Alteration of ganglioside synthesis by GM3 synthase knockout in murine embryonic fibroblasts.

To probe the functions of membrane gangliosides, the availability of ganglioside-depleted cells would be a valuable resource. To attempt to identify a useful genetic model of ganglioside depletion, we assessed ganglioside metabolism in murine GM3 synthase (GM3S)-/- knockout primary embryonic fibroblasts (MEF), because normal fibroblast gangliosides (GM3, GM2, GM1, and GD1a), all downstream products of GM3S, should be absent. We found that heterozygote MEF (GM3S+/-) did have a 36% reduced content of qualitatively normal gangliosides (7.0+/-0.8 nmol LBSA/mg cell protein; control: 11+/-1.6 nmol). However, two unexpected findings characterized the homozygous (GM3-/-) MEF. Despite complete knockout of GM3S, (i) GM3-/- MEF retained substantial ganglioside content (21% of normal or 2.3+/-1.1 nmol) and (ii) these gangliosides were entirely different from those of wild type MEF by HPTLC. Mass spectrometry identified them as GM1b, GalNAc-GM1b, and GD1alpha, containing both N-acetyl and N-glycolylneuraminic acid and diverse ceramide structures. All are products of the 0 pathway of ganglioside synthesis, not normally expressed in fibroblasts. The results suggest that complete, but not partial, inhibition of GM3 synthesis results in robust activation of an alternate pathway that may compensate for the complete absence of the products of GM3S.

Hydrotherapy as a possible neuroleptic and sedative treatment.

Psychotic symptoms such as delusions and hallucinations can have a devastating effect on a patient's social functioning. Since psychosis is rarely congenital, it is possible that lifestyle factors play a role in its etiology. This paper offers a hypothesis that some of these factors could be: (a) A lifestyle lacking evolutionarily conserved stressors such as frequent exposure to heat and/or cold, resulting in a lack of "thermal exercise" which could lead to malfunctioning of the brain. (b) Partial retention and absorption of toxic waste in the colon, as described in more detail below. (c) Genetic makeup that makes a person vulnerable to the above conditions. To test the hypothesis, three types of hydrotherapy are proposed (to be tested separately) as a putative neuroleptic treatment: head-out hot showers, adapted cold showers (twice daily each), and colon hydrotherapy (every 3-12 weeks, which also includes a dietary change according to Harvard's Healthy Eating Pyramid). The following is supporting evidence: Dopaminergic transmission in the mesolimbic pathway is involved in central processing of pain and negative stimuli (e.g. stress-induced analgesia) in addition to its role in the pathophysiology of psychosis. It is also known that if a neural pathway can perform two different functions, then the execution of one function will often suppress the other (e.g. gate control theory of pain). Thus, a pain-based therapy, such as a moderately hot shower, could have a "crowding out" effect on pathological processes within the mesolimbic system. In addition, hyperthermia is known to induce fatigue and depress activity of the frontal cortex (the sedative effect). As described previously, an adapted cold shower could work as a mild electroshock applied to the sensory cortex and, therefore, it might have an antipsychotic effect similar to that of electroconvulsive therapy. Additionally, a cold shower is a vivid example of stress-induced analgesia and would also be expected to "crowd out" or suppress psychosis-related neurotransmission within the mesolimbic system. Human and bacterial toxic waste can sometimes be partially retained in the colon and it is known that many high-molecular-weight compounds can be absorbed there. Most narcotics can cause intoxication if administered rectally and there is also significant comorbidity of schizophrenia with intestinal illnesses. Additionally, there is indirect evidence that colon cleansing can significantly improve mental state. Therefore, it is possible that chronic intoxication with yet unknown components of partially retained waste could be one of the unrecognized organic causes of psychosis.

Adapted cold shower as a potential treatment for depression.

