Accuracy, Reproducibility, and Interpretation of Fatty Acid Methyl Ester Profiles of Model Bacterial Communities

We determined the accuracy and reproducibility of whole-community fatty acid methyl ester (FAME) analysis with two model bacterial communities differing in composition by using the Microbial ID, Inc. (MIDI), system. The biomass, taxonomic structure, and expected MIDI-FAME profiles under a variety of environmental conditions were known for these model communities a priori. Not all members of each community could be detected in the composite profile because of lack of fatty acid “signatures” in some isolates or because of variations (approximately fivefold) in fatty acid yield across taxa. MIDI-FAME profiles of replicate subsamples of a given community were similar in terms of fatty acid yield per unit of community dry weight and relative proportions of specific fatty acids. Principal-components analysis (PCA) of MIDI-FAME profiles resulted in a clear separation of the two different communities and a clustering of replicates of each community from two separate experiments on the first PCA axis. The first PCA axis accounted for 57.1% of the variance in the data and was correlated with fatty acids that varied significantly between communities and reflected the underlying community taxonomic structure. On the basis of our data, community fatty acid profiles can be used to assess the relative similarities and differences of microbial communities that differ in taxonomic composition. However, detailed interpretation of community fatty acid profiles in terms of biomass or community taxonomic composition must be viewed with caution until our knowledge of the quantitative and qualitative distribution of fatty acids over a wide variety of taxa and the effects of growth conditions on fatty acid profiles is more extensive.     

Treatment with tumour infiltrating lymphocytes and interleukin-2 in patients with metastatic melanoma: A pilot study

Tumour infiltrating lymphocytes (TIL) were isolated and expanded from biopsy samples of 4 patients with metastatic melanoma. The patients were treated with autologous expanded TIL and continuous or bolus infusion of Interleukin 2 (IL-2) at a dose of 18 × 10⁶ International Units/m²/day for 5 days starting 36–48 hours after administration of cyclophosphamide at a dose of 1 g/m². The number of TIL infused ranged from 10¹⁰ to 5,56 × 10¹⁰ cells. Two patients had stable disease (SD) lasting for 2 1/2 and 4 months respectively and they died 24 and 13 months after therapy. One patient died during therapy due to a pseudomonas septicaemia and another patient developed progressive disease (PD). He died 3 months after the start of therapy. The side effects were substantial but most of them were reversible upon cessation of the treatment.The majority of the expanded TIL of all patients were of the CD8+ phenotype. Cutaneous metastases from two patients, removed after treatment with IL-2 and TIL, showed moderate lymphocytic infiltration also mainly of CD8+ T cells.The treatment with IL-2 and TIL is feasible, but further investigations should continue in an attempt to improve the efficacy of the therapy, to reduce toxicity and to diminish the costs and labour of the culture methods.     

AUTORADIOGRAPHIC INVESTIGATION ON THE CELL KINETICS OF CRYPT EPITHELIA IN THE JEJUNUM OF THE MOUSE °CONFIRMATION OF STEADY STATE GROWTH WITH CONSTANT FREQUENCY DISTRIBUTION OF CELLS THROUGHOUT THE CYCLE

Cell kinetic parameters of cells in the crypt of the jejunum of the mouse were obtained autoradiographically. A number of different methods used in cell proliferation studies were applied to the same animal strain kept under constant conditions. In order to avoid effects of geometrical factors, squashes of isolated crypts were used.
The generation time was determined by the per cent labelled mitoses method of Quastler, modified by double labelling with ³H- and ¹⁴C-TdR. This modified method permits a more exact determination of the generation time. The duration of the cycle was 14 hr.
Double labelling experiments in which an injection of ³H-TdR was followed by an injection of ¹⁴C-TdR after 1 hr showed that the cell flux was 7.0%/hr at the beginning of the S-phase and 7.68%/hr at the end. Assuming steady state growth a constant cell flux of 7.15%/hr within the whole cycle can be derived from the measured generation time of 14 hr. These results clearly show that the crypt epithelia constitute a steady state system with constant frequency distribution of the cells throughout the cycle.
The per cent labelled mitoses method after a single injection of ³H-TdR as well as double labelling experiments with ³H- and ¹⁴C-TdR give an estimate of the S-phase of 8.0 or 7.4 hr respectively. Double determinations lead to a value of 0.54 or 0.52 hr respectively for the duration of mitosis and to values of 77% and 72%     

