abstract: A report from Microbial Genomes, a joint conference of the American Society for Microbiology and the Institute for Genomic Research, Monterey, California, USA, 28-31 January, 2001.
author: Gary K Schoolnik
date: 2001
institute: 1Departments of Medicine and of Microbiology and Immunology, Stanford Medical School, Beckman Center, Stanford, CA 94305, USA. E-mail: email@example.com
title: The accelerating convergence of genomics and microbiology

The rapid use of available genome sequences by biologists was brilliantly evident in many of this meeting's plenary lectures. The most pervasive theme was the relationship between genome sequence and adaptation of a microbe to a specialized niche. It is particularly instructive in this regard to contrast the largest and smallest bacterial genomes discussed.

The complex and resourceful life style of Myxobacteria as free-living soil organisms (Dale Kaiser, Stanford University, USA) includes organized social behavior during food-seeking excursions and, in periods of scarcity, the formation of fruiting bodies containing spores that can be carried by the wind to richer habitats. Fruiting body development has attracted the attention of developmental biologists because it provides an opportunity to study spatial differentiation in a genetically tractable and relatively simple organism. Recent information about the surprisingly small size of the human genome notwithstanding, it is reasonable to assume that complexity and genome size should be related. And so it seems to be: Barry Goldman (Monsanto, St Louis, USA) reported that *Myxococcus xanthus* has a genome of 9.5 Mb, making it the largest bacterial genome so far sequenced, and containing an estimated 7,500 open reading frames (ORFs).

By contrast, the genome of the obligate aphid endosymbiont *Buchnera aphidicola*, discussed by Siv Andersson (Uppsala University, Sweden), comprises only 583 ORFs, of which 579 are related to *Escherichia coli* genes. Thus, *B. aphidicola* probably evolved from a genetically well-endowed, free-living ancestor and, while occupying its niche within the cytoplasm of a eukaryotic cell, drastically contracted its genome size. Insight into how this occurred comes from comparison of the sequences of two closely related *Buchnera* strains that co-evolved with their corresponding aphid hosts over the past 50 million years. On average, one gene has been eliminated each 5-10 million years. As the size of a genome is the sum of the rates at which genes are lost and gained, the on-going loss of *Buchnera* DNA was not compensated for by the acquisition of horizontally transferred DNA or by the duplication of indigenous genes. *Buchnera* probably illustrates a late stage in a multistep evolutionary process from free-living microbe, through the intermediate stages of facultative and then obligate intracellular parasitism, to its current status as an endosymbiont.

Some (but not all) microbiologists consider the cytoplasm to be an extreme environment and thus inhabited by extremophiles. None would doubt, however, that the capacity of, or indeed the requirement for, *Halobacterium* sp. NRC-1 to grow in 3-5 M NaCl would so qualify. The completion of its sequence, reported by Shiladitya DasSarma (University of Massachusetts, Amherst, USA), shows that *Halobacterium* contains 91 copies of insertion sequences and that its 2,682 ORFs are distributed between one large chromosome and two related minichromosomes. Transposition of its many IS repetitive elements would provide a mechanism for genetic plasticity through the fusion of smaller replicons. In turn, this may have led to the acquisition of essential genes by megaplasmids and hence to the possibility of competition between plasmid and chromosome. A second undisputed adaptation to an extreme environment is the capacity of hyperthermophilic archaea to grow at high temperature. One such organism, *Pyrococcus furiosus*, grows optimally at 100?C and is resistant to ionizing radiation owing, in part, to its capacity to reassemble chromosome fragments. Jocelyn DiRuggiero (University of Maryland Biotechnology Institute, Baltimore, USA) discussed the initiation of microarray hybridization experiments to identify genes that are differentially expressed during exposure of this organism to ? irradiation and to sublethal temperatures.

The oceans encompass surprisingly diverse environments, where different depths within the water column provide distinctive habitats within which adaptive evolution occurs. One such depth-dependent variable is light. Edward DeLong and colleagues (Monterey Bay Aquarium Research Institute, California, USA) used large-insert bacterial artificial chromosome (BAC) libraries to characterize uncultured marine bacterioplankton. Among the genes recovered was the coding sequence for a bacterial rhodopsin. Although this gene was found in the genome of an uncultivated ? proteobacterium, it is most similar in amino-acid sequence to archaeal rhodopsins, indicating its possible acquisition by horizontal gene transfer. Expression of this gene in *E. coli* and functional analysis of the corresponding protein showed a ?~max~ corresponding to the wavelength of light available in the upper water column. By contrast, deeper-water rhodopsins were found to be blue-shifted, indicating that spectral tuning had occurred during the evolution of this protein family within bacteria living at different depths.

Evolutionary divergence by closely related strains living at different ocean depths is nowhere better demonstrated than in *Prochlorococcus*, a marine cyanobacterium whose photosynthetic activity contributes significantly to primary production in the oceans. Gabrielle Rocap (Massachusetts Institute of Technology, Cambridge, USA) contrasted the sequence of two highly related strains of *Prochlorococcus* that exhibit strikingly different optimal light levels that adapt one for growth at the ocean surface and the other for growth in a deep water environment. Despite their closely related 16S rRNA sequences, the two genomes are substantially different with respect to size, GC content and codon usage. The strain living in the nitrogen-poor waters of the ocean surface lacks genes for nitrite and nitrate reductases, whereas the strain living in nitrogen-rich, deeper water possesses genes predicted to confer both activities. Thus, the sequencing of closely related strains living in different environments led to the identification of two genomic 'eco-types' that reflect both the constraints and advantages of their respective niches. How this diversity arose will be a matter of much future study, but surely one such mechanism is lateral gene transfer between natural populations of marine bacteria, as discussed by John Paul (University of South Florida, St Petersburg, USA).

Three fascinating genome sequence works-in-progress were presented. Steven Slater (Cereon Genomics LLC, Cambridge, USA) discussed *Agrobacterium tumefaciens*, a plant pathogen and the etiological agent of crown gall disease; Martin Odom (DuPont, Wilmington, USA) discussed the status and initial findings of the *Methylomonas* project, a highly specialized aerobic eubacterium that can use only C-1 compounds (methanol/methane) for carbon and energy; and Malcolm Gardner (The Institute for Genome Research (TIGR), Rockville, USA) discussed *Theileria parva*, a tick-borne intracellular protozoan parasite of domesticated ruminants that induces malignant transformation of lymphocytes leading to fatal lymphosarcomas in afflicted animals.

The use of genome sequences to study microbial pathogenesis and epidemiology or to develop new vaccines and diagnostic assays was the focus of five talks. I presented my laboratory's microarray expression-profiling study of *Mycobacterium tuberculosis* growing within the macrophage phagosome and its transcriptional response to the nutrients and reactive oxygen and nitrogen intermediates that characterize this niche. Timothy Palzkill (Baylor College of Medicine, Houston, USA) described a phage-display method to express protein products corresponding to each of the 1,030 ORFs of the *Treponema pallidum* genome. This is a particularly worthwhile goal because this bacterium, the agent of syphilis, cannot be propagated *in vitro* and there is a compelling need for improved serological tests. The emerging drug resistance of *Staphylococcus aureus* is a major threat to all hospitalized patients and thus it was encouraging to learn that Uwe von Ahsen and colleagues (Intercell GmBH, Vienna, Austria) had used patients' sera and *in vivo* and *in vitro* methods to display all the proteins encoded by the staphylococcal genome, to identify genes coding for antigenic proteins expressed during infection and accessible to the immune system.

The movement of a genetically diverse infectious agent through a population can be tracked with the highest resolution by the combined use of conventional and molecular epidemiology. For the latter, Andreas Duesterhoeft (Qiagen GmBH, Dusseldorf, Germany) described a method for genotyping microorganisms by detecting single-nucleotide polymorphisms, and its use to study the epidemiology of tuberculosis. Genetic diversity can also occur during a chronic infection and can lead to mutants that are particularly well adapted to the host; these may be more virulent or more able to persist. Evgeni Sokurenko (University of Washington, Seattle, USA) addressed this important phenomenon by describing a method that can identify point mutations in total genomic DNA and is suitable for the analysis of genetic variability in bacteria isolated from infected tissues.

Genomics now and in the foreseeable future will both require and spawn novel analytical, data-mining and sequencing tools. Sung-Hou Kim (University of California, Berkeley, USA) forcefully admonished attendees that a crucial distinction exists between protein functions that are predicted from homology searches of databases, the proven role of a protein within a cellular pathway, and its molecular function as discerned by structure-function analysis. This is not merely the gap between genomics and proteomics - and the need to bridge it - as the latter term generally refers only to the 'proteome', that is, the complete protein expression capacity of the genome. Rather, as discussed by Kim, it will probably require innovative use of the rapidly expanding crystal-structure database to compile an encyclopedia of all protein folds and the development of tools that identify the set of folds that comprise the inferred protein products of newly sequenced genomes.


abstract: Macrophages play an important role in modulating the immune function of the human body, while foam cells differentiated from macrophages with subsequent fatty streak formation play a key role in atherosclerosis. We hypothesized that nanotopography modulates the behavior and function of macrophages and foam cells without bioactive agent. In the present study, nanodot arrays ranging from 10‐ to 200‐nm were used to evaluate the growth and function of macrophages and foam cells. In the quantitative analysis, the cell adhesion area in macrophages increased with 10- to 50-nm nanodot arrays compared to the flat surface, while it decreased with 100- and 200-nm nanodot arrays. A similar trend of adhesion was observed in foam cells. Immunostaining, specific to vinculin and actin filaments, indicated that a 50-nm surface promoted cell adhesion and cytoskeleton organization. On the contrary, 200-nm surfaces hindered cell adhesion and cytoskeleton organization. Further, based on quantitative real-time polymerase chain reaction data, expression of inflammatory genes was upregulated for the 100- and 200-nm surfaces in macrophages and foam cells. This suggests that nanodots of 100‐ and 200‐nm triggered immune inflammatory stress response. In summary, nanotopography controls cell morphology, adhesions, and proliferation. By adjusting the nanodot diameter, we could modulate the growth and expression of function-related genes in the macrophages and foam cell system. The nanotopography-mediated control of cell growth and morphology provides potential insight for designing cardiovascular implants.
author: Mohammed Mohiuddin; Hsu-An Pan; Yao-Ching Hung; Guewha Steven Huang
date: 2012
institute: 1Department of Material Science and Engineering, National Chiao Tung University, 1001 University Road, Hsinchu 300, Taiwan; 2Section of Gynecologic Oncology, Department of Obstetrics and Gynecology, China Medical University and Hospital, 91 Hsueh Shih Road, Taichung 404, Taiwan; 3College of Medicine, China Medical University, Taichung 404, Taiwan
references:
title: Control of growth and inflammatory response of macrophages and foam cells with nanotopography

# Background

Recent fabrication of nanostructured materials with different surface properties has generated a great deal of interest for developing implant materials, i.e., cardiovascular, dental, orthopedic, percutaneous, subcutaneous, and auditory \[1-5\]. The interface between nanostructured materials and biological tissues is likely to vary dependent upon the surface properties of the nanomaterial. Understanding the degree of toxicity induced by the unique cellular interaction of nanostructured materials is a major concern before utilization in biomedical applications \[6-8\]. Therefore, fabricating biocompatible materials which are designed to perform specific functions within living organisms has become a key component for generating nanodevices for biomedical applications, including implants.

