Human Milk Protein Production in Xenografts of Genetically Engineered Bovine Mammary Epithelial Stem Cells

BACKGROUND: In the bovine species milk production is well known to correlate with mammary tissue mass. However, most advances in optimizing milk production relied on improvements of breeding and husbandry practices. A better understanding of the cells that generate bovine mammary tissue could facilitate important advances in milk production and have global economic impact. With this possibility in mind, we show that a mammary stem cell population can be functionally identified and isolated from the bovine mammary gland. We also demonstrate that this stem cell population may be a promising target for manipulating the composition of cow's milk using gene transfer. METHODS AND FINDINGS: We show that the in vitro colony-forming cell assay for detecting normal primitive bipotent and lineage-restricted human mammary clonogenic progenitors are applicable to bovine mammary cells. Similarly, the ability of normal human mammary stem cells to regenerate functional bilayered structures in collagen gels placed under the kidney capsule of immunodeficient mice is shared by a subset of bovine mammary cells that lack aldehyde dehydrogenase activity. We also find that this activity is a distinguishing feature of luminal-restricted bovine progenitors. The regenerated structures recapitulate the organization of bovine mammary tissue, and milk could be readily detected in these structures when they were assessed by immunohistochemical analysis. Transplantation of the bovine cells transduced with a lentivirus encoding human β-CASEIN led to expression of the transgene and secretion of the product by their progeny regenerated in vivo. CONCLUSIONS: These findings point to a common developmental hierarchy shared by human and bovine mammary glands, providing strong evidence of common mechanisms regulating the maintenance and differentiation of mammary stem cells from both species. These results highlight the potential of novel engineering and transplant strategies for a variety of commercial applications including the production of modified milk components for human consumption

Cell Hierarchy and Lineage Commitment in the Bovine Mammary Gland

The bovine mammary gland is a favorable organ for studying mammary cell hierarchy due to its robust milk-production capabilities that reflect the adaptation of its cell populations to extensive expansion and differentiation. It also shares basic characteristics with the human breast, and identification of its cell composition may broaden our understanding of the diversity in cell hierarchy among mammals. Here, Lin− epithelial cells were sorted according to expression of CD24 and CD49f into four populations: CD24medCD49fpos (putative stem cells, puStm), CD24negCD49fpos (Basal), CD24highCD49fneg (putative progenitors, puPgt) and CD24medCD49fneg (luminal, Lum). These populations maintained differential gene expression of lineage markers and markers of stem cells and luminal progenitors. Of note was the high expression of Stat5a in the puPgt cells, and of Notch1, Delta1, Jagged1 and Hey1 in the puStm and Basal populations. Cultured puStm and Basal cells formed lineage-restricted basal or luminal clones and after re-sorting, colonies that preserved a duct-like alignment of epithelial layers. In contrast, puPgt and Lum cells generated only luminal clones and unorganized colonies. Under non-adherent culture conditions, the puPgt and puStm populations generated significantly more floating colonies. The increase in cell number during culture provides a measure of propagation potential, which was highest for the puStm cells. Taken together, these analyses position puStm cells at the top of the cell hierarchy and denote the presence of both bi-potent and luminally restricted progenitors. In addition, a population of differentiated luminal cells was marked. Finally, combining ALDH activity with cell-surface marker analyses defined a small subpopulation that is potentially stem cell- enriched

Isolation of Clostridium bifermentans from oil mill wastewaters converting cinnamic acid to 3-phenylpropionic acid and emendation of the species

