Self-metalation of 2H-tetraphenylporphyrin on Cu(111): an x-ray spectroscopy study.

The bonding and the temperature-driven metalation of 2H-tetraphenylporphyrin (2H-TPP) on the Cu(111) surface under ultrahigh vacuum conditions were investigated by a combination of x-ray photoelectron spectroscopy (XPS) and near-edge x-ray absorption fine structure (NEXAFS) spectroscopy with density functional theory calculations. Thin films were prepared by organic molecular beam epitaxy and subsequent annealing. Our systematic study provides an understanding of the changes of the spectroscopic signature during adsorption and metalation. Specifically, we achieved a detailed peak assignment of the 2H-TPP multilayer data of the C1s and the N1s region. After annealing to 420 K both XPS and NEXAFS show the signatures of a metalloporphyrin, which indicates self-metalation at the porphyrin-substrate interface, resulting in Cu-TPP. Furthermore, for 2H-TPP monolayer samples we show how the strong influence of the copper surface is reflected in the spectroscopic signatures. Adsorption results in a strongly deformed macrocycle and a quenching of the first NEXAFS resonance in the nitrogen edge suggesting electron transfer into the LUMO. For Cu-TPP the spectroscopic data indicate a reduced interaction of first-layer molecules with the substrate as demonstrated by the relaxed macrocycle geometry.

Temperature dependence of conformation, chemical state, and metal-directed assembly of tetrapyridyl-porphyrin on Cu(111).

We present a combined scanning tunneling microscopy (STM), near-edge x-ray-absorption fine-structure, and x-ray photoemission spectroscopy (XPS) study on the bonding and ordering of tetrapyridyl-porphyrin molecules on the Cu(111) surface in the 300-500 K temperature range. Following deposition at 300 K the molecules are adsorbed with a pronounced conformational adaptation of the anchored species featuring a saddle-shaped macrocycle and terminal groups pointing toward the substrate. Upon moderate annealing supramolecular chains evolve that are stabilized by metal-ligand interactions between the mesopyridyl substituents and copper adatoms resulting in twofold copper coordination. Annealing to temperatures exceeding 450 K strongly alters the molecular appearance in high-resolution STM data. This modification was also induced by controlled voltage pulses and related to a deprotonation of the molecule by XPS. Under appropriate conditions a novel binding motif leads to honeycomb structures coexisting with the chain segments. The conformation withstands annealing without large modification.

The chemical class of quinazoline compounds provides a core structure for the design of anticytomegaloviral kinase inhibitors.

HCMV is a member of the family Herpesviridae and represents a worldwide distributed pathogen with seropositivity rates in the adult population ranging between 40% and 90%. Notably, HCMV infection is a serious, sometimes life-threatening medical problem for newborns and immunosuppressed individuals, including transplant recipients and patients under antitumoral chemotherapy. Current standard therapy with valganciclovir has the disadvantage of inducing drug-resistant virus mutants and toxicity-related side effects. Our analysis stresses the earlier finding that kinase inhibitors of the quinazoline class exert an antiviral response by targeting the viral protein kinase pUL97 without inducing resistance. Therefore, quinazolines have been used as a core structure to gain insight in the mode of inhibitor-kinase interaction. Here, we demonstrate that (i) the novel quinazolines Vi7392 and Vi7453 are highly active against HCMV laboratory and clinically relevant strains including maribavir- and ganciclovir-resistant variants, (ii) antiviral activity is not cell-type specific and was also detected in a placental explant tissue model using a genetically intact HCMV strain (iii) the viral kinase pUL97 represents a target of the anticytomegaloviral activity of these compounds, (iv) induction of pUL97-conferring drug resistance was not detectable under single-step selection, thus differed from the induction of ganciclovir resistance, and (v) pUL97 drug docking simulations enabled detailed insights into specific drug-target binding properties providing a promising basis for the design of optimized kinase inhibitors. These novel findings may open new prospects for the future medical use of quinazoline drug candidates and the use of drug-target dynamic simulations for rational design of antivirals.

