Patient experience from a doctor's perspective: A case report concerning treatment, fracture healing and rehabilitation of multiple complex injuries due to a high energy motor vehicle collision.

A 35-year old healthy male trauma surgery chief resident, suffered a high-speed motor vehicle collision. The patient sustained the following injuries: a Gustilo-Anderson grade 2 open comminuted intra-articular fracture of the left distal femur (AO 33C3.3), a Hawkins 1A neck fracture of the right talus (AO 81.2A), an undisplaced Lisfranc injury of the right foot comprising avulsion fractures at the base of the 1st, 2nd and 5th metatarsal as well as the cuboid bone suggesting ligament injury and 2nd to 5th carpometacarpal dislocations of the right (non-dominant) hand with comminuted fractures of the capitate, hamate, trapezoid and the base of the fifth metacarpal bone. A staged-treatment approach ensued. An external fixator (ex-fix) was placed over the left knee, followed by definitive fixation of the distal femoral fracture using a Qwix screw, Non-Contact Bridging (NCB) plate and Locking Compression Plate (LCP). An ex-fix was placed over the right wrist, followed by open reduction and k-wire fixation. The talar fracture of the right foot was treated with a single lag screw and the Lisfranc injury was treated non-operatively with four weeks of non-weight bearing cast immobilization. An intensive clinical rehabilitation program was started, including early use of Continuous Passive Motion (CPM), daily non-weightbearing swimming pool exercises, hand, physical and recreational therapy. One year after the injury the patient was rehabilitated and resumed his surgical residency. Two years after the injury, limited flexion and pain in the left leg remains, possibly related to partial union of the femoral fracture. Range of motion (ROM) of the right ankle and wrist remains limited, not causing significant functional impairment. Lessons learned from a patient experience combined with detailed descriptions of injuries, rehabilitation and long term outcomes can be used as a reference for treating patients with comparable injuries.

Biofabrication of a shape-stable auricular structure for the reconstruction of ear deformities.

Bioengineering of the human auricle remains a significant challenge, where the complex and unique shape, the generation of high-quality neocartilage, and shape preservation are key factors. Future regenerative medicine-based approaches for auricular cartilage reconstruction will benefit from a smart combination of various strategies. Our approach to fabrication of an ear-shaped construct uses hybrid bioprinting techniques, a recently identified progenitor cell population, previously validated biomaterials, and a smart scaffold design. Specifically, we generated a 3D-printed polycaprolactone (PCL) scaffold via fused deposition modeling, photocrosslinked a human auricular cartilage progenitor cell-laden gelatin methacryloyl (gelMA) hydrogel within the scaffold, and cultured the bioengineered structure in vitro in chondrogenic media for 30 days. Our results show that the fabrication process maintains the viability and chondrogenic phenotype of the cells, that the compressive properties of the combined PCL and gelMA hybrid auricular constructs are similar to native auricular cartilage, and that biofabricated hybrid auricular structures exhibit excellent shape fidelity compared with the 3D digital model along with deposition of cartilage-like matrix in both peripheral and central areas of the auricular structure. Our strategy affords an anatomically enhanced auricular structure with appropriate mechanical properties, ensures adequate preservation of the auricular shape during a dynamic in vitro culture period, and enables chondrogenically potent progenitor cells to produce abundant cartilage-like matrix throughout the auricular construct. The combination of smart scaffold design with 3D bioprinting and cartilage progenitor cells holds promise for the development of clinically translatable regenerative medicine strategies for auricular reconstruction.

Hydrogel-based reinforcement of 3D bioprinted constructs.

Progress within the field of biofabrication is hindered by a lack of suitable hydrogel formulations. Here, we present a novel approach based on a hybrid printing technique to create cellularized 3D printed constructs. The hybrid bioprinting strategy combines a reinforcing gel for mechanical support with a bioink to provide a cytocompatible environment. In comparison with thermoplastics such as [Formula: see text]-polycaprolactone, the hydrogel-based reinforcing gel platform enables printing at cell-friendly temperatures, targets the bioprinting of softer tissues and allows for improved control over degradation kinetics. We prepared amphiphilic macromonomers based on poloxamer that form hydrolysable, covalently cross-linked polymer networks. Dissolved at a concentration of 28.6%w/w in water, it functions as reinforcing gel, while a 5%w/w gelatin-methacryloyl based gel is utilized as bioink. This strategy allows for the creation of complex structures, where the bioink provides a cytocompatible environment for encapsulated cells. Cell viability of equine chondrocytes encapsulated within printed constructs remained largely unaffected by the printing process. The versatility of the system is further demonstrated by the ability to tune the stiffness of printed constructs between 138 and 263 kPa, as well as to tailor the degradation kinetics of the reinforcing gel from several weeks up to more than a year.