A strictly anaerobic, Gram-positive, motile and spore-forming bacterium, designated strain TYR6, was isolated from olive mill wastewaters on cinnamic acid. Cells were straight rods and occurred singly, in pairs or in short chains. Strain TYR6 utilized a number of carbohydrates (glucose, fructose, mannose, maltose, sorbitol, myo-inositol, and ribose) and peptides (yeast extract, Casamino acids, Biotrypcase, peptone, and gelatin), grew optimally at 37°C and at pH 7, and had a G+C content of 29.1 mol%. Strain TYR6 was phylogenetically related to Clostridium bifermentans (similarity of 99%) with which it bears a high phenotypic similarity and DNA homology (80%). In addition, strain TYR6 shares with C. bifermentans ATCC 638T and DSM 630 the ability to metabolize cinnamic acid (but not any of its derivatives), with or without glucose supplementation, via the reduction of the double bond of the C3-aliphatic side chain to 3-phenylpropionic (hydrocinnamic) acid. Addition of glucose accelerated the rate of growth and increased markedly the conversion yield of cinnamic acid to 3-phenylpropionic acid. Based on the results, we propose to emend the description of C. bifermentans

Isolation of a cinnamic acid-metabolizing Clostridium glycolicum strain from oil mill wastewaters and emendation of the species description

A strictly anaerobic, gram-positive, motile, sporulated bacterium, designated strain CIN5, was isolated from olive mill wastewaters after enrichment on cinnamic acid. The rod-shaped cells were slightly curved (0.4-1.1 x 2.0-15 μm) and occurred singly or in pairs. Strain CIN5 utilized a limited number of carbohydrates (glucose, fructose, maltose, sorbitol), grew optimally at 37 °C and at pH 7.3-7.5 and had a DNA G+C content of 29.1+/-0.3 mol%. Strain CIN5 was very closely related to Clostridium glycolicum DSM 1288T. Both strain CIN5 and the type strain of C. glycolicum transformed cinnamic acid to hydrocinnamic acid and a wide range of other cinnamic acid derivatives, including o-, m- and p-coumaric, o-, m- and p-methoxycinnamic, p-methylcinnamic, caffeic, ferulic and isoferulic acids, to their corresponding 3-phenylpropionic acids by reducing the double bond of the side chain. Glucose supplementation increased the rate of conversion markedly. The emendation of the description of C. glycolicum is proposed to include these new characteristics

Isolation from a shea cake digester of a tannin-degrading Streptococcus gallolyticus strain that decarboxylates protocatechuic and hydroxycinnamic acids, and emendation of the species

A facultatively anaerobic, mesophilic, non-motile, non-sporulating bacterium, designated strain B7, was isolated from an anaerobic digester fed with shea cake rich in tannins and aromatic compounds, after enrichment on tannic acid. The coccoid cells (less than 2 microm in diameter) occurred in pairs or short chains and stained gram-positive. Strain B7 fermented a wide range of carbohydrates (cellobiose, fructose, galactose, glucose, lactose, maltose, mannitol, melibiose, raffinose and trehalose), grew optimally at pH 7.0 and had a G+C content of 40.4+/-0.3 mol%. Strain B7 was closely related to Streptococcus gallolyticus ACM 3611T, a member of the Streptococcus bovis rRNA cluster, with a sequence similarity of 98% and a DNA hybridization value of 86 mol%. Isolate B7 hydrolysed tannic acid and decarboxylated gallic acid to pyrogallol, traits also observed in S. gallolyticus ACM 3611T. In addition, both strains decarboxylated protocatechuic acid to catechol, p-coumaric acid to 4-vinylphenol, caffeic acid to 4-vinylcatechol and ferulic acid to 4-vinylguaiacol. An unsubstituted para-hydroxyl group on the benzene ring was required for decarboxylation. Glucose addition markedly increased the conversion rate. As these traits were not described previously, emendation of the description of the species Streptococcus gallolyticus is proposed