Hepatitis B virus surface antigen as a reporter of promoter activity.

The coding sequence for the hepatitis B virus surface antigen (HBsAg) was used as a new reporter gene for studies on eukaryotic promoter activity and upstream regulatory sequences. The data observed in transfection assays were comparable to results obtained with conventional chloramphenicol acetyltransferase (CAT) assays, as was demonstrated using various transcriptional regulation sequences. The expression of HBsAg as a reporter protein offered some advantages: (i) In transient expression assays, a time course of promoter activity depending on variable culture conditions could be monitored over a period of time, since the HBsAg was secreted into the culture supernatant. (ii) Evaluation of HBsAg from supernatant aliquots and quantification of the corresponding promoter activities could be performed easily, using the very sensitive and readily available diagnostic HBsAg kits. (iii) In contrast to the conventional CAT assay, the cells remained available for further tests, e.g., Western blot, immunofluorescence or transcript analysis. Characteristics of several Epstein-Barr virus (EBV) promoters, depending on the virus state of EBV-positive B-cells (latency, chemical induction, lytic superinfection, trans-activation), were assayed using the HBsAg reporter system.

The persistent variant of influenza C virus carries one characteristic point mutation in RNA segment 1.

Influenza C/ Ann Arbor/1/50-pi(C/AA-pi) virus causes persistent infections in MDCK and Wi38 cells, but sets limited, wild-type like infections in other cells. Concluding that persistence itself it dependent on the host environment, we determined the nucleotide sequence of the C/AA-pi analogous gene to the basic polymerase 2 (PB2) of influenza A virus, which is known to be a determinant for the host range. C/AA-pi and the parental wild-type virus (C/AA-wt) have 16 nucleotides in common that are different to a previously published PB2 sequence (C/JJ/50). These variations, which are probably due to divergent passages histories, are scattered along the sequence and are partially found in another published isolate (C/Berlin/1/85). One single mutation, however, is unique to the persistent variant. Nucleotide 28 mutated from T to C which leads to a change of amino acid 3 from Leu to Phe. This substitution is stably associated with the persistent phenotype throughout multiple passages in different cells lines and eggs and cannot be found in any other influenza C viruses.

A persistent variant of influenza C virus fails to interact with actin filaments during viral assembly.

C/AA-pi virus, a variant of influenza C/Ann Arbor/1/50 virus, establishes persistent infections in MDCK cells, characterized by low levels of progeny production. During viral assembly, nucleoprotein (NP) was found homogeneously distributed over cytoplasmic and nuclear compartments and matrix (M) protein was likewise localized in a barely structured fashion. In contrast, infections with nonpersistent influenza A, B and C viruses produced cytoplasmic granular structures, which typically consisted of colocalized NP and M proteins. Studies on the in vitro interaction between NP and M proteins revealed identical binding capacities comparing influenza C wild-type virus with the persistent variant. Cytochalasin D treatment of infected cells demonstrated that NP protein of the wild-type virus, but not of the persistent variant, was distinctly associated with cellular actin filaments. Moreover, the assembly characteristics of wild-type virus were modulated in the presence of recombinant persistent-type NP protein towards a behaviour similar to persistent infection. Cell type specificity was particularly illustrated in C/AA-pi virus-infected Vero cells, which did not support viral persistence, but produced granular wild-type-like complexes. Thus, interaction between NP, M and actin proteins (i) is a basic part of the viral assembly process, (ii) is dominantly modulated by NP protein and (iii) is specifically altered in the case of persistent infection.

Persistent infection with an influenza C virus variant is dominantly established in the presence of the parental wild-type virus.

