Evolutionary trade-offs, Pareto optimality, and the geometry of phenotype space.

Biological systems that perform multiple tasks face a fundamental trade-off: A given phenotype cannot be optimal at all tasks. Here we ask how trade-offs affect the range of phenotypes found in nature. Using the Pareto front concept from economics and engineering, we find that best-trade-off phenotypes are weighted averages of archetypes--phenotypes specialized for single tasks. For two tasks, phenotypes fall on the line connecting the two archetypes, which could explain linear trait correlations, allometric relationships, as well as bacterial gene-expression patterns. For three tasks, phenotypes fall within a triangle in phenotype space, whose vertices are the archetypes, as evident in morphological studies, including on Darwin's finches. Tasks can be inferred from measured phenotypes based on the behavior of organisms nearest the archetypes.

Dynamic proteomics of individual cancer cells in response to a drug.

Why do seemingly identical cells respond differently to a drug? To address this, we studied the dynamics and variability of the protein response of human cancer cells to a chemotherapy drug, camptothecin. We present a dynamic-proteomics approach that measures the levels and locations of nearly 1000 different endogenously tagged proteins in individual living cells at high temporal resolution. All cells show rapid translocation of proteins specific to the drug mechanism, including the drug target (topoisomerase-1), and slower, wide-ranging temporal waves of protein degradation and accumulation. However, the cells differ in the behavior of a subset of proteins. We identify proteins whose dynamics differ widely between cells, in a way that corresponds to the outcomes-cell death or survival. This opens the way to understanding molecular responses to drugs in individual cells.

The incoherent feed-forward loop accelerates the response-time of the gal system of Escherichia coli.

Complex gene regulation networks are made of simple recurring gene circuits called network motifs. One of the most common network motifs is the incoherent type-1 feed-forward loop (I1-FFL), in which a transcription activator activates a gene directly, and also activates a repressor of the gene. Mathematical modeling suggested that the I1-FFL can show two dynamical features: a transient pulse of gene expression, and acceleration of the dynamics of the target gene. It is important to experimentally study the dynamics of this motif in living cells, to test whether it carries out these functions even when embedded within additional interactions in the cell. Here, we address this using a system with incoherent feed-forward loop connectivity, the galactose (gal) system of Escherichia coli. We measured the dynamics of this system in response to inducing signals at high temporal resolution and accuracy by means of green fluorescent protein reporters. We show that the galactose system displays accelerated turn-on dynamics. The acceleration is abolished in strains and conditions that disrupt the I1-FFL. The I1-FFL motif in the gal system works as theoretically predicted despite being embedded in several additional feedback loops. Response acceleration may be performed by the incoherent feed-forward loop modules that are found in diverse systems from bacteria to humans.

Random urine calcium/osmolality in the assessment of calciuria in children with decreased muscle mass.

BACKGROUND: Random urine Ca/creatinine (UCa/Cr) is used to estimate 24-hour Ca excretion. However, due to decreased urine creatinine excretion in children with decreased muscle mass (DMM), UCa/Cr overestimates their Ca excretion. OBJECTIVE: To evaluate whether in children with DMM random urine Ca/osmolality (UCa/Osm) can accurately predict hypercalciuria (24-hour urine Ca > 4.0 mg/kg) and at which "cutoff" value. METHODS: 19 children with DMM and 29 with normal muscle mass (NMM), ages 6 - 17 years, were studied. DMM was diagnosed based on clinical findings and decreased serum creatinine, and confirmed by low urine creatinine excretion. Over 24 hours, subjects collected each void separately. After each sample was analyzed, samples of each participant were combined to form a 24-hour specimen from which an aliquot (AL) was obtained; 24-hour urine Ca was first correlated with the corresponding AL Ca/Cr and Ca/Osm. As an internal control, a similar assessment ofproteinuria was conducted. In the next step, AL data were compared with individual urine samples to identify the time of day when a random sample best correlates with AL. RESULTS: The correlation coefficient between 24-hour Ca and AL Ca/Cr in all children was 0.61, in NMM 0.96, and in DMM 0.69 (in all p < 0.001). The correlation coefficient between 24-hour urine Ca and AL Ca/Osm in all children was 0.90, in NMM 0.90, and in DMM 0.91 (in all p < 0.001). In children with DMM, the correlation coefficient of 24-hour protein with AL protein/Cr was 0.75, and with protein/Osm 0.98 (both p < 0.001). Receiver operating characteristic curves showed UCa/Cr as a better predictor of 24-hour Ca > 4.0 mg/kg in NMM, whereas UCa/Osm was a better predictor of hypercalciuria in DMM patients. In NMM, UCa/Cr ratio > 0.20 had sensitivity of 88% and specificity of 96% in detecting 24-hour Ca > 4.0 mg/kg, whereas in those with DMM UCa/Osm (x 10) ratio of > 0.25 had sensitivity of 100% and specificity of 93% in detecting hypercalciuria. It was further found that random urine specimens collected between 9:00 a.m. and 2:00 p.m. best represented 24-hour urine data. CONCLUSION: In children with DMM, UCa/Osm can successfully replace UCa/Cr as a screening tool for hypercalciuria.

