Spatial dynamics of multistage cell lineages in tissue stratification

In developing and self-renewing tissues, terminally differentiated (TD) cell types are typically specified through the actions of multistage cell lineages. Such lineages commonly include a stem cell and multiple progenitor (transit-amplifying) cell stages, which ultimately give rise to TD cells. As the tissue reaches a tightly controlled steady-state size, cells at different lineage stages assume distinct spatial locations within the tissue. Although tissue stratification appears to be genetically specified, the underlying mechanisms that direct tissue lamination are not yet completely understood. Herein, we use modeling and simulations to explore several potential mechanisms that can be utilized to create stratification during developmental or regenerative growth in general systems and in the model system, the olfactory epithelium of mouse. Our results show that tissue stratification can be generated and maintained through controlling spatial distribution of diffusive signaling molecules that regulate the proliferation of each cell type within the lineage. The ability of feedback molecules to stratify a tissue is dependent on a low TD death rate: high death rates decrease tissue lamination. Regulation of the cell cycle lengths of stem cells by feedback signals can lead to transient accumulation of stem cells near the base and apex of tissue.     

Different regulation of cigarette smoke induced inflammation in upper versus lower airways

Background

Cigarette smoke (CS) is known to initiate a cascade of mediator release and accumulation of immune and inflammatory cells in the lower airways. We investigated and compared the effects of CS on upper and lower airways, in a mouse model of subacute and chronic CS exposure.

Methods

C57BL/6 mice were whole-body exposed to mainstream CS or air, for 2, 4 and 24 weeks. Bronchoalveolar lavage fluid (BAL) was obtained and tissue cryosections from nasal turbinates were stained for neutrophils and T cells. Furthermore, we evaluated GCP-2, KC, MCP-1, MIP-3α, RORc, IL-17, FoxP3, and TGF-β1 in nasal turbinates and lungs by RT-PCR.

Results

In both upper and lower airways, subacute CS-exposure induced the expression of GCP-2, MCP-1, MIP-3α and resulted in a neutrophilic influx. However, after chronic CS-exposure, there was a significant downregulation of inflammation in the upper airways, while on the contrary, lower airway inflammation remained present. Whereas nasal FoxP3 mRNA levels already increased after 2 weeks, lung FoxP3 mRNA increased only after 4 weeks, suggesting that mechanisms to suppress inflammation occur earlier and are more efficient in nose than in lungs.

Conclusions

Altogether, these data demonstrate that CS induced inflammation may be differently regulated in the upper versus lower airways in mice. Furthermore, these data may help to identify new therapeutic targets in this disease model.     

Impact of engineering flow conditions on plasmid DNA yield and purity in chemical cell lysis operations

Chemical lysis of bacterial cells using an alkaline solution containing a detergent may provide an efficient scalable means for selectively removing covalently closed circular plasmid DNA from high-molecular-weight contaminating cellular components including chromosomal DNA. In this article we assess the chemical lysis of E. coli cells by SDS in a NaOH solution and determine the impact of pH environment and shear on the supercoiled plasmid and chromosomal DNA obtained. Experiments using a range of plasmids from 6 kb to 113 kb determined that in an unfavorable alkaline environment, where the NaOH concentration during lysis is greater than 0.15 +/- 0.03 M (pH 12.9 +/- 0.2), irreversible denaturation of the supercoiled plasmid DNA occurs. The extent of denaturation is shown to increase with time of exposure and NaOH concentration. Experiments using stirred vessels show that, depending on NaOH concentration, moderate to high mixing rates are necessary to maximize plasmid yield. While NaOH concentration does not significantly affect chromosomal DNA contamination, a high NaOH concentration is necessary to ensure complete conversion of chromosomal DNA to single-stranded form. In a mechanically agitated lysis reactor the correct mixing strategy must balance the need for sufficient mixing to eliminate potential regions of high NaOH concentrations and the need to avoid excessive breakage of the shear sensitive chromosomal DNA. The effect of shear on chromosomal DNA is examined over a wide range of shear rates (10(1)-10(5) s(-1)) demonstrating that, while increasing shear leads to fragmentation of chromosomal DNA to smaller sizes, it does not lead to significantly increased chromosomal DNA contamination except at very high shear rates (about 10(4)-10(5) s(-1)). The consequences of these effects on the choice of lysis reactor and scale-up are discussed.     

Hematology and serum chemistry comparisons between free-ranging and rehabilitated harbor seal (Phoca vitulina richardsi) pups

The objectives of this study were to compare the hematology and serum chemistry values between free-ranging and stranded harbor seal (Phoca vitulina richardsi) pups and to ascertain how blood values of stranded pups changed during the rehabilitation process. Coincident with these comparisons, reference values were obtained for free-ranging pups. Stranded harbor seal pups (n = 28) recovered from areas between Pebble Beach and Moss Landing, California (USA) were admitted to The Marine Mammal Center, Sausalito, from March to May 1995, 1996, and 1998. Blood samples were collected from harbor seal pups before and after rehabilitation. As a control group, wild harbor seal pups were captured at Pebble Beach and Elkhorn Slough (n = 42) during the 1995, 1996, and 1998 pupping seasons. Mean eosinophil and calcium values of wild pups were significantly greater than those of newly admitted pups, whereas mean bands, aspartate aminotransferase, alanine aminotransferase, total bilirubin, and chloride values were significantly lower (P < or = 0.05). Mean neutrophil, band, lymphocyte, eosinophil, basophil, calcium, phosphorus, blood urea nitrogen, potassium, total protein, and globulin values of rehabilitated pups increased significantly after 2-3 mo in captivity, whereas, mean red blood cell, hemoglobin, hematocrit, cholesterol, and total bilirubin values decreased significantly (P < or = 0.05).