Two influenza C viruses were used for double-infection experiments to investigate the dominance of their phenotypes. The wild-type virus (C/AA-wt) had been characterized by its short-lived productive cycle, whereas a distinct variant derived from it (C/AA-pi) was demonstrated to persist in long-term passages of infected MDCK cultures. Here we show that the persistent virus C/AA-pi is capable of replicating in the presence of abundant amounts of wild-type virus: the persistent virus could be diluted to 10(-9) within wild-type inoculum, still developing a stable form of persistence. This behaviour was reflected by permanent virus release and by continuous enzymatic activity of the viral HEF glycoprotein in infected cells. All long-term cultures tested remained positive for viral NS protein and vRNA. On the vRNA level, it was shown that viral segments originated from the persistent-type genome, while wild-type vRNAs were not maintained after double-infection. Thus, the genotype of the persistent variant was dominantly selected in serial passages. These results indicate a specific intracellular advantage of persistent influenza C virus over the parental wild-type.

Inhibition of influenza C viruses by human MxA protein.

Human MxA protein was analyzed for its ability to inhibit the replication of different influenza C viruses. Three laboratory derivatives of viral strain C/Ann Arbor/1/50 were investigated, namely the parental wild-type virus C/AA-wt, the persistent variant C/AA-pi and the highly cytopathogenic variant C/AA-cyt. In addition, strain C/Paris/214/91 isolated from an influenza patient was used. Multiplication of all four viruses was suppressed in MxA-expressing Vero cells, as indicated by a decrease in viral RNA synthesis, viral protein synthesis, virion production and induction of a cytopathic effect. Inhibition correlated with the level of MxA expression. Furthermore, inhibition was independent of cell clone-specific differences in expression of virus receptors, as demonstrated by receptor reconstitution experiments. Thus, human MxA protein has antiviral activity against influenza C viruses.

Repair of right ventricular free wall defect with a pedicled muscle flap.

A unique case of repair of a full-thickness cardiac defect and simultaneous reconstruction of an infected median sternotomy wound is presented. A right ventricular defect, 6 cm in diameter, was closed with a fascia lata graft and reinforced with a rectus abdominis muscle flap. The superior portion of the mediastinum was obliterated with a pectoralis major muscle flap. The patient tolerated the procedure well and remains free of cardiac symptoms seven months postoperatively, with no evidence of residual infection.

Nucleotide-specific PCR for molecular virus typing.

Nucleotide sequence studies detected a double-point mutation in the genomic RNA segment 4 (nt 871 and 872) of the persistent variant C/AA-pi of influenza C/Ann Arbor/1/50 virus. The 3'-end-points of two distinct PCR primers were positioned exactly at this genome location and thereby adjusted the priming determinant complementary to the varied strain or to its wild-type counterpart. Consequently, positive RT-PCR products strictly referred to one of the two viruses examined, in both cases, using either virion or infected-cell RNA templates. Artificial virus mixtures could easily be distinguished by this method in a subsequent qualitative gel analysis. PCR annealing conditions and control reactions were optimized, for the monitoring of influenza virus isolates throughout multifold passages. Thus, sequence diversity in just two neighbouring nucleotides is sufficient to determine whether or not successful PCR amplification takes place, and this method can be used as a reliable means of virus strain differentiation.

Postoperative mydriasis after repair of orbital floor fracture.

Mydriasis after operative repair of orbital floor fracture has been attributed to manipulation of the inferior oblique muscle. We treated two patients with mydriasis, one with an isolated mydriatic pupil and the other with a tonic pupil, which followed posterior orbital floor injuries and repair. The posterior location of the fractures suggests that surgical manipulation of or near the ciliary ganglion may account for these phenomena. Patients should be warned before posterior orbital floor repair about possible mydriatic or tonic pupils as a complication.

Lower limb salvage by microvascular free-tissue transfer in patients with homozygous sickle cell disease.

