Isolation and Characterisation of a Recombinant Antibody Fragment That Binds NCAM1-Expressing Intervertebral Disc Cells

Degeneration of the intervertebral discs (IVD) is a leading cause of neck and low back pain. Degeneration begins in the central nucleus pulposus region, leading to loss of IVD osmotic properties. Regeneration approaches include administration of matrix-mimicking scaffolds, cells and/or therapeutic factors. Cell-targeting strategies are likely to improve delivery due to the low cell numbers in the IVD. Single-chain antibody fragments (scFvs) that bind IVD cells were isolated for potential delivery of therapeutics to degenerated IVD. The most cell-distal domain of neural cell adhesion molecule 1 (NCAM1) was cloned and expressed in Escherichia coli. Phage display technology was used to isolate a human scFv against the recombinant domain by panning a scFv library on the immobilised protein. The isolated scFv bound cultured rat astrocytes, as well as bovine nucleus pulposus and annulus fibrosus cells in immunocytochemical studies. The scFv also labelled cells in bovine spinal cord and six-month and two-year old bovine IVD sections by immunohistochemistry. Antibody fragments can provide cell-binding moieties at improved cost, time, yield and functionalisation potential over whole antibodies. The described scFv has potential application in delivery of therapeutics to NCAM1-expressing cells in degenerated IVD.     

Abundance and Production of Riparian Trees in the Lowland Floodplain of the Queets River, Washington

Riparian zones associated with alluvial rivers are spatially dynamic, forming distinct vegetative mosaics that exhibit sharp contrasts in structure and processes related to the underlying biophysical template. The productivity of riparian plants, especially trees, influences streamside community characteristics as well as the forms and fluxes of organic matter to adjacent streams – thereby strongly impacting patterns of channel morphology, water flow, sedimentation, and habitat in rivers. As part of a comprehensive investigation of riparian dynamics in coastal rain forest rivers of the Pacific Northwest (USA), we examined riparian tree abundance (density, basal area, and biomass) and rates of production (basal area growth [BAI] and bole wood biomass increase [P]) of seven common species – red alder (Alnus rubra), Sitka spruce (Picea sitchensis), bigleaf maple (Acer macrophyllum), western hemlock (Tsuga heterophylla), black cottonwood (Populus trichocarpa), vine maple (Acer circinatum) and willow (Salix spp.) – in the lowland floodplain of the Queets River (Olympic National Park), Washington. Measurements were made annually for three years (1999 – 2001) in 16 permanent plots on three biophysical templates that formed a toposequence – active floodplain, young terrace and mature terrace. Stem density was highest in the active floodplain (∼27,000 stems/ ha), decreasing in the young terrace (∼2,700 stems /ha) and the mature terrace (∼500 stems/ha). Basal area and total stem biomass were lowest in the active floodplain (∼16 m2/ha and ∼18 Mg dry weight/ha, respectively) and higher on the young terrace (∼32 m2/ha and ∼134 Mg dry weight/ha) and on the mature terrace (∼69 m2/ha and ∼540 Mg dry weight /ha). Total plot-scale BAI was not significantly different among the physical templates with mean values ranging from approximately 1.4 (low terrace) to approximately 2.8 m2/ha/y (active floodplain). In contrast, P was significantly higher on the mature terrace (10.3 Mg/ha) than the active floodplain (3.2 Mg/ha) but there was no significant difference between young terrace (6.5 Mg/ha) and mature terrace. For the entire Queets River floodplain (57 km² over 77 km of river length), the mature terrace contributed 81% of the total annual production (28,764 Mg) whereas the active floodplain and young terrace accounted only for 5 and 14%, respectively. Overall, we show that riparian trees grow quickly in this coastal Pacific Northwest system and that the older riparian forests on mature terraces are the main contributors to stem production at the plot and floodplain scales for at least 350 years after stand initiation. This suggests that, in combination with the rapid lateral migrations of many alluvial rivers, the older riparian forests on those terraces are important and sustained sources of organic matter (especially large woody debris, LWD) that, over decades to centuries, shape the character of coastal rivers in the Pacific Northwest.     

A SNF2-like protein facilitates dynamic control of DNA methylation

DRD1 is a SNF2-like protein previously identified in a screen for mutants defective in RNA-directed DNA methylation of a seed promoter in Arabidopsis. Although the initial study established a role for DRD1 in RNA-directed DNA methylation, it did not address whether DRD1 is needed for de novo or maintenance methylation, or whether it is required for methylation of other target sequences. We show here that DRD1 is essential for RNA-directed de novo methylation and acts on different target promoters. In addition, an unanticipated role for DRD1 in erasure of CG methylation was shown when investigating maintenance methylation after segregating away the silencing trigger. DRD1 is unique among known SNF2-like proteins in facilitating not only de novo methylation of target sequences in response to RNA signals, but also loss of methylation when the silencing inducer is withdrawn. The opposing roles of DRD1 could contribute to the dynamic regulation of DNA methylation.     