Macrophages play a critical role during innate and acquired immune responses through the phagocytosis of foreign material. During an immune response, macrophages are typically the first cell type to respond and will secrete proteins (cytokines and chemokines) in order to recruit more immune cells to the site of injury. Atherosclerosis is a pathological process that takes place in the major arteries and is the underlying cause of heart attacks, stroke, and peripheral artery disease. The earliest detectable lesions, called fatty streaks, contain macrophage foam cells that are derived from recruited monocytes. The formation of these foam cells correlates to inflammatory responses \[9-11\]. In particular, immune cells such as monocytes and macrophages play a key role in mediating host tissue response to implants in the foreign body reaction.One study demonstrated that the macrophage receptor with collagenous structure (MARCO) displayed limited expression in healthy cells but increased in expression around the synovial fluid following hip replacements \[12\]. This study indicated that the presence of a foreign body can generate an immune response, and the continued presence of the foreign body can potentially lead to macrophage buildup and production of foam cells.

Recent reports have shown that microscaled landscapes are able to direct shape and migration of cultured cells. When cultured on ridges and grooves of nanoscale dimensions, cells migrate more extensively to the ridges than into the grooves. Cell shape is aligned and extended in the direction of the groove \[13\]. Osteoblasts grown on a fibrous matrix composed of multiwalled carbon nanofibers (100-nm in diameter) exhibit increased proliferation compared to those on flat glass surfaces \[14-16\]. Nanodots larger than 100-nm in diameter induced an apoptosis-like morphology for NIH-3T3 fibroblast cells \[17\]. Breast epithelial cells proliferate and form multicellular spheroids on interwoven polyamide fibers fabricated using electrospinning polymer solution onto a glass slide \[18\]. A 3-D nanofibrillar surface covalently modified with tenascin-C-derived peptides enhances neuronal growth *in vitro*\[19\]. The cardiomyoblast H9c2 shows induced cell adhesion and cytoskeleton organization on nanodot arrays smaller than 50-nm \[20\].

Recently, arrays of nanodots with defined diameter and depth have been fabricated using aluminum nanopores as a template during oxidation of tantalum thin films \[21\]. The pore size of aluminum oxide is controllable and uniformly distributed; the depth of dots depends on the voltage applied; thus, it can serve as a convenient mold to fabricate tantalum into a nanodot array of specific diameter and depth. The structure containing nanodots of uniform size could serve as a comparable nanolandscape to probe cellular response at the molecular level. Although many implant surface topographies are commercially available, there is generally a lack of detailed comparative histological studies at the nano-interface that document how these surfaces interact with living cells, in particular immune cells. In the present study, different sizes of nanodot arrays ranging from 10- to 200-nm were used to evaluate the growth and inflammatory response of macrophages and foam cells.

# Methods

## Chemicals

Dulbecco's modified Eagle medium (DMEM), FBS, antibiotics, and all other tissue culture reagents were obtained from Gibco (Life Technologies, Carlsbad, CA, USA). Glutaraldehyde and osmium tetroxide were purchased from Electron Microscopy Sciences (Hatfield, PA, USA). Anti-vinculin mouse antibody was purchased from Abcam (Cambridge, MA, USA). Alexa Fluor 594 phalloidin and Alexa Fluor 488 goat anti-mouse IgG were purchased from Invitrogen (Carlsbad, CA, USA). Trypsin was purchased from Sigma-Aldrich (St. Louis, MO, USA). CuSO4, KBr, thiobarbituric acid, trichloroacetic acid, and other commonly used chemicals were purchased from Sigma-Aldrich or Merck (Whitehouse Station, NJ, USA).

## Isolation of mouse peritoneal macrophages and formation of foam cells

Resident peritoneal macrophages were isolated and cultured from five mice (20 g each) and were washed once with DMEM and once with DMEM containing 10% fetal bovine serum. The cells were seeded on to different nanodot arrays, ranging from 10- to 200-nm, in DMEM containing 10% fetal bovine serum and 100 pg/ml penicillin and cultured for 3 to 4 h at 37°C in an incubator containing 5% CO~2~ with 90% humidity. The nonadherent cells were removed, and the monolayers were then placed in DMEM containing 10% fetal bovine serum supplemented with 100 μg/ml oxidized low-density lipoprotein (ox-LDL) or acetyl LDL; plates were further incubated for an additional 72 h.

## Oil red O staining

Monolayers of foam cells prepared on nanodot surface were fixed with 10% formaldehyde in phosphate-buffered saline (PBS) (pH 7.4) for 10 min at room temperature, then stained with Oil Red O, and counterstained with hematoxylin for 10 min \[22\].

## Fabrication and characterization of nanodot arrays

Nanodot arrays were fabricated by anode aluminum oxide processing as described previously \[21\]. A tantalum nitride (TaN) thin film of 150-nm thickness was sputtered onto a 6-in. silicon wafer followed by deposition of a 3-μm-thick aluminum onto the top of a TaN layer. Anodization was carried out in 1.8 M sulfuric acid at 5 V for the 10-nm nanodot array or in 0.3 M oxalic acid at 25, 60, and 100 V for 50-, 100-, and 200-nm nanodot arrays, respectively. Porous anodic alumina was formed during the anodic oxidation. The underlying TaN layer was oxidized into tantalum oxide nanodots using the alumina nanopores as a template. The porous alumina was removed by immersing in 5% (*w*/*v*) H~3~PO~4~ overnight.

The dot diameters were 15 ± 2.8-nm, 58.1 ± 5.6-nm, 95.4 ± 9.2-nm, and 211.5 ± 30.6-nm for the 10-, 50-, 100-, and 200-nm dot arrays. The average heights were 11.3 ± 2.5-nm, 51.3 ± 5.5-nm, 101.1 ± 10.3-nm, and 154.2 ± 27.8-nm, respectively. Dot-to-dot distances were 22.8 ± 4.6-nm, 61.3 ± 6.4-nm, 108.1 ± 2.3-nm, and 194.2 ± 15.1-nm, respectively. The dimension and homogeneity of nanodot arrays were measured and calculated from images taken using a JEOL JSM-6500 TFE-SEM (JEOL Ltd., Akishima, Tokyo, Japan).

## The cell viability assay

Cells were harvested and fixed with 4% formaldehyde in PBS for 30 min followed by PBS wash for three times. The membrane was permeated by incubating in 0.1% Triton X-100 for 10 min, followed by PBS wash for three times. The sample was incubated with 4′,6-diamidino-2-phenylindole (DAPI) and phalloidin for 15 min at room temperature. The samples were mounted and imaged using a Leica TSC SP2 confocal microscope (Leica Microsystems Ltd., Milton Keynes, UK). The number of viable cells was counted using ImageJ software (National Institutes of Health, Bethesda, MA, USA) and expressed in terms of cell density.

## Scanning electron microscopy

The harvested cells were fixed with 1% glutaraldehyde in PBS at 4°C for 20 min, followed by post-fixation in 1% osmium tetroxide for 30 min. Dehydration was performed through a series of ethanol concentrations (10-min incubation each in 50%, 60%, 70%, 80%, 90%, 95%, and 100% ethanol) and air-dried. The specimen was sputter-coated with platinum and examined using a JEOL JSM-6500 TFE-SEM at an accelerating voltage of 5 keV.

## Immunostaining

Cells were harvested and fixed with 4% paraformaldehyde in PBS for 15 min followed by PBS wash for three times. Cell membrane was permeated by incubating in 0.1% Triton X-100 for 10 min, followed by PBS wash for three times, and blocked by 1% bovine serum albumin (BSA) in PBS for 1 h and PBS wash for three times. The sample was incubated with anti-vinculin antibody (properly diluted in 1% BSA) and phalloidin for 1 h, followed by incubation with Alexa Fluor 488 goat anti-mouse antibody for 1 h, and then followed by PBS wash for three times. Samples were mounted and imaged by using a Leica TSC SP2 confocal microscope.

