Telomere Architecture Correlates with Aggressiveness in Multiple Myeloma.

The prognosis of multiple myeloma (MM), an incurable B-cell malignancy, has significantly improved through the introduction of novel therapeutic modalities. Myeloma prognosis is essentially determined by cytogenetics, both at diagnosis and at disease progression. However, for a large cohort of patients, cytogenetic analysis is not always available. In addition, myeloma patients with favorable cytogenetics can display an aggressive clinical course. Therefore, it is necessary to develop additional prognostic and predictive markers for this disease to allow for patient risk stratification and personalized clinical decision-making. Genomic instability is a prominent characteristic in MM, and we have previously shown that the three-dimensional (3D) nuclear organization of telomeres is a marker of both genomic instability and genetic heterogeneity in myeloma. In this study, we compared in a longitudinal prospective study blindly the 3D telomeric profiles from bone marrow samples of 214 initially treatment-naïve patients with either monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), or MM, with a minimum follow-up of 5 years. Here, we report distinctive 3D telomeric profiles correlating with disease aggressiveness and patient response to treatment in MM patients, and also distinctive 3D telomeric profiles for disease progression in smoldering multiple myeloma patients. In particular, lower average intensity (telomere length, below 13,500 arbitrary units) and increased number of telomere aggregates are associated with shorter survival and could be used as a prognostic factor to identify high-risk SMM and MM patients.

Near-field infrared nanospectroscopy and super-resolution fluorescence microscopy enable complementary nanoscale analyses of lymphocyte nuclei.

Recent super-resolution fluorescence microscopy (3D-Structured Illumination Microscopy, 3D-SIM) studies have revealed significantly altered nuclear organization between normal lymphocyte nuclei and those of classical Hodgkin's Lymphoma. Similar changes have been found in Multiple Myeloma (MM) nuclei, as well as in a premalignant condition, Monoclonal Gammopathy of Unknown Significance (MGUS). Using 3D-SIM, an increase in DNA-poor and DNA-free voids was evident in reconstructed 3D-SIM images of diseased nuclei at 40 nm pixel resolution (x,y: 40 nm, z: 80 nm). At best, far-field FTIR imaging yields spatially resolved images at ∼500 nm spatial resolution; however, near-field infrared imaging breaks the diffraction limit at a scale comparable to that of 3D-SIM, providing details on the order of 30 nm spatial resolution. We report here the first near-field IR imaging of lymphocyte nuclei, and far-field IR imaging results of whole lymphocytes and nuclei from normal human blood. Cells and nuclei were mounted on infrared-compatible substrates, including CaF2, undoped silicon wafers, and gold-coated silicon wafers. Thermal source far-field FTIR images were obtained with an Agilent-Cary 620 microscope, 15× objective, 0.62 NA and 64 × 64 array Focal Plane Array detector (University of Manitoba), or with a similar microscope equipped with both 15× and 25× (0.81 NA) objectives, 128 × 128 FPA and either thermal source or synchrotron source (single beam) infrared light at the Advanced Light Source (ALS), LBNL, Berkeley CA. Near-field IR spectra were acquired at the ALS, on the in-house SINS equipment, as well as with a Neaspec system, both illuminated with synchrotron light. Finally, some near-field IR spectra and images were acquired at Neaspec GmbH, Germany. Far-field IR spectra of normal cells and nuclei showed the characteristic bands of DNA and proteins. Near-field IR spectra of nuclei showed variations in bands assigned to protein and nucleic acids including single and double-stranded DNA. Near-field IR images of nuclei enabled visualization of protein and DNA distribution in spatially-resolved chromosome territories and nuclear pores.

Analysis of carbohydrate-active enzymes in Thermogemmatispora sp. strain T81 reveals carbohydrate degradation ability.

