Protoplast formation and regeneration in <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Method for production and regeneration of Lactobacillus delbrueckii protoplasts are described. The protoplasts were obtained by treatment with a mixture of lysozyme and mutanolysin in protoplast buffer at pH 6.5 with different osmotic stabilizers. The protoplasts were regenerated on deMan, Rogosa and Sharpe (MRS) with various osmotic stabilizers. Maximum protoplast formation was obtained in protoplast buffer with sucrose as an osmotic stabilizer using a combination of lysozyme (1 mg/ml) and mutanolysin (10 μg/ml). Maximum protoplast regeneration was obtained on MRS medium with sucrose (0.5 M) as an osmotic stabilizer. The regeneration medium was also applicable to other species of lactobacilli as well. This is, to our knowledge, the first report on protoplast formation and efficient regeneration in case of L. delbrueckii.     

Effects of substratum morphology on cell physiology

Among the host of substratum properties that affect animal cell behavior, surface morphology has received relatively little attention. The earliest effect of surface morphology on animal cells was discovered almost a century ago when it was found that cells became oriented in response to the underlying topography. This phenomenon is now commonly known as contact guidance. From then until very recentrly, little progress has been made in understanding the role of surface morphology on cell behavior, primarily due to a lack of defined surfaces with uniform morphologies. This problem has been solved recently with the development of photolithographic techniques to prepare substrata with well defined and uniform surface morphologies. Availability of such surfaces has facilitated systematic in vitro experiments to study influence of surface morphology on diverse cell physiological aspects such as adhesion, growth, and function. For example, these studies have shown that surfaces with uniform multipls parallel grooves can enhance cell adhesion by confining cells in grooves and by mechanically interlocking them. Several independent studies have demosterated that cell shape is a major determinant of cell growth and function. Because surface morphology has been shown to modulate the extent of cell spreading and cell shape, its effects on cell growth and function appear to be mediated via this biological coupling between cell shape and function. New evidence in the cell biology literature is emerging to suggest that surface morphology could affect other cell behavioral properties such as post-translational modifications. Further elucidation of such effects will enable better designs for implant and cell culture substrata.     

The Arf GAP CNT-2 regulates the apoptotic fate in C. elegans asymmetric neuroblast divisions

During development, all cells make the decision to live or die. Although the molecular mechanisms that execute the apoptotic program are well defined, less is known about how cells decide whether to live or die. In C.?elegans, this decision is linked to how cells divide asymmetrically [1, 2]. Several classes of molecules are known to regulate asymmetric cell divisions in metazoans, yet these molecules do not appear to control C.?elegans divisions that produce apoptotic cells [3]. We identified CNT-2, an Arf GTPase-activating protein (GAP) of the AGAP family, as a novel regulator of this type of neuroblast division. Loss of CNT-2 alters daughter cell size and causes the apoptotic cell to adopt the fate of its sister cell, resulting in extra neurons. CNT-2's Arf GAP activity is essential for its function in these divisions. The N terminus of CNT-2, which contains?a GTPase-like domain that defines the AGAP class of Arf GAPs, negatively regulates CNT-2's function. We provide evidence that CNT-2 regulates receptor-mediated endocytosis and consider the implications of its role in asymmetric cell divisions.     

Taste representations in the Drosophila brain

Drosophila taste compounds with gustatory neurons on many parts of the body, suggesting that a fly detects both the location and quality of a food source. For example, activation of taste neurons on the legs causes proboscis extension or retraction, whereas activation of proboscis taste neurons causes food ingestion or rejection. We examined whether the features of taste location and taste quality are mapped in the fly brain using molecular, genetic, and behavioral approaches. We find that projections are segregated by the category of tastes that they recognize: neurons that recognize sugars project to a region different from those recognizing noxious substances. Transgenic axon labeling experiments also demonstrate that gustatory projections are segregated based on their location in the periphery. These studies reveal the gustatory map in the first relay of the fly brain and demonstrate that taste quality and position are represented in anatomical projection patterns.     