Increases in myeloperoxidase levels after exercise in myocardial perfusion scintigraphy are not induced by myocardial ischemia.

BACKGROUND: Increased systemic levels of myeloperoxidase (MPO) have been reported in patients with acute myocardial ischemia. We studied the association between exercise-induced myocardial ischemia measured by myocardial perfusion scintigraphy (MPS) and the magnitude and time course of changes in MPO levels in humans. METHODS: One hundred and twenty six patients underwent symptom limited exercise MPS. Myocardial ischemia was assessed semi-quantitatively. Plasma samples were taken before the start of exercise (baseline), at maximum exercise and every hour up to 6 h after maximum exercise. RESULTS: Myocardial ischemia was present in 42 (33%) patients. MPO levels rapidly increased during exercise in patients with and without ischemia (to 131% (106-172%) and 145% (121-199%) of baseline, respectively). MPO levels and absolute changes in MPO did not differ between patients with and without ischemia at any time point. None of the patient characteristics, including presence of ischemia, was independently predictive of the absolute increase in MPO levels during exercise. CONCLUSIONS: Exercise related immediate increases in MPO levels do not reflect myocardial ischemia.

One-pot two-step enzymatic coupling of pyrimidine bases to 2-deoxy-D-ribose-5-phosphate. A new strategy in the synthesis of stable isotope labeled deoxynucleosides.

The enzymatic synthesis of thymidine from 2-deoxy-D-ribose-5-phosphate is achieved, in a one-pot two-step reaction using phosphoribomutase (PRM) and commercially available thymidine phosphorylase (TP). In the first step the sugar-5-phosphate is enzymatically rearranged to alpha-2-deoxy-D-ribose-1-phosphate. Highly active PRM is easily obtained from genetically modified overproducing E. coli cells (12,000 units/84 mg protein) and is used without further purification. In the second step thymine is coupled to the sugar-1-phosphate. The thermodynamically unfavorable equilibrium is shifted to the product by addition of MnCl(2) to precipitate inorganic phosphate. In this way the overall yield of the beta-anomeric pure nucleoside increases from 14 to 60%. In contrast to uracil, cytosine is not accepted by TP as a substrate. Therefore, 2'-deoxy-cytidine is obtained by functional group transformations of the enzymatically prepared 2'-deoxy-uridine. The method has been demonstrated by the synthesis of [2',5'-(13)C(2)]- and [1',2',5'-(13)C(3)]thymidine as well as [1',2',5'-(13)C(3)]2'-deoxyuridine and [3',4'-(13)C(2)]2'-deoxycytidine. In addition the nucleoside bases thymine and uracil are tetralabeled at the (1,3-(15)N(2),2,4-(13)C(2))-atomic positions. All compounds are prepared without any scrambling or dilution of the labeled material and are thus obtained with a very high isotope enrichment (96-99%). In combination with the methods that have been developed earlier it is concluded that each of the (13)C- and (15)N-positions and combination of positions of the pyrimidine deoxynucleosides can be efficiently labeled starting from commercially available and highly (13)C- or (15)N-enriched formaldehyde, acetaldehyde, acetic acid, potassium cyanide, methylamine hydrochloride, and ammonia.

Spt4 modulates Rad26 requirement in transcription-coupled nucleotide excision repair.