The phylum Chloroflexi is phylogenetically diverse and is a deeply branching lineage of bacteria that express a broad spectrum of physiological and metabolic capabilities. Members of the order Ktedonobacteriales, including the families Ktedonobacteriaceae, Thermosporotrichaceae, and Thermogemmatisporaceae, all have flexible aerobic metabolisms capable of utilizing a wide range of carbohydrates. A number of species within these families are considered cellulolytic and are capable of using cellulose as a sole carbon and energy source. In contrast, Ktedonobacter racemifer, the type strain of the order, does not appear to possess this cellulolytic phenotype. In this study, we confirmed the ability of Thermogemmatispora sp. strain T81 to hydrolyze cellulose, determined the whole-genome sequence of Thermogemmatispora sp. T81, and using comparative bioinformatics analyses, identified genes encoding putative carbohydrate-active enzymes (CAZymes) in the Thermogemmatispora sp. T81, Thermogemmatispora onikobensis, and Ktedonobacter racemifer genomes. Analyses of the Thermogemmatispora sp. T81 genome identified 64 CAZyme gene sequences belonging to 57 glycoside hydrolase families. The genome of Thermogemmatispora sp. T81 encodes 19 genes for putative extracellular CAZymes, similar to the number of putative extracellular CAZymes identified in T. onikobensis (17) and K. racemifer (17), despite K. racemifer not possessing a cellulolytic phenotype. These results suggest that these members of the order Ktedonobacteriales may use a broader range of carbohydrate polymers than currently described.

Comparative Genomics of Core Metabolism Genes of Cellulolytic and Non-cellulolytic Clostridium Species.

Microbial production of fuels such as ethanol, butanol, hydrogen (H2), and methane (CH4) from waste biomass has the potential to provide sustainable energy systems that can displace fossil fuel consumption. Screening for microbial diversity and genome sequencing of a wide-range of microorganisms can identify organisms with natural abilities to synthesize these alternative fuels and/or other biotechnological applications. Clostridium species are the most widely studied strict anaerobes capable of fermentative synthesis of ethanol, butanol, or hydrogen directly from waste biomass. Clostridium termitidis CT1112 is a mesophilic, cellulolytic species capable of direct cellulose fermentation to ethanol and organic acids, with concomitant synthesis of H2 and CO2. On the basis of 16S ribosomal RNA (rRNA) and chaperonin 60 (cpn60) gene sequence data, phylogenetic analyses revealed a close relationship between C. termitidis and C. cellobioparum. Comparative bioinformatic analyses of the C. termitidis genome with 18 cellulolytic and 10 non-cellulolytic Clostridium species confirmed this relationship, and further revealed that the majority of core metabolic pathway genes in C. termitidis and C. cellobioparum share more than 90% amino acid sequence identity. The gene loci and corresponding amino acid sequences of the encoded enzymes for each pathway were correlated by percentage identity, higher score (better alignment), and lowest e-value (most significant "hit"). In addition, the function of each enzyme was proposed by conserved domain analysis. In this chapter we discuss the comparative analysis of metabolic pathways involved in synthesis of various useful products by cellulolytic and non-cellulolytic biofuel and solvent producing Clostridium species. This study has generated valuable information concerning the core metabolism genes and pathways of C. termitidis CT1112, which is helpful in developing metabolic engineering strategies to enhance its natural capacity for better industrial applications.

Abdominal lymphatic malformation: Spectrum of imaging findings.

Lymphatic malformations are congenital vascular malformations with lymphatic differentiation. Although the most common locations for lymphatic malformation are the neck and axilla, they can occur at several locations in the body including the abdomen. The abdominal location is rather rare and accounts for approximately 5% of all lymphatic malformation. Abdominal lymphatic malformation can arise from mesentery, omentum, gastrointestinal tract, and retroperitoneum. Clinical presentation includes an abdominal lump, vague abdominal discomfort, and secondary complications including intestinal obstruction, volvulus, ischemia, and bleeding. There is a broad spectrum of radiological manifestation. In the present review, we discuss the imaging appearance of abdominal lymphatic malformation. The diagnosis of lymphatic malformation in our series was based on the histopathological examination (in cases who underwent surgery) and fine needle aspiration cytology.

Biliary Fasciola gigantica infestation in a nonendemic area--An intraoperative surprise.

