Poly(ethylenimine)-mediated transfection: a new paradigm for gene delivery

Poly(ethylenimine) (PEI) is a synthetic polycation that has been used successfully for gene delivery both in vitro and in vivo due, in theory, to a form of protection that is afforded to the carried plasmids. In this study the stability of PEI/DNA complexes was demonstrated using deoxyribonuclease (DNase) 1 and DNase 2, various levels of pH, and increasing exposure times. DNA that was complexed with PEI was not degraded when exposed to at least 25 Units of either enzyme for 24 h while uncomplexed forms of the same plasmid were digested when exposed to 0.010 Units of DNase 1 for 0.05 h or 0. 003 Units of DNase 2 for 1 h. For further comparison, the stability of complexes made with poly(L-lysine) (PLL) and DNA was examined and found to be lower than that of PEI/DNA complexes; PLL-complexed DNA was digested on exposure to 1.25 Units of DNase 1 for 3 min. Cells were transfected with PEI/DNA complexes and, by using a pH indicator and optical recording techniques, it was found that the normal lysosomal pH value of 5.0 was not altered, bringing into question PEI's hypothesized lysosomal entry. Confocal microscopy showed that PEI/DNA complexes and lysosomes do not merge during transfection (although PLL/DNA complexes do). The lack of lysosomal involvement in PEI-mediated transfection is surprising because it goes against the conventional wisdom that has attempted to explain how PEI functions during transfection. PEI forms a stable complex with DNA, which moves from endocytosis to nuclear entry without significant cellular obstacles.     

A new paradigm for human stick balancing: a suspended not an inverted pendulum

We studied 14 skilled subjects balancing a stick (a television antenna, 52 cm, 34 g) on their middle fingertip. Comprehensive three-dimensional analyses revealed that the movement of the finger was 1.75 times that of the stick tip, such that the balanced stick behaved more like a normal noninverted pendulum than the inverted pendulum common to engineering models for stick balancing using motors. The average relation between the torque applied to the stick and its angle of deviation from the vertical was highly linear, consistent with simple harmonic motion. We observed clearly greater rotational movement of the stick in the anteroposterior plane than the mediolateral plane. Despite this magnitude difference, the duration of stick oscillatory cycles was very similar in both planes, again consistent with simple harmonic motion. The control parameter in balancing was the ratio of active torque applied to the stick relative to gravitational torque. It determined both the pivot point and oscillatory cycle period of the pendulum. The pivot point was located at the radius of gyration (about the centre of mass) of the stick from its centre of mass, showing that the subjects attuned to the gravitational dynamics and mass distribution of the stick. Hence, the key to controlling instability here was mastery of the physics of the unstable object. The radius of gyration may—similar to centre of mass—contribute to the kinesthesis of rotating limb segments and control of their gravitational dynamics.     

Pathogen inactivation: a new paradigm for preventing transfusion-transmitted infections

Remarkable improvements have been made in blood safety since the onset of the HIV epidemic. However, the current paradigm does not prevent all transfusion-transmitted infections and is reactive to new agents, thus accepting that some patients may be harmed before preventive measures are introduced. Several methods are now available that selectively damage DNA and RNA, thus inactivating pathogens contaminating blood components while not damaging the cells or plasma proteins of the blood component. Clinical trials have been completed and pathogen-inactivated platelets and plasma are widely used in Europe. A recent consensus conference recommended implementation of pathogen inactivation when a feasible and fe method is available that inactivates a broad spectrum of pathogens. The shortcomings of our present paradigm for preventing transfusion-transmitted diseases are described, along with a summary of the status of pathogen inactivation.     

Genetics of sexual development: a new paradigm

The classical paradigm of human and mammalian sexual development, largely based on work of Alfred Jost, depicts the genetic factor(s) that determine(s) sex as influencing only the fate of the gonad. A maleness factor produces testes (Primary Sex Determination). These organs secrete hormones which cause male Secondary Sexual Differentiation. In absence of the maleness factor, by default the gonad becomes an ovary, and the absence of testicular hormones leads to female secondary differentiation. In this article a new paradigm is proposed, to accommodate recent findings. Sexual dimorphism precedes gonadal development, in a Pregonadal Stage. Furthermore, female development is not by default-both male (Y) and female (X) sex-chromosomal primary sex-determining mechanisms probably exist. The human/mammalian male Y-chromosomal sex-determining gene is now known (SRY/Sry), and a candidate for a non-inactivated, X-linked, female determining factor, is under study. However, the proximate gonad-determining genes are probably on autosomes. Pathways between the primary factors and the proximate gonad-determining genes are indirect and complex. A hypothetical gene Z has been proposed, that inhibits the testis determiner and is itself the target of suppression by SRY/Sry. Candidates for proximate testis and ovary-determining factors and for Z also exist. The "default" concept has also been superseded with respect to secondary sexual differentiation. Absence of testicular hormones does not produce a normal female phenotype; ovarian genes and hormones are necessary. Finally, sex-chromosomal sex-determining genes influence the development not only of non-gonadal organs of secondary sexual development, but also of organs outside of the reproductive system.     

