Location, allocation, relocation: isolating adult tissue stem cells in three dimensions

The literature on isolation of adult tissue stem cells is vast and disparate. To better organize the field, we redefine 'isolation', re-expressing it as the sum of three component vectors: location, allocation and relocation. Location is the isolation of stem cells in situ by anatomical features. Allocation is physical isolation by cell sorting. Relocation is isolation of the functional properties of a stem cell to regenerate its normal progeny when relocated to a new environment. Techniques for the allocation and relocation of stem cells from certain tissues (e.g. hematopoietic) are currently better defined than their location, whereas those of other tissues (e.g. mammary glands) have had recent advances along all three vectors. Yet another group (e.g. gastric glands), have stem cells with well characterized location, emerging techniques for allocation but still rudimentary techniques for relocation.     

Trunk, abdomen, and pressure sore reconstruction

LEARNING OBJECTIVES: After reading this article, the participant should be able to: 1. Describe the principles of wound closure, torso reconstruction, and pressure sore reconstruction. 2. Outline standard options to treat defects of the chest, abdomen, and back and pressure ulcers in all anatomical areas. 3. Manage and prevent pressure ulcers. SUMMARY: Chest wall reconstruction is indicated following tumor resection, radiation wound breakdown, or intrathoracic sepsis. Principles of wound closure and chest wall stabilization, where indicated, are discussed. Principles of abdominal wall reconstruction continue to evolve with the introduction of newer bioprosthetics and the application of functional concepts for wound closure. The authors illustrate these principles using commonly encountered clinical scenarios and guidelines to achieve predictable results. Pressure ulcers continue to be devastating complications to patients' health and a functional hazard when they occur in the bedridden, in patients with spinal cord injuries, and in patients with neuromuscular disease. Management of pressure ulcers is also very expensive. The authors describe standard options to treat defects of the chest, abdomen, and back and pressure ulcers in all anatomical areas. A comprehensive understanding of principles and techniques will allow practitioners to approach difficult issues of torso reconstruction and pressure sores with a rational confidence and an expectation of generally satisfactory outcomes. With pressure ulcers, prevention remains the primary goal. Patient education and compliance coupled with a multidisciplinary team approach can reduce their occurrence significantly. Surgical management includes appropriate patient selection, adequate débridement, soft-tissue coverage, and use of flaps that will not limit future reconstructions if needed. Postoperatively, a strict protocol should be adapted to ensure the success of the flap procedure. Several myocutaneous flaps commonly used for the surgical management of pressure are discussed. Commonly used flaps in chest and abdominal wall reconstruction are discussed and these should be useful for the practicing plastic surgeon.     

Eliminating expression of erucic acid-encoding loci allows the identification of “hidden” QTL contributing to oil quality fractions and oil content in <Emphasis Type="Italic">Brassica juncea</Emphasis> (Indian mustard)

Oil content and oil quality fractions (viz., oleic, linoleic and linolenic acid) are strongly influenced by the erucic acid pathway in oilseed Brassicas. Low levels of erucic acid in seed oil increases oleic acid content to nutritionally desirable levels, but also increases the linoleic and linolenic acid fractions and reduces oil content in Indian mustard (Brassica juncea). Analysis of phenotypic variability for oil quality fractions among a high-erucic Indian variety (Varuna), a low-erucic east-European variety (Heera) and a zero-erucic Indian variety (ZE-Varuna) developed by backcross breeding in this study indicated that lower levels of linoleic and linolenic acid in Varuna are due to substrate limitation caused by an active erucic acid pathway and not due to weaker alleles or enzyme limitation. To identify compensatory loci that could be used to increase oil content and maintain desirable levels of oil quality fractions under zero-erucic conditions, we performed Quantitative Trait Loci (QTL) mapping for the above traits on two independent F1 doubled haploid (F1DH) mapping populations developed from a cross between Varuna and Heera. One of the populations comprised plants segregating for erucic acid content (SE) and was used earlier for construction of a linkage map and QTL mapping of several yield-influencing traits in B. juncea. The second population consisted of zero-erucic acid individuals (ZE) for which, an Amplified Fragment Length Polymorphism (AFLP)-based framework linkage map was constructed in the present study. By QTL mapping for oil quality fractions and oil content in the ZE population, we detected novel loci contributing to the above traits. These loci did not co-localize with mapped locations of the fatty acid desaturase 2 (FAD2), fatty acid desaturase 3 (FAD3) or fatty acid elongase (FAE) genes unlike those of the SE population wherein major QTL were found to coincide with mapped locations of the FAE genes. Some of the new loci identified in the ZE population could be detected as ‘weak’ contributors (with LOD < 2.5) in the SE population in which their contribution to the traits was “masked” due to pleiotropic effects of erucic acid genes. The novel loci identified in this study could now be used to improve oil quality parameters and oil content in B. juncea under zero-erucic conditions.     