Absence of BiP Co-chaperone DNAJC3 Causes Diabetes Mellitus and Multisystemic Neurodegeneration

Diabetes mellitus and neurodegeneration are common diseases for which shared genetic factors are still only partly known. Here, we show that loss of the BiP (immunoglobulin heavy-chain binding protein) co-chaperone DNAJC3 leads to diabetes mellitus and widespread neurodegeneration. We investigated three siblings with juvenile-onset diabetes and central and peripheral neurodegeneration, including ataxia, upper-motor-neuron damage, peripheral neuropathy, hearing loss, and cerebral atrophy. Exome sequencing identified a homozygous stop mutation in DNAJC3. Screening of a diabetes database with 226,194 individuals yielded eight phenotypically similar individuals and one family carrying a homozygous DNAJC3 deletion. DNAJC3 was absent in fibroblasts from all affected subjects in both families. To delineate the phenotypic and mutational spectrum and the genetic variability of DNAJC3, we analyzed 8,603 exomes, including 506 from families affected by diabetes, ataxia, upper-motor-neuron damage, peripheral neuropathy, or hearing loss. This analysis revealed only one further loss-of-function allele in DNAJC3 and no further associations in subjects with only a subset of the features of the main phenotype. Our findings demonstrate that loss-of-function DNAJC3 mutations lead to a monogenic, recessive form of diabetes mellitus in humans. Moreover, they present a common denominator for diabetes and widespread neurodegeneration. This complements findings from mice in which knockout of Dnajc3 leads to diabetes and modifies disease in a neurodegenerative model of Marinesco-Sjögren syndrome.     

Bacterial and Archaeal Phylogenetic Diversity of a Cold Sulfur-Rich Spring on the Shoreline of Lake Erie,Michigan