Topological generalizations of network motifs.

Biological and technological networks contain patterns, termed network motifs, which occur far more often than in randomized networks. Network motifs were suggested to be elementary building blocks that carry out key functions in the network. It is of interest to understand how network motifs combine to form larger structures. To address this, we present a systematic approach to define "motif generalizations": families of motifs of different sizes that share a common architectural theme. To define motif generalizations, we first define "roles" in a subgraph according to structural equivalence. For example, the feedforward loop triad--a motif in transcription, neuronal, and some electronic networks--has three roles: an input node, an output node, and an internal node. The roles are used to define possible generalizations of the motif. The feedforward loop can have three simple generalizations, based on replicating each of the three roles and their connections. We present algorithms for efficiently detecting motif generalizations. We find that the transcription networks of bacteria and yeast display only one of the three generalizations, the multi-output feedforward generalization. In contrast, the neuronal network of C. elegans mainly displays the multi-input generalization. Forward-logic electronic circuits display a multi-input, multi-output hybrid. Thus, networks which share a common motif can have very different generalizations of that motif. Using mathematical modeling, we describe the information processing functions of the different motif generalizations in transcription, neuronal, and electronic networks.

Efficient sampling algorithm for estimating subgraph concentrations and detecting network motifs.

SUMMARY: Biological and engineered networks have recently been shown to display network motifs: a small set of characteristic patterns that occur much more frequently than in randomized networks with the same degree sequence. Network motifs were demonstrated to play key information processing roles in biological regulation networks. Existing algorithms for detecting network motifs act by exhaustively enumerating all subgraphs with a given number of nodes in the network. The runtime of such algorithms increases strongly with network size. Here, we present a novel algorithm that allows estimation of subgraph concentrations and detection of network motifs at a runtime that is asymptotically independent of the network size. This algorithm is based on random sampling of subgraphs. Network motifs are detected with a surprisingly small number of samples in a wide variety of networks. Our method can be applied to estimate the concentrations of larger subgraphs in larger networks than was previously possible with exhaustive enumeration algorithms. We present results for high-order motifs in several biological networks and discuss their possible functions. AVAILABILITY: A software tool for estimating subgraph concentrations and detecting network motifs (mfinder 1.1) and further information is available at http://www.weizmann.ac.il/mcb/UriAlon/

The coherent feedforward loop serves as a sign-sensitive delay element in transcription networks.

Recent analysis of the structure of transcription regulation networks revealed several "network motifs": regulatory circuit patterns that occur much more frequently than in randomized networks. It is important to understand whether these network motifs have specific functions. One of the most significant network motifs is the coherent feedforward loop, in which transcription factor X regulates transcription factor Y, and both jointly regulate gene Z. On the basis of mathematical modeling and simulations, it was suggested that the coherent feedforward loop could serve as a sign-sensitive delay element: a circuit that responds rapidly to step-like stimuli in one direction (e.g. ON to OFF), and at a delay to steps in the opposite direction (OFF to ON). Is this function actually carried out by feedforward loops in living cells? Here, we address this experimentally, using a system with feedforward loop connectivity, the L-arabinose utilization system of Escherichia coli. We measured responses to step-like cAMP stimuli at high temporal resolution and accuracy by means of green fluorescent protein reporters. We show that the arabinose system displays sign-sensitive delay kinetics. This type of kinetics is important for making decisions based on noisy inputs by filtering out fluctuations in input stimuli, yet allowing rapid response. This information-processing function may be performed by the feedforward loop regulation modules that are found in diverse systems from bacteria to humans.

Structure and function of the feed-forward loop network motif.