## Quantitative real-time RT-PCR

Total RNA was extracted from macrophages and foam cells using TRI-reagent (Talron Biotech, Rehovot, Israel) according to the manufacturer's specifications. The RNA was isolated using chloroform extraction and isopropanol precipitation. The crude RNA extract was immediately purified with an RNeasy Mini Kit (Qiagen, Venlo, Netherlands) to remove impurities and unwanted organics. Purified RNA was resuspended in DEPC water and quantified by OD~260~. The OD~260~ to OD~280~ ratio usually exceeded 2.0 at this stage. For cDNA synthesis, 1 μg of total RNA was annealed with 1 μg of oligo-dT primer, followed by reverse transcription using Super Script® III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) in a total volume of 50 μl. Between 0.2 and 0.5 μl of the reverse transcription reactions was used for quantitative polymerase chain reaction (qPCR) using SYBR Green I on an iCycler iQ5 (Bio-Rad Laboratories, Hercules, CA, USA). Cycling conditions were as follows: 1× (5 min at 95°C) and 50× (20 s at 95°C, 20 s at 55°C, and 40 s at 72°C). Fluorescence was measured after each 72°C step. Expression levels were obtained as threshold cycles (Ct), which were determined by the iCycler iQ Detection System software (Bio-Rad Laboratories, Hercules, CA, USA). Relative transcript quantities were calculated using the ΔΔCt method. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as a reference gene and was amplified from the same cDNA samples. Due to the difference in threshold cycles of the sample mRNA relative to the GAPDH, mRNA was defined as ΔCt. The difference between the ΔCt of the untreated control and the ΔCt of the SMF-treated sample was defined as ΔΔCt. The fold change in mRNA expression was expressed as 2^ΔΔCt^. The results were expressed as the mean ± standard deviation (SD) of six experiments.

# Results and discussion

## Nanotopography-modulated morphology and cell spread of macrophages and foam cells

To characterize how the macrophages and foam cells interact with the aforementioned nanodot arrays, the cells were cultured for 72 h on nanodot arrays of different diameters including flat substrate as a control. The morphological appearance of adhered cells was imaged by both optical image microscopy and scanning electron microscopy (SEM) (Figures 1 and 2). Macrophages and foam cells grown on the flat surface and 10-nm nanodot arrays exhibited flat and extended conformation during the course of 3 days. Cells grown on the 50-nm nanodot arrays showed more extended morphology than those on the flat surface with apparently larger surface area for each cell. Cells grown on the 100-nm nanodot arrays exhibited a distorted morphology with shrinking surface area and increased length of lamellipodia. Apoptosis-like appearance and reduction in surface area with extended lamellipodia were seen with cells seeded on the 200-nm nanodot arrays.

Formation of focal adhesions reflected by the attachment of filopodia and lamellipodia to the substratum indicates healthy growth for cultured cells. SEM images showed that the lamellar body of migrating cells, seeded on 50-nm nanodot arrays, exhibited wide and thick characters with a large number of filopodia (Figure 2). Cells seeded on flat and 10-nm nanodot arrays showed comparable lamellipodia. However, the cells seeded on 100- and 200-nm nanodot arrays were mounted with extended length and narrow-size lamellipodia. Figure 3a shows the correlation between cell spread area and dot size for macrophages and foam cells . Cell surface area representing the percent adhesion area of viable cells relative to cells cultured on a flat surface was calculated and plotted against the nanodot diameter. Based on quantitative analysis, the cell spread area of macrophages increased significantly between 21.6% and 37.9% with increasing dot sizes of 10- to 50-nm, respectively. Interestingly, as dot diameter increased from 100- to 200-nm, there was a significant reduction in cell surface area of 22.7% and 43.2%, respectively. A similar biphasic trend, increasing cell surface area from 10- to 50-nm and decreasing from 100- to 200-nm, was observed with foam cells. This trend of change correlated with qualitative analysis as shown in Figure 1. Although the precise reason for such differential growth pattern is not known, since increasing the dot diameter above 100-nm stimulates an apoptosis-like growth and a significant reduction in the surface area, these results demonstrate that nanodot arrays larger than 100-nm are less biocompatible to cells.

## Nanotopography-modulated cell adhesion and cytoskeleton organization of macrophages and foam cells

To evaluate cell adhesion and cytoskeleton reorganization, immunostaining specific to vinculin and actin filaments was performed on nanodot arrays (Figure 4). The amount of vinculin staining in foam cells was significantly less than that in macrophages for all nanodot sizes. Foam cells had fewer focal adhesion molecules than macrophages when grown on nanodot arrays and might have the destiny of apoptosis. Vinculin staining was well distributed for macrophages grown on the flat surface and on the 10- to 100-nm nanodot arrays, with the highest density of vinculin for cells grown on 50-nm nanodot arrays. Nevertheless, the amount of vinculin staining decreased for cells grown on 200-nm nanodot arrays (Figure 4). For foam cells, vinculin staining had the same trend as that for macrophages: increasing from flat to 50-nm nanodot arrays, becoming gradually lost for cells grown on 100-nm nanodot arrays, and completely disappearing for 200-nm nanodot arrays.

Immunostaining of actin filaments indicated a well-organized cytoskeleton in macrophages grown on flat, 10-, and 50-nm nanodot arrays, but it is gradually lost for 100-nm nanodot arrays and has completely disappeared for 200-nm nanodot arrays. For foam cells, cytoskeleton arrangement had the same trend as macrophages: increasing from flat to 50-nm nanodot arrays, becoming gradually lost for cells grown on 100-nm nanodot arrays, and completely disappearing for 200-nm nanodot arrays.

Immunostaining indicated that nanodot arrays in the range of 10- to 100-nm promoted cell adhesion and cytoskeleton organization for macrophages and foam cells (Figure 3b). Best adhesion occurred at 50-nm nanodots, whereas nanodots of 200-nm retarded the formation of focal adhesions and inhibited the organization of the cytoskeleton.

## Nanotopography-modulated cell density

To evaluate the viability of macrophages and foam cells on varied nanodot arrays, cells were seeded on nanodot arrays, ranging from 10- to 200-nm including flat control. Macrophages and foam cells were cultured for 72 h, and then, DAPI staining was performed to verify viable cells on each nanodot array and flat surface (Figure 3c). For macrophages, compared to the flat surface, there were 10.6%, 149.1%, and 26.5% increases in the number of viable cells for 10-, 50-, and 100-nm nanodot arrays, respectively, but 41.2% reduction was observed on 200-nm nanodot arrays. For foam cells, a 110% increase in the number of viable cells was observed for 50-nm nanodot arrays, and 28.6% reduction occurred for 200-nm nanodot arrays.

## Effect of nanotopography on the expression of genes related to inflammation and circulatory repair

The cytokine gene expression profiles related to inflammation of macrophages and foam cells cultured on nanodot arrays were measured at 72 h, using qPCR. The cytokines examined were TNF-α, IL-6, IL-10, CCL-2, and CCL-3 (Figure 5a,b). In addition, genes important for the development of the circulatory system and repair were also evaluated (Figure 5c,d): PAI-1, VEGF, and PECAM.

TNF-α is a pro-inflammatory cytokine and is responsible for activation and positive regulation of the NFκB pathway, which is a key regulator of the immune response \[23\]. There was a five-fold increase in TNF-α found at 50-nm nanodot arrays in macrophages, while a five- to six-fold increase was demonstrated in foam cells at 50- to 100-nm nanodot arrays (Figure 5a,b). Similarly, IL-6 encodes a pro-inflammatory cytokine that is critical for activating an acute inflammatory response and is responsible for recruiting adaptive immune cells to the site of injury or infection. There was a significant three-fold increase of IL-6 in macrophages cultured on 200-nm nanodot arrays, while there was about a one-fold increase at 50- to 100-nm nanodot arrays. In contrast, the foam cells responded differently than the macrophages, with a three- to five-fold increase of IL-6 when cultured on 10- to 100-nm nanodot arrays and a two-fold increase for 200-nm nanodot arrays. In comparison, IL-10 is an anti-inflammatory cytokine responsible for blocking the synthesis of pro-inflammatory cytokines and negatively regulates NFκB activation. There was an increasing trend of IL-10 gene expression in the macrophages and the foam cells (Figure 5a,b). For the CCL-2 chemokine, which functions to recruit monocytes and other immune cells, there was no significant change in any of the macrophage topography conditions. However, in foam cells, there was a 2.5-fold increase at 200-nm nanodot arrays, while there was less than one-fold increase at 10- to 100-nm nanodot arrays (Figure 5b). Another common chemokine is CCL-3. This protein is responsible for recruiting and activating leukocytes to aid in an immune response. Figure 5a showed less than 1% increase in CCL-3 in macrophages at 10- to 200-nm nanodot arrays, while a six-fold increase at 200-nm nanodot arrays in foam cells was observed (Figure 5b).

In addition to the immune response, genes associated with circulatory repair were also investigated. PAI-1 is a gene involved in the prevention of blood clots. There was a five-fold increase in PAI-1 observed at 200-nm nanodot arrays in macrophages (Figure 5c), whereas foam cells displayed an inconsistent pattern of increase of PAI-1 (Figure 5d). Furthermore, VEGF is known to play a key role in the creation of new blood vessels during development as well as repair during injury. There was an inconsistent increase in VEGF for the macrophages, while foam cells demonstrated a significant increase at the 50-nm topography (Figure 5d). PECAM is responsible for removing old neutrophils from the site of injury and preventing buildup of immune cell debris. In the macrophages, there was no change in PECAM genes; however, the foam cells displayed significant increases for the 50- to 200-nm topographies. In summary, the differences in relative gene expressions shown here are topography induced and normalized to flat surface.

When evaluating the gene expression trends observed in the macrophages, there was a highly significant increase in TNF-α expression for the 50-nm topography. For all of the topographies, there was limited impact on the expression of CCL-3 and CCL-2, whereas, there was a mild elevation in IL-6 expression. Taken together, there was a limited induction of an inflammatory response for these topographies. The expression of PAI-1, VEGF, and PECAM were also evaluated, and there were low levels of expression for all genes in all topographies except for the 200-nm topography, which displayed significant increases in PAI-1 expression. Since PAI-1 helps to prevent blood clots and the buildup of cells, in conjunction with data from the gene expression studies where limited immune responses were observed, the 200-nm topography might assist in the prevention of foam cell formation.

When evaluating the foam cells directly, there were high levels of the early-response pro-inflammatory cytokines IL-6 and TNF-α for the 50- and 100-nm topographies with mild increases in expression for the 10- and 200-nm topographies. Furthermore, the 200-nm topography demonstrated high levels of expression for the pro-inflammatory cytokine CCL-3. In addition, when the circulatory repair genes were assessed, the 100-nm topography demonstrated a significant increase in the PECAM gene which functions to remove leukocyte debris from cells. Since foam cells are derived from macrophages that are not cleared from the system, this 100-nm topography could greatly aid in the prevention or removal of foam cells from the body.

