Proteomic analysis of a sea-ice diatom: salinity acclimation provides new insight into the dimethylsulfoniopropionate production pathway.

Dimethylsulfoniopropionate (DMSP) plays important roles in oceanic carbon and sulfur cycling and may significantly impact climate. It is a biomolecule synthesized from the methionine (Met) pathway and proposed to serve various physiological functions to aid in environmental stress adaptation through its compatible solute, cryoprotectant, and antioxidant properties. Yet, the enzymes and mechanisms regulating DMSP production are poorly understood. This study utilized a proteomics approach to investigate protein changes associated with salinity-induced DMSP increases in the model sea-ice diatom Fragilariopsis cylindrus (CCMP 1102). We hypothesized proteins associated with the Met-DMSP biosynthesis pathway would increase in relative abundance when challenged with elevated salinity. To test this hypothesis axenic log-phase cultures initially grown at a salinity of 35 were gradually shifted to a final salinity of 70 over a 24-h period. Intracellular DMSP was measured and two-dimensional gel electrophoresis was used to identify protein changes at 48 h after the shift. Intracellular DMSP increased by approximately 85% in the hypersaline cultures. One-third of the proteins increased under high salinity were associated with amino acid pathways. Three protein isoforms of S-adenosylhomo-cysteine hydrolase, which synthesizes a Met precursor, increased 1.8- to 2.1-fold, two isoforms of S-adenosyl Met synthetase increased 1.9- to 2.5-fold, and S-adenosyl Met methyltransferase increased by 2.8-fold, suggesting active methyl cycle proteins are recruited in the synthesis of DMSP. Proteins from the four enzyme classes of the proposed algal Met transaminase DMSP pathway were among the elevated proteins, supporting our hypothesis and providing candidate genes for future characterization studies.

Preliminary study: treatment with intramuscular interferon beta-1a results in increased levels of IL-12Rß2+ and decreased levels of IL23R+ CD4+ T - Lymphocytes in multiple sclerosis.

BACKGROUND: There are a lack of biomarkers which can be used to predict clinical outcomes for multiple sclerosis (MS) patients receiving interferon beta (IFN-ß). Thus the objective of this study was to characterize changes in CD4+ T-lymphocyte expression in an unbiased manner following initiation of intramuscular (IM) IFN-ß-1a treatment, and then to verify those findings using marker-specific assays. METHODS: Peripheral blood specimens were collected from twenty MS patients before and after treatment with intramuscular (IM) IFN-ß-1a and were used for isolation of mononuclear cells (PBMCs). mRNA expression patterns of negatively-selected CD4+ T-cells from the PBMCs were analyzed using microarray gene expression technology. IL-12 and IL-23 receptor levels on PBMC-derived CD4+ T-cells were analyzed by flow cytometry. The phosphorylation status of Stat4 was measured by performing densitometry on western blots. RESULTS: Microarray analyses demonstrated that mRNA expression of the IL-12Rß2 gene was uniformly up-regulated in response to IFN-ß-1a treatment and was associated with an increased number of IL-12Rß2+ CD4+ T-cells by flow cytometry in 4 of 6 patients. This finding was substantiated by demonstrating that Stat4 phosphorylation, a transcription factor for IL-12, was increased after treatment. Conversely, the number of IL-23R+ CD4+ T-cells was decreased following treatment. CONCLUSIONS: The IL-12 receptor shares a common subunit, the IL-12Rß2, with the IL-23 receptor. Both of these receptors have a probable role in regulating IL-17 and TH-17 cells, important mediators of inflammation in multiple sclerosis (MS). Thus, the changes in the numbers of CD4+ T-cells expressing these receptors in response to IFN-ß-1a treatment may point to an important mechanism of action for this drug, but further large scale studies are needed to confirm these preliminary observations.

MT1 melatonin receptor internalization underlies melatonin-induced morphologic changes in Chinese hamster ovary cells and these processes are dependent on Gi proteins, MEK 1/2 and microtubule modulation.