Depression is a debilitating mood disorder that is among the top causes of disability worldwide. It can be characterized by a set of somatic, emotional, and behavioral symptoms, one of which is a high risk of suicide. This work presents a hypothesis that depression may be caused by the convergence of two factors: (A) A lifestyle that lacks certain physiological stressors that have been experienced by primates through millions of years of evolution, such as brief changes in body temperature (e.g. cold swim), and this lack of "thermal exercise" may cause inadequate functioning of the brain. (B) Genetic makeup that predisposes an individual to be affected by the above condition more seriously than other people. To test the hypothesis, an approach to treating depression is proposed that consists of adapted cold showers (20 degrees C, 2-3 min, preceded by a 5-min gradual adaptation to make the procedure less shocking) performed once or twice daily. The proposed duration of treatment is several weeks to several months. The following evidence appears to support the hypothesis: Exposure to cold is known to activate the sympathetic nervous system and increase the blood level of beta-endorphin and noradrenaline and to increase synaptic release of noradrenaline in the brain as well. Additionally, due to the high density of cold receptors in the skin, a cold shower is expected to send an overwhelming amount of electrical impulses from peripheral nerve endings to the brain, which could result in an anti-depressive effect. Practical testing by a statistically insignificant number of people, who did not have sufficient symptoms to be diagnosed with depression, showed that the cold hydrotherapy can relieve depressive symptoms rather effectively. The therapy was also found to have a significant analgesic effect and it does not appear to have noticeable side effects or cause dependence. In conclusion, wider and more rigorous studies would be needed to test the validity of the hypothesis.

Possible use of repeated cold stress for reducing fatigue in chronic fatigue syndrome: a hypothesis.

BACKGROUND: Physiological fatigue can be defined as a reduction in the force output and/or energy-generating capacity of skeletal muscle after exertion, which may manifest itself as an inability to continue exercise or usual activities at the same intensity. A typical example of a fatigue-related disorder is chronic fatigue syndrome (CFS), a disabling condition of unknown etiology and with uncertain therapeutic options. Recent advances in elucidating pathophysiology of this disorder revealed hypofunction of the hypothalamic-pituitary-adrenal axis and that fatigue in CFS patients appears to be associated with reduced motor neurotransmission in the central nervous system (CNS) and to a smaller extent with increased fatigability of skeletal muscle. There is also some limited evidence that CFS patients may have excessive serotonergic activity in the brain and low opioid tone. PRESENTATION OF THE HYPOTHESIS: This work hypothesizes that repeated cold stress may reduce fatigue in CFS because brief exposure to cold may transiently reverse some physiological changes associated with this illness. For example, exposure to cold can activate components of the reticular activating system such as raphe nuclei and locus ceruleus, which can result in activation of behavior and increased capacity of the CNS to recruit motoneurons. Cold stress has also been shown to reduce the level of serotonin in most regions of the brain (except brainstem), which would be consistent with reduced fatigue according to animal models of exercise-related fatigue. Finally, exposure to cold increases metabolic rate and transiently activates the hypothalamic-pituitary-adrenal axis as evidenced by a temporary increase in the plasma levels of adrenocorticotropic hormone, beta-endorphin and a modest increase in cortisol. The increased opioid tone and high metabolic rate could diminish fatigue by reducing muscle pain and accelerating recovery of fatigued muscle, respectively. TESTING THE HYPOTHESIS: To test the hypothesis, a treatment is proposed that consists of adapted cold showers (20 degrees Celsius, 3 minutes, preceded by a 5-minute gradual adaptation to make the procedure more comfortable) used twice daily. IMPLICATIONS OF THE HYPOTHESIS: If testing supports the proposed hypothesis, this could advance our understanding of the mechanisms of fatigue in CFS.

Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously.

A procedure for precise assembly of linear DNA constructs as long as 20 kb is proposed. The method, which we call long multiple fusion, has been used to assemble up to four fragments simultaneously (for a 10.8 kb final product), offering an additional improvement on the combination of long PCR and overlap extension PCR. The method is based on Pfu polymerase mix, which has a proofreading activity. We successfully assembled (and confirmed by sequencing) seven different linear constructs ranging from 3 to 20 kb, including two 20 kb products (from fragments of 11, 1.7 and 7.5 kb), two 10.8 kb constructs, and two constructs of 6.1 and 6.2 kb, respectively. Accuracy of the PCR fusion is greater than or equal to one error per 6.6 kb, which is consistent with the expected error rate of the PCR mix. The method is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as somatic cell knockout in human cells or creation of whole genomes of viruses for vaccine research.

Mathematical model describing erythrocyte sedimentation rate. Implications for blood viscosity changes in traumatic shock and crush syndrome.