A 7year old girl infected with the zoonotic trematode, Fasciola gigantica is reported because of the extreme rarity of this condition in our region. Because of the overlap in symptomatology and radiological features, the more common amebic/pyogenic liver abscess in the initial hepatic migratory phase and later choledochal cyst/biliary ascariasis when the parasite was finally located in the extrahepatic bile ducts, were thought of delaying effective treatment. The diagnosis was confirmed only by surgical exploration. The characteristic contrast enhanced computed tomography scan features retrospectively identified were multiple clustered hypodense lesions in the liver with peripheral enhancement in the acute hepatic migratory phase, and periportal tracking in the previously affected areas of the liver with biliary dilatation and a linear hypointense lesion within the common bile duct in the chronic phase. Although a known association, she did not have eosinophilia. This child, who became symptomatic at the age of 5.5years, also appears to be one of the youngest patients reported with Fasciola gigantica.

Draft Genome Sequence of the Hydrogen- and Ethanol-Producing Bacterium Clostridium intestinale Strain URNW.

Here, we report the draft genome sequence of Clostridium intestinale strain URNW, which can convert biomass to useful products such as biofuels (hydrogen or ethanol) and other soluble end products.

Draft Genome Sequence of the Cellulolytic, Mesophilic, Anaerobic Bacterium Clostridium termitidis Strain CT1112 (DSM 5398).

Here, we report the draft genome sequence of Clostridium termitidis strain CT1112 (DSM 5398), a mesophilic, cellulolytic bacterium that can utilize a variety of sugars, as well as pure cellulose, as a sole carbon source; it also synthesizes fermentation end products with potential industrial applications.

The Paenibacillus polymyxa species is abundant among hydrogen-producing facultative anaerobic bacteria in Lake Averno sediment.

Lake Averno sediment was used to isolate the facultative anaerobic bacteria having the potential for H(2) production. Twenty-five out of 35 isolates recovered from the sediment sample produced hydrogen under anaerobic conditions from glucose with yields ranging from 0.1 to 0.49 mol H(2)/mol glucose. Identification based on 16S rRNA gene sequence analysis revealed that most of them belong to the Firmicutes group, with a prevalence of the Paenibacillus polymyxa species. Seven distinct genomic fingerprints among the 11 P. polymyxa isolates were obtained using the random amplified polymorphic DNA (RAPD) technique. Glucose fermentation by P. polymyxa isolates was investigated. Glucose was totally consumed after 3 days of fermentation. The fermentation products were hydrogen (0.18-0.47 mol H(2)/mol glucose), ethanol (0.1-0.5 mol ethanol/mol glucose), and 2,3-butanediol (0.1 mol 2,3-butanediol/mol glucose). Lower amounts of acetic, butyric, formic, lactic, and propionic acids were detected. All metabolic data concerning P. polymyxa isolates were analyzed by cluster analysis to reveal similarities and/or differences with clustering based on RAPD profiles. Despite the high metabolic similarity among almost all P. paenibacillus isolates, results of cluster analyses of metabolic and genetic data do not match completely.

Phylogeny in aid of the present and novel microbial lineages: diversity in Bacillus.

Bacillus represents microbes of high economic, medical and biodefense importance. Bacillus strain identification based on 16S rRNA sequence analyses is invariably limited to species level. Secondly, certain discrepancies exist in the segregation of Bacillus subtilis strains. In the RDP/NCBI databases, out of a total of 2611 individual 16S rDNA sequences belonging to the 175 different species of the genus Bacillus, only 1586 have been identified up to species level. 16S rRNA sequences of Bacillus anthracis (153 strains), B. cereus (211 strains), B. thuringiensis (108 strains), B. subtilis (271 strains), B. licheniformis (131 strains), B. pumilus (83 strains), B. megaterium (47 strains), B. sphaericus (42 strains), B. clausii (39 strains) and B. halodurans (36 strains) were considered for generating species-specific framework and probes as tools for their rapid identification. Phylogenetic segregation of 1121, 16S rDNA sequences of 10 different Bacillus species in to 89 clusters enabled us to develop a phylogenetic frame work of 34 representative sequences. Using this phylogenetic framework, 305 out of 1025, 16S rDNA sequences presently classified as Bacillus sp. could be identified up to species level. This identification was supported by 20 to 30 nucleotides long signature sequences and in silico restriction enzyme analysis specific to the 10 Bacillus species. This integrated approach resulted in identifying around 30% of Bacillus sp. up to species level and revealed that B. subtilis strains can be segregated into two phylogenetically distinct groups, such that one of them may be renamed.

