ability of most of these candidate alternative cell sources to engraft and repopulate the liver of preclinical animal models with the repopulation ability found with primary human hepatocytes. We will discuss the current shortcomings of the different cell types, and some of the next steps that we believe need to be taken to create alternative hepatocyte progeny capable of regenerating the failing liver.status: publishe

Current Status and Challenges of Human Induced Pluripotent Stem Cell-Derived Liver Models in Drug Discovery

The pharmaceutical industry is in high need of efficient and relevant in vitro liver models, which can be incorporated in their drug discovery pipelines to identify potential drugs and their toxicity profiles. Current liver models often rely on cancer cell lines or primary cells, which both have major limitations. However, the development of human induced pluripotent stem cells (hiPSCs) has created a new opportunity for liver disease modeling, drug discovery and liver toxicity research. hiPSCs can be differentiated to any cell of interest, which makes them good candidates for disease modeling and drug discovery. Moreover, hiPSCs, unlike primary cells, can be easily genome-edited, allowing the creation of reporter lines or isogenic controls for patient-derived hiPSCs. Unfortunately, even though liver progeny from hiPSCs has characteristics similar to their in vivo counterparts, the differentiation of iPSCs to fully mature progeny remains highly challenging and is a major obstacle for the full exploitation of these models by pharmaceutical industries. In this review, we discuss current liver-cell differentiation protocols and in vitro iPSC-based liver models that could be used for disease modeling and drug discovery. Furthermore, we will discuss the challenges that still need to be overcome to allow for the successful implementation of these models into pharmaceutical drug discovery platforms