Studies of sulfidic springs have provided new insights into microbial metabolism, groundwater biogeochemistry, and geologic processes. We investigated Great Sulphur Spring on the western shore of Lake Erie and evaluated the phylogenetic affiliations of 189 bacterial and 77 archaeal 16S rRNA gene sequences from three habitats: the spring origin (11-m depth), bacterial-algal mats on the spring pond surface, and whitish filamentous materials from the spring drain. Water from the spring origin water was cold, pH 6.3, and anoxic (H₂, 5.4 nM; CH₄, 2.70 μM) with concentrations of S²⁻ (0.03 mM), SO₄²⁻ (14.8 mM), Ca²⁺ (15.7 mM), and HCO₃⁻ (4.1 mM) similar to those in groundwater from the local aquifer. No archaeal and few bacterial sequences were >95% similar to sequences of cultivated organisms. Bacterial sequences were largely affiliated with sulfur-metabolizing or chemolithotrophic taxa in Beta-, Gamma-, Delta-, and Epsilonproteobacteria. Epsilonproteobacteria sequences similar to those obtained from other sulfidic environments and a new clade of Cyanobacteria sequences were particularly abundant (16% and 40%, respectively) in the spring origin clone library. Crenarchaeota sequences associated with archaeal-bacterial consortia in whitish filaments at a German sulfidic spring were detected only in a similar habitat at Great Sulphur Spring. This study expands the geographic distribution of many uncultured Archaea and Bacteria sequences to the Laurentian Great Lakes, indicates possible roles for epsilonproteobacteria in local aquifer chemistry and karst formation, documents new oscillatorioid Cyanobacteria lineages, and shows that uncultured, cold-adapted Crenarchaeota sequences may comprise a significant part of the microbial community of some sulfidic environments.Cold, sulfidic springs upwelling into caves (1, 16-19) or exposed at the land surface (14, 15, 31, 39, 47, 50, 51) have recently been shown to harbor unique microbial communities, reflective of the aqueous sulfur chemistry of the upwelling groundwater or of unique cave conditions. Within these spring and cave ecosystems, new and unique Epsilonproteobacteria 16S rRNA gene sequences associated with a limited number of cultured isolates that carry out oxidation of sulfur compounds have been discovered (7). The abundance of Epsilonproteobacteria sequences in these settings and associated biogeochemical research have led to new interest in the role of microbially mediated sulfuric acid speleogenesis as an important limestone dissolution process that may contribute to the development of karst features in limestone bedrock (19). Additionally, in streamlets from sulfidic springs, unique symbioses between uncultured Euryarchaeota, Crenarchaeota, and Epsilonproteobacteria spp. that grow in whitish, macroscopically visible filaments have been described (31, 51). Sulfur cycling was identified as a major means of energy production and maintenance of microbial communities in cold, saline, perennial springs emanating from permafrost in the Arctic (47). Studies of cold, sulfidic springs have therefore provided new insights into microbial metabolism, ecology, and evolution as well as groundwater biogeochemistry and geologic processes.All studies of sulfidic springs to date have focused on terrestrial landscapes typically associated with limestone (CaCO₃) bedrock. Limestone is one of several carbonate sedimentary rocks deposited by ancient seas, which may contain significant amounts of gypsum (CaSO₄·2H₂O) as well as pockets of hydrocarbon deposits, both a source of sulfur. Water that moves for long distances through such rocks evolves through sequential dissolution and precipitation reactions to a geochemistry that bears little resemblance to freshwater. SO₄²⁻ becomes available for microbial reduction to sulfide in aquifer zones where conditions are appropriate. Where spring waters rich in CaCO₃, CO₂, and sulfide emerge at the surface, carbonate deposition and microbially mediated sulfide oxidation occur. These processes result in tufa deposits and the whitish crusts often noted in sulfidic spring outflows (12, 13). Carbonate bedrock underlies large portions of the lower Laurentian Great Lakes. Caves in contact with lake water occur on islands in Lake Erie and along the Bruce Peninsula in Ontario, Canada. A cold, sulfidic spring is located in Ancaster, Ontario, about 5 km from the Lake Ontario shoreline (13). Recently, plumes of high-conductivity sulfidic groundwater, surrounded by whitish filamentous materials and variously colored microbial mats, were reported to occur at a 93-m depth in Lake Huron (2, 49). However, there have been few molecular surveys of Bacteria or Archaea in any Great Lakes environment, and no reports focusing on the molecular phylogenetic diversity of microorganisms associated with these Great Lakes sulfidic environments.Along the western shoreline of Lake Erie and within Monroe County, MI, sinkholes and springs are abundant in the Silurian-Devonian carbonate bedrock, and Ca²⁺ and Mg²⁺ with SO₄²⁻ or HCO₃⁻ dominate groundwater composition (43). In some areas of Monroe County, sulfide in groundwater prohibits its use as a drinking water source. Great Sulphur Spring (GSS) was first described by Sherzer in 1900 (53) and was named for its sulfide-rich water. The spring arises from Silurian-Devonian carbonate bedrock within 0.5 km of the Lake Erie shoreline and is a convenient location for accessing sulfide-rich groundwater and for exploring potential interactions between groundwater and lake water. As part of a larger study of nearshore groundwater interactions with Lake Erie (27) and to better understand the potential role of microorganisms in sulfur chemistry of nearshore groundwater, we evaluated the chemistry and bacterial and archaeal 16S rRNA gene diversity of GSS. Our study documents a unique microbial community for the Laurentian Great Lakes, comprised in large part of new lineages and uncultivated members of the Archaea, Deltaproteobacteria, Epsilonproteobacteria, and Cyanobacteria. These sequences suggest a microbial community structure driven by (possibly H₂S-based) carbon fixation and chemolithotrophy of reduced compounds such as H₂, H₂S, or reduced nitrogen compounds, all consistent with spring geochemistry.     

Mechanisms of haplotype divergence at the <Emphasis Type="Italic">RGA08</Emphasis> nucleotide-binding leucine-rich repeat gene locus in wild banana <Emphasis Type="Italic">(Musa balbisiana)</Emphasis>

Background  

Comparative sequence analysis of complex loci such as resistance gene analog clusters allows estimating the degree of sequence conservation and mechanisms of divergence at the intraspecies level. In banana (Musa sp.), two diploid wild species Musa acuminata (A genome) and Musa balbisiana (B genome) contribute to the polyploid genome of many cultivars. The M. balbisiana species is associated with vigour and tolerance to pests and disease and little is known on the genome structure and haplotype diversity within this species. Here, we compare two genomic sequences of 253 and 223 kb corresponding to two haplotypes of the RGA08 resistance gene analog locus in M. balbisiana "Pisang Klutuk Wulung" (PKW).     

