Modeling a hydropic recipient twin in twin-twin transfusion syndrome

We developed a mathematical model of twin-twin transfusion syndrome (TTTS) that includes a hydropic recipient twin, adding interstitial and intracellular fluid compartments, fetal congestive cardiac failure, and the dynamics of renin-angiotensin system (RAS) mediators to our previous TTTS model. Ten differential equations for each twin, coupled by the net fetofetal transfusion of blood and blood components, i.e., colloids, osmoles, and RAS mediators, describe the development of fetal arterial and venous blood volumes, blood osmolality and colloid osmotic pressure (COP), interstitial fluid volume and COP, intracellular fluid volume, amniotic fluid volume and osmolality, and RAS mediator concentration. We included varying placental anastomoses, placental sharing, and amnionicity. The 20 differential equations were solved numerically from 0 to 40 wk with a 0.6-s time step. Consistent with clinical experience, model predictions are as follows. Unidirectional arteriovenous anastomoses and arteriovenous anastomoses inadequately compensated by oppositely directed anastomoses cause severe TTTS that includes a hydropic recipient. Adequately compensated arteriovenous anastomoses simulated TTTS without hydrops. The probability that oppositely directed anastomoses prevent onset of a hydropic recipient after TTTS onset, i.e., the largest interval between onset of TTTS and onset of hydrops in the recipient, was best for a venovenous anastomosis, closely followed by an arterioarterial and finally an oppositely directed arteriovenous anastomosis. Hydropic recipients have decreased amniotic fluid volume. Unequal placental sharing and amnionicity modify hydrops onset. In conclusion, our model simulates a sequence of events that results in a hydropic recipient twin in severe TTTS. The model may allow an assessment of the efficacy of current therapeutic interventions for TTTS cases that include a hydropic recipient twin.     

Acellularization-Induced Changes in Tensile Properties Are Organ Specific - An In-Vitro Mechanical and Structural Analysis of Porcine Soft Tissues

Introduction

Though xenogeneic acellular scaffolds are frequently used for surgical reconstruction, knowledge of their mechanical properties is lacking. This study compared the mechanical, histological and ultrastructural properties of various native and acellular specimens.

Materials and Methods

Porcine esophagi, ureters and skin were tested mechanically in a native or acellular condition, focusing on the elastic modulus, ultimate tensile stress and maximum strain. The testing protocol for soft tissues was standardized, including the adaption of the tissue’s water content and partial plastination to minimize material slippage as well as templates for normed sample dimensions and precise cross-section measurements. The native and acellular tissues were compared at the microscopic and ultrastructural level with a focus on type I collagens.

Results

Increased elastic modulus and ultimate tensile stress values were quantified in acellular esophagi and ureters compared to the native condition. In contrast, these values were strongly decreased in the skin after acellularization. Acellularization-related decreases in maximum strain were found in all tissues. Type I collagens were well-preserved in these samples; however, clotting and a loss of cross-linking type I collagens was observed ultrastructurally. Elastins and fibronectins were preserved in the esophagi and ureters. A loss of the epidermal layer and decreased fibronectin content was present in the skin.

Discussion

Acellularization induces changes in the tensile properties of soft tissues. Some of these changes appear to be organ specific. Loss of cross-linking type I collagen may indicate increased mechanical strength due to decreasing transverse forces acting upon the scaffolds, whereas fibronectin loss may be related to decreased load-bearing capacity. Potentially, the alterations in tissue mechanics are linked to organ function and to the interplay of cells and the extracellular matrix, which is different in hollow organs when compared to skin.     

Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl:adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterases.

We have deduced the nucleotide sequence of the genes encoding the three components of 4-chlorobenzoate (4-CBA) dehalogenase from Pseudomonas sp. CBS-3 and examined the origin of these proteins by homology analysis. Open reading frame 1 (ORF1) encodes a 30-kDa 4-CBA-coenzyme A dehalogenase related to enoyl-coenzyme A hydratases functioning in fatty acid beta-oxidation. ORF2 encodes a 57-kDa protein which activates 4-CBA by acyl adenylation/thioesterification. This 4-CBA:coenzyme A ligase shares significant sequence similarity with a large group of proteins, many of which catalyze similar chemistry in beta-oxidation pathways or in siderophore and antibiotic synthetic pathways. These proteins have in common a short stretch of sequence, (T,S)(S,G)G(T,S)(T,E)G(L,X)PK(G,-), which is particularly highly conserved and which may represent an important new class of "signature" sequence. We were unable to find any proteins homologous in sequence to the 16-kDa 4-hydroxybenzoate-coenzyme A thioesterase encoded by ORF3. Analysis of the chemistry and function of the proteins found to be structurally related to the 4-CBA:coenzyme A ligase and the 4-CBA-coenzyme A dehalogenase supports the proposal that they evolved from a beta-oxidation pathway.     