Role of the host cell cycle in theAgrobacterium-mediated genetic transformation ofPetunia: Evidence of an S-phase control mechanism for T-DNA transfer

Chimeric -glucuronidase (GUS) gene expression in an efficientAgrobacterium-mediated transformation system utilising mesophyll cells ofPetunia hybrida synchronized with cell cycle phase-specific inhibitors (mimosine and colchicine) was used to show the absolute requirement of S-phase for transfer and/or integration of the transferred DNA (T-DNA). Flow-cytometric analysis of nuclear DNA content and immunohistological detection of bromodeoxyuridine (BrdUrd) incorporation showed that, prior to phytohormone treatment, most (98%) mesophyll cells were at GO-Gl-phase (quiescent phase) and no cell division was occurring. After 48 h and 72 h of phytohormone treatment, there was a rapid increase in S-G2-M-phase populations (> 75%) and a concomitant decrease (down to 24%) in G0–-G1-phase cells. Assays of GUS showed that maximum transformation (> 95% of explants) also occurred after this period. Our data showed that mimosine and colchicine blocked the mesophyll cells at late Gl-phase and M-phase, respectively. No transformation (= GUS expression) was observed in phytohormone-treated cells inhibited in late G1 by mimosine. However, after removal of mimosine, 82% of the explants were transformed, indicating the non-toxic and reversible effect of the inhibitor. On the other hand, a relatively high transformation frequency (65% of explants) was observed after blocking the cell cycle at M-phase with colchicine. However, only transient, but no stable, gene expression (= kanamycin-resistant callus formation) was observed in colchicine-treated M-phase-arrested cells. Similarly, endoreduplication of nuclear DNA, which occurred during the 48 h of phytohormone treatment in some mesophyll cells and cells located along the minor veins in the leaf explants, resulted in transient GUS expression only. These observations indicate a direct correlation between endoreduplication and transient GUS gene expression. Obviously, for stable GUS gene expression, cell division and proliferation are required, indicating that both DNA duplication (S-phase) and cell division (M-phase) are strongly related to stable transformation. We propose that the present system should facilitate further dissection of the process of T-DNA integration in the host genome and therefore should aid in developing new strategies for transformation of recalcitrant plants.Abbreviations BAP 6-benzylaminopurine - BM basal medium - BrdUrd bromodeoxyuridine - GUS -glucuronidase - Km^R kanamycin resistant - T-DNA transferred DNA     

Location and identification of the genes for adenovirus type 2 early polypeptides

Virus-specific RNA was prepared from cells early after adenovirus type 2 infection and fractionated by hybridization to specific fragments of viral DNA. The viral mRNA was used to program cell-free protein synthesis, and the products were analyzed by electrophoresis. The genes for the early polypeptides of apparent molecular weight 44,000, 15,000, 72,000, 15,500, 19,000, and 11,000 daltons were located, respectively, between positions 0–4.1, 4.1–16.7, 58.5–70.7, 75.9–83.4, 89.7–98.6, and 89.7–98.6 of the conventional adenovirus DNA map. The polypeptide of molecular weight 72,000 daltons was shown to be the single-strand DNA-binding protein described by others. RNAs from three different adeno-transformed cell lines each program the synthesis in vitro of predominantly the 15K polypeptide, as well as variable amounts of the polypeptide of molecular weight 44,000 daltons. The genes for these two polypeptides are located in the portion of DNA known to be required for transformation of rodent cells by adenovirus.     

Waist Circumference,BMI, and Visceral Adipose Tissue in White Women and Women of African Descent

Although waist circumference (WC) is a marker of visceral adipose tissue (VAT), WC cut‐points are based on BMI category. We compared WC‐BMI and WC‐VAT relationships in blacks and whites. Combining data from five studies, BMI and WC were measured in 1,409 premenopausal women (148 white South Africans, 607 African‐Americans, 186 black South Africans, 445 West Africans, 23 black Africans living in United States). In three of five studies, participants had VAT measured by computerized tomography (n = 456). Compared to whites, blacks had higher BMI (29.6 ± 7.6 (mean ± s.d.) vs. 27.6 ± 6.6 kg/m², P = 0.001), similar WC (92 ± 16 vs. 90 ± 15 cm, P = 0.27) and lower VAT (64 ± 42 vs. 101 ± 59 cm², P < 0.001). The WC‐BMI relationship did not differ by race (blacks: β (s.e.) WC = 0.42 (.01), whites: β (s.e.) WC = 0.40 (0.01), P = 0.73). The WC‐VAT relationship was different in blacks and whites (blacks: β (s.e.) WC = 1.38 (0.11), whites: β (s.e.) WC = 3.18 (0.21), P < 0.001). Whites had a greater increase in VAT per unit increase in WC. WC‐BMI and WC‐VAT relationships did not differ among black populations. As WC‐BMI relationship did not differ by race, the same BMI‐based WC guidelines may be appropriate for black and white women. However, if WC is defined by VAT, race‐specific WC thresholds are required.     