Induction of cellular immune responses against carcinoembryonic antigen in patients with metastatic tumors after vaccination with altered peptide ligand-loaded dendritic cells

Purpose: Dendritic cells (DCs) are characterized by their extraordinary capacity to induce T-cell responses, providing the opportunity of DC-based cancer vaccination protocols. In the present study, we conducted a phase I/II clinical trial to determine the capability of DCs differentiated from immunomagnetically isolated CD14+ monocytes and pulsed with a carcinoembyonic antigen-derived altered peptide (CEAalt) to induce specific CD8+ T cells in cancer patients. Experimental design: Nine patients with CEA-positive colorectal cancer (n=7) or lung cancer (n=2) were enrolled in this study. Autologous CD14+ monocytes were isolated by large-scale immunomagnetic separation and differentiated to mature DCs in sufficient numbers and at high purity. After incubation with the CEAalt peptide and keyhole limpet hemocyanin, DCs were administered to patients intravenously at dose levels of 1×10⁷ and 5×10⁷ cells. Patients received four immunizations every second week. Results: ELISPOT analysis revealed a vaccine-induced increase in the number of CEAalt peptide-specific Interferon (IFN)-gamma producing CD8+ T cells in five of nine patients and of CD8+ T lymphocytes recognizing the native CEA peptide in three of nine patients. In addition, CD8+ T lymphocytes derived from one patient exhibiting an immunological response after vaccination efficiently lysed peptide-loaded T2 cells and tumor cells. Immunization was well tolerated by all patients without severe signs of toxicity. Conclusion: Vaccination with CEAalt-pulsed DCs derived from immunomagnetically isolated CD14+ monocytes efficiently expand peptide-specific CD8+ T lymphocytes in vivo and may be a promising alternative for cancer immunotherapy.     

A derivatization method for the simultaneous detection of glucosinolates and isothiocyanates in biological samples

Various analytical methods have been established to quantify isothiocyanates (ITCs) that derive from glucosinolate hydrolysis. However, to date there is no valid method applicable to pharmacokinetic studies that detects both glucosinolates and ITCs. A specific derivatization procedure was developed for the determination of ITCs based on the formation of a stable N-(tert-butoxycarbonyl)-l-cysteine methyl ester derivative, which can be measured by high-performance liquid chromatography with ultraviolet detection after extraction with ethylacetate. The novel method, which is also applicable to the indirect determination of glucosinolates after their hydrolysis by myrosinase, was established for the simultaneous determination of glucoraphanin and sulforaphane. By derivatization, the sensitivity of ITC detection was increased 2.5-fold. Analytical recoveries from urine and plasma were greater than 75% and from feces were approximately 50%. The method showed intra- and interday variations of less than 11 and 13%, respectively. Applicability of the method was demonstrated in mice that received various doses of glucoraphanin or that were fed a glucoraphanin-rich diet. Besides glucoraphanin and sulforaphane, glucoerucin and erucin were detected in urine and feces of mice. The novel method provides an essential tool for the analysis of bioactive glucosinolates and their hydrolysis products and, thus, will contribute to the elucidation of their bioavailability.     

Renal denervation modulates angiotensin receptor expression in the renal cortex of rabbits with chronic heart failure

Excessive sympathetic drive is a hallmark of chronic heart failure (HF). Disease progression can be correlated with plasma norepinephrine concentration. Renal function is also correlated with disease progression and prognosis. Because both the renal nerves and renin-angiotensin II system are activated in chronic HF we hypothesized that excessive renal sympathetic nerve activity decreases renal blood flow in HF and is associated with changes in angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) expression. The present study was carried out in conscious, chronically instrumented rabbits with pacing-induced HF. We found that rabbits with HF showed a decrease in mean renal blood flow (19.8±1.6 in HF vs. 32.0±2.5 ml/min from prepace levels; P<0.05) and an increase in renal vascular resistance (3.26±0.29 in HF vs. 2.21±0.13 mmHg·ml(-1)·min in prepace normal rabbits; P<0.05) while the blood flow and resistance was not changed in HF rabbits with the surgical renal denervation. Renal AT1R expression was increased by ～67% and AT2R expression was decreased by ～87% in rabbits with HF; however, kidneys from denervated rabbits with HF showed a near normalization in the expression of these receptors. These results suggest renal sympathetic nerve activity elicits a detrimental effect on renal blood flow and may be associated with alterations in the expression of angiotensin II receptors.