Design of a targeted peptide nucleic acid prodrug to inhibit hepatic human microsomal triglyceride transfer protein expression in hepatocytes

In this study, we present the design and synthesis of an antisense peptide nucleic acid (asPNA) prodrug, which displays an improved biodistribution profile and an equally improved capacity to reduce the levels of target mRNA. The prodrug, K(GalNAc)(2)-asPNA, comprised of a 14-mer sequence complementary to the human microsomal triglyceride transfer protein (huMTP) gene, conjugated to a high-affinity tag for the hepatic asialoglycoprotein receptor (K(GalNAc)(2)). The prodrug was avidly bound and rapidly internalized by HepG2s. After iv injection into mice, K(GalNAc)(2)-asPNA accumulated in the parenchymal liver cells to a much greater extent than nonconjugated PNA (46% +/- 1% vs 3.1% +/- 0.5% of the injected dose, respectively). The prodrug was able to reduce MTP mRNA levels in HepG2 cells by 35-40% (P < 0.02) at 100 nM in an asialoglycoprotein receptor- and sequence-dependent fashion. In conclusion, hepatocyte-targeted PNA prodrugs combine a greatly improved tropism with an enhanced local intracellular availability and activity, making them attractive therapeutics to lower the expression level of hepatic target genes such as MTP.     

Reported infections after human tissue transplantation before and after new food and drug administration (FDA) regulations, United States, 2001 through June, 2010

Processors distributed about 1.5?million human tissue allografts in the U.S. in 2007. The potential for transmitting infections through allografts concerns clinicians and patients. In 2005, FDA implemented Current Good Tissue Practice (CGTP) rules requiring tissue establishments to report to FDA certain serious infections after allograft transplantations. We describe infection reports following tissue transplants received by FDA from 2005 through June, 2010, and compare reporting before and after implementation of CGTP rules. We identified reports received by FDA from January 2001 through June, 2010, for infections in human tissue recipients, examining the reports by tissue type, organism, time to onset, severity, and reporter characteristics. Among 562 reports, 83 (20.8/year) were received from 2001?C2004, before the CGTP rules, 43 in the 2005 transition year, and 436 (96.9/year) from 2006 through June, 2010, after the rules. Tissue processors accounted for 84.2% of reports submitted after the rules, compared to 26.5% previously. Bacterial infections were the most commonly reported organisms before (64.6%) and after (62.2%) the new rules. Afterward, 2.5% (11) of reports described deaths, and 33.7% (147) involved hospitalizations. Before the rules, 13% (11) described deaths, and another 72% involved hospitalizations. Reports received by the FDA quadrupled since 2005, suggesting that CGTP regulations have contributed to increased reporting and improved tissue safety surveillance. However, these data do not confirm that the reported infections were caused by suspect tissues; most reports may represent routine post-surgical infections not actually due to allografts.     

Substance P analogues function as bombesin receptor antagonists and inhibit small cell lung cancer clonal growth

Human small cell lung cancer (SCLC) produces and secretes BN/GRP (bombesin/gastrin releasing peptide). Because BN stimulates the growth of SCLC cells and these cells have receptors for BN-like peptides, it is important to define agents which disrupt this self-promoting autocrine growth cycle. Here, substance P analogues were evaluated as BN receptor antagonists using SCLC cell lines. (D-Arg¹, D-Pro², D-Trp^7,9, Leu¹¹) substance P [(APTTL)SP] was one of the more potent analogues tested in inhibiting BN-like peptide receptor binding with an IC₅₀ value of 1 μM. Micromolar concentrations of (APTTL)SP antagonized BN receptor mediated elevation of cytosolic Ca²⁺ levels and decreased the colony formation in soft agarose. These data suggest that SP analogues function as SCLC BN receptor antagonists and may be useful in disrupting the autocrine growth function of BN-like peptides.     