After 72 h, macrophages showed an increase in inflammatory gene expression on 10- and 200-nm nanodot arrays, while the difference was not significant compared to the flat. Foam cells showed the most inflammatory gene expression on 200-nm nanodot arrays. The common acute inflammation gene expression of CCL-3 responded significantly to the topography of 100- and 200-nm nanodot arrays for foam cells. However, the macrophages showed the most acute inflammation on the 10-nm nanodot arrays. Thus, the topographical effect on the PAI-1 gene expression was difficult to discern.

Recently, it has been shown that three-dimensional surface topography (size, shape, and surface texture) is one of the most important parameters that influence cellular reactions \[24,25\]. Other studies have demonstrated that the difference in cellular response correlates with a modulation of the concentration of serum proteins on the surface \[26,27\]. Many studies have shown that cell biomaterial interactions can activate macrophages which results in the synthesis of pro-inflammatory agents such as TNFα, IFNγ, IL-1, and IL-6 \[28,29\] Most likely, the surface properties, such as material surface chemistry and topography, can modulate the expression of pro-inflammatory cytokines and chemokines by macrophages in a time-dependent manner \[30\].

Although many studies have investigated cellular reaction to different surface patterns, the behavior of immune cells, such as macrophages and foam cells, cultured on different diameters of nanodots has not been studied thoroughly. Our study suggests that topography may modulate the phenotypes of macrophages and foam cells in the context of foreign body response. The response to topography in the form of nanodot arrays in the range of 10- to 200-nm has revealed a distinctive pattern, and topography indeed affects cell morphology, density, adhesion, and cytokine expression compared to flat controls. The changes in cell morphology are observed in four different sizes of nanodot arrays, indicating that the findings in this study are topography-mediated. Using topography-induced change in macrophages and foam cell behavior, it is possible to influence phenotypic response, such as cell activation, motility, and maturation in the foreign body response. While there is a mild induction of the inflammatory response for the 100- and 200-nm topographies, these growth conditions also supported expressions of genes that would be responsible in the prevention of foam cell formation and the removal of foam cells, suggesting potential benefits. Furthermore, it might be possible to treat the cells with antioxidants or other anti-inflammatory mediators to prevent the inflammatory response while benefiting from the increase in PAI-1 and PECAM.

# Conclusion

We have shown topologic modulation of cell growth, cell density, cell spreading area, and immune functions. Our results demonstrated that 50-nm nanodots displayed a biocompatible surface compared to 100- and 200-nm nanodots in terms of macrophage and foam cell growth. In addition, based on qPCR data, 100- and 200-nm surface-induced inflammatory gene expression in macrophages and foam cells suggest that nanostructured materials (100- and 200-nm) trigger the immune inflammatory stress response. The role of topography in modulating implant tissue reaction would require further elucidation. This study suggests that nanotopography may be beneficial for the design of cardiovascular implants.

# Competing interests

The authors declare that they have no competing interests.

# Authors' contributions

MM and HAP are responsible for concept and design. MM carried out the experiments, analyzed the data, and wrote the paper under the close supervision of YCH and GSH. All authors read and approved the final manuscript.

# Authors' information

MM and HAP are doctoral degree students at the Department of Material Science and Engineering, National Chiao Tung University. GSH is a professor at the Department of Material Science and Engineering, National Chiao Tung University. YCH is a professor at the Department of Obstetrics and Gynecology, China Medical University and Hospital and also professor at the College of Medicine, China Medical University.

## Acknowledgment

This study was supported in part by the National Science Counsel Grant 100-2923-B-009-001-MY3 and by the 'Aim for the Top University Plan of the National Chiao' Tung University and Ministry of Education, Taiwan, R.O.C.


abstract: A report of the seventh annual meeting on Systems Biology: Global Regulation of Gene Expression, 23-27 March 2010, Cold Spring Harbor, USA.
author: Manolis Kellis; John L Rinn
date: 2010
institute: 1Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA; 2Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; 3Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA; 4Department of Pathology, Harvard Medical School, Boston, MA 02115, USA
title: Sequences to systems

# Meeting report

From a chemical standpoint, a cell can seem as a densely intertwined mess of molecules, but their emergent systems-level properties exhibit structure, coordination and order, leading to the orchestrated complexity we call life. As the seventh annual meeting on Systems Biology: Global Regulation of Gene Expression at the Cold Spring Harbor Laboratory showcased, systems biology aims to bridge the vast gap of knowledge between the pairwise molecular interactions of individual cellular components and the large-scale behavior of cellular circuits that result from their synergism. The talks given at the meeting exemplified a new definition of systems biology, where genomic technologies can revolutionize our understanding of classical problems by enabling molecular interactions to be linked to systems behavior in a mechanistic way.

A large number of definitions have been proposed for systems biology, including 'the study of a single pathway by someone with an attention-deficit disorder' humorously offered by Irv Bernstein and quoted in a keynote address by Steve Henikoff (Fred Hutchinson Cancer Research Center, Seattle, USA). Henikoff went on to demonstrate that Trithorax and Polycomb response elements that trigger cellular differentiation could be recognized by their patterns of histone turnover. He argued that histone variant H2A.Z is mutually exclusive with DNA methylation, drawing a novel connection between histones, their dynamics and the epigenetic inheritance of DNA methylation.

The importance of epigenetic information in mediating the stable inheritance of cellular states was underscored in the context of human disease by the second keynote speaker, Kevin Struhl (Havard Medical School, Boston, USA). By dissecting the transcriptional circuits responding to Src signaling that can yield stably transformed cells, Struhl uncovered a feedback loop mediated by the transcription factor NFκB that is sustainable for many generations in the absence of further signaling. Struhl went further to demonstrate that this pathway is targetable using FDA-approved drugs, opening up novel avenues of cancer therapeutics.

# Parsing the genome

The study of any system begins with a comprehensive list of parts and their interactions, and so a major theme of the meeting was the mapping of the building blocks of gene regulation: the control regions, the DNA words within them and their sentence structure.

On one hand, several talks focused on defining sites of transcriptional factor binding, histones and sites of DNA methylation at ever-increasing resolution and complexity. For example, two talks by Barbara Wold (California Institute of Technology) and Stephen Tapscott (Fred Hutchinson Cancer Research Center) presented data revealing thousands of binding sites for the master regulator of muscle differentiation MyoD, concluding that numerous additional functions and or combinatorics result in cell-fate decisions such as muscle differentiation. One of us (MK), presented a new algorithm specifically for revealing biologically meaningful combinations of chromatin marks, and their corresponding chromatin states, that reflect rich information about the underlying biological function of the associated genomic regions. The algorithms have distinguished more than 50 chromatin states, including 11 different classes of promoter regions, associated with different functional categories, and several classes of enhancer regions associated with different expression levels for downstream genes.

On the other hand, several talks focused on the DNA sequences predictive of regulatory activity. Eric Mendenhall (Massachusetts General Hospital, Boston, USA) presented remarkable data that distinct types of DNA sequences can recruit key chromatin factors such as Polycomb. Through multiple transgenic studies Mendenhall showed that he could insert DNA sequence motifs, even those 'that have never seen Polycomb before', into the mouse genome that subsequently recruited Polycomb binding, suggesting a model of sequence-based recruitment of this complex. Eileen Furlong (European Molecular Biology Laboratory, Heidelberg, Germany) demonstrated the importance of combinatorial binding events in determining cell fate decisions, using a support vector machine to model transcription factor binding data and reveal the *cis*-regulatory motifs and motif combinations that define mesodermal fate in *Drosophila melanogaster*. A major conclusion was clear evidence that a single expression pattern could result from several different combinatorial codes of transcription factors and their dynamics. Arend Sidow (Stanford University, Stanford, USA) presented interesting insight into what determines nucleosome occupancy during the differentiation of distinct blood cell types both *in vitro* and *in vivo*. He presented clear evidence that *in vitro* nucleosomes associate with sequence-specific motifs, such as GC motifs, and exclude others, such as AT sites, whereas *in vivo* these sequence preferences are largely overridden. Moreover, Sidow demonstrated that nucleosome positioning is globally remodeled across different states of cellular differentiation.

Collectively these studies demonstrate the power of chromatin-level and DNA-level information for predicting regulatory activity, the importance of word combinations and mark combinations, and the interplay of static sequences and dynamic cell-type specific changes in defining the processes of differentiation and, more generally, gene regulation.

# Unconventional interactions of the secondary type

The late Ed Lewis famously said 'Progress will still need to be driven by the logic of genetics and by further increases in abstraction'. This sentiment was echoed at this year's meeting, most prominently in the unveiling of numerous unconventional protein-DNA and protein-RNA interactions.

A dramatic example was presented by Heng Zhu (Johns Hopkins Medical Center, Baltimore, USA), who has used human proteome arrays to map global DNA-binding interactions. His analysis identified a striking and atypical interaction between kinases and DNA. Specifically, Zhu identified a carboxy-terminal domain of the kinase ERK2 that binds to DNA with nanomolar affinity and through this interaction serves as a transcriptional repressor. He also presented initial evidence for more than 300 unconventional DNA-binding proteins, including unusual suspects such as RNA-binding proteins and mitochondrial proteins.