BACKGROUND: The erythrocyte sedimentation rate (ESR) is a simple and inexpensive laboratory test, which is widespread in clinical practice, for assessing the inflammatory or acute response. This work addresses the theoretical and experimental investigation of sedimentation a single and multiple particles in homogeneous and heterogeneous (multiphase) medium, as it relates to their internal structure (aggregation of solid or deformed particles). METHODS: The equation system has been solved numerically. To choose finite analogs of derivatives we used the schemes of directional differences. RESULTS: (1) Our model takes into account the influence of the vessel wall on group aggregation of particles in tubes as well as the effects of rotation of particles, the constraint coefficient, and viscosity of a mixture as a function of the volume fraction. (2) This model can describe ESR as a function of the velocity of adhesion of erythrocytes; (3) Determination of the ESR is best conducted at certain time intervals, i.e. in a series of periods not exceeding 5 minutes each; (4) Differential diagnosis of various diseases by means of ESR should be performed using the aforementioned timed measurement of ESR; (5) An increase in blood viscosity during trauma results from an increase in rouleaux formation and the time-course method of ESR will be useful in patients with trauma, in particular, with traumatic shock and crush syndrome. CONCLUSION: The mathematical model created in this study used the most fundamental differential equations that have ever been derived to estimate ESR. It may further our understanding of its complex mechanism.

Blunt trauma to large vessels: a mathematical study.

BACKGROUND: Blunt trauma causes short-term compression of some or all parts of the chest, abdomen or pelvis and changes hemodynamics of the blood. Short-term compression caused by trauma also results in a short-term decrease in the diameter of blood vessels. It has been shown that with a sudden change in the diameter of a tube or in the direction of the flow, the slower-moving fluid near the wall stops or reverses direction, which is known as boundary layer separation (BLS). We hypothesized that a sudden change in the diameter of elastic vessel that results from compression may lead not only to BLS but also to other hemodynamic changes that can damage endothelium. METHODS: We applied Navier-Stokes, multiphase and boundary layer equations to examine such stress. The method of approximation to solve the BL equations was used. Experiments were conducted in an aerodynamic tube, where incident flow velocity and weight of carriage with particles before and after blowing were measured. RESULTS: We found that sudden compression resulting from trauma leads to (1) BLS on the curved surface of the vessel wall; (2) transfer of laminar boundary layer into turbulent boundary layer. Damage to the endothelium can occur if compression is at least 25% and velocity is greater than 2.4 m/s or if compression is at least 10% and velocity is greater than 2.9 m/s. CONCLUSION: Our research may point up new ways of reducing the damage from blunt trauma to large vessels. It has the potential for improvement of safety features of motor vehicles. This work will better our understanding of the precise mechanics and critical variables involved in diagnosis and prevention of blunt trauma to large vessels.

Possible stimulation of anti-tumor immunity using repeated cold stress: a hypothesis.

BACKGROUND: The phenomenon of hormesis, whereby small amounts of seemingly harmful or stressful agents can be beneficial for the health and lifespan of laboratory animals has been reported in literature. In particular, there is accumulating evidence that daily brief cold stress can increase both numbers and activity of peripheral cytotoxic T lymphocytes and natural killer cells, the major effectors of adaptive and innate tumor immunity, respectively. This type of regimen (for 8 days) has been shown to improve survival of mice infected with intracellular parasite Toxoplasma gondii, which would also be consistent with enhanced cell-mediated immunity. PRESENTATION OF THE HYPOTHESIS: This paper hypothesizes that brief cold-water stress repeated daily over many months could enhance anti-tumor immunity and improve survival rate of a non-lymphoid cancer. The possible mechanism of the non-specific stimulation of cellular immunity by repeated cold stress appears to involve transient activation of the sympathetic nervous system, hypothalamic-pituitary-adrenal and hypothalamic-pituitary-thyroid axes, as described in more detail in the text. Daily moderate cold hydrotherapy is known to reduce pain and does not appear to have noticeable adverse effects on normal test subjects, although some studies have shown that it can cause transient arrhythmias in patients with heart problems and can also inhibit humoral immunity. Sudden immersion in ice-cold water can cause transient pulmonary edema and increase permeability of the blood-brain barrier, thereby increasing mortality of neurovirulent infections. TESTING THE HYPOTHESIS: The proposed procedure is an adapted cold swim (5-7 minutes at 20 degrees Celsius, includes gradual adaptation) to be tested on a mouse tumor model. Mortality, tumor size, and measurements of cellular immunity (numbers and activity of peripheral CD8+ T lymphocytes and natural killer cells) of the cold-exposed group would be compared to those of control groups (warm swim and no treatment). Cold-water stress would be administered twice a day for the duration of several months. IMPLICATIONS OF THE HYPOTHESIS: If the hypothesis is supported by empirical studies and the method is shown to be safe, this could lead to the development of an adjunctive immunotherapy for some (non-lymphoid) cancers, including those caused by viral infections.