The link between the PDL1 costimulatory pathway and Th17 in fetomaternal tolerance

Fetomaternal tolerance has been shown to depend both on regulatory T cells (Tregs) and negative signals from the PD1-PDL1 costimulatory pathway. More recently, IL-17-producing T cells (Th17) have been recognized as a barrier in inducing tolerance in transplantation. In this study, we investigate the mechanisms of PDL1-mediated regulation of fetomaternal tolerance using an alloantigen-specific CD4(+) TCR transgenic mouse model system (ABM-tg mouse). PDL1 blockade led to an increase in embryo resorption and a reduction in litter size. This was associated with a decrease in Tregs, leading to a lower Treg/effector T cell ratio. Moreover, PDL1 blockade inhibited Ag-specific alloreactive T cell apoptosis and induced apoptosis of Tregs and a shift toward higher frequency of Th17 cells, breaking fetomaternal tolerance. These Th17 cells arose predominantly from CD4(+)Foxp3(-) cells, rather than from conversion of Tregs. Locally in the placenta, similar decrease in regulatory and apoptotic markers was observed by real-time PCR. Neutralization of IL-17 abrogated the anti-PDL1 effect on fetal survival rate and restored Treg numbers. Finally, the adoptive transfer of Tregs was also able to improve fetal survival in the setting of PDL1 blockade. This is to our knowledge the first report using an alloantigen-specific model that establishes a link between PDL1, Th17 cells, and fetomaternal tolerance.     

Study of liquid culture system for micropropagation of the medicinal plant Solanum nigrum L. and its effect on antioxidant property

Solanum nigrum Linn., known for hepatoprotective and antioxidant properties, is extensively harvested from the wild. Supply is far short of demand, necessitating requirement of efficient in vitro propagation protocols. Nodal explants from wild S. nigrum plants were cultured in vitro in MS medium supplemented with 3.0 mg L^?1 IAA, 0.5 mg L^?1 BAP and gelled with 0.8 % agar. After 30 days, shoots buds were transferred to liquid MS medium of same composition. Subsequent subculture was carried out every 6 days. Shoot doubling time in solid and liquid media was calculated. Total phenolics, proanthocyanidin and flavonoid contents, ABTS^.+ and hydrogen peroxide radical scavenging antioxidant capacity of wild and in vitro aqueous leaf extracts were estimated and compared. In MS agar, 18 shoot buds were produced per explant after 30 days of culture, with shoot doubling time of 7.11 days. In liquid media, 21 shoots per explant were produced in 6 days, with a 5-fold higher multiplication rate and shoot doubling time of 1.37 days. Leaves were morphologically similar to those of wild plants. Total phenolics, proanthocyanidin and flavonoid contents of in vitro leaf extracts was 5–10 times higher than that of wild plants while ABTS^.+ and H₂O₂ radical scavenging activity was similar in both extracts. Liquid media is better suited for in vitro propagation of S. nigrum since enhanced multiplication rate was observed with shorter subculture intervals. Moreover, plants retained normal morphology and antioxidant property.     

Novel Binding Motif and New Flexibility Revealed by Structural Analyses of a Pyruvate Dehydrogenase-Dihydrolipoyl Acetyltransferase Subcomplex from the Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex

The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly.     

The Interplay of Angle Strain and Aromaticity: Molecular and Electronic Structures of [0n]Paracyclophanes

The belt-like polyphenylenes, [0_n]paracyclophanes, (n = 5 and 6), have been investigated using semi-empirical, ab initio and DFT methods. The molecular structure, rotational barrier on twisting a single phenyl ring and the aromatic character within each ring as well as in the whole molecule have been evaluated. [0₅]Paracyclophane is predicted to have a quinonoid structure. In contrast, the equatorial pentaphenyl fragment found in C₇₀ as well as the hexagons of the less strained [0₆]paracyclophane have benzenoid character. Approximate band structures have been derived for larger cycles of [0_n] paracyclophanes.Electronic Supplementary Material available.     

Bacteriophage-like particle of Rochalimaea henselae

An extracellular particle approximately 40 nM in diameter was detected in culture supernatant from the fastidious bacterium Rochalimaea henselae. This particle has at least three associated proteins and contains 14kbp linear DNA segments that are heterogeneous in sequence. The 14kbp DNA was also present in R. henselae cells as an extrachromosomal element for all 14 strains tested. Despite attempts to induce lysis of R henselae, plaque formation was not observed. A similar particle, also containing 14 kbp DNA, was observed in Bartonella bacilliformis, and may be analogous to a bacteriophage that has been described elsewhere for B. bacilliformis.     

Cloning and characterization of fxp, the flexible pilin gene of Aeromonas hydrophila

The flexible pilus of Aeromonas hydrophila is a morphologically and biochemically unique organelle which binds eukaryotic cell surfaces and whose expression is induced by specific physiochemical conditions. fxp, the structural gene coding for the flexible pilus subunit, was localized on a 7.6kb plasmid of A. hydrophila strain AH26. A putative Shine-Dalgarno sequence and -10 and -35 regions were identified, a signal peptide sequence delineated, and the coding sequence compared with other bacterial sequences and found to be unique. Plasmid and chromosomal DNA was prepared from 66 other Aeromonas strains and 12 strains from other bacterial genera and examined by Southern blot hybridization using a labelled fxp oligonucleotide and the 7.6kb plasmid as probes. No hybridizing sequences were identified except in the original strain, AH26. It is proposed that fxp codes for a highly evolved organelle, possibly widely distributed in nature, but that it is carried on a genetic element that is rapidly lost from most strains upon in vitro cultivation and storage.