Howard Chang (Stanford University, Stanford, USA) presented a comprehensive study demonstrating modular binding of the large intergenic noncoding RNA (lincRNA) HOTAIR to chromatin-modifying complexes such as PRC2, the histone demethylase LSD1, and COREST, a DNA-binding protein. Chang presented a model, based on functional assays, whereby HOTAIR binds all three of these unconventional RNA-binding proteins through a different specific structured domain and in this way can guide them specifically to their target genomic loci. Thus, lincRNAs could serve as adaptors to facilitate the formation of specific RNA-protein interactions. One of us (JR) presented evidence that such mediation is a global phenomenon that extends beyond HOTAIR and includes hundreds of lincRNAs interacting with chromatin-modifying complexes and other DNA-binding proteins involved in cancer. Overall, the genome could be replete with unorthodox RNA-binding interactions serving as RNA adaptors to facilitate the establishment and or maintenance of specific epigenetic states. Tom Gingeras (Cold Spring Harbor Laboratory, New York, USA) expanded the horizons of unorthodox RNA functions, presenting a rich diversity of noncoding RNA molecules that localize to distinct cellular compartments and are derived from unconventional processes, emphasizing the further need for abstraction in our understanding of the mammalian transcriptome and the potential regulatory roles of noncoding RNAs.

# Close encounters of the three-dimensional type

At this year's meeting the genome came to life in three dimensions, as a result of 'proximity ligation' techniques that define the three-dimensional neighbors of a given genomic sequence relative to all others in the genome. This reveals potentially important regulatory interactions mediated by DNA looping and compartmentalization.

Job Dekker (University of Massachusetts, Worcester, USA) described work using several variants of chromatin-conformation capture (3C) to reveal these interactions. Dekker and his colleagues have used carbon-copy 3C (5C) to resolve the three-dimensional structure of the beta-globin locus in both the activated and repressed states. He proposed a model by which a large genomic-linker region serves to open up or compact the locus upon activation or repression, respectively. Erez Lieberman-Aiden (Broad Institute, Cambridge, USA) has expanded the technique to take on the entire genome, and reported that megabases of DNA fold into a dense, highly organized, and knot-free structure known as a 'fractal globule', thus providing a glimpse of the three-dimensional genome.

Using a different proximity ligation approach called ChIA-PET, Yijun Ruan (Genome Institute of Singapore, Singapore) has revealed three-dimensional genomic architecture in the context of transcriptional regulation. Ruan specifically demonstrated tertiary DNA interactions mediated by transcription factors and RNA polymerase II. An interesting connection to this finding was presented by Martin Hemberg (Children's Hospital, Harvard Medical School, Boston, USA) where he described that numerous enhancers, which often result from long-range DNA interactions, are associated with RNA polymerase II and are transcribed at low levels.

Collectively these studies have added a new dimension to systems biology and provide novel insights into the three-dimensional architecture of the human genome.

# Systematic evolution

The evolutionary dimension of gene regulation was addressed in regard to the selective pressures and plasticity of regulatory regions and elements. Ollie Rando (University of Massachusetts, Worcester, USA) presented an evolutionary analysis of nucleosome positioning in 12 ascomycete species. Perhaps most notably, the nucleosome positioning corresponded to specific gene sets, such as mitochondrial genes or splicing genes, and differed in physiologically relevant ways in different species.

Continuing the theme of evolution and binding localization, Mike Snyder (Stanford University, Stanford, USA) presented an unprecedented dataset comprising the binding sites for several key transcription factors across ten different human individuals. Snyder showed that genetic variation between individuals had resulted in the loss and gain of binding sites for key transcriptional players such as RNA polymerase II and the transcription factor NFκB. Thus, individual variation results in unique binding patterns of key factors, which should provide potential insights for personalized medicine and disease susceptibility.

Alexander Stark (IMP, Vienna, Austria) presented comparative work on the DNA binding of transcriptional regulators of mesoderm development in six *Drosophila* species at a range of evolutionary distances from each other. He showed that not only were the locations of the peaks representing binding-site occupancy conserved, but also the intensity of the peaks, suggesting that lower-occupancy sites are also functional and are selectively maintained. He presented evidence that these sites were subsequently occupied at later developmental stages, suggesting that lower occupancy may be associated with priming for subsequent activation.

# Cell circuits and dynamics

The ultimate goal in systems biology is to define cellular circuits and their resulting cellular outputs. This year's meeting reported unprecedented progress in mapping cell circuits in response to specific stimuli, perturbing them and monitoring their cellular responses. A comprehensive example was presented by Aviv Regev (Broad Institute, Cambridge, USA), who described work with a model mammalian cell system - mouse dendritic cells - which trigger distinct inflammatory or antiviral responses depending on the input stimuli. Regev's strategy was to monitor the complete transcriptional response to each stimulus, identify the key regulators (transcription factors, chromatin-modifying complexes and RNA-binding proteins) in each pathway, and then individually perturb the actions of hundreds of these regulators via stable gene knockdown and monitor the resulting transcriptional changes. This not only identified numerous new regulators across these critical cellular pathways, but also revealed the cooption of cell-cycle circuits in the antiviral response. This approach to systems biology could also be applied to numerous other cell-based models.

Underlying seemingly stable cell circuits are multiple additional layers of modulation, which include both stochastic single-cell variability in transcript levels and regulated processes of post-transcriptional inhibition and splicing. Alexander Van Oudenaarden (Massachusetts Institute of Technology, Cambridge, USA) demonstrated that cell-fate decisions in *Caenorhabditis elegans* intestinal specification arise from an underlying process of variable transcription levels. These stochastic processes resulted in threshold-dependent ON or OFF decisions of master regulators that guide cellular differentiation. David Bartel (Whitehead Institute, Cambridge, USA) described how the dramatic effects of post-transcriptional regulation by microRNAs (miRNAs) can result from even the slightest fine-tuning of transcript levels and translation efficiency. In a potential reversal of the dominant dogma in miRNA regulation, he also presented evidence from mammalian cells that for two miRNAs at least, the major effect of targeting appears to be transcript degradation rather than translational inhibition. Chris Burge (Massachusetts Institute of Technology, Cambridge, USA) presented studies that revealed a remarkable complexity in mRNA transcript splicing and how these isoforms are altered in cancer.

Collectively, this year's meeting revealed new insight into genomic sequences, binding sites and structures that has moved systematic study of gene regulation to new dimensions of both understanding and further abstraction in systems biology.


abstract: A look into the future of a biologist: daily activities governed by presidential mandates and acts.
author: Gregory A Petsko
date: 2007
institute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA
title: A day in the life of a genome biologist in the not-too-distant future

**Megalopolis, United States of Western Hemisphere, 18 June 2273.** The following fragment was uncovered in an underwater archaeological dig, on the site now generally believed to be the location of the almost mythical engineering school, Screwloose La Tech. Ever since global warming led to the flooding of the entire Eastern Seaboard of North America in 2138, artifacts from the Cambrian Period, as the period of the great universities in Cambridge is called, have been very hard to come by. This document is therefore of great historical significance. Nothing is known about the writer, except that similarities of style suggest that he may actually have been the same as the master of the *Genome Biology* columns, whose work has recently been challenged as fraudulent.

Pursuant to Federal Paperwork Reduction Act 3,671 of 2016, herewith is submitted daily activities report for Saturday, 3 June 2030.

**7:44 am to 8:02 am.** Arrived shuttle parking lot number 4, located 11.25 miles from campus. Turned off motor on electric car and detached mandatory passive restraint devices (seat belt, shoulder harness, neck brace, ankle restraints and wrist locks) before exiting. Plugged battery cord into recharger built into parking meter. Inserted \$255 in five dollar coins to cover daily parking and battery charging costs. \[Personal note: must remember to get more five dollar coins from bank. Ever since university raised off-campus parking fees for faculty to \$41,000 per year, have been lugging around more bags of coins than an armored car.\] Waited 30 minutes for shuttle bus to campus.

**8:46 am to 9:27 am.** Arrived at office. Couldn't unlock door because minor head cold has altered appearance of eyes and eyeball-scanner security device couldn't recognize them. Waited 30 minutes for campus swat team to respond to security alarm and unlock door.

**9:28 am to 11:11 am.** Turned on computer and checked email. Only 1,459 new messages since last night. Ran new Microsoft anti-spam program, which automatically deleted 832 of them, including message from mother. Ran newer Microsoft anti-spam bug fixer, which restored message from mother and other 831 junk messages, plus 204 new junk messages it found from somewhere. Switched to personal examination of messages. Found that only two messages actually were important; all others were spam, including message from mother.

**11:12 am to 11:54 am.** Surprise inspection by agents from Government Office for Toxicity Checking and Hazard Assessment (GOTCHA). Examined all labs-on-chips at every student desk-bench. Found four minor violations (two chips were not dusted and the dust from two others was not properly collected and stored). Ever since scientific responsibility for wrongdoing (SCREW) mandate of 2020, signed into law by President George IJK Bush IV, scientist responsible for laboratory must pay fines for any GOTCHA violations immediately from personal funds. Presented debit card to agents for fines of \$4,400 (\$1,100 per violation). Oh well, still a lot better than last week.

**11:55 am to 12:39 pm.** Met with two students from advanced genomic, cell, molecular and psychological neurosociobiology course. Listened to complaints about their midterm grade. Students argued that grade of A+++, well below class average, severely hampered their ability to gain admission to medical school and therefore constituted grievous psychological harm. Threatened to inflict equivalent grievous bodily harm unless I changed grades. Ever since mandatory American national interpersonal armament concealment (MANIAC) statute law, signed by President George LMN Bush V, required that all citizens carry concealed weapons at all times, such student-faculty disputes have become easier to resolve. Started reaching for pocket grenade launcher but then considered that odds of 2-against-1 dictated non-violent response. Changed grades to class average of Super-A+++++++.

**12:40 pm to 12:45 am.** Ate lunch, which as always consisted of trans-fat sandwich on tobacco leaf bread. Paused to reflect how amazing that earlier generations of scientists actually considered this unhealthy - exactly the opposite of what careful research has now shown.

**12:45 pm to 1:56 am.** Reviewed results of latest genome sequencing project in the lab. Sequence of gerbil genome, begun at 12 noon, now complete. Immediately entered file of results into Microsoft Office 2025, which immediately crashed after erasing file. Restored file from backup microdot and relaunched program, which proceeded to write paper describing results, including changing spelling of my name to something it liked better. Attempted to change name back only to be told by the program that it was smarter than me, always would be smarter than me, and that I should go away and let my betters do their job. \[Personal note: visit district court and change spelling of my name to that used by program\].

