Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice^1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates⁴.Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient⁵.Herein, we provide additional details of this technique to supplement the video.     

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient’s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.     

Chondrolaryngoplasty for appearance

Prominent thyroid cartilage (pomus Adamus) is frequently a constant embarrassment to the male transsexual as well as to the asthenic male. The demand for reduction and contouring of the pomus Adamus continues to increase in our societies today. Since our first presentation of this procedure, we have been able to follow 31 patients over a 17-year period. We present a review of our technique of chondrolaryngoplasty, with anatomic details and a follow-up of 31 patients ranging from 4 months to 17 years. The results are effective and satisfying, with few complications. Those which do occur tend to be transient, with the most frequent being a temporary mild voice weakness. We believe this operation has a place among the techniques of plastic surgeons.     

FISH diagnostics

For over two decades banding has remained the "gold standard" of cytogenetic analysis, providing the first genome-wide screen for abnormalities. However, conventional cytogenetic banding techniques are limited to the detection of rearrangements involving more than 2 Mb of DNA. In addition,the identification of de novo unbalanced chromosome rearrangements provides a particular challenge for chromosome banding to decipher. In recent years a number of techniques based on FISH have evolved, all of which complement the conventional banding approaches and which have steadily increased the accuracy of cytogenetic diagnosis. FISH is now the method of choice because of the increased sensitivity, and speed with which it can be applied to a variety of cellular targets. In this article we try to highlight the technical aspects of FISH and the practical application of this technique on different tumors (soft tissue tumors, breast carcinomas, renal cell carcinomas, bladder tumors and germ cell tumors).     

Applications of diagonal chromatography for proteome-wide characterization of protein modifications and activity-based analyses

Numerous gel-free proteomics techniques have been reported over the past few years, introducing a move from proteins to peptides as bits of information in qualitative and quantitative proteome studies. Many shotgun proteomics techniques randomly sample thousands of peptides in a qualitative and quantitative manner but overlook the vast majority of protein modifications that are often crucial for proper protein structure and function. Peptide-based proteomic approaches have thus been developed to profile a diverse set of modifications including, but not at all limited, to phosphorylation, glycosylation and ubiquitination. Typical here is that each modification needs a specific, tailor-made analytical procedure. In this minireview, we discuss how one technique - diagonal reverse-phase chromatography - is applied to study two different types of protein modification: protein processing and protein N-glycosylation. Additionally, we discuss an activity-based proteome study in which purine-binding proteins were profiled by diagonal chromatography.     

mRNA on the Move: The Road to Its Biological Destiny

Cells have evolved to regulate the asymmetric distribution of specific mRNA targets to institute spatial and temporal control over gene expression. Over the last few decades, evidence has mounted as to the importance of localization elements in the mRNA sequence and their respective RNA-binding proteins. Live imaging methodologies have shown mechanistic details of this phenomenon. In this minireview, we focus on the advanced biochemical and cell imaging techniques used to tweeze out the finer aspects of mechanisms of mRNA movement.     

A worm's eye view of the immune system: consequences for evolution of human autoimmune disease

Humans and the many parasites that we can host have co-evolved over millions of years. This has been compared to an arms race in which the immune armoury of the human has evolved to deal with potential pathogens and the pathogen has evolved strategies to evade, and in some cases use, the immune system of the human host. Recently, there have been marked changes in the exposure of individuals in the developed world to both microorganisms and metazoan parasites, so the immune stimuli such organisms provide no longer have a role in our lives. As we discuss here, this is a marked perturbation, and the absence of the associated immunomodulation might have led to the increased emergence of autoimmune diseases.     

Starch Granulomatosis of the Peritoneum

Starch glove powder is used extensively by surgeons in Britain and is generally considered innocuous so that precautions to prevent granuloma formation, previously taken when talc glove powder was in use, are now neglected. Reported here are five cases of starch granulomatosis of the peritoneum occurring over a period of a few months. This condition requires reoperation within a limited time for its diagnosis and may be confused macroscopically with disseminated malignant disease or tuberculosis or may simply cause adhesions. Recognition is dependent on a high degree of suspicion by both surgeon and histopathologist, as special histological techniques may be necessary. Consequently, it is only in a minority of the florid cases that a diagnosis is made, and the condition would appear to be much more common than is generally realized. A plea is made for scrupulous care to avoid starch powder contamination of the operative field.     

