Predicting Protein-Protein Interaction by the Mirrortree Method: Possibilities and Limitations

Molecular co-evolution analysis as a sequence-only based method has been used to predict protein-protein interactions. In co-evolution analysis, Pearson''s correlation within the mirrortree method is a well-known way of quantifying the correlation between protein pairs. Here we studied the mirrortree method on both known interacting protein pairs and sets of presumed non-interacting protein pairs, to evaluate the utility of this correlation analysis method for predicting protein-protein interactions within eukaryotes. We varied metrics for computing evolutionary distance and evolutionary span of the species analyzed. We found the differences between co-evolutionary correlation scores of the interacting and non-interacting proteins, normalized for evolutionary span, to be significantly predictive for proteins conserved over a wide range of eukaryotic clades (from mammals to fungi). On the other hand, for narrower ranges of evolutionary span, the predictive power was much weaker.     

Detection of Autophagy in Tissue by Standard Immunohistochemistry: Possibilities and Limitations

Transmission electron microscopy (TEM) is currently the standard method to monitor autophagy in tissue. Because TEM is labor intensive, we recently questioned whether marker proteins could be found for unambiguous detection of autophagy in tissue using standard immunohistochemical techniques. Our findings indicated that the identification of autophagy-specific biomarkers for tissue is highly compromised due to lack of differential gene expression. In this respect, TEM remains an indispensable technique for evaluation of autophagy in situ. Nevertheless, immunohistochemical staining of microtubule-associated protein 1 light chain 3 (LC3) appeared to be a valuable technique to detect autophagosome formation in tissue but only when this protein is overexpressed, e.g. in GFP-LC3 transgenic animals. Furthermore, demonstration of granular cytoplasmic ubiquitin inclusions by immunohistochemistry may be an attractive technique to measure autophagic cell degeneration in some human pathologies such as neurodegenerative diseases, heart failure and atherosclerosis.

Addenda to:

In Situ Detection of Starvation-Induced Autophagy

W. Martinet, G.R.Y. De Meyer, L. Andries, A.G. Herman and M.M. Kockx

J Histochem Cytochem 2005; In press     

z-VAD-fmk-Induced Non-Apoptotic Cell Death of Macrophages: Possibilities and Limitations for Atherosclerotic Plaque Stabilization

Macrophages play a pivotal role in atherosclerotic plaque destabilization in contrast to smooth muscle cells (SMCs). As a consequence, removal of macrophages from plaques via selective induction of cell death represents a promising approach to stabilize non-obstructive, rupture-prone atherosclerotic lesions. However, the mechanisms to initiate cell death in macrophages but not in other cell types of the plaque, in particular SMCs, are unknown. Recently, we have shown that the pan-caspase inhibitor z-VAD-fmk induces autophagy and necrotic cell death in J774A.1 and RAW264.7 macrophages as well as in IFN-gamma primed primary mouse peritoneal macrophages, but not in vascular SMCs or C2C12 myoblasts. The different sensitivity to z-VAD-fmk is largely based on differential expression of receptor-interacting protein 1 (RIP1). This finding suggests that caspase inhibition activates RIP1 which in turn initiates autophagy, although other explanations should be taken into account. z-VAD-fmk-treated J774A.1 macrophages overexpress and secrete several chemokines and cytokines, including TNFa. The combination of z-VAD-fmk and TNFalpha, but not TNFalpha alone, induces SMCs necrosis. In this regard, z-VAD-fmk is detrimental and not beneficial for atherosclerotic plaque stability due to stimulation of inflammatory responses and indirect induction of SMC death. Future work is needed to determine the mechanism(s) that selectively trigger nonapoptotic cell death in plaque macrophages without evoking inflammation and SMC death.