Ecology and biotechnological potential of Paenibacillus polymyxa: a minireview.

Microbial diversity is a major resource for biotechnological products and processes. Bacteria are the most dominant group of this diversity which produce a wide range of products of industrial significance. Paenibacillus polymyxa (formerly Bacillus polymyxa), a non pathogenic and endospore-forming Bacillus, is one of the most industrially significant facultative anaerobic bacterium. It occurs naturally in soil, rhizosphere and roots of crop plants and in marine sediments. During the last two decades, there has been a growing interest for their ecological and biotechnological importance, despite their limited genomic information. P. polymyxa has a wide range of properties, including nitrogen fixation, plant growth promotion, soil phosphorus solubilisation and production of exopolysaccharides, hydrolytic enzymes, antibiotics, cytokinin. It also helps in bioflocculation and in the enhancement of soil porosity. In addition, it is known to produce optically active 2,3-butanediol (BDL), a potentially valuable chemical compound from a variety of carbohydrates. The present review article aims to provide an overview of the various roles that these microorganisms play in the environment and their biotechnological potential.

Hydrogen and polyhydroxybutyrate producing abilities of microbes from diverse habitats by dark fermentative process.

Thirty five bacterial isolates from diverse environmental sources such as contaminated food, nitrogen rich soil, activated sludges from pesticide and oil refineries effluent treatment plants were found to belong to Bacillus, Bordetella, Enterobacter, Proteus, and Pseudomonas sp. on the basis of 16S rRNA gene sequence analysis. Under dark fermentative conditions, maximum hydrogen (H(2)) yields (mol/mol of glucose added) were recorded to be 0.68 with Enterobacter aerogenes EGU16 followed by 0.63 with Bacillus cereus EGU43 and Bacillus thuringiensis EGU45. H(2) constituted 63-69% of the total biogas evolved. Out of these 35 microbes, 18 isolates had the ability to produce polyhydroxybutyrate (PHB), which varied up to 500 mg/l of medium, equivalent to a yield of 66.6%. The highest PHB yield was recorded with B. cereus strain EGU3. Nine strains had high hydrolytic activities (zone of hydrolysis): lipase (34-38 mm) -Bacillus sphaericus strains EGU385, EGU399 and EGU542; protease (56-62 mm) -Bacillus sp. strains EGU444, EGU447 and EGU445; amylase (23 mm) -B. thuringiensis EGU378, marine bacterium strain EGU409 and Pseudomonas sp. strain EGU448. These strains with high hydrolytic activities had relatively low H(2) producing abilities in the range of 0.26-0.42 mol/mol of glucose added and only B. thuringiensis strain EGU378 had the ability to produce PHB. This is the first report among the non-photosynthetic microbes, where the same organism(s) -B. cereus strain EGU43 and B. thuringiensis strain EGU45, have been shown to produce H(2) - 0.63 mol/mol of glucose added and PHB - 420-435 mg/l medium.

Phylogeny vs genome reshuffling: horizontal gene transfer.

The evolutionary events in organisms can be tracked to the transfer of genetic material. The inheritance of genetic material among closely related organisms is a slow evolutionary process. On the other hand, the movement of genes among distantly related species can account for rapid evolution. The later process has been quite evident in the appearance of antibiotic resistance genes among human and animal pathogens. Phylogenetic trees based on such genes and those involved in metabolic activities reflect the incongruencies in comparison to the 16S rDNA gene, generally used for taxonomic relationships. Such discrepancies in gene inheritance have been termed as horizontal gene transfer (HGT) events. In the post-genomic era, the explosion of known sequences through large-scale sequencing projects has unraveled the weakness of traditional 16S rDNA gene tree based evolutionary model. Various methods to scrutinize HGT events include atypical composition, abnormal sequence similarity, anomalous phylogenetic distribution, unusual phyletic patterns, etc. Since HGT generates greater genetic diversity, it is likely to increase resource use and ecosystem resilience.