Arch Pediatr

AIM: To investigate the clinical, laboratory, electrophysiological, and imaging features associated with death or neurological impairment at 1 year of age in term neonates with hypoxic-ischemic encephalopathy (HIE) treated by therapeutic hypothermia (TH). METHODS: This was a single-center retrospective and descriptive study conducted over a period of 2 years. We included consecutive term newborns with moderate or severe HIE who were treated by TH initiated within the sixth hour after birth and continued for 72 h,. For all patients, brain magnetic resonance imaging (MRI) was performed before the eighth day and a score was established; furthermore, at least two electroencephalograms were recorded. RESULTS: Among the 33 patients included, 20 neonates had a favorable outcome and 13 had an unfavorable outcome. Early clinical seizures (15% vs. 53.8%, p = 0.047), the persistence of a poor prognosis according to the electroencephalogram pattern after TH (0% vs. 69.2%, p = 0.0001), and an elevated score on the early brain MRI (2 vs. 11, p < 0.001) combined with a high lactate/N-acetyl-aspartate ratio (0.52 vs. 1.33, p = 0.008) on spectroscopy were associated with death and a poor outcome. CONCLUSION: A combination of tools can help the medical team to establish the most reliable prognosis for these full-term neonates, to guide care, and to inform parents most appropriately and sincerely

Basin-scale analysis of rainfall and runoff in Peru (1969-2004) : Pacific, Titicaca and Amazonas drainages

According to the Peruvian agricultural ministry, the Pacific watersheds where the great cities and intense farming are located only benefit from 1% of the available freshwater in Peru. Hence a thorough knowledge of the hydrology of this region is of particular importance. In the paper, analysis of this region and of the two other main Peruvian drainages, the Titicaca and Amazonas are reported. Rainfall and runoff data collected by the Peruvian National Service of Meteorology and Hydrology (SENAMHI) and controlled under the Hydrogeodynamics of the Amazon Basin (HyBAm) project is the basis of this basin-scale study that covers the 1969-2004 period. Beyond the strong contrasting rainfall conditions that differentiate the dry coastal basins and the wet eastern lowlands, details are given about in situ runoff and per basin rainfall distribution in these regions, and about their different altitude-rainfall relationships. Rainfall and runoff variability is strong in the coastal basins at seasonal and inter-annual time scales, and related to extreme El Nino events in the Pacific Ocean. However, rainfall and runoff are more regular in the Andes and Amazonas at the inter-annual time scale. Warm sea-surface temperatures in the northern tropical Atlantic tend to produce drought in the southern Andes basins. Moreover, significant trends and change-points are observed in the runoff data of Amazonas basins where rainfall and runoff decrease, especially after the mid-1980s and during the low-stage season. Almost all the coastal basins show some change in minimum runoff during the last 35 years while no change is observed in rainfall. This means that human activity may have changed runoff in this region of Peru, but this hypothesis deserves more study

Trends in rainfall and temperature in the Peruvian Amazon-Andes basin over the last 40 years (1965-2007)

The hydroclimatology of the Peruvian Amazon-Andes basin (PAB) which surface corresponding to 7% of the Amazon basin is still poorly documented. We propose here an extended and original analysis of the temporal evolution of monthly rainfall, mean temperature (T-mean), maximum temperature (T-max) and minimum temperature (T-min) time series over two PABs (Huallaga and Ucayali) over the last 40years. This analysis is based on a new and more complete database that includes 77 weather stations over the 1965-2007 period, and we focus our attention on both annual and seasonal meteorological time series. A positive significant trend in mean temperature of 0.09 degrees C per decade is detected over the region with similar values in the Andes and rainforest when considering average data. However, a high percentage of stations with significant T-mean positive trends are located over the Andes region. Finally, changes in the mean values occurred earlier in T-max (during the 1970s) than in T-min (during the 1980s). In the PAB, there is neither trend nor mean change in rainfall during the 1965-2007 period. However, annual, summer and autumn rainfall in the southern Andes presents an important interannual variability that is associated with the sea surface temperature in the tropical Atlantic Ocean while there are limited relationships between rainfall and El Nino-Southern Oscillation (ENSO) events. On the contrary, the interannual temperature variability is mainly related to ENSO events