Engineered systems are often built of recurring circuit modules that carry out key functions. Transcription networks that regulate the responses of living cells were recently found to obey similar principles: they contain several biochemical wiring patterns, termed network motifs, which recur throughout the network. One of these motifs is the feed-forward loop (FFL). The FFL, a three-gene pattern, is composed of two input transcription factors, one of which regulates the other, both jointly regulating a target gene. The FFL has eight possible structural types, because each of the three interactions in the FFL can be activating or repressing. Here, we theoretically analyze the functions of these eight structural types. We find that four of the FFL types, termed incoherent FFLs, act as sign-sensitive accelerators: they speed up the response time of the target gene expression following stimulus steps in one direction (e.g., off to on) but not in the other direction (on to off). The other four types, coherent FFLs, act as sign-sensitive delays. We find that some FFL types appear in transcription network databases much more frequently than others. In some cases, the rare FFL types have reduced functionality (responding to only one of their two input stimuli), which may partially explain why they are selected against. Additional features, such as pulse generation and cooperativity, are discussed. This study defines the function of one of the most significant recurring circuit elements in transcription networks.

Subgraphs in random networks.

Understanding the subgraph distribution in random networks is important for modeling complex systems. In classic Erdos networks, which exhibit a Poissonian degree distribution, the number of appearances of a subgraph G with n nodes and g edges scales with network size as approximately N(n-g). However, many natural networks have a non-Poissonian degree distribution. Here we present approximate equations for the average number of subgraphs in an ensemble of random sparse directed networks, characterized by an arbitrary degree sequence. We find scaling rules for the commonly occurring case of directed scale-free networks, in which the outgoing degree distribution scales as P(k) approximately k(-gamma). Considering the power exponent of the degree distribution, gamma, as a control parameter, we show that random networks exhibit transitions between three regimes. In each regime, the subgraph number of appearances follows a different scaling law, approximately Nalpha, where alpha=n-g+s-1 for gamma<2, alpha=n-g+s+1-gamma for 2gamma(c), where s is the maximal outdegree in the subgraph, and gamma(c)=s+1. We find that certain subgraphs appear much more frequently than in Erdos networks. These results are in very good agreement with numerical simulations. This has implications for detecting network motifs, subgraphs that occur in natural networks significantly more than in their randomized counterparts.

Biological networks: the tinkerer as an engineer.

This viewpoint comments on recent advances in understanding the design principles of biological networks. It highlights the surprising discovery of "good-engineering" principles in biochemical circuitry that evolved by random tinkering.

Detailed map of a cis-regulatory input function.

Most genes are regulated by multiple transcription factors that bind specific sites in DNA regulatory regions. These cis-regulatory regions perform a computation: the rate of transcription is a function of the active concentrations of each of the input transcription factors. Here, we used accurate gene expression measurements from living cell cultures, bearing GFP reporters, to map in detail the input function of the classic lacZYA operon of Escherichia coli, as a function of about a hundred combinations of its two inducers, cAMP and isopropyl beta-d-thiogalactoside (IPTG). We found an unexpectedly intricate function with four plateau levels and four thresholds. This result compares well with a mathematical model of the binding of the regulatory proteins cAMP receptor protein (CRP) and LacI to the lac regulatory region. The model is also used to demonstrate that with few mutations, the same region could encode much purer AND-like or even OR-like functions. This possibility means that the wild-type region is selected to perform an elaborate computation in setting the transcription rate. The present approach can be generally used to map the input functions of other genes.

Network motifs: simple building blocks of complex networks.

Complex networks are studied across many fields of science. To uncover their structural design principles, we defined "network motifs," patterns of interconnections occurring in complex networks at numbers that are significantly higher than those in randomized networks. We found such motifs in networks from biochemistry, neurobiology, ecology, and engineering. The motifs shared by ecological food webs were distinct from the motifs shared by the genetic networks of Escherichia coli and Saccharomyces cerevisiae or from those found in the World Wide Web. Similar motifs were found in networks that perform information processing, even though they describe elements as different as biomolecules within a cell and synaptic connections between neurons in Caenorhabditis elegans. Motifs may thus define universal classes of networks. This approach may uncover the basic building blocks of most networks.

Urinary tract infection in childhood. Review of guidelines and recommendations.