Detection of human papillomavirus in sanitary napkins: a new paradigm in cervical cancer screening

Human papillomavirus was successfully detected by polymerase chain reaction (PCR) in menstrual blood or vaginal discharge collected in sanitary napkins in 100% of 17 women having koilocytosis, cervical intraepithelial neoplasia, or squamous carcinoma. We advocate this form of cervical cancer screening because of its high sensitivity and acceptance by patients.     

Learning to fear suffocation: a new paradigm for interoceptive fear conditioning

The present study aimed to establish a new interoceptive fear conditioning paradigm. The conditioned stimulus (CS) was a flow resistor that slightly obstructs breathing; the unconditional stimulus (US) was a breathing occlusion. The paired group (N = 21) received 6 acquisition trials with paired CS–US presentations. The unpaired group (N = 19) received 6 trials of unpaired CS–US presentations. In the extinction phase, both groups were administered 6 CS‐only trials. Measurements included startle eyeblink response, electrodermal responses, and self‐reported US expectancy. In the paired group, startle blink responses were larger during CS compared to intertrial interval during acquisition and extinction. Electrodermal and US expectancies were larger for the paired than for the unpaired group during acquisition, but not during extinction. The present paradigm successfully established interoceptive fear conditioning with panic‐relevant stimuli.     

The immune tolerance network: a new paradigm for developing tolerance-inducing therapies

Immune tolerance therapies are designed to reprogram immune cells in a highly specific fashion to eliminate pathogenic responses while preserving protective immunity. A concept that has tantalized immunologists for decades, the development of tolerance-inducing therapies, would revolutionize the management of a wide range of chronic and often debilitating diseases by obviating the need for lifelong immunosuppressive regimens. The advances of the past decade have provided a more detailed understanding of the molecular events associated with T-cell recognition and activation. Building on these advances, immunologists have demonstrated the feasibility of various tolerance-inducing approaches in small- and large-animal models of autoimmunity, allergy, and transplant graft rejection. Unprecedented opportunities to test these approaches in a variety of human diseases have now emerged. To capitalize on these advances, the National Institutes of Health recently established the Immune Tolerance Network (ITN), an international consortium of more than 70 basic and clinical immunologists dedicated to the evaluation of novel tolerance-inducing therapies and associated studies of immunologic mechanisms. By using a unique interactive approach to accelerate the development of clinical tolerance therapies, the ITN is partnering with the biotechnology and pharmaceutical industries to examine innovative tolerogenic approaches in a range of allergic and autoimmune diseases and to prevent graft rejection after transplantation. Two years since its inception, the ITN now has approximately 2 dozen clinical trials or tolerance assays studies ongoing or in later stages of protocol development. This report summarizes the rationale for emphasizing clinical research on immune tolerance and highlights the progress of the ITN.     

Drug development strategies for asthma: in search of a new paradigm

The spiraling costs of asthma treatment seem set to continue rising, given the equivocal performance of the latest generation of specific anti-inflammatory drugs in trials in adult asthmatics. We argue that the continuation of this trend is inevitable unless there is a substantial realignment of entrenched drug development policy in the pharmaceutical industry and a parallel shift in licensing policy by regulatory authorities to encourage the development of drugs capable of halting the progression from acute to chronic asthma when the disease first manifests in childhood. The theoretical framework for such an approach, including proof-of-principle data from studies in children with early-stage disease and a range of candidate drugs, already exists. What is needed is informed debate on the risks versus potential benefits of this approach.     

Carbohydrate mimicry: a new paradigm of autoimmune diseases

Molecular mimicry of microbial components by self components is thought to be the mechanism that accounts for the antigen and tissue specificity of immune responses in post-infectious autoimmune diseases. Little direct evidence exists, and research in this area has focused principally on T cell mediated anti-peptide responses, rather than on humoral responses to carbohydrate structures. Guillain-Barré syndrome, the most frequent cause of acute neuromuscular paralysis, sometimes occurs after Campylobacter jejuni enteritis. Recent studies have revealed that carbohydrate mimicry of the bacterial lipo-oligosaccharide by the human ganglioside is an important cause of the syndrome. This new concept that carbohydrate mimicry can cause an autoimmune disease provides a clue to inducing the resolution of pathogenesis of other immune-mediated diseases.     

Human body frequency modulation by 0.9% sodium chloride solutions: a new paradigm and perspective for human health

This case study demonstrates that the normal human body frequency, which can be disturbed by electromagnetic influences of the environment, can be modulated by 0.9% sodium chloride solutions (physiological saline) and that occurrence of allergic reactions have subsequently been suppressed as a result of this modulation. The use of distilled water as control showed no effect on occurrence of allergic reactions. Further observations on the growth of various plants in a greenhouse exposed to various geomagnetic fields support the previous observations on humans. The neutralization of electromagnetic influences on humans using 0.9% sodium chloride solution or by enclosure of plants within a copper wire Faraday cage resulting in a normal and uniform growth of plants as compared with disturbed and irregular growth in unenclosed controls, is demonstrated. These original observations propose a new strategy to suppress or prevent allergic reactions and possibly other effects observed in various human pathologies in relation to a disturbance of human body frequencies. It is hypothesized that the double helix structure of desoxyribonucleic acid (DNA) could be modified by environmental electromagnetic fields and that disresonance between the two chains of DNA could lead to the expression of specific pathology.