Computational and experimental investigation of flow and fluid mixing in the roller bottle bioreactor

The fully three-dimensional velocity field in a roller bottle bioreactor is simulated for two systems (creeping flow and inertial flow conditions) using a control volume-finite element method, and validated experimentally using particle imaging velocimetry. The velocity fields and flow patterns are described in detail using velocity contour plots and tracer particle pathline computations. Bulk fluid mixing in the roller bottle is then examined using a computational fluid tracer program and flow visualization experiments. It is shown that the velocity fields and flow patterns are substantially different for each of these flow cases. For creeping flow conditions the flow streamlines consist of symmetric, closed three-dimensional loops; and for inertial flow conditions, streamlines consist of asymmetric toroidal surfaces. Fluid tracers remain trapped on these streamlines and are unable to contact other regions of the flow domain. As a result, fluid mixing is greatly hindered, especially in the axial direction. The lack of efficient axial mixing is verified computationally and experimentally. Such mixing limitations, however, are readily overcome by introducing a small-amplitude vertical rocking motion that disrupts both symmetry and recirculation, leading to much faster and complete axial mixing. The frequency of such motion is shown to have a significant effect on mixing rate, which is a critical parameter in the overall performance of roller bottles.     

Continuity of Microblade Technology in the Indian Subcontinent Since 45 ka: Implications for the Dispersal of Modern Humans

We extend the continuity of microblade technology in the Indian Subcontinent to 45 ka, on the basis of optical dating of microblade assemblages from the site of Mehtakheri, (22° 13'' 44″ N Lat 76° 01'' 36″ E Long) in Madhya Pradesh, India. Microblade technology in the Indian Subcontinent is continuously present from its first appearance until the Iron Age (~3 ka), making its association with modern humans undisputed. It has been suggested that microblade technology in the Indian Subcontinent was developed locally by modern humans after 35 ka. The dates reported here from Mehtakheri show this inference to be untenable and suggest alternatively that this technology arrived in the Indian Subcontinent with the earliest modern humans. It also shows that modern humans in Indian Subcontinent and SE Asia were associated with differing technologies and this calls into question the “southern dispersal” route of modern humans from Africa through India to SE Asia and then to Australia. We suggest that modern humans dispersed from Africa in two stages coinciding with the warmer interglacial conditions of MIS 5 and MIS 3. Competitive interactions between African modern humans and Indian archaics who shared an adaptation to tropical environments differed from that between modern humans and archaics like Neanderthals and Denisovans, who were adapted to temperate environments. Thus, while modern humans expanded into temperate regions during warmer climates, their expansion into tropical regions, like the Indian Subcontinent, in competition with similarly adapted populations, occurred during arid climates. Thus modern humans probably entered the Indian Subcontinent during the arid climate of MIS 4 coinciding with their disappearance from the Middle East and Northern Africa. The out of phase expansion of modern humans into tropical versus temperate regions has been one of the factors affecting the dispersal of modern humans from Africa during the period 200–40 ka.     

Development of biocatalysts for production of commodity chemicals from lignocellulosic biomass

Lignocellulosic biomass is recognized as potential sustainable source for production of power, biofuels and variety of commodity chemicals which would potentially add economic value to biomass. Recalcitrance nature of biomass is largely responsible for the high cost of its conversion. Therefore, it is necessary to introduce some cost effective pretreatment processes to make the biomass polysaccharides easily amenable to enzymatic attack to release mixed fermentable sugars. Advancement in systemic biology can provide new tools for the development of such biocatalysts for sustainable production of commodity chemicals from biomass. Integration of functional genomics and system biology approaches may generate efficient microbial systems with new metabolic routes for production of commodity chemicals. This paper provides an overview of the challenges that are faced by the processes converting lignocellulosic biomass to commodity chemicals. The critical factors involved in engineering new microbial biocatalysts are also discussed with more emphasis on commodity chemicals.