Children with urinary tract infection continue to be an important part of the pediatric practice. New uroradiologic imaging techniques like cortical radionuclide scanning and prenatal ultrasonography improved our understanding of the etiology, effect of treatment and outcome of these patients. Evidently, most kidneys at risk are those which already sustained intrauterine damage by obstruction or vesicoureteral reflux. It is the pediatrician's role to minimize ex-utero damage caused by bacterial infection by early diagnosis and appropriate intervention. The introduction of new potent oral antimicrobials limits the need for hospitalization only to the very young infant and the very seriously ill child. Whereas the roles of routine renal ultrasound and cortical radionuclide scan are debatable, all young children and select older children have to be investigated by cystography for possible vesicoureteral reflux. In children with vesicoureteral reflux, long-term antibiotic prophylaxis is required in most children but in a few surgical correction might be indicated. Young siblings of the propositus with vesicoureteral reflux have to be investigated as well for possible reflux. This review covers these and other guidelines and recommendations of diagnosis and treatment of UTI in children at the beginning of the third millennium.

Pneumonia-associated acute glomerulonephritis.

OBJECTIVE: Post-infectious glomerulonephritis typically occurs 7-14days after an infection. However, in several children we observed acute glomerulonephritis (AGN) to develop concurrently with pneumonia. The objective of the study was to delineate the clinical course and outcome of pneumonia-associated AGN. STUDY DESIGN: The hospital database was searched from 1984 - 1999 for c+hildren admitted with both acute pneumonia and AGN, each diagnosis having been made within 72 hours of each other. RESULTS: 11 boys, age 3.8- 12.7 years, were identified. Ten children had lobar pneumonia and I had an interstitial infiltrate. All responded to antibiotic therapy with resolution of fever and respiratory symptoms. Only I child developed an empyema. The mean +/- SD hospital stay was 5.9 +/- 3.9 days. All patients had an abnormal urinalysis with hematuria (gross hematuria in 5), proteinuria and cellular casts. At presentation, 7 children had a serum creatinine > 1.0 mg/dl and creatinine clearance < or = 80 ml/min/1.73 m2; in all, serum creatinine returned to normal and the creatinine clearance was > 80 ml! min/1.73 m2 on follow-up. Nine of the 11 children had a low serum complement C3, 3 of whom also had low complement C4. Anti-streptolysin-O (ASO) titers were elevated in all 10 children tested. Six children developed hypertension and received antihypertensive medications. Only I child was severely oliguric requiring peritoneal dialysis for 4 days. He underwent a kidney biopsy, which showed acute proliferative glomerulonephritis without crescents. Neither a biopsy nor dialysis was performed in the other children. At follow-up, blood pressure, urinalysis and serum complements had normalized in the 9 children in whom follow-up was available. CONCLUSION: Children with pneumonia who are found to have abnormal urinalysis. hypertension, azotemia or oliguria should be evaluated for concomitant glomerulonephritis. In most children, pneumonia-associated AGN runs a benign course and has a good prognosis, however, in some short-term medical intervention may be necessary.

Preservation of bone mass in pediatric dialysis and transplant patients.

Renal osteodystrophy continues to be a major challenge to the physician treating the child with end-stage renal disease (ESRD). The gold standard for the assessment of bone status is bone histomorphometry, which divides bone pathology into 3 main types; high-turnover, low-turnover, and mixed disease. The high-turnover disease, related to hyperparathyroidism, has been the one most extensively investigated; however, optimal therapy, especially in the growing child, is yet unclear. Overzealous treatment might result in adynamic bone disease (an extreme example of low-turnover disease), and further interference with statural growth. Pre-existent bone disease after kidney transplantation seems to worsen immediately, probably because of the high dose of corticosteroids used. In children who attain normal kidney function in the allograft, bone status seems to improve over time. Little is known about bone in transplanted patients with reduced glomerular filtration rate (GFR). The correlation between bone histology and its main surrogates, bone remodeling markers and bone mineral density, is yet unclear, but it might serve to follow the progress of an individual patient. New therapeutic modalities aimed at suppressing hyperparathyroidism, and consequently bone resorption, as well as agents directly attenuating bone resorption, should be further investigated for their effect on bone in patients with ESRD or after transplantation. Similarly, agents stimulating bone formation, particularly growth hormone, require further attention for their potential to improve bone status. Bone health and the child's somatic growth at ESRD or after kidney transplantation are closely related, and therapy should be aimed at achieving optimal results for both.

Antacid-induced rickets in infancy.