Epstein-Barr viral BNLF2a protein hijacks the tail-anchored protein insertion machinery to block antigen processing by the transport complex TAP

Virus-infected cells are eliminated by cytotoxic T lymphocytes, which recognize viral epitopes displayed on major histocompatibility complex class I molecules at the cell surface. Herpesviruses have evolved sophisticated strategies to escape this immune surveillance. During the lytic phase of EBV infection, the viral factor BNLF2a interferes with antigen processing by preventing peptide loading of major histocompatibility complex class I molecules. Here we reveal details of the inhibition mechanism of this EBV protein. We demonstrate that BNLF2a acts as a tail-anchored protein, exploiting the mammalian Asna-1/WRB (Get3/Get1) machinery for posttranslational insertion into the endoplasmic reticulum membrane, where it subsequently blocks antigen translocation by the transporter associated with antigen processing (TAP). BNLF2a binds directly to the core TAP complex arresting the ATP-binding cassette transporter in a transport-incompetent conformation. The inhibition mechanism of EBV BNLF2a is distinct and mutually exclusive of other viral TAP inhibitors.     

Male baldness: immediate single-stage rotation of very long arterialized temporo-parieto-occipital flaps

Over a period of 5 years we have performed 62 rotations of long temporo-parieto-occipital flaps in a single stage without delay for both frontal and occipital male baldness. The technique and indications are reported and discussed. The good results obtained permit us to affirm that this surgical technique is safe, simple to perform, and rapidly effective. It has clear advantages over techniques involving delay, including guaranteed survival and vitality of the rotated flap, speedier execution, and few, if not minimal, complications.     

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 1973¹ by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram ² with a further advancement coming from the same laboratory in 1999 ³. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others ^4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.     

True grit: A microwear experiment

Recently we noted the effects of experimental diets on microscopic dental wear in the American opossum and concluded that it might prove difficult to distinguish the microwear produced by an insectivorous diet from that produced by some kinds of herbivorous ones. We also noted that wear caused by gritty diets and those containing plant opal, although they might be confused with one another, are easily distinguished from other sorts of dietary wear. Our conclusions have been challenged on the basis that possibly we did not allow sufficient time in the experiments for diagnostic wear patterns to emerge. Additional data reported here show that this is not so. Even in our n “control” animals, fed a relatively soft unabrasive diet, enough time elapsed to produce significant dental wear. A new technique is described which for the first time allows the study of changing patterns of microscopic wear in a living animal over a period of time, thus allowing each animal to serve as its own control. A solution containing a broad-spectrum proteolytic enzyme when applied to the teeth of an anesthetized animal removes the proteinaceous coat (pellicle) which will otherwise obscure wear scratches. Precision dental impressions can then be made which reveal the details of the pattern of microwear on the teeth.     

Protein-lipid interactions of bacteriophage M13 major coat protein

During the past years, remarkable progress has been made in our understanding of the replication cycle of bacteriophage M13 and the molecular details that enable phage proteins to navigate in the complex environment of the host cell. With new developments in molecular membrane biology in combination with spectroscopic techniques, we are now in a position to ask how phages carry out this delicate process on a molecular level, and what sort of protein-lipid and protein-protein interactions are involved. In this review we will focus on the molecular details of the protein-protein and protein-lipid interactions of the major coat protein (gp8) that may play a role during the infection of Escherichia coli by bacteriophage M13.     

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.     

A nonadaptive scenario explaining the genetic predisposition to obesity: the "predation release" hypothesis

The "thrifty gene hypothesis" suggests we evolved genes for efficient food collection and fat deposition to survive periods of famine and that now that food is continuously available, these genes are disadvantageous because they make us obese in preparation for a famine that never comes. However, famines are relatively infrequent modern phenomena that involve insufficient mortality for thrifty genes to propagate. I suggest here that early hominids would have been subjected to stabilizing selection for body fatness, with obesity selected against by the risk of predation. Around two million years ago predation was removed as a significant factor by the development of social behavior, weapons, and fire. The absence of predation led to a change in the population distribution of body fatness due to random mutations and drift. Because this novel hypothesis involves random drift, rather than directed selection, it explains why, even in Western society, most people are not obese.     