The nucleotide excision repair machinery can be targeted preferentially to lesions in transcribed sequences. This mode of DNA repair is referred to as transcription-coupled repair (TCR). In yeast, the Rad26 protein, which is the counterpart of the human Cockayne syndrome B protein, is implicated specifically in TCR. In a yeast strain genetically deprived of global genome repair, a deletion of RAD26 renders cells UV sensitive and displays a defect in TCR. Using a genome-wide mutagenesis approach, we found that deletion of the SPT4 gene suppresses the rad26 defect. We show that suppression by the absence of Spt4 is specific for a rad26 defect and is caused by reactivation of TCR in a Rad26-independent manner. Spt4 is involved in the regulation of transcription elongation. The absence of this regulation leads to transcription that is intrinsically competent for TCR. Our findings suggest that Rad26 acts as an elongation factor rendering transcription TCR competent and that its requirement can be modulated by Spt4.

The derivation of knee joint types from the geometry of the cruciate ligament four-bar system.

The system of the anterior (a) and posterior (p) cruciate ligaments and their distances between attachments to femur (f) and tibia (t) as found in the knee joint of tetrapods is considered as a planar crossed four-bar linkage. The shape of the femoral articulating surfaces (condyles) can be calculated starting from a flat or curved tibial articulating surface and known bar-lengths (Menschik, 1974 Z. Orthop. 112, 481-495; Huson 1974 Orthopäde 3, 119-126). Regression analysis of the dimensions of the cruciate ligament four-bar system of 11 species of mammal and one species of bird revealed a general ratio of (a): (t): (p): (f) = (7.1): (7.9): (10.0): (6.1). These data differ from the results obtained by Badoux (1984 Acta Anat. 119, 60-64) who examined only dog and horse. Our data of the dog agree with those of Badoux, i.e. (a): (t): (p): (f) approximately equal to (10): (8): (10): (4). Based on these ratios between bar-lengths, two types of knee joint shapes were distinguished. The shape of the dog's joint ("type A") has a very large femoral condyle compared with the tibial articulating surface. Maximum knee angulation is 170-180 degrees. Sliding between the articulating surfaces of this joint is distributed approximately uniformly over the whole angulation range. The general shape obtained from the regression analysis ("type R") has a relatively small femoral condyle and an angulation range of about 174 degrees. Uniformly distributed sliding occurs with this range over an angle less than 90 degrees. Theoretically derived, limiting requirements concerning maximum angulation range (delta gamma max < or = 180 degrees), stabilization (e.g. avoidance of a perpendicular position of the cruciate ligaments to the articulating surfaces; delta gamma 78.5 max > or = 90 degrees) and uniformly distributed sliding (delta gamma s > or = 30 degrees) lead to at least two different possible knee joint shapes. These shapes correspond to the two real knee joint shapes found from the statistical analysis mentioned above. This was verified by studying quantitative characteristics obtained from the derivation of knee joint shapes from the bar lengths and vice versa. The bird (Ardea) possessed a knee joint shape, very different from the shapes described above (i.e. f > t, type D1).

Cloning of Schizosaccharomyces pombe rph16+, a gene homologous to the Saccharomyces cerevisiae RAD16 gene.

The RAD16 gene is involved in the nucleotide excision repair of UV damage in the transcriptional silenced mating type loci (Terleth et al., 1990 and Bang et al., 1992) and in non-transcribed stands of active genes in Saccharomyces cerevisiae (Verhage et al., 1994). Using touchdown-PCR with primers derived from various domains of the S. cerevisiae Rad 16 protein, a specific Schizosaccharomyces pombe probe was isolated. This probe was used to obtain the complete RAD16 homologous gene from a S. pombe chromosomal bank. DNA sequence analysis of the rph16+ gene revealed an open reading frame of 854 amino acids. Comparison of the amino acid sequences of the Rhp16 and Rad16 proteins showed a high level of conservation: 68% similarity. The Rhp16 protein sequence contains the two Zn-finger motifs and the putative helicase domains as found in the Rad16 protein. Like the RAD16, the rph16+ gene is UV-inducible (Bang et al., 1995). In analogy with the rad16 mutant, the rhp16 disruption mutant is viable and grows normally, indicating that the gene does not have an essential function. The rhp16 disruption mutant is not sensitive for UV but is sensitive for cisplatin. The rhp16+ gene cloned behind the GAI 1 promoter partially complements the UV sensitivity and the defect in the non-transcribed strand DNA repair of a S. cerevisiae rad16 mutant, indicating functional homology between the rhp16+ and RAD16 genes. The structural and functional homology between the two genes suggests that the RAD16 dependent subpathway of NER for the repair of non-transcribed DNA is evolutionary conserved.

