Noninvasive measurement of effective diffusivities in cell immobilization gels through use of near-infrared spectroscopy.

Encapsulated cell systems provide some advantages over typical suspension cell cultivations as higher cell densities may be obtained; however, the supply of nutrients to the cells often is a limiting factor in productivity. In this study, we describe the development of a new approach to characterize the effective diffusivity of nutrients in immobilized cell materials. Near-infrared spectroscopy is employed to measure nutrient concentrations within a specially designed diffusion chamber that permits noninvasive sampling at ten spatial positions and multiple timepoints. To demonstrate this technique, we measured the effective diffusivity of glutamine in a cell-free 3% (w/w) agarose gel and determined the effective diffusivity (D(eff)) = 6.46 x 10(-10) m(2)/s, which is in good agreement with theoretical values.

Rapid calibration of near-infrared spectroscopic measurements of mammalian cell cultivations.

Near-infrared (NIR) spectroscopy is a flexible method that can be employed to noninvasively monitor the concentrations of multiple nutrients and wastes in mammalian cell bioreactors. Development of suitable calibrations can be a labor- and time-intensive process that must be repeated when process conditions are altered significantly. To address this difficulty, we have produced a new approach for generating NIR spectroscopic calibrations that requires significantly less time compared with standard calibration schemes. This method reduces development time from the present level of several weeks to several hours. A small number of experimentally collected spectra serve as inputs to a computational procedure that yields a large number of simulated spectra, each containing both analyte-specific and analyte-independent information. Such simulated spectra may be employed as a calibration set for quantifying analytes in experimentally collected spectra. Spectroscopic measurements of the concentrations of five components (ammonia, glucose, glutamate, glutamine, and lactate) can be accomplished with levels of error similar to those obtained with full experimental calibrations. A key to this process is the utilization of random numbers, which randomizes the influence of natural variations, present in each experimentally collected spectrum, on the resultant composite spectrum. This approach may increase the feasibility of employing NIR spectroscopy to monitor bioreactors and other biological processes subjected to varying operating conditions.

Minimally invasive aortic valve replacement.

During a consecutive 12-month period from January 1996 to January 1997 inclusive, 108 aortic valve replacements were performed by one group of surgeons in two community hospitals The majority of the valve replacements were done in combination with other procedures or were redo surgeries. Thirty-one patients had primary isolated aortic valve replacement. Fourteen patients underwent aortic valve replacement via a standard sternotomy, and seventeen patients underwent aortic valve replacement using a minimally invasive parasternal approach, as described by Dr. Cosgrove. There were no operative deaths in either group; however there was one hospital death in each of the two groups. Blood loss and postoperative pain were less in the minimally invasive group. Although the cross-clamp times were longer in the minimally invasive group, with a mean of 82.7 min as compared with 63.1 min in the standard group, the length of stay was shortened, with a median of 5 days in the minimally invasive group as compared to 7 days in the sternotomy group. In the follow-up which ranges from 4-15 months, all patients in the minimally invasive group were New York Heart Class I or II. Patients with the parasternal incisions are permitted to return to work much earlier than those with a standard sternotomy incision. The decreased blood loss and postoperative pain, combined with the anticipated ease of re-entry via a median sternotomy in the future (should redo aortic valve replacement become necessary), make this approach our procedure of choice in isolated primary aortic valve replacement.

mtDNA sequences suggest a recent evolutionary divergence for Beringian and northern North American populations.

Conventional descriptions of the pattern and process of human entry into the New World from Asia are incomplete and controversial. In order to gain an evolutionary insight into this process, we have sequenced the control region of mtDNA in samples of contemporary tribal populations of eastern Siberia, Alaska, and Greenland and have compared them with those of Amerind speakers of the Pacific Northwest and with those of the Altai of central Siberia. Specifically, we have analyzed sequence diversity in 33 mitochondrial lineages identified in 90 individuals belonging to five Circumpolar populations of Beringia, North America, and Greenland: Chukchi from Siberia, Inupiaq Eskimos and Athapaskans from Alaska, Eskimos from West Greenland, and Haida from Canada. Hereafter, we refer to these five populations as "Circumarctic peoples." These data were then compared with the sequence diversity in 47 mitochondrial lineages identified in a sample of 145 individuals from three Amerind-speaking tribes (Bella Coola, Nuu-Chah-Nulth, and Yakima) of the Pacific Northwest, plus 16 mitochondrial lineages identified in a sample of 17 Altai from central Siberia. Sequence diversity within and among Circumarctic populations is considerably less than the sequence diversity observed within and among the three Amerind tribes. The similarity of sequences found among the geographically dispersed Circumarctic groups, plus the small values of mean pairwise sequence differences within Circumarctic populations, suggest a recent and rapid evolutionary radiation of these populations. In addition, Circumarctic populations lack the 9-bp deletion which has been used to trace various migrations out of Asia, while populations of southeastern Siberia possess this deletion. On the basis of these observations, while the evolutionary affinities of Native Americans extend west to the Circumarctic populations of eastern Siberia, they do not include the Altai of central Siberia.