Chronic, excruciatingly painful ulcerations of the lower extremities in patients with homozygous sickle cell anemia (HbSS) present a frustrating clinical problem for the reconstructive surgeon. Despite adequate wound care and skin grafting, there is a dismally high incidence of recurrence. Furthermore, there is a paucity of reliable locoregional fasciocutaneous, muscle, and myocutaneous flaps in the ankle region. Free-tissue transfer has become the procedure of choice for reconstruction of the lower third of the leg. However, in sickle cell anemia, does the obligate period of flap ischemia inherent in free-tissue transfer inevitably doom a flap to failure? We present our multidisciplinary experience over 55 months with five free flaps in four homozygous sickle cell anemia patients 21 to 38 years old who had chronic nonhealing leg ulcerations. Special perioperative measures included exchange transfusion to lower hemoglobin S to below 30 percent, maintaining the hematocrit at 31 to 35 percent, intraoperative flap washout and perfusion with warm heparinized saline-dextran solution, administration of dextran and aspirin intraoperatively and postoperatively, prophylactic topical and systemic anti-Pseudomonas antibiotics, supplemental oxygen, and warm ambient room temperature. Flaps included the latissimus dorsi muscle (two patients), the temporoparietal fascia (one patient), and "split" omentum for bilateral lower limb salvage (one patient). Successful free-tissue transfer was accomplished in all patients. One patient suffered gradual partial occlusion of the microcirculation by sickled erythrocytes following a transient hypothermic, hypotensive episode. Sufficient flap tissue survived to permit skin grafting with an excellent result. Pseudomonas infection occurred in two patients (three flaps).(ABSTRACT TRUNCATED AT 250 WORDS)

Early, aggressive management of postoperative oropharyngocutaneous fistulas.

Oropharyngocutaneous fistulas remain a serious and potentially lethal complication. Advantages from surgical repair and the use of musculocutaneous flaps have been demonstrated. Timing of the procedure, however, has not been adequately addressed or emphasized. This report presents our experience with early, aggressive management of postoperative orocutaneous fistulas. Patients were reoperated at an average of 12 days after the initial surgery and underwent exploration, debridement of all devitalized tissues, and closure by reelevation of previously used flaps or with additional flaps. All wounds healed without further problem. We conclude that as long as the patient's general condition permits, early, aggressive management of fistulas should be the procedure of choice to reduce hospital stay and costly wound care and to avoid maceration and partial or complete necrosis of flaps and the potential rupture of the carotid artery. Timely radiotherapy can then be delivered, and quality of life can be significantly improved.

Chronic wounds and delusions of parasitosis in the drug abuser.

Plastic surgeons are routinely consulted about the care of difficult or chronic wounds. Most of these wounds have a physiologic basis and are treated according to basic wound-healing principles. Patients with factitious wounds are difficult to treat and present diagnostic dilemmas. We present a case and review of the literature of drug-induced delusions of parasitosis to inform plastic surgeons of this important entity and for consideration in the differential diagnosis of chronic wounds.

The role of tissue expansion in the reconstruction of secondary burn defects.

Tissue expansion has become firmly established as an acceptable and useful modality in the treatment of various soft tissue defects. This article relates our experience with this technique for the reconstruction of secondary burn deformities in 26 patients. Details of expander placement and use are discussed, as well as the results and complications in this series. The advantages of this method, including a superior quality reconstruction, limited donor site morbidity, and cost-effectiveness, are discussed. The disadvantages of several operative steps, multiple office visits, and the objectionable appearance during expansion are enumerated. Guidelines for patient selection and technique refinements are also considered to reduce complications and to achieve an optimal functional and aesthetic result.

Experimental infection with a persistent influenza C virus variant leads to prolonged genome detection in the chicken lung.

A persistent variant of influenza C virus was used to infect chickens by intraamniotic (i.a.) inoculation. The infected hatchings were analyzed for virus production in different tissues and for the continuous presence of viral RNA genomes. The permissiveness for infection was demonstrated primarily for the chicken lung, besides other organs. Viral antigens could be detected by indirect immunofluorescence staining for a period of 8 days and reisolates were obtained mainly at early time points post infection (p.i.). Nested reverse transcription-polymerase chain reaction (RT-PCR) directed to 3 genomic sequences was positive at least until day 53, whereby no distinct end point was determined. These experiments provide first evidence for the long-term stability of influenza C virus RNA segments in vivo.