A 3-month-old premature infant presented with a "soft skull." Clinical and radiologic findings confirmed the diagnosis of rickets. Biochemistry revealed normal serum parathyroid hormone (PTH) and undetectable urine phosphate. These findings combined with a history of 5-6 weeks' treatment with high-dose aluminum-rich antacid established the diagnosis of antacid-induced rickets. Discontinuation of the medicine combined with phosphate and vitamin D supplementation resulted in quick resolution of all clinical, radiologic, and biochemical abnormalities. Our patient demonstrates that in premature infants antacid-induced rickets can develop within a few weeks; normal serum PTH concentration and hypophosphaturia are highly indicative of the diagnosis, and contrary to the situation in adults in whom hypercalciuria has been often described, in infants hypocalciuria is more commonly observed. Pediatricians should avoid or minimize the use of aluminum-containing antacids, and when used, carefully monitor mineral metabolism.

Ordering genes in a flagella pathway by analysis of expression kinetics from living bacteria.

The recent advances in large-scale monitoring of gene expression raise the challenge of mapping systems on the basis of kinetic expression data in living cells. To address this, we measured promoter activity in the flagellar system of Escherichia coli at high accuracy and temporal resolution by means of reporter plasmids. The genes in the pathway were ordered by analysis algorithms without dependence on mutant strains. The observed temporal program of transcription was much more detailed than was previously thought and was associated with multiple steps of flagella assembly.

Measurement of urinary concentration: a critical appraisal of methodologies.

The measurement of urine concentration provides information concerning the kidney's ability to appropriately respond to variations in fluid homeostasis. It also assists in the interpretation of other tests performed on the same urine specimen. The gold standard of estimating urinary concentration is the measurement of its osmolality; however, this procedure is not readily available to the practicing physician. Therefore, urine concentration is usually determined by measurement of its specific gravity (SG), which provides a fair estimate of urine osmolality. Over the years numerous tests have been developed to measure urine SG in a simple, quick, reliable and easily available method. These tests measure SG either directly (e.g., gravimetry) or by indirect methods (e.g., refractometry and reagent strip). All these tests have certain limitations based on their underlying physical principles. Specific gravity as measured by refractometry is influenced by proteinuria, such that for each 10 g/l protein the SG increases by 0.003. SG is also influenced by glucosuria such that it increases by approximately 0.002 per 10 g/l glucose when compared with urinary osmolality. Unlike osmolality, which is only affected by the number of particles, refractometry is affected by number, mass and chemical structure of the dissolved particles; hence large molecules like radiographic contrast or mannitol will increase SG relative to osmolality. The reagent strip is minimally affected by glucose, mannitol or radiographic contrast. However, it is affected by urinary pH such that only urine in the pH range of 7.0-7.5 can be correctly interpreted. The measurement of SG by reagent strip is based on the ionic strength of the urine and thus is significantly affected by the ionic composition of the urine and by proteins which have an electric charge in solution. In our experience, SG measured by the refractometer is consistently more accurate than the reagent strip. For the clinician who is interpreting urine SG results, it is important to be aware of these limitations and understand the reasons for possible potential errors of each particular method.

Transcriptional gene expression profiles of colorectal adenoma, adenocarcinoma, and normal tissue examined by oligonucleotide arrays.

Using an oligonucleotide array containing sequences complementary to approximately 3200 full-length human cDNAs and 3400 expressed sequence tags (GeneChip, Affymetrix), mRNA expression patterns were probed in 18 colon adenocarcinomas and 4 adenomas. Paired normal tissue was available and analyzed for each of the tumors. Relatively few changes in transcript expression are associated with colon cancer. Nineteen transcripts (0.48% of those detected) had at least 4-10.5-fold higher mRNA expression in carcinoma compared with paired normal samples, whereas 47 transcripts (1.3% of those detected) had at least 4-38-fold or lower expression in the tumor tissue compared with the normal samples. Some of these differences were confirmed by reverse transcription-PCR. Many of these transcripts were already known to be abnormally expressed in neoplastic tissue in general, or colon cancer in particular, and several of these differences were also observed in premalignant adenoma samples. A two-way hierarchical clustering algorithm successfully distinguished adenoma from adenocarcinoma and normal tissue, generating a phylogenetic tree that appropriately represented the clinical relationship between the three tissue types included in the analysis. This supports the concept that genome-wide expression profiling may permit a molecular classification of solid tumors.