The in vitro more efficiently repaired cisplatin adduct cis-Pt.GG is in vivo a more mutagenic lesion than the relative slowly repaired cis-Pt.GCG adduct.

The toxic effect and the mutagenicity of two differentially repaired site-specific cis-diamminedichloroplatinum(II) (cis-DDP) lesions were investigated. Detailed analysis of the UvrABC-dependent repair of the two lesions in vitro showed a more efficient repair of the cis-Pt.GG adduct compared to that of the cis-Pt.GCG adduct (Visse et al., 1994). Furthermore, previously, a dependency of cis-DDP mutagenesis on UvrA and UvrB, but not on UvrC was found (Brouwer et al., 1988). To possibly relate survival and mutagenesis to repair, plasmids containing the same site-specific cis-DDP lesions as those that were used in the detailed repair studies were transformed into Escherichia coli. The results indicate that both lesions are very efficiently bypassed in vivo. Mutation analysis was performed using a denaturing gradient gel electrophoresis technique, which allows identification of mutations without previous selection. Although the cis-Pt.GG adduct is in vitro more efficiently repaired than the cis-Pt.GCG adduct, it appeared to be more mutagenic. We present a model in which this result is related to the previously observed dependency of the mutagenicity of cis-DDP lesions on the Uvr A and B proteins.

The actual incision determines the efficiency of repair of cisplatin-damaged DNA by the Escherichia coli UvrABC endonuclease.

The UvrABC endonuclease from Escherichia coli repairs a broad spectrum of DNA lesions with variable efficiencies. The effectiveness of repair is influenced by the nature of the lesion, the local DNA sequence, and/or the topology of the DNA. To get a better understanding of the aspects of this multistep repair reaction that determine the effectiveness of repair, we compared the incision efficiencies of linear DNA fragments containing either a site-specific cis-[Pt(NH3)2(d(GpG)-N7(1),-N7(2)]] or a cis- Pt(NH3)2[d(GpCpG)-N7(1),-N7(3)]] adduct. Overall the DNA with the cis-PtGG adduct was incised about 3.5 times more efficiently than the cis-Pt.GCG-containing DNA. The rate of UvrB-DNA preincision complex formation for both lesions was similar and high in relation to the incision. DNase I footprints, however, showed that the local structure of the two preincision complexes is different. An assay was developed to measure the binding of UvrC to the preincision complexes and it was found that the binding rate of UvrC to the more slowly incised cis-Pt.GCG preincision complex was higher than to the cis-Pt.GG preincision complex. This most likely reflects a qualitative difference in preincision complex structures. For both lesions the binding of UvrC to the preincision complex was fast compared to the kinetics of actual incision. Apparently, direct incision of cisplatin damage requires an additional conformational change after the binding of UvrC.

The first zinc-binding domain of UvrA is not essential for UvrABC-mediated DNA excision repair.

Specific mutations in uvrA were introduced to analyze the role of the zinc-binding domains of the protein in DNA excision repair. Zinc-coordinating cysteines were substituted into non-coordinating serine or glycine residues. Mutations leading to changes in the second zinc-binding domain had a profound effect on UV survival in vivo; however these mutant proteins could not be isolated for in vitro analyses. Amino acid substitutions in the first zinc-binding domain had very little effect on UV survival in vivo. In vitro analyses showed that although this domain no longer coordinates zinc, ATPase activity, helicase activity, DNA binding, incision of damaged DNA and DNA repair synthesis appeared to be normal. Therefore it seems that the first zinc-binding domain of UvrA is not essential for DNA excision repair.

Analysis of UvrABC endonuclease reaction intermediates on cisplatin-damaged DNA using mobility shift gel electrophoresis.

