You are what you eat and drink. Steak can sit in your stomach or orange juice wind through your intestines, but they only become part of your body once they're taken up by your cells. First, foods must be reduced to a soup of proteins, fats, sugars, and so on. But even then, getting these materials into a cell isn't as simple as sticking them in your mouth. For one, there's the membrane enclosing a cell. Simply puncturing a hole in the membrane would spill the cell's contents, harming or killing the cell.Instead, all eukaryotes—organisms whose cells have nuclei—use a carefully orchestrated process called endocytosis to bring materials into their cells. Eukaryotic cells first form cavities in their cell membrane that surround nearby particles or fluid. These pockets seal shut and bud off into the cell to form small membrane-bound sacs called vesicles.When taking in fluids, eukaryotic cells use two distinct mechanisms—to take tiny sips or huge gulps. With one process, called pinocytosis, cells continually form small pockets in the cell membrane that enclose small droplets of fluid in vesicles called pinosomes. These newly formed vesicles, called early endosomes, bud off from the membrane and fuse with other early endosomes. In one form of pinocytosis, the vesicles are encaged by a protein called clathrin that tightly constrains their size. These carriers incorporate membrane constituents (for example, growth factors) with very high selectivity. In macropinocytosis, on the other hand, large ruffles in the membrane engulf mass quantities of fluid in vesicles known as macropinosomes.Beyond taking in nutrients, these processes are essential to the function of many organs—from the brain, where nerve cells receive other cells' chemical signals by pinocytosis, to the kidney, where cells use macropinocytosis to take in waste fluids for processing. Macropinocytosis is also relevant to cancer cells; it has long been known that oncogenes dramatically induce this endocytic process, affecting the signaling status of these cells. But compared with other types of endocytosis, molecular biologists know surprisingly little of the mechanisms behind macropinocytosis. They do know that the Rab5 protein—an enzyme that coordinates a complex network of other proteins, called effectors—is crucial for both pinocytosis and macropinocytosis.PLoS Biology, Marino Zerial and colleagues have found a new protein, which they named Rabankyrin-5, that forms a further link between these two mechanisms for fluid uptake. The protein is necessary for macropinocytosis, and its levels control the rate of this process. In addition, Rabankyrin-5 helps regulate endosome trafficking and coordinates this mechanism with macropinocytosis.Now, as reported in this issue of In two commonly used human and mouse cell lines, the researchers found the protein Rabankyrin-5 along with Rab5 on both types of pinosomes, early endosomes and macropinosomes. The early endosomes usually fuse with one another inside the cell, but when the researchers blocked Rabankyrin-5 activity, this fusion fell sharply. Suppressing Rabankyrin-5 activity also stifled macropinocytosis; overexpressing the effector, on the other hand, sent macropinocytosis into overdrive.The researchers also looked at endocytosis in mouse kidney and canine kidney cell lines. Inside the kidney, fluid-carrying ducts are lined with epithelial cells that take up liquids through their exposed surface. The researchers found Rabankyrin-5 predominately on vesicles at this surface, and as in the other experiments, overexpression of the protein promoted macropinocytosis. Together, these findings suggest Rabankyrin-5 plays a role in regulating this form of fluid uptake and plays a role in kidney function. The discovery of Rabankyrin-5 involvement in macropinocytosis also has implications for other physiological and pathological mechanisms such as the immune system response, defense against pathogens, and hyperactivation of signaling pathways in cancer cells.Rabankyrin-5 contains various regions that bind other proteins and also lipids found in cell membranes, suggesting the protein plays a mechanical role in forming vesicles. The protein also has regions found on other proteins that are involved in signaling and development, so it may help direct vesicles' traffic within the cell. The protein also has regions characteristic of proteins involved in clathrin-dependent endocytosis, which fits with the researchers' finding that Rabankyrin-5 affects pinocytosis.All told, Rabankyrin-5 appears to form a bridge between two distinct mechanisms, pinocytosis and macropinocytosis, that cells use to take in fluids. While the details of how Rabankyrin-5 functions are still unclear, these findings give researchers a new handle for grasping how macropinocytosis works and how cells control when and how much they drink in their surroundings.