Addendum to:

Macrophages but not Smooth Muscle Cells Undergo Benzyloxycarbonyl-Val-Ala-DL-Asp(O-methyl)-fluoromethylketone-Induced Non-Apoptotic Cell Death Depending on Receptor-Interacting Protein 1 Expression: Implications for the Stabilization of Macrophage-Rich Atherosclerotic Plaques

W. Martinet, G.R.Y. De Meyer, J.-P. Timmermans, A.G. Herman, M.M. Kockx

J Pharmacol Exp Ther 2006; 317:1356-64     

The Measurement of Plant Elongation Rates by Means of Holographic Interferometry: Possibilities and Limitations

Holographic interferometry is a method of measuring very smallmovements and deformations of solid, opaque objects. It wouldappear at first sight to be an ideal technique to use for themonitoring of plant elongation. This paper examines the possibilitiesof this application of holography, reports on the problems ofusing living plants as holographic subjects, and suggests thatthe technique may have some limited application in this fieldin measuring growth rates over very short times.     

Nematode Autofluorescence and Its Use as an Indicator of Viability

Representatives of 15 nematode genera were viewed with 450-490-nm epi-illumination and found to autofluoresce. The autofluorescence was limited to 1-5-μm-d globules in the intestinal cells of live nematodes. When adult Pratylenchus penetrans or Caenorhabditis elegans were killed with formaldehyde, freezing, or heat, autofluorescence dispersed throughout the body. Mixed stages of P. penetrans were killed by freezing at several different temperatures. Estimates of survival based on autofluorescence dispersal matched estimates based on mobility more closely than did estimates based on the vital stain, eosin-y.     

Basic Fuchsin as an Indicator in Endo's Medium

The writer has made an investigation of various samples of basic fuchsin for use in the Endo medium for differentiating the bacteria of the colon-typhoid group. Various different concentrations of the fuchsin samples have been used in making the media. The conclusions are as follows:

American made fuchsins differ markedly in their alcohol solubility properties. They contain materials which are very readily soluble in 95% alcohol, but which are precipitated by sodium sulphite.

This precipitation may be prevented by increasing the dilution of the fuchsin in alcohol.

In order to secure more dependable results in the use of decolorized basic fuchsin as an indicator in Endo Agar, it is advisable to test the fuchsin in different dilutions in alcohol in order to secure a completely decolorized solution. It is also advisable to carefully test those fuchsins which decolorize only in high dilutions with a known organism in Endo agar before relying on it as a satisfactory indicator for the presence of sewage organisms.     

Aging as an Event of Proteostasis Collapse

Aging cells accumulate damaged and misfolded proteins through a functional decline in their protein homeostasis (proteostasis) machinery, leading to reduced cellular viability and the development of protein misfolding diseases such as Alzheimer’s and Huntington’s. Metabolic signaling pathways that regulate the aging process, mediated by insulin/IGF-1 signaling, dietary restriction, and reduced mitochondrial function, can modulate the proteostasis machinery in many ways to maintain a youthful proteome for longer and prevent the onset of age-associated diseases. These mechanisms therefore represent potential therapeutic targets in the prevention and treatment of such pathologies.     

Culturability as an Indicator of Succession in Microbial Communities

Successional theory predicts that opportunistic species with high investment of energy in reproduction and wide niche width will be replaced by equilibrium species with relatively higher investment of energy in maintenance and narrower niche width as communities develop. Since the ability to rapidly grow into a detectable colony on nonselective agar medium could be considered as characteristic of opportunistic types of bacteria, the percentage of culturable cells may be an indicator of successional state in microbial communities. The ratios of culturable cells (colony forming units on R2A agar) to total cells (acridine orange direct microscopic counts) and culturable cells to active cells (reduction of 5-cyano-2,3-ditolyl tetrazolium chloride) were measured over time in two types of laboratory microcosms (the rhizosphere of hydroponically grown wheat and aerobic, continuously stirred tank reactors containing plant biomass) to determine the effectiveness of culturabilty as an index of successional state. The culturable cell:total cell ratio in the rhizosphere decreased from approximately 0.25 to less than 0.05 during the first 30-50 days of plant growth, and from 0.65 to 0.14 during the first 7 days of operation of the bioreactor. The culturable cell:active cell ratio followed similar trends, but the values were consistently greater than the culturable cell:total cell ratio, and even exceeded I in early samples. Follow-up studies used a cultivation-independent method, terminal restriction fragment length polymorphisms (TRFLP) from whole community DNA, to assess community structure. The number of TRFLP peaks increased with time, while the number of culturable types did not, indicating that the general decrease in culturability is associated with a shift in community structure. The ratio of respired to assimilated C-14-labeled amino acids increased with the age of rhizosphere communities, supporting the hypothesis that a shift in resource allocation from growth to maintenance occurs with time. Results from this work indicate that the percentage of culturable cells may be a useful method for assessing the successional state of microbial communities.     