Melatonin induces cellular differentiation in numerous cell types. Data show that multiple mechanisms are involved in these processes that are cell-type specific and may be receptor dependent or independent. The focus of this study was to specifically assess the role of human MT1 melatonin receptors in cellular differentiation using an MT1-Chinese hamster ovary (CHO) model; one that reproducibly produces measurable morphologic changes in response to melatonin. Using multiple approaches, we show that melatonin induces MT1-CHO cells to hyperelongate through a MEK 1/2, and ERK 1/2-dependent mechanism that is dependent upon MT1 receptor internalization, Gi protein activation, and clathrin-mediated endocytosis. Using immunoprecipitation analysis, we show that MT1 receptors form complexes with Gi(alpha) 2,3, Gq(alpha), beta-arrestin-2, MEK 1/2, and ERK 1/2 in the presence of melatonin. We also show that MEK and ERK activity that is induced by melatonin is dependent on Gi protein activation, clathrin-mediated endocytosis and is modulated by microtubules. We conclude from these studies that melatonin-induced internalization of human MT1 melatonin receptors in CHO cells is responsible for activating both MEK 1/2 and ERK 1/2 to drive these morphologic changes. These events, as mediated by melatonin, require Gi protein activation and endocytosis mediated through clathrin, to form MT1 receptor complexes with beta-arrestin-2/MEK 1/2 and ERK 1/2. The MT1-CHO model is invaluable to mapping out signaling cascades as mediated through MT1 receptors especially because it separates out MEK/ERK 1/2 activation by MT1 receptors from that of receptor tyrosine kinases.

Identification and Characterization of msf, a Novel Virulence Factor in Haemophilus influenzae.

Haemophilus influenzae is an opportunistic pathogen. The emergence of virulent, non-typeable strains (NTHi) emphasizes the importance of developing new interventional targets. We screened the NTHi supragenome for genes encoding surface-exposed proteins suggestive of immune evasion, identifying a large family containing Sel1-like repeats (SLRs). Clustering identified ten SLR-containing gene subfamilies, each with various numbers of SLRs per gene. Individual strains also had varying numbers of SLR-containing genes from one or more of the subfamilies. Statistical genetic analyses of gene possession among 210 NTHi strains typed as either disease or carriage found a significant association between possession of the SlrVA subfamily (which we have termed, macrophage survival factor, msf) and the disease isolates. The PittII strain contains four chromosomally contiguous msf genes. Deleting all four of these genes (msfA1-4) (KO) resulted in a highly significant decrease in phagocytosis and survival in macrophages; which was fully complemented by a single copy of the msfA1 gene. Using the chinchilla model of otitis media and invasive disease, the KO strain displayed a significant decrease in fitness compared to the WT in co-infections; and in single infections, the KO lost its ability to invade the brain. The singly complemented strain showed only a partial ability to compete with the WT suggesting gene dosage is important in vivo. The transcriptional profiles of the KO and WT in planktonic growth were compared using the NTHi supragenome array, which revealed highly significant changes in the expression of operons involved in virulence and anaerobiosis. These findings demonstrate that the msfA1-4 genes are virulence factors for phagocytosis, persistence, and trafficking to non-mucosal sites.

Multi-channeled biodegradable polymer/CultiSpher composite nerve guides.

Innovative methods to fabricate porous, biodegradable conduits were developed to produce nerve guides with multiple longitudinally aligned channels. The geometry of the nerve guide's channels was designed to be appropriate for harboring neurite extension. Both the coated mandrel and mandrel adhesion techniques permit flexibility in the number of channels, channel organization, and channel diameters. In this study, the composite nerve guides were comprised of poly(caprolactone) (PCL) and porous collagen-based beads (CultiSphers). The incorporation of the collagenous beads results in enhanced cortical neuron adhesion, viability, and neurite extension as compared to PCL alone. Additionally, Schwann cell studies indicated that the PCL/CultiSpher composite is a suitable substrate for cell adhesion. Mechanical properties of the PCL/CultiSpher material and in vitro degradation rates indicate the potential usefulness of this novel composite for use in the fabrication of nerve guides.

Geographically separate outbreaks of shigellosis in Auckland, New Zealand, linked by molecular subtyping to cases returning from Samoa.