In Search of Drug Targets for Mycobacterium tuberculosis.

Mycobacterium tuberculosis is the etiological agent for tuberculosis in humans. The studies related to survival of this pathogen in the human host and development of drugs against reveal that the organism uses a complex physiology to adapt to the host environment. Many studies were targeted to key enzymes that allow this pathogen to either survive or remain latent within the host. Most of the models, which address the survival of pathogen, have evaluated limited dissolved oxygen and prevailing stress conditions. Hence, the truncated citric acid cycle, with the glyoxylate shunt was suggested as an option for survival of the pathogen and pathogenesis. We propose that the precursors to support this pathway could also be generated via enzymatic conversion involving poly-beta-hydroxybutyrate (PHB). We have used available genome sequence data and analyzed for the possible enzymatic conversions that can generate glyoxylate, acetyl CoA, and other enolases that can also be useful for various fatty acid transformations. The enzymes for accumulation and further hydrolysis of PHB were examined in sequence data analysis. The target enzymes were searched for in the genome using identified conserved domains. Using M. tuberculosis H37Rv as a model bacterium a supportive pathway has been envisaged and integrated with glyoxylate cycle to provide a complete option to pathogen for sustainable consumption of available carbon source(s). The study proposes that the enzymes of PHB synthesis and hydrolysis are possible targets for drug design, and that this should be considered when evaluating isocitrate lyase and malate synthase as targets.

Insight in to the phylogeny of polyhydroxyalkanoate biosynthesis: horizontal gene transfer.

Polyhydroxyalkanoates (PHAs) are gaining more and more importance the world over due to their structural diversity and close analogy to plastics. Their biodegradability makes them extremely desirable substitutes for synthetic plastics. PHAs are produced in organisms under certain stress conditions. Here, we investigated 253 sequenced (completely and unfinished) genomes for the diversity and phylogenetics of the PHA biosynthesis. Discrepancies in the phylogenetic trees for phaA, phaB and phaC genes of the PHA biosynthesis have led to the suggestion that horizontal gene transfer (HGT) may be a major contributor for its evolution. Twenty four organisms belonging to diverse taxa were found to be involved in HGT. Among these, Bacillus cereus ATCC 14579 and Xanthomonas axonopodis pv. citri str. 306 seem to have acquired all the three genes through HGT events and have not been characterized so far as PHA producers. This study also revealed certain potential organisms such as Streptomyces coelicolor A3(2), Staphylococcus epidermidis ATCC 12228, Brucella suis 1330, Burkholderia sp., DSMZ 9242 and Leptospira interrogans serovar lai str. 56601, which can be transformed into novel PHA producers through recombinant DNA technology.

Combing databases reveals potential antibiotic producers.

The large-scale, persistent use of antibiotics has provoked microbes to evolve mechanisms to evade them. Pharmaceutical companies have found this to be counterproductive to their business economics. To maintain company interest to invest in this sector, innovative alternatives are needed. The availability of metabolic and genomic databases has opened up an avenue for such discoveries. Using these databases, potential producers of penicillin and cephalosporin have been traced. In addition, organisms that can be transformed from their present 'non'-producer status to antibacterial producers by supplementing their missing gene(s) by recombinant DNA technology have been revealed.

Mining genomic databases to identify novel hydrogen producers.

The realization that fossil fuel reserves are limited and their adverse effect on the environment has forced us to look into alternative sources of energy. Hydrogen is a strong contender as a future fuel. Biological hydrogen production ranges from 0.37 to 3.3 moles H(2) per mole of glucose and, considering the high theoretical values of production (4.0 moles H(2) per mole of glucose), it is worth exploring approaches to increase hydrogen yields. Screening the untapped microbial population is a promising possibility. Sequence analysis and pathway alignment of hydrogen metabolism in complete and incomplete genomes has led to the identification of potential hydrogen producers.