Hensen's node,but not other biological signallers,can induce supernumerary digits in the developing chick limb bud

Summary The purpose of this study was to determine whether the organizer regions of early avian and amphibian embryos could induce supernumerary (SN) wing structures to develop when they were grafted to a slit in the anterior side of stage 19–23 chick wing buds. Supernumerary digits developed in 43% of the wings that received anterior grafts of Hensen's node from stage 4–6 quail or chick embryos; in addition, 16% of the wings had rods of SN cartilage, but not recognizable SN digits. The grafted quail tissue did not contribute to the SN structures. When tissue anterior or lateral to Hensen's node or lateral pieces of the area pellucida caudal to Hensen's node were grafted to anterior slits, the wings usually developed normally. No SN structures developed when Hensen's nodes were grafted to posterior slits in chick wing buds. Wings developed normally when pieces of the dorsal lip of the blastopore from stage 10–11.5 frog (Xenopus laevis and Rana pipiens) embryos were grafted to anterior slits. No SN digits developed when other tissues that have limb-inducing activity in adult urodele amphibians [chick otic vesicle, frog (Rana pipiens) lung and kidney] or that can act as heteroinductors in neural induction (rat kidney, lung, submaxillary gland and urinary bladder; mouse liver and submaxillary gland) were grafted to anterior slits in chick wing buds. SN digits also failed to develop following preaxial grafts of chick optic vesicles. These results suggest that although the anteroposterior polarity of the chick wing bud can be influenced by factors other than the ZPA (e.g., Hensen's node, retinoids), the wing is not so labile that it can respond to a wide variety of inductively-active tissues.     

Caspase inhibition blocks cell death and results in cell cycle arrest in cytokine-deprived hematopoietic cells

Cytokine deprivation has been classically used to study molecular processes of apoptosis. Following interleukin (IL)-3 withdrawal in FL5.12 cells, Bax undergoes a conformational change that results in its mitochondria targeting, cytochrome c release, activation of caspase-9, and apoptosis. Cells overexpressing Casp9DN (dominant negative caspase-9) or treated with the caspase inhibitor Q-VD-OPh increased viability but failed to increase clonogenic survival. We find that caspase-inhibited cells had a significant fraction of viable cells (herein termed "rescued" cells) that failed to initiate cell division after IL-3 add back. The "rescued" cells had reduced mitochondrial potential, stained for active Bax, and had reduced staining with dihydroethidium, an agent sensitive to superoxide levels. Readdition of IL-3 after deprivation demonstrated that Bax activation was reversed, whereas altered 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide and dihydroethidium staining persisted for days. Furthermore, the "rescued" cells were resistant to rotenone, an inhibitor of mitochondrial respiration. The cells were highly sensitive to 2-deoxyglucose, an inhibitor of glycolysis and proposed anti-cancer agent. We conclude that the inhibition of caspase-9 allows cells to retain viability, but cells have prolonged mitochondrial dysfunction and enter a unique nondividing state that shares some properties with malignant cells.     

Chromosome 17 abnormalities and lack of TP53 mutations in paediatric central nervous system tumours

Central nervous system (CNS) tumours are the most common solid tumours in children. Cytogenetic and molecular genetic studies of these neoplasms have previously shown abnormalities of chromosome 17, implicating genes on this autosome in tumorigenesis. To identify mutations in the TP53 tumour suppressor gene (17p13.1), we have sequenced the five highly conserved regions of this gene in 29 mixed paediatric CNS tumors. No mutations were detected by this analysis. In order to identify other candidate disease loci on chromosome 17, we have carried out a detailed deletion mapping analysis using 16 polymorphic DNA markers on 19 of the above tumours and an additional four cases. Abnormalities of chromosome 17 occurred in nine cases (39%), six of which were primitive neuroectodermal tumour (PNET)-medulloblastomas. These findings suggest that it is unlikely that the TP53 gene is directly involved in the development of common paediatric brain tumours. This is in contrast to findings from adult brain and other tumour types. Moreover, the frequency of chromosome 17 aberrations, especially in PNET-medulloblastomas, suggests that other genes on this chromosome contribute to tumourigenesis.     

Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI=3.5) and a basic variant (pI=10.2)

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 degrees C in 0.1M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge-dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds.A primary objective in this study was to investigate the influence of charge-charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the alpha-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge-dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge-charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pK(Sa)-pK(5K)) calculated using a simple model based on Coulomb's Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulomb's Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson-Boltzmann equation, which considers desolvation and charge-dipole interactions in addition to charge-charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge-charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.