Quantitative measurement of proteins by western blotting with Cy5-coupled secondary antibodies

The concentration of proteins in cells is an important parameter that determines how a protein will interact with other proteins or pharmacological agents. Recent developments in Western blotting techniques have now made this a method of choice to measure protein concentration in complex solutions such as total cell extracts. We show that detection of Cy5-coupled secondary antibodies by PhosphorImager analysis produces signals that approach linearity with respect to protein concentration over a 20-fold range. We used this technique to estimate cellular levels of zyxin, which is an important protein component of the actin cytoskeleton in mammalian cells. By producing specific protein standards based on sequences that are available from public databases, it is now possible to estimate the concentration of almost any protein by this technique.     

Penicillin binding proteins: key players in bacterial cell cycle and drug resistance processes

Bacterial cell division and daughter cell formation are complex mechanisms whose details are orchestrated by at least a dozen different proteins. Penicillin-binding proteins (PBPs), membrane-associated macromolecules which play key roles in the cell wall synthesis process, have been exploited for over 70 years as the targets of the highly successful beta-lactam antibiotics. The increasing incidence of beta-lactam resistant microorganisms, coupled to progress made in genomics, genetics and immunofluorescence microscopy techniques, have encouraged the intensive study of PBPs from a variety of bacterial species. In addition, the recent publication of high-resolution structures of PBPs from pathogenic organisms have shed light on the complex intertwining of drug resistance and cell division processes. In this review, we discuss structural, functional and biological features of such enzymes which, albeit having initially been identified several decades ago, are now being aggressively pursued as highly attractive targets for the development of novel antibiotherapies.     

Serological potential for fungal identification

POLONELLI, L. & MORACE, G., 1989. Serological potential for fungal identification. Specific antigens are valuable for the identification of fungal cultures. Early attempts to immunoidentify fungi were hampered by heterogeneity of antigens, antibody preparations and use of improper serological procedures. In recent years, the double diffusion exoantigcn technique has proved to be the most effective method for immunological identification of mycelial fungus cultures. Additional advances in perfecting methods occurred with the adoption of improved reference antisera obtained either through absorption or by immunizing animals with selected immunoelectrophoretic arcs or precipitin bands (reference antigens). Preliminary studies have shown that serodiagnostically important antigens may be used for accurately and rapidly identifying hyaline as well as dematiaceous fungi. Agglutination techniques consisting oflatex particles sensitized with rabbit anti- Cryptococcus neoformans globulin or Candida monospecific antisera permit the detection of specific yeast antigens in a few minutes. In spite of the great success obtained with the antigen test methods, some limitations in these procedures are apparent. The major problem derives from the occurrence of extensive cross reactions among congeneric species.
Hybridoma technology permits the production of uniform and standardized antisera (monoclonal antibodies) reacting with species-specific or strain-specific antigenic determinants (Western blotting technique) and the availability of functional pure epitopes (affinity chromatography). The current value and limitations as well as further avenues for the advance of the different procedures are reported.     

重组酶聚合酶扩增技术：一种新的核酸扩增策略

重组酶聚合酶扩增 (recombinase polymerase amplification, RPA)是近年来兴起的一种等温核酸扩增技术,它比聚合酶链式反应(polymerase chain reaction, PCR)及其它等温扩增技术更快速、便捷、高效。本文将详细介绍RPA这项新颖的技术,并对其在医疗诊断、农业、食品、生物安全等方面的研究及应用进展进行综述。期望这项技术得到更多的关注,使其发展更加完善,将来在更多的领域充分发挥作用,甚至书写核酸检测历史新篇章。     

Mass spectrometry: m/z 1983-2008

While definitely not a new technology, mass spectrometry (MS) has seen incredible growth over the past 25 years. Mass spectrometry has rapidly evolved to the forefront of analytical techniques; its ability to analyze proteins is the major driving force in the field of proteomics. MS instrumentation has increased approximately 5-fold in sensitivity every three years. The level of performance that is achievable with MS today allows scientists to study proteins in ways that were inconceivable a quarter century ago. This review of the history of MS over the past 25 years is timely in that it encompasses two of the biggest developments, electrospray and matrix-assisted laser desorption/ionization (MALDI), which have enabled many of the uses of this technology today.