Extensive mitochondrial diversity within a single Amerindian tribe.

Sequencing of a 360-nucleotide segment of the mitochondrial control region for 63 individuals from an Amerindian tribe, the Nuu-Chah-Nulth of the Pacific Northwest, revealed the existence of 28 lineages defined by 26 variable positions. This represents a substantial level of mitochondrial diversity for a small local population. Furthermore, the sequence diversity among these Nuu-Chah-Nulth lineages is greater than 60% of the mitochondrial sequence diversity observed in major ethnic groups such as Japanese or sub-Saharan Africans. It was also observed that the majority of the mitochondrial lineages of the Nuu-Chah-Nulth fell into phylogenetic clusters. The magnitude of the sequence difference between the lineage clusters suggests that their origin predates the entry of humans into the Americas. Since a single Amerindian tribe can contain such extensive molecular diversity, it is unnecessary to presume that substantial genetic bottlenecks occurred during the formation of contemporary ethnic groups. In particular, these data do not support the concept of a dramatic founder effect during the peopling of the Americas.

Genetic variation in an inbred plant: variation in tissue cultures of soybean [Glycine max (L.) Merrill].

Although soybean [Glycine max (L.) Merrill] grows as an inbreeding, generally homozygous, plant, the germplasm of the species contains large amounts of genetic variation. Analysis of soybean DNA has indicated that variation of RFLP (restriction fragment length polymorphism) markers within the species usually entails only two alleles at any one locus and that mixtures of such dimorphic loci account for virtually all of the restriction fragment variation seen in soybean (G. max), and in its ancestors, G. soja and G. gracilis. We report here that tissue cultures prepared from root tissue of individual soybean plants develop RFLP allelic differences at various loci. However, these newly generated alleles are almost always the same as ones previously found and characterized in other varieties of cultivated soybean (cultivars). This repeated generation of particular alleles suggests that much of the genetic variation seen in soybean could be the consequence of specific, relatively frequently employed, recombinational events. Such a mechanism would allow inbred cultivars to generate genetic variation (in the form of alternative alleles) in a controlled manner, perhaps in response to stress.

Chemotaxis in Escherichia coli: construction and properties of lambda tsr transducing phage.

The tsr gene of Escherichia coli, located at approximately 99 min on the chromosomal map, encodes a methyl-accepting protein that serves as the chemoreceptor and signal transducer for chemotactic responses to serine and several repellents. To determine whether any other chemotaxis or motility genes were located in the tsr region, we constructed and characterized two lambda tsr transducing phages that each contain about 12 kilobases of chromosomal material adjacent to tsr. lambda tsr70 carries sequences from the promoter-proximal side of tsr; lambda tsr72 carries sequences from the promoter-distal side of tsr. Restriction maps of the bacterial inserts in these phages and Southern hybridization analyses of the bacterial chromosome indicated that the tsr gene is transcribed in the counterclockwise direction on the genetic map. Insert deletions were isolated in lambda tsr70 and transferred into the host chromosome to examine the null phenotype of tsr. All such strains exhibited wild-type swimming patterns and chemotactic responses to a variety of stimuli, but were specifically defective in serine taxis and other Tsr-mediated responses. In addition, UV programming experiments demonstrated that Tsr and several of its presumptive degradation products were the only bacterial proteins encoded by lambda tsr70 and lambda tsr72 that required host FlbB/FlaI function for expression. These findings indicate that there are probably no other chemotaxis-related genes in the tsr region. A series of tsr point mutations were isolated by propagating lambda tsr70 on a mutD host and used to construct a fine-structure map of the tsr locus. These mutations should prove valuable in exploring structure-function relationships in the Tsr transducer.

Comparative hemodynamic properties of vein and mammary artery in coronary bypass operations.

To understand better the observed differences in bypass flows between vein and internal mammary artery (IMA) grafts, a technique was devised for anastomosing both vein and IMA to the same anterior descending coronary artery in 14 patients. In the stable postperfusion state, flows in the two bypass conduits were simultaneously recorded as well as pressure relationships in both grafts and the left ventricle. The supply/demand ratio for left ventricular performance was calculated with respect to the diastolic pressure-time index/tension-time index (DPTI/TTI) for each bypass independently and simultaneously and then compared. The DPTI/TTI ratio was nearly two times greater with the vein bypass than with the IMA. This difference was further confirmed by the flow studies, in which blood flow through the vein ranged 2 to 3 times higher than IMA flow to the same coronary bed. By present criteria the DPTI/TTI ratio for IMA grafts to the left ventricle was inadequate in the majority of patients studied, and atrial pacing markedly lowered the DPTI/TTI ratio of the IMA. The choice of vein or IMA as a bypass is a critical determinant of the resultant bypass-left ventricular DPTI/TTI ratio. Vein bypasses exhibited far superior hemodynamic capability in the resting state, and the effect of atrial pacing on the DPTI/TTI ratio in IMA-vein-left ventricle bypasses confirms this point.