Enlargement of Cerebral Ventricles as an Early Indicator of Encephalomyelitis

Inflammatory disorders of the central nervous system such as multiple sclerosis and acute disseminated encephalomyelitis involve an invasion of immune cells that ultimately leads to white matter demyelination, neurodegeneration and development of neurological symptoms. A clinical diagnosis is often made when neurodegenerative processes are already ongoing. In an attempt to seek early indicators of disease, we studied the temporal and spatial distribution of brain modifications in experimental autoimmune encephalomyelitis (EAE). In a thorough magnetic resonance imaging study performed with EAE mice, we observed significant enlargement of the ventricles prior to disease clinical manifestation and an increase in free water content within the cerebrospinal fluid as demonstrated by changes in T₂ relaxation times. The increase in ventricle size was seen in the lateral, third and fourth ventricles. In some EAE mice the ventricle size started returning to normal values during disease remission. In parallel to this macroscopic phenomenon, we studied the temporal evolution of microscopic lesions commonly observed in the cerebellum also starting prior to disease onset. Our data suggest that changes in ventricle size during the early stages of brain inflammation could be an early indicator of the events preceding neurological disease and warrant further exploration in preclinical and clinical studies.     

P-Aminodimethylaniline Monohydrochloride as an Indicator of Microbial Action on Fats

Flooding p-aminodimethylaniline monohydrochloride on fat emulsion agar inoculated with certain types of microorganisms frequently results in marked color changes in the fat globules. It is shown in this paper that the colors result from the increased solubility in fat and fatty acids of this dye as it becomes oxidized. Some of the acids oxidize the dye on contact and therefore color very quickly; fats become colored only when some other agent oxidizes the dye. The characteristic color reactions with certain fats and fatty acids are described for various degrees of oxidation of the dye; this suggests the explanation for the colors observed in the inoculated globules flooded with this dye. A table is included showing the colors in globules of oil that were inoculated with 39 pure cultures of bacteria.     