AIM: To investigate simultaneous outbreaks of Shigella sonnei gastroenteritis occurring in February 2001 at a health camp for socially deprived children and an elderly care facility. METHODS: Those with symptoms were interviewed using a standardised questionnaire. Cases were defined as having at least three loose stools over a 24 hour period and stool samples requested. A case-control study investigating routes of transmission was performed at the health camp. Environmental investigations of food safety and hygiene were conducted at each facility. RESULTS: At the camp, 15 (37%) students and 15 (28%) staff met case criteria. Contact with human faeces (OR 4.0; 95% confidence interval 1.0-16.3; p = 0.05) and, for staff, eating camp food (OR 6.9; 1.0-5.0; p = 0.06) were shown to be independent risk factors for illness. At the elderly care facility, four (19%) residents and four (25%) staff met case criteria. Molecular subtyping confirmed that the outbreaks were related to each other and to other cases in travellers returning from Samoa to Auckland and other New Zealand cities over a four month period. CONCLUSION: Molecular subtyping is of considerable use in communicable disease investigation, providing strong evidence for links between outbreaks. With expanded technological capability, New Zealand could perform routine molecular subtyping of selected organisms to improve the detection and the investigation of regional and inter-regional outbreaks of infection.

Collagenous microbeads as a scaffold for tissue engineering with adipose-derived stem cells.

BACKGROUND: Standard approaches to soft-tissue reconstruction include autologous tissue flaps and alloplastic implants. Both of these approaches have disadvantages, including donor-site morbidity, implant migration, and foreign body reaction. Autologous fat transplantation, with a minimally invasive cannula harvest, has lower donor-site morbidity than tissue flaps do, but there is an unpredictable degree of resorption of the transplanted fat over time. Adipose-derived stem cells isolated from harvested fat are better able to withstand the mechanical trauma from the suction cannula and may allow for improved cell survival and generation of new fat tissue after transfer to another anatomic site. The authors hypothesized that porous collagenous microbeads (CultiSphers; Sigma, St. Louis, Mo.) could be useful as injectable cell delivery vehicles for adipose-derived stem cells. This strategy would allow induction of differentiation ex vivo and precise placement of cells and scaffold in a tissue bed. The objective of this study was to assess the ability of the stem cells to proliferate and differentiate on these microbeads. METHODS: Adipose-derived stem cells were isolated from discarded human adipose tissue and cultured on porous collagenous microbeads in a stirred bioreactor (spinner flask). The cells attached and proliferated on the microbeads and maintained high viability over several weeks of culture. RESULTS: When exposed to adipogenic or osteogenic medium, the cells differentiated into adipocytes and osteoblasts, respectively, while attached to the microbeads. CONCLUSION: Collagenous microbeads are a favorable scaffold for adipose-derived stem cells, allowing ex vivo proliferation and differentiation on particles that are small enough to be injected.

Scientific basis for the use of hypotonic solutions with ultrasonic liposuction.

BACKGROUND: A number of plastic surgeons have advocated using hypotonic solution in ultrasound lipoplasty, theorizing that induced adipocyte swelling increases membrane susceptibility to ultrasonic disruption. Additionally, it has been theorized that potassium increases membrane permeability. This study aimed to determine the effect of solution osmolality on adipocyte diameter, the time course of hypotonic solution action, and the effect of potassium addition on adipocyte diameter. METHODS: Base solutions with three different osmolalities were prepared: normal saline (NS) (154 mOsm/l), 1/2NS (77 mOsm/l), and 1/4NS (38.5 mOsm/l). Each solution was modified to contain 0, 5, and 10 mEq/l of potassium and adjusted to starting osmolality. Adipocytes of six patients were suspended in the nine solutions, and diameters were determined at 0, 15, 30, and 45 min. Diameters were measured using imaging software (Kodak ID 3.6). RESULTS: At time 0, the average adipocyte diameter was 79 +/- 8 microm, and no difference was seen in any of the solutions. Cells in the NS group showed no significant increase in diameter over 45 min. The 1/2NS group achieved an 8% +/- 1.9% increase in diameter at 45 min (p < 0.05). The 1/4NS group showed an increase by 14% +/- 2.4% (p < 0.01) at 15 min, and 15% +/- 2.3% (p < 0.01) at 45 min. Potassium had no independent effect on cell diameter. CONCLUSIONS: Hypotonic solution can significantly increase human adipocyte diameter. The findings showed that 1/2NS had a significant effect within 15 min. Tumescent solutions with an osmolality of 1/4NS may be useful in facilitating ultrasonic lipoplasty.