**1:56 am to 2:39 pm.** Submitted finished manuscript, with Bill Gates III first author, as he is on all papers everywhere, to *Nature Gerbil*, *Gerbil Cell*, *Gerbil Genome Biology*, *PloS Gerbil*, and *Current Gerbil Biology*. (Ever since famous Supreme Court decision in case of J Craig Venter *vs Nature*, *Science* and *Cell*, scientists have had the right to submit their papers simultaneously to as many journals as they like, and to have the same paper published in up to five journals at once.) Since the Harold Varmus overthrow of capitolism (HAVOC) Act of 2011 made it illegal for journals to reject papers or to make a profit, all journals now accept all papers automatically so paper was immediately accepted by all five. Should appear on-line sometime in 2037 if the backlog doesn't get worse.

**2:39 am to 3:55 pm.** Attended mandatory daily sensitivity training workshop, required of all faculty by the Office of Self-Esteem, which has controlled all US universities since the Political Correctness Wars of 2014. Was reminded yet again that any idea, no matter how idiotic, must be respected since to do otherwise would stifle student creativity and self-expression.

**3:55 pm to 4:00 pm.** Thought about meaning of results of research.

**4:00 pm to 6:15 pm.** Realized I had forgotten to check e-mail for several hours. Found 2,651 new messages. Not bad. Deleted all except offer from ex-Nigerian government official who wants to give me \$24 billion in return for promise to think happy thoughts about him. Must look into this tomorrow.

**6:15 pm to 7 pm.** Packed up to go home. Waited 45 minutes for shuttle bus to parking lot. While waiting, checked latest news on iPhone. Still no progress on ending war in Iraq.


date: 2000
title: Draft Heidelberg Collaboration Statement of Purpose (22/9/2000)

*Warning - this is an unedited draft, which will be replaced by a final version mid of October \[will be posted at this URL\].*

Following a meeting convened to discuss the EU-funded MedCERTAIN project on improving information quality on the Internet, a broad group of individuals from 4 continents and 21 countries, drawn from a wide range of sectors including industry, leading Web sites, governmental and intergovernmental bodies, academia, consumer organisations, standards and ethical code bodies and publishers agree that:

1\. Despite the enormous value for the public, patients and professionals of the health and medical Internet, the participants are concerned about its potential for harm. However, it is also important not to limit freedom of expression and use of the internet for health related purposes.

2\. The participants wish to explore in an ongoing collaborative effort opportunities for helping people, patients and professionals to identify health information useful to them, addressing certain key issues, including:

-   What is an appropriate way to describe the characteristics of the full range of Internet health and medical sites ?

-   How can the descriptions be summarised as metadata for different purposes and users of health information ?

-   How do the identity, training and other factors (eg. self reporting vs. external audit) influence the description and summary ?

-   Can criteria be developed to indicate quality for different users and purposes ?

-   How can the potential of information and communications technology, such as the MedCERTAIN project, be harnessed in a consistent and cost-effective way ?

-   What are the legal and other implications of trustmarks and other devices or mechanisms to denote quality ?

-   How to establish and motivate a worldwide, representative network of evaluators?

-   What are the areas of potential overlap with other initiatives and organisations?

3\. The participants resolve to work together, building on experience and expertise of this group, key quality improvement initiatives such as Discern, eHealth ethics code, HONcode, Hi-Ethics code, OMNI and others, and working towards a methodological clarification and resolution of the above and other related issues in the near future, providing constructive support to the MedCERTAIN and other projects and continuing to build together trust on the health Internet.

*Participants:*

Lucas M. Bachmann (Horten Zentrum für praxisorientierte Forschung und Wissenstransfer, Switzerland), Carl R. Blesius (MedCERTAIN, Germany), Markus Blume (mymedia GmbH, Germany), Carl J. Brandt (NetDoktor.com, Denmark), Dan Brickley (MedCERTAIN, United Kingdom), Alejandro Cacherosky (Buenos Aires Health Secretariat, Argentina), Ken Campell (Leukaemia Research Fund, United Kingdom), Richard Cleland (Federal Trade Comission, U.S.A.), Phil Cross (MedCERTAIN, United Kingdom), Elenice de Castro (PAHO/WHO Pan American Health Organization / World Health Organization, Brazil), Guy de Roy (Ordre des Medecins Conseil National, Belgium), Tony Delamothe (BMJ, United Kingdom), Martin D. Denz (Swiss Medical Informatics Association / Universitätsspital Zürich, Switzerland), Persephone Doupi (Erasmus University, Netherlands), Joan Dzenowagis (WHO World Health Organization, Switzerland), Christer Edling (The Swedish Society of Medicine, Sweden), Gunther Eysenbach (University of Heidelberg / MedCERTAIN, Germany), Gerard Freriks (TNO, Netherlands), Franz Frühwald (Internationales Büro der Österreichischen Ärztekammer, Austria), Lisa Gray (BIOME, United Kingdom), Pelle Gustafsson (Swedish Medical Association, Sweden), Gerhard Heine (European Commission, Luxembourg), Katrin Hörner (Arztpartner / Almeda, Germany), Robert Hsiung (University of Chicago, U.S.A.), Jostein Ingulfsen (Norwegian Board of Health, Norway), Thomas Isenberg (German Association of Consumer Organisations - Arbeitsgemeinschaft der Verbraucherverbände, Germany), Edward Jacob (KNMG, Netherlands), Alex R. Jadad (Canadian Cochrane Centre, Canada), Jacobo Kelber (ensalud.net, Mexico), Hugo Kitzinger (mymedia GmbH, Germany), Inge Kokot (Deutsches Grünes Kreuz e.V., Germany), Hans-Joachim Koubenec (Stiftung Warentest, Germany), Michel Labrecque (Université Laval Québec, Canada, Canada), Kristian Lampe (Finnish Office for Health Technology Assessment / MedCERTAIN, Finland), Stephane Lejeune (Swiss Cancer League, Switzerland), Leonard B. Lerer (INSEAD, France), Odile Leroy (PasteurMed, France), Nicolas Lienert (Medgate AG, Switzerland), Pål Lindström (LocusMedicus, Sweden), Sándor Lipp (Hungarian Ministry of Health, Hungary), Leena Lodenius (Finnish Duodecim Medical Society, Finland), Antti Malmivaara (Finnish Institute of Occupational Health, Finland), Miquel Angela Mayer Pujadas (Official Medical College of Barcelona, Spain), Peter Mills (EncycloMedica Ltd, United Kingdom), Cesar Molinero (Planet Medica, Belgium), Marc Muret (Zürcher Aerzte für Klassische Homöopathie, Switzerland), Wolfgang Nagel (GesundheitScout24 GmbH, Germany), Tim Nater (Health on the Net Foundation HON, Switzerland), Joerg Nitzsche (Zentralbibiliothek für Medizin Koeln, Germany), Debra O´Connor (La Trobe University, Australia), Gert Purkert (Arztpartner / Almeda, Germany), Ramon Sarrias Ramis (Official Medical College of Barcelona, Spain), Christine Reuter (A MedWorld AG, Germany), Ahmad Risk (Internet Healthcare Coalition, United Kingdom), Carl Bénédict Roth (GetWellness, Switzerland), Sebastian Schmid (Arztpartner / Almeda, Germany), Christiane Schmitz (Heinrich Heine Universität, Germany), Luk Schoonbaert (Belgacom Multimedia Ventures, Belgium), Frank Schuler (gfp Kommunikation Köln, Germany), Ulrich Schwanke (DocCheck, Germany), Sasha Shepperd (Imperial College School of Medicine, United Kingdom), Myra Sidrassi (MedCERTAIN, Germany), Chris Sigouin (McMaster University, Canada), Chris Silagy (Monash Medical Centre, Australia), Denise Silber (Internet Healthcare Coalition, U.S.A), Martin Sonderegger (Universitätsspital Zürich, Switzerland), Anke Steckelberg (Universität Hamburg, Germany), Frederik Tautz (Heinrich Heine Universität, Germany), Nicolas P. Terry (Center for Health Law Studies, St. Louis University, U.S.A.), Christian Thomeczek (Ärztliche Zentralstelle Qualitätssicherung, Germany), Wouter Tukker, (Praha Communications, Czech Republic), Gerard H. van der Zanden (The Netherlands Institute for Health Promotion and Disease Prevention NIGZ, Netherlands), Georg von Below (Swiss Medical Assoc./FMH, Switzerland), Frank von Danwitz (Deutsches Diabetes-Forschungsinstitut, Germany), C.-Peter Waegemann (Medical Records Institute, U.S.A.), Thomas Wetter (Dept. Of Medical Informatics, Universität Heidelberg, Germany), Petra Wilson, (European Commission, Belgium), Margaret A. Winker (JAMA, U.S.A.), Jeremy Wyatt (University College London, United Kingdom), Gabriel Yihune (MedCERTAIN, Germany)


abstract: Does identifying genetic differences between groups itself constitute an act of racism?
author: Gregory A Petsko
date: 2004
institute: 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA
title: Color blind

By the time you read this, the result of the 2004 US Presidential election will be known - assuming, of course, that there isn't a repeat of the travesty of 2000, when the Supreme Court of the United States, by a vote that split along the lines of which political party had appointed which particular judge, awarded the election to George W. Bush by the slimmest of margins (less than 1,000 votes) in the state of which his brother was governor. Contested ballots notwithstanding, I never cease to be amazed when I cast my vote on election day. After the cacophony of the seemingly endless campaign, on election day a curious quiet descends on the country. No bands play. No troops march. People waiting in line to vote tend to speak in hushed tones, almost as though they were in church. I didn't see a single cell phone in use. The transfer of power in what is arguably the most powerful nation in the history of the world happens softly, like a whisper in the dark. That's the wonder of democracy.