Outcome study: periareolar mammaplasty patients' perspective

Residual scars on occasion compromise patient satisfaction with breast reduction procedures. Periareolar breast reduction was proposed to minimize the scarring produced by the operation. This technique was criticized predominantly for producing a breast with low projection, for recurrence of ptosis after surgery, and for widening of the periareolar scar. The purpose of this study was to evaluate patient satisfaction from this technique. In the first branch of the study, 11 patients who had periareolar or circumareolar breast reduction were compared with 13 patients who had vertical scar reduction mammaplasty. The groups were matched for patient's age, size of reduction, and follow-up period. The patients were contacted by phone and requested to comment on four aspects of their operation: breast size, symmetry, quality of scars, and appearance and position of the nipple-areola complex. The replies were converted to numerical values and analyzed statistically. A panel of three women, prospective patients for breast reduction, were presented with standardized five-view before-and-after photographs and requested to score the results using the same criteria. Their responses were analyzed in the same manner. Both phases of the study produced similar results. Breast reduction done through a periareolar scar scored higher than a vertical scar technique. The operation did not differ with respect to shape, symmetry, or nipple-areola shape but did with respect to dissatisfaction with scars, which affected the overall result. This experimental model of assessing results of cosmetic operations is proposed as a tool to assess the patient's rather than the surgeon's perspective of a surgical technique, and it could find application in assessing other cosmetic operations.     

Rapid identification and interpretation of gene–environment associations using the new R.SamBada landscape genomics pipeline

sam βada is a genome–environment association software, designed to search for signatures of local adaptation. However, pre‐ and postprocessing of data can be labour‐intensive, preventing wider uptake of the method. We have now developed R.SamBada, an r ‐package providing a pipeline for landscape genomic analysis based on sam βada , spanning from the retrieval of environmental conditions at sampling locations to gene annotation using the Ensembl genome browser. As a result, R.SamBada standardizes the landscape genomics pipeline and eases the search for candidate genes of local adaptation, enhancing reproducibility of landscape genomic studies. The efficiency and power of the pipeline is illustrated using two examples: sheep populations from Morocco with no evident population structure and Lidia cattle from Spain displaying population substructuring. In both cases, R.SamBada enabled rapid identification and interpretation of candidate genes, which are further discussed in the light of local adaptation. The package is available in the r CRAN package repository and on GitHub (github.com/SolangeD/R.SamBada).     

Prenatal nicotine affects catecholamine gene expression in newborn rat carotid body and petrosal ganglion

Nicotine exposure modifiesthe expression of catecholamine and opioid neurotransmitter systemsinvolved in attenuation of hypoxic chemosensitivity. We used insitu hybridization histochemistry to determine the effect of prenataland early postnatal nicotine exposure on tyrosine hydroxylase (TH),dopamine -hydroxylase (DH), preproenkephalin (PPE), andD₂-dopamine receptor mRNA levels in the rat carotid bodyand petrosal ganglion during postnatal development. In the carotidbody, nicotine increased TH mRNA expression in animals at 0 and 3 postnatal days (both, P < 0.05 vs. control) withoutaffecting TH mRNA levels at 6 and 15 days. At 15 postnatal days, DHmRNA levels were increased in the carotid body of nicotine-exposed animals. Dopamine D₂-receptor mRNA levels in the carotidbody increased with postnatal age but were unaffected by nicotineexposure. PPE was not expressed in the carotid body at any of the agesstudied in control or treated animals. In the petrosal ganglion,nicotine increased the number of ganglion cells expressing TH mRNA inanimals at 3 days (P < 0.01 vs. control). DH mRNAexpression was not induced nor was PPE mRNA expression increased in thepetrosal ganglion in treated animals. Prenatal nicotine exposureupregulates mRNAs involved in the synthesis of two inhibitoryneuromodulators, dopamine and norepinephrine, in peripheral arterialchemoreceptors, which may contribute to abnormalities incardiorespiratory control observed in nicotine exposed animals.