One of the least understood steps in the UvrABC mediated excision repair process is the recognition of lesions in the DNA. The isolation of different reaction intermediates is of vital importance for the unraveling of the mechanism. A mobility shift gel electrophoresis assay is described which visualizes such intermediates. After incubation of a DNA substrate containing a specific cisplatin adduct with UvrA alone or with UvrA and UvrB, UvrA.DNA, UvrAB.DNA and UvrB.DNA complexes were observed which could be identified using specific antibodies. At low UvrA concentrations in the presence of UvrB only the UvrB.DNA complex is observed. Bands corresponding to the UvrAB.DNA complex and also other nonspecific bands are found at relatively high UvrA concentrations. The DNase-I footprint for the UvrAB.- and UvrB.DNA complex are very similar and protect about 20 bases. Both complexes are incised in the presence of UvrC with comparable efficiency. The UvrAB.- and the UvrB.DNA complex were both incised at the 8th phosphodiester bond 5' to a specific cisplatin adduct. In addition the UvrAB.DNA complex could also be incised at the 15th phosphodiesterbond 5' to the damaged site. The results suggest that the UvrB.DNA complex is the natural substrate for UvrC-induced incision.

Uvr excision repair protein complex of Escherichia coli binds to the convex side of a cisplatin-induced kink in the DNA.

The Escherichia coli UvrABC endonuclease is capable of initiating the repair of a wide variety of DNA damages. To study the binding of the UvrAB complex to the DNA at the site of a lesion we have constructed a synthetic DNA fragment with a defined cis-diamminedichloroplatinum(II) (cis-Pt).GG adduct. The cis-Pt.GG is the major adduct after treatment of DNA with the antitumor agent cisplatin. Binding to the DNA at the site of the defined lesion was studied with DNase I and MPE.Fe(II) hydroxyl radical footprinting. The results indicate that the UvrAB complex binds to the convex side of the kink in the DNA caused by the cis-Pt.GG adduct. Concerted incisions of the damaged strand by the UvrABC endonuclease were at the 8th phosphodiester bond 5' to and at the 4th bond 3' of the adjacent guanines. An additional incision was found at the 15th phosphodiester bond 5' to the damaged site. This extra incision was stimulated by a high concentration of UvrC.

Identification of the uvrA6 mutation of Escherichia coli.

The uvrA6 mutation has been cloned on a multicopy plasmid by using a chloramphenicol resistance marker introduced next to the uvrA gene in the Escherichia coli chromosome. The mutation was shown to reside in the N-terminal part of the uvrA gene. Sequencing part of this region of the mutant gene revealed a frameshift mutation at positions 207 to 209, which leads to a stop codon at position 262. A marker rescue experiment showed that this frameshift is the only mutation responsible for the UV-sensitive phenotype of the UvrA6 mutant. The method presented is suitable for the cloning of every chromosomal uvrA mutation and can be useful for the study of the functional domains of the UvrA protein.

Radiation-induced changes in the cell membrane of cultured human endothelial cells.

We investigated the effect of irradiation on the kinetic characteristics of amino acid and glucose transport, and the effect on the activity of the cell membrane-bound enzyme 5'-nucleotidase and on the receptor-mediated stimulation of cyclic adenosine monophosphate synthesis by prostaglandin E1. Irradiation inhibited the sodium-dependent amino acid transport by a reduced binding of the amino acid to the transport unit. The transport of glucose, which appeared to be a sodium-independent process, was temporarily stimulated by increased maximal velocity of the transport. No effect was found on the binding to the transport unit. Irradiation increased the 5'-nucleotidase activity and decreased the prostaglandin E1-stimulated cyclic adenosine monophosphate synthesis 48 h after exposure to 20 Gy. It is concluded that irradiation decreases sodium-dependent transport by impairment of the transport unit, does not impair a sodium-independent process, and has opposite effects on membrane-bound enzyme activity and a receptor-mediated process.

Studies on the determination of extracellular galactosyltransferase in human intestinal tissue.

The determination of extracellular galactosyl transferase (EC 2.4.1.38) activity in human intestinal tissue by assessment of the incorporation of label after incubation with UDP[3H]galactose was evaluated. Intestinal biopsy specimens were incubated with membrane-permeable L-[1-14C]fucose and non-permeable UDP-D-[6-3H]galactose (UDP[3H]Gal). Comparison of the amounts of 3H- and 14C-label incorporated into subcellular fractions showed uptake and incorporation of galactose formed by the hydrolysis of UDP[3H]Gal by brush-border enzymes. The results indicate that incorporation of galactose after incubation of the tissue with UDP[3H]Gal is not exclusively attributable to extracellular galactosyl transferase.