Contrary to what many Americans think, democracy wasn't invented here. It has a long history, going back at least to ancient Greece. And, especially if we have another disputed election this time, I would have trouble arguing that it was perfected here either. But if not the practice of democracy, I think the concept of democracy may have reached its zenith on these shores, in the city of Philadelphia, in the month of June, in the year 1776. At that time and place, Thomas Jefferson composed these words for the document in which the thirteen former British colonies declared their independence: "all men are created equal". As a weapon in a war, which it was, that phrase was powerful. It was meant to convince and to confound, and it did both. Then it developed a life of its own, and began to reverberate around the world like the sound of an exploding volcano. It echoed in the French Revolution thirteen years later. It could be heard in 1848, the year of revolutions in Europe. It was still echoing more than a hundred years later when India broke from Britain. It can still be heard today, as loud and potent as ever. Not bad for an idea that is biological nonsense.

Basketball star Michael Jordan could jump through the roof of a building; I never could. Footballer David Beckham can bend a corner kick around a goalpost; I can barely kick a soccer ball straight. I can't run a marathon in under three hours and I never would be able to no matter how hard and long I trained. Neither could most people, nor do they expect to. Implicit in the meritocracy of sport is the idea that, although practice and coaching are important, there are vast differences in talent among individuals. Such limitations are understood in other areas as well: most people cannot do high-level mathematics and probably never could no matter how long they studied. I took piano lessons for five years, but Horowitz is a pianist; I'm not, and never will be. We all know these things, and accept them. But taken literally, "all men are created equal" could imply that such facts aren't true. Of course, the phrase was never meant to refer to physical abilities, or talent for music or abstract reasoning. It meant that all men (alas, the Founding Fathers probably really did mean only men - there were no Founding Mothers to set them straight) should be equal in the eyes of the law, and the government. Since I live in a country where the wealthy and influential often receive preferential treatment by both, I understand that this is an ideal not always achieved in practice.

But the extreme interpretation is always there, in any democracy. It's responsible for the contempt of 'the elite' or 'intellectuals' that one hears, especially around election time. It underlies our iconoclastic tendencies. Somewhere, in some inner place that is hard to reach, a lot of us harbor the secret belief that we're just as good as anybody, in just about everything. Maybe this delusion is harmless, but I doubt it. I think it contributes to the streak of anti-intellectualism that has a foothold in Western culture right now. I think it also makes it easier for populists and demagogues to coax people into voting against their self-interests. But even if I'm wrong about these consequences, there is one area in which the notion of absolute equality is certainly dangerous: the area of medicine.

This month, a report appeared in *The New England Journal of Medicine* (**351**:2049-2055; issue of 11 November 2004) entitled "Combination of Isosorbide Dinitrate and Hydralazine in Blacks with Heart Failure". The article was written by Anne L. Taylor MD, Susan Ziesche RN, and a number of other authors, on behalf of the African-American Heart Failure Trial Investigators. The paper presents the results of a study of 1,050 black patients with advanced heart failure. Half received a placebo, while the other half received BiDil, a combination of two generic drugs (isosorbide dinitrate, which stimulates the production of nitric oxide, and hydralazine, an antioxidant and vasodilator that appears to prevent the nitric oxide from deactivating). The results were so striking that the study was terminated prematurely so that all remaining patients could be given BiDil. Over the two years of the study, 6.2% of the patients given the drug died, compared with 10.2% of those given the standard treatment, a 43% improvement. First hospitalizations were also reduced by 33%. Ray Gibbons, a cardiologist at the Mayo Clinic and spokesman for the American Heart Association, says "When you have this level of risk reduction in a high-stakes disease like heart failure, it's at least a home run." The study was initiated by NitroMed, Inc., a biotechnology company in Lexington, Massachusetts. Combined use of the two generic drugs would allow NitroMed to receive a new patent for the joint formulation. An earlier trial of the mixture, by Dr Jay Cohn of the University of Minnesota in the 1980s, had been disappointing: in a test against the general population, the drugs fared worse than angiotensin converting enzyme (ACE) inhibitors. But Dr Cohn reanalyzed the data when it became clear years later that one reason African-Americans die from heart failure at 2.5 times the rate of Caucasians was because ACE inhibitors appear to be much less effective in black patients than in non-blacks. Sure enough, in the 395 black patients in the original study, the drug combination appeared better than the standard therapy. The new trial, exclusively in African-Americans, was begun as a consequence.

So why isn't everybody cheering? Many are, including the Association of Black Cardiologists, who cosponsored the trial. But others are not. In a perspective in the same issue of *The New England Journal of Medicine*, Dr M Gregg Bloche of Georgetown University Law Center asks, "Are we moving into a new era of race-based therapeutics?" He believes the answer is yes, especially since the US Food and Drug Administration has already indicated that if the drug is approved, it will be labeled as indicated for patients of African-American descent, an unprecedented situation. (Although at least 29 drugs are known to have different efficacies between blacks and Caucasians, none of them is labeled in this way.) JudyAnn Bigby, director of the Office for Women, Family and Community Programs at Brigham and Women's Hospital in Boston, fears that "if people get one little inkling that there's a biological basis to race, we could potentially lose ground in understanding racial differences in disease. Biology could be an excuse for not looking at the social basis" of diseases like heart disease, which is known to be affected by stress, income, diet and access to health care, among other non-genetic factors. "You have the federal government giving its imprimatur, its stamp of approval, to using race as a biological category," warns Jonathan Kahn, of Hamline University, who wrote a paper in the *Yale Journal of Health Policy, Law, and Ethics* on the danger of approving BiDil. "To my mind, it's the road to hell being paved with good intentions."

This concern is understandable given the sorry history of the US when it comes to matters of race. Yet we have known for half a century that sickle-cell anemia is overwhelmingly a disease of blacks, that Tay-Sachs Disease is overwhelmingly a disease of Ashkenazi Jews, and that cystic fibrosis is overwhelmingly a disease of Caucasians. Somehow that knowledge has not set us on the road to hell. Why not? I think that main reason is that 'race' in these diseases is only a surrogate marker: in each case the specific genetic abnormality is known. Blacks do not suffer disproportionally from sickle-cell anemia because they are genetically inferior to whites, they suffer from it because the sickle cell mutation is more commonly found in people of African origin, and because the presence of the mutation in such individuals appears to confer protection against infection by the malaria parasite. But there are 'non-blacks' who have the same mutation and suffer from the same disease. Race isn't the issue; the issue is who has the relevant genetic trait. Once the affected gene is known, anybody can be tested for the disease-causing mutation. Race becomes irrelevant.

BiDil works in African-Americans because there is some gene - possibly more than one, but probably no more than a few - that differs between most people of African-American descent and those of, say, Indo-European descent. The tools of genomics can be used to find that gene or genes, and when that is done it will be possible to test everyone who suffers from heart failure for the mutations that confer susceptibility to BiDil therapy. When that happens, it will certainly become clear that many non-blacks would also benefit from BiDil. Since this would increase the market for the drug considerably above, say, the 375,000 African-Americans who suffer from moderate to severe heart failure, it would be in the interest of NitroMed to support the search for those genetic differences. Then the drug could be labeled the right way: according to its molecular targets, not the color of the patient's skin.

Last spring, The Magic Theater in San Francisco presented a play by Cassandra Medley entitled *'Relativity'*. It concerns an African-American woman scientist who is asked by her mother to disprove that all racial groups are genetically similar (you can read a synopsis of it in Sandra Soo-Jin Lee's thoughtful review in *PloS Biology* 2004, **2**:1263-1264). The play raises the question: does identifying genetic differences between groups itself constitute an act of racism? Obviously I don't think so, but I do realize that any statement of differences between races can never be neutral. That is why we must shift the discussion away from color and towards those things that make us different as individuals, not as members of some group.

Genomic data indicate that all humans share about 99.9% of our genetic make-up. As more data accumulate and it becomes clear that the concept of race is meaningless biologically, we should be able to agree that all 'men' are almost equal biologically, and to shift our focus once and for all to those genetic differences that are meaningful, such as those that determine our susceptibility to disease and our responses - beneficial and adverse - to therapy. One of the great future benefits of the Human Genome Project may be that, in years to come, when someone is described as being a credit to their race, it will be understood by everyone that what is meant is the human race.


date: 2004
title: Editorial

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abstract: Bioinformatics-based searches for correlations between subcellular localization and pI or charge distribution of proteins have failed to detect meaningful correlations. Recent work published in *BMC Biology* finds that a physicochemical metric of charge distribution correlates better with subcellular pH than does pI.
  .
  See research article <http://www.biomedcentral.com/1741-7007/7/69>
author: Bertrand Garcia-Moreno
date: 2009
institute: 1Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA
references:
title: Adaptations of proteins to cellular and subcellular pH

The need for tight regulation of intracellular pH is one of the most important and constant organizing principles of living systems. It is essential because the fundamental energy transduction machinery of cells runs mainly on H^+^ gradients and proton-coupled electron transfer reactions. It is also necessary because pH determines the charge state of weak acids and bases (for example, side chains of Asp, Glu, His, Lys and Arg, and so on), and the charge state in turn affects many physical and physiological properties of biological molecules, especially proteins and RNAs (Figure 1). Regulation of intracellular pH is so central to the living state that even the most primitive cells capable of energy transduction must have been able to control intracellular pH by expelling H^+^ produced from the hydrolysis of organic compounds.

How tightly is pH regulated in cells and organisms? Consider that the normal pH of human arterial blood is 7.40. Depression below pH 7.35 leads to acidosis, a condition that requires medical attention, and elevation to pH 7.50 results in alkalosis, responsible for the unpleasant symptoms of altitude sickness. Similarly small changes of 0.1 pH units in intracellular pH or within subcellular compartments can have physiological consequences. What is remarkable is that despite the need for very tight regulation, the pH in various cellular compartments varies significantly. In organs such as the human stomach, it can be extreme.

In cells, pH is nearly neutral in the cytoplasm, in the endoplasmic reticulum and in mitochondria. It is more acidic in vacuoles, lysosomes (as low as pH 5) and in the Golgi. It is more basic in the nucleus and in peroxisomes (as high as pH 8) \[1\]. Proteins can harness these differences in cellular and subcellular pH for physiological purposes. For example, the influenza virus requires exposure to the slightly acidic conditions in the lysosome to become activated \[2\]. Human hemoglobin acts similarly as a pH sensor that targets exercised tissue for delivery of oxygen by responding to local acidity \[2\].