Pepper Mild Mottle Virus as an Indicator of Fecal Pollution

Accurate indicators of fecal pollution are needed in order to minimize public health risks associated with wastewater contamination in recreational waters. However, the bacterial indicators currently used for monitoring water quality do not correlate with the presence of pathogens. Here we demonstrate that the plant pathogen Pepper mild mottle virus (PMMoV) is widespread and abundant in wastewater from the United States, suggesting the utility of this virus as an indicator of human fecal pollution. Quantitative PCR was used to determine the abundance of PMMoV in raw sewage, treated wastewater, seawater exposed to wastewater, and fecal samples and/or intestinal homogenates from a wide variety of animals. PMMoV was present in all wastewater samples at concentrations greater than 1 million copies per milliliter of raw sewage. Despite the ubiquity of PMMoV in human feces, this virus was not detected in the majority of animal fecal samples tested, with the exception of chicken and seagull samples. PMMoV was detected in four out of six seawater samples collected near point sources of secondary treated wastewater off southeastern Florida, where it co-occurred with several other pathogens and indicators of fecal pollution. Since PMMoV was not found in nonpolluted seawater samples and could be detected in surface seawater for approximately 1 week after its initial introduction, the presence of PMMoV in the marine environment reflects a recent contamination event. Together, these data demonstrate that PMMoV is a promising new indicator of fecal pollution in coastal environments.Existing wastewater treatment practices are not always effective at removing the large number of pathogens (bacteria, protists, and viruses) present in human feces (17, 42, 47-49, 51). Therefore, wastewater discharges into the environment can have a negative impact on human health. Recreational waters throughout the United States are monitored for the presence of fecal pollution as a means of limiting public exposure to pathogens in areas impacted by wastewater discharges (44). The presence of pathogenic viruses in aquatic environments is an important parameter to consider in the evaluation of water quality. However, the bacterial indicators currently used to detect fecal contamination, such as fecal coliforms and enterococci, often do not correlate with the presence of feces-associated viruses and other pathogens (5, 10, 26, 33, 37, 51). In response, several researchers have proposed the use of viral indicators as a more effective method for monitoring wastewater contamination and the associated risks to public health (11, 14, 31).To date, the majority of the proposed viral indicators of fecal pollution are enteric viruses transmitted via the fecal-oral route (4). Enteric viruses present in raw sewage (including members of the families Adenoviridae, Caliciviridae, Picornaviridae, and Reoviridae and of the genus Anellovirus) have been used in several previous studies to identify fecal pollution in the environment (7, 8, 11, 12, 13, 18, 19, 27, 28, 32-36, 38, 50, 51). Of the enteric viruses that have been used as indicators, only the adenoviruses were ubiquitously found in raw sewage samples collected throughout the United States (41). Picobirnaviruses and Torque teno virus are abundant in raw sewage from some regions and thus have also been proposed as indicator viruses (15, 41). However, one potential problem with the use of human viruses as indicators is that their abundance in wastewater depends on the degree of infection and shedding in the human population at any given time.In addition to viruses infecting humans, other viruses shed in feces may be useful for indicating wastewater pollution. The plant pathogen Pepper mild mottle virus (PMMoV) was the most abundant virus found in a metagenomic survey of RNA viruses from human feces (52). PMMoV is a positive-sense, single-stranded RNA virus that belongs to the Tobamovirus genus and infects hot, bell, and ornamental peppers (Capsicum spp.) (9). The nonenveloped, rod-shaped PMMoV virions are extremely stable (9) and have been demonstrated to retain their infectivity for plants after passage through the human gut (52). PMMoV originates from processed pepper products (e.g., hot sauce and curry) and is excreted in human feces at concentrations of 1 million to 1 billion viruses per g (dry weight) (52). Since the presence of PMMoV in human feces is dietary in origin, this plant pathogen may be more abundant in the healthy human population than viruses that cause human disease.This study analyzed the presence of PMMoV in raw sewage and treated wastewater samples collected from wastewater treatment facilities throughout the coastal United States. To determine if PMMoV is a human-specific indicator useful for tracking the source of fecal pollution, fecal samples from numerous animals were tested for this virus. Finally, the presence of PMMoV in marine environments exposed to wastewater was determined and compared to that of other microbial indicators. The results of this work demonstrate that PMMoV is a promising indicator of fecal pollution.     

Copeptin as an Indicator of Hemodynamic Derangement and Prognosis in Liver Cirrhosis

Background

Advanced liver cirrhosis is associated with systemic hemodynamic derangement leading to the development of severe complications associated with increased mortality. Copeptin is a stable cleavage product of the precursor of arginine vasopressin, a key-regulator in hemodynamic homeostasis. Copeptin is currently considered a reliable prognostic marker in a wide variety of diseases other than cirrhosis. The present study aimed to assess copeptin, both experimentally and clinically, as a potential biomarker of hemodynamic derangement and to evaluate its prognostic significance in cirrhosis.

Materials and Methods

Two studies were executed: 1) in 18 thioacetamide-induced cirrhotic rats and 5 control rats, plasma copeptin and hemodynamic measurements were performed, 2) in 61 cirrhotic patients, serum copeptin concentration was measured in samples collected at time of registration at the waiting list for liver transplantation. In 46 patients, also a second copeptin measurement was performed during follow-up while registered at the waiting list for liver transplantation. To determine the association of serum copeptin and clinical data with outcome, Cox proportional hazard regression analysis and Kaplan Meier analysis were performed.

Results

Plasma copeptin concentration was significantly higher in cirrhotic rats than in controls (1.6 ± 0.5 vs. 0.9 ± 0.1 pmol/L, p< 0.01) and was negatively correlated to the mean arterial blood pressure (r = -0.574, p = 0.013). In cirrhotic patients, serum copeptin concentration was high [11.0 (5.2–24.0) pmol/L] and increased significantly during the time of registration at the waiting list for liver transplantation. MELD and MELD-sodium score were significantly correlated to serum copeptin [MELD: (r = 0.33, p = 0.01), MELD-sodium: (r = 0.29, p = 0.02)], also at time of the second copeptin measurement [MELD and MELD-sodium: r = 0.39, p< 0.01]. In cirrhotic humans, serum copeptin concentration was significantly associated with outcome, independently of the MELD and MELD-sodium score. Patients with a low serum copeptin concentration at time of registration at the liver transplant waiting list had significantly better transplant-free survival rates at 3, 6 and 12 months of follow-up as compared to those with a high serum copeptin concentration (Log-rank: p< 0.01, p< 0.01 and p = 0.02 respectively).

Conclusions

Circulating copeptin levels are elevated in rats and humans with cirrhosis. Copeptin is independently associated with outcome in cirrhotic patients awaiting liver transplantation.     

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.     

Modeling the Entire Human Mortality Curve: A Regulatory Model of Aging

Biophysics - Abstract—Modeling of the human aging process was performed based on the relationship between overall viability and the processes of growth and self-renewal of tissues, which is...     

Serum Calcium Concentration as an Indicator of Intervertebral Disk Degeneration Prognosis

This study aims to investigate the relationship between serum macro- and trace element contents and the degree of disk degeneration in patients with intervertebral disk herniation (IDH). This study was carried out on 69 subjects (30 women and 39 men) diagnosed with IDH. Blood samples of the subjects were collected, and serum concentrations of the elements that include macroelements, such as calcium, phosphorus, potassium, sodium, and magnesium, and trace elements, such as zinc, iron, copper, and selenium, were determined using inductively coupled plasma–atomic emission spectrometry. Magnetic resonance imaging (MRI) examination of the entire lumbar region of the vertebral column was conducted using a 1.5-T MRI scanner. The degree of disk degeneration was classified into three categories. Correlation analysis between the degree of disk degeneration and the serum element was performed using SPSS 16.0. In the correlation analysis between the degree of disk degeneration and the element contents, only calcium was found to be negatively correlated with the degree of disk degeneration (r?=??0.332, P?<?0.01). Comparison results between male and female groups showed no significant difference in the element content and in the degree of disk degeneration (P?>?0.05). Moreover, the serum calcium content showed a significant correlation with the degree of disk degeneration, suggesting that the serum calcium concentration can be used as an indicator of intervertebral disk degeneration prognosis.     

Rate-Temperature Curves as an Unambiguous Indicator of Biological Activity in Soil

Experiments are described in which we used a mass spectrometer to monitor O₂ uptake of enclosed soil samples as a function of temperature. We found that an Arrhenius plot of the rate of O₂ uptake showed pronounced local maxima attributable to biological activity, whereas similar plots of rates obtained with abiotic soils yielded straight lines. This procedure thus provides a basis for distinguishing biological from chemical activity for reactions, such as O₂ uptake, that can occur via either biological or chemical pathways.