The tight regulation of cellular and subcellular pH might imply that charged residues of proteins have been tailored for structural or functional purposes under specific conditions of pH. For example, the isoelectric point (pI) of proteins, which describes the balance between acidic and basic residues, might have co-evolved with the pH of the organelles in which they exist to enable or to optimize function \[3\]. Learning to recognize these adaptations would be useful for the annotation of proteomes and for understanding protein function and evolution. Previous bioinformatics-based searches for correlations between subcellular localization and pI or charge distribution have yielded interesting nuggets but have failed to detect a meaningful correlation \[4\]. The problem is that the pI is a relatively insensitive global metric of the number and types of ionizable groups; the distribution of pI values in a proteome is inherently bimodal owing to the normal differences in the p*K*~a~ (the acid dissociation constant) of the dominant acidic (Asp and Glu) and basic (Lys and Arg) residues \[4,5\]. Now Chan and Warwicker \[5\] have examined the correlation of intracellular pH with calculated electrostatic contributions to stability. Using this more physical metric of charge distribution, they found that the average pH of maximal stability for proteins in a subcellular compartment correlates better with subcellular pH than does pI. The properties of histidine residues underlie this correlation.

# Effects of charge distribution on properties of proteins

Identifying further meaningful correlations between subcellular localization or subcellular pH with the number, types, distribution and properties of ionizable groups in proteins, starting from either sequence or structure, will be interesting but challenging. The problem is that the charge properties of proteins have evolved under pressure to satisfy a large number of physical and biological constraints (Figure 1). Trying to identify trends or adaptations by focusing on one or two among the many constraints is akin to examining a piece of music through statistical analysis of musical notes or their values (that is, their duration), ignoring all other attributes of the music. Averaged over their entire oeuvre, the works of Bach and Beethoven might indeed show statistically significant differences in their predilection for some notes, but it is unlikely that this will be reflected in any individual piece, that the level of discrimination will be sufficient to identify a composer from among a list of hundreds, or that this approach will improve our understanding or appreciation of music. A meaningful search for adaptations of proteins to subcellular pH should consider simultaneously some of the following structural, biological and solution properties of proteins, all of which are influenced by charged residues.

## Spatial localization

The distribution of charges on a protein can be biased by the protein's location within a subcellular compartment. For example, membrane proteins tend to be more basic than cytosolic ones \[4\]. This might be an adaptation to facilitate interactions between positive charges on the protein and the predominantly negatively charged polar head groups of membrane phospholipids. However, to illustrate how deceptive general trends can be, consider that some proteins that interact strongly and irreversibly with membranes actually use Ca^2+^ bridges that require post-translational modifications that add negative charges to the proteins \[2\].

## Stability

The pH dependence of stability of proteins is governed by differences in p*K*~a~ values of ionizable groups in folded and unfolded forms. Using structure-based continuum electrostatics calculations, Alexov \[6\] showed previously that the pI and the pH optimum for stability can be quite different. Some proteins, such as proteases in the lysosome, are clearly adapted for maximal stability and activity in the relatively low pH of this compartment. Chan and Warwicker \[5\] show that this correlation of the pH of maximal stability with intracellular pH is only evident when the properties of many proteins within a subcellular compartment are averaged. Their study illustrates the power of quantitative physicochemical approaches in the analysis of proteomes. The effectiveness of this approach is also illustrated by other studies of charge contributions to the stability of thermophilic proteins. Proteins from thermophilic organisms usually have a higher number of charges and ion pairs than their mesophilic homologs \[7\]. An analysis of the number and types of charges would conclude that charges in thermophilic proteins were selected to enhance the stability of proteins through Coulomb interactions. However, a structure-based study of electrostatic contributions to stability using a physicochemical model failed to detect any correlation between the excess charges in thermophilic proteins and increased stability \[7\].

## Solubility

Solubility is a critical factor in the evolution of protein sequences and folds \[8\]. In general, charges in globular proteins are surrounded by charges of the opposite sign \[9\]. This would seem to reflect evolutionary tuning of surface charges to maximize stabilizing Coulomb interactions in the folded state, but it is more likely to be an adaptation to enhance solubility. Charges affect the solubility of proteins; therefore, the adaptation of charges to tune any property of a protein for a specific subcellular pH could affect solubility. Because there is no theory of protein solubility, it is impossible to determine how a specific distribution of charges affects solubility. In general, solubility correlates with pI (proteins tend to be least soluble at their pI); therefore, given the lack of correlation between pI and subcellular pH, solubility probably does not correlate well with subcellular pH. A large number of ionizable residues, especially basic ones, can enhance the solubility of a protein. However, unexpected factors can also influence solubility. For example, the solubility of folded proteins is affected by stability; a large energy gap between the fully folded, soluble form and partially unfolded, less soluble forms is needed to prevent population of aggregation-prone states. Solubility can in fact be affected by single mutations that do not alter the number of charges in the protein.

## Interactions

Charges are essential both to prevent and to stabilize complexes of proteins with macromolecules and with small molecules. A correlation has been found between the pH optimum of stability of monomeric proteins and of their complexes \[10\]. This suggests that pH-dependent properties of the monomers and of the complexes coevolved at the same pH. The presence of a specific constellation of charges for functional interactions can bias the distribution and properties of charges in proteins. For example, regions of high density of positive charge are usually found in the faces of proteins that interact with nucleic acids. Any correlation between nuclear pH and the distribution of charges in nuclear proteins in a eukaryotic cell is likely to be a secondary consequence of these functional adaptations.

## Enzymatic activity

The ionizable groups that control enzymatic catalysis usually titrate with highly perturbed p*K*~a~ values tuned for catalysis under the appropriate pH conditions. Just one of these groups with perturbed p*K*~a~ can have a dramatic influence on the pH dependence of protein stability \[11\]. This might be the reason that the pH optimum of enzymatic reactions is, in general, not correlated with the pH of maximum stability. The adaptation of some proteins to subcellular pH might involve tuning of the p*K*~a~ values of active-site residues. There are no computational tools that reproduce the properties of internal ionizable groups accurately, and so this essential adaptation to subcellular pH cannot yet be examined quantitatively.

## Environmental conditions

Previous attempts to identify protein adaptations to subcellular pH have not considered that charges and their contribution to stability, solubility, dynamics, conformation, function, and so on are sensitive to physical variables (temperature, pressure), and to the chemical composition of their milieu (osmolytes, ionic composition, metabolites). Chan and Warwicker \[5\] point out that differences in the distribution of charges in proteins from extremophiles and mesophiles illustrate the important roles of charges in proteins for adaptation to specific environmental conditions. The influence of the ionic milieu on adaptations to intracellular pH deserves special mention because subcellular pH is coupled to ion homeostasis (that is, changes in intracellular pH are coupled to changes in concentration of other ions). Protein adaptations to unique ionic environments are already known. For example, hemoglobin, which is regulated physiologically by the large anion 2,3-bis-phosphglycerate, has a cleft with a high concentration of positively charged residues where the di-anion binds \[2\]. Another example is that of extracellular proteins or proteins that exist in vacuoles or other compartments that can have high Ca^2+^ concentrations. These proteins might be expected to be more acidic to maximize interactions of Asp and Glu residues with Ca^2+^ to enhance stability or for other purposes. Attempts to identify protein adaptations to subcellular pH should consider how the ionic milieu characteristic of a subcellular compartment might have influenced these adaptations.

## Buffer capacity

The regulation of subcellular pH is partly achieved through the buffer capacity of metabolites and macromolecules. This is the reason that a correlation of protein pI with subcellular pH has been sought. However, the ability of a protein to act as a buffer in a subcellular environment will depend not just on the number of ionizable groups and their p*K*~a~ values, but also on the concentration of protein in the compartment. A meaningful correlation between pI and subcellular pH might yet be found if protein concentration and compartment volume were taken into consideration.

## Biological considerations

The distribution of charges in proteins can be biased by a number of biological factors that are not linked to intra-cellular pH in an obvious way. Evolutionary history, mutational bias, AT nucleotide bias, level of gene expression and optimization of translational efficiency are some factors that will have to be examined closely.

## Physicochemical considerations

Chan and Warwicker \[5\] have raised other interesting issues that will have to be considered when searching for protein adaptations to subcellular pH. The adaptations might not be tuned for function in the steady state intracellular pH, but rather to the transient changes in pH that might be experienced in an intracellular compartment. Chan and Warwicker also made the interesting suggestion that the dependence of the pH of maximum stability on subcellular pH might reflect the need to minimize spontaneous fluctuations in H^+^ concentration within a compartment \[5\]. There is clear need for a more detailed inventory of H^+^ in subcellular compartments based on the known number and calculated p*K*~a~ of weak acids and bases, and on emerging data of protein localization and concentrations. There is also need for improved understanding of the diffusion properties of protons in the osmotically complex intracellular environment, where there is no water that is not under the influence of solute, and where, formally speaking, the concept of pH is not valid.

# Conclusions

Many examples of regulatory adaptations of proteins for stability or function at a specific pH are known. If charges in proteins have indeed been optimized for specific functional purposes, quite possibly they also display structural adaptations to specific subcellular conditions of pH and ionic composition. These adaptations will not be easily identified from bioinformatics analysis of proteomes using global metrics of charge distribution (such as pI). Although in general, the mean properties of proteins might be less informative than the deviations from the mean, in practice the only correlations that have been found with intra-cellular pH are with electrostatic properties calculated with physical models, averaged over many different types of proteins in a given subcellular compartment. To identify further adaptations of proteins to subcellular pH it will be useful to analyze proteomes with physicochemical models, and to consider simultaneously many of the physical and biological constraints that guided the evolution and the adaptation of proteins to the pH and ionic properties of their physiological milieu. This more integrative and physical approach might begin to reveal how different elements are combined in harmony to constitute the symphony of the cell.