Wireless local area networks (WLANs) are considered the next generation of clinical data network. They open the possibility for capturing clinical data in a prehospital setting (e.g., a patient's home) using various devices, such as personal digital assistants, laptops, digital electrocardiogram (EKG) machines, and even cellular phones, and transmitting the captured data to a physician or hospital. The transmission rate is crucial to the applicability of the technology in the prehospital setting.We created two separate WLANs to simulate a virtual local are network environment such as in a patient's home or an emergency room (ER). The effects of different methods of data transmission, number of clients, and roaming among different access points on the file transfer rate were determined.The present results suggest that it is feasible to transfer small files such as patient demographics and EKG data from the patient's home to the ER at a reasonable speed. Encryption, user control, and access control were implemented and results discussed.Implementing a WLAN in a centrally managed and multiple-layer-controlled access control server is the key to ensuring its security and accessibility. Future studies should focus on product capacity, speed, compatibility, interoperability, and security management. The development of the Internet has encouraged doctors to use computers and hospitals to use wireless communications , since wAt present, patient data such as electrocardiograms (EKG) and demographics are seldom sent from the prehospital environment to the ER before the ambulance arrives . As a reThe objective of this study was to assess the ability of wireless technology to facilitate data communication between a prehospital setting and an ER . The idea was for all the data collected by paramedical personnel to be transmitted to an ER server from the patient's home, on the way to the ER, or upon arrival at the ER. Thus the transmission rate is crucial to the usefulness and applicability of the technology. To test the feasibility of wireless data transmission under the various scenarios, two separate WLANs were created, one around our office and another in a house. In this report, we discuss our testing of the wireless technology, and its potentials and limitations in simulated prehospital settings.The WLAN products used (Aironet 340 and 350 series wireless client adaptors and access points (APs); Cisco) offered 11-megabits-per-second (Mbps) transmission rates, built-in security features (including 40- to 128-bit encryption) and Web-based management. The transmission rates of files of different sizes were measured with different APs, patch antennae, clients, and transfer methods. The security of patient data was ensured using a centrally managed Access Control Server (ACS). Other issues such as standards, roaming, and cost are also discussed here.Within a WLAN, data are transmitted between a server and its wireless clients via an AP antenna. Both workstations and laptops were used here as servers, and file transfer rates were measured for both systems. We used Gateway Select series, Dell Inspiron series, and a Toshiba Satellite laptop computer as clients. The computers had CPU operating at 0.8–1.2 GHz, 256–1024 MB of RAM, and 10–40 GB hard drives, and all ran the Microsoft Windows 2000 Professional operating system. PDAs (Ipaq Pocket PCs, models 3550 and 3570, 200-MHz CPU, 32–64 MB RAM, Compaq) were also tested as wireless clients. The wireless coverage was tested using two APs (Aironet 340 and 350 series, Cisco) and a patch antenna (S2406P, Cushcraft Corporation). The feasibility of using cellular phones (StarTac 7868, Motorola) in data transmission in the area not covered by the WLAN was also tested.The various software used in the study for wireless client management, file transfer, and access control included the Aironet Client Utility (Cisco), Link Status Meter (LSM, Cisco), the ACS (V3.0, Cisco), Phone Tools (BVRP Software) for faxing, and file transfer protocol (FTP) for measuring the file transmission rate. The software LSM classifies the link status as the percentage of maximum signal strength and quality: "excellent" (>75%), "good" (40–75%), "fair" (20–40%), or "poor" (<20%); where signal strength and quality refer to the client adapter's radio signal at the time packets are being received, quantified as bytes transmitted and received and the errors that occur. Detailed descriptions of the mentioned software are available from the manuals provided by the vendors.The Aironet 340 and 350 series APs were tested by a two-step approach. In the first step, one AP was connected directly with the server that was not connected to the campus Ethernet backbone. In the second step, the AP was assigned a public Internet Protocol (IP) address and connected to the Ethernet backbone in the Susan Mott Webb Nutrition (Webb) Building at the University of Alabama at Birmingham. The IP address was assigned to an AP through either HyperTerminal or a Web console using a Web browser. An administrator ID and password were then created to enhance the Web console security. The client computer required a type II PCMCIA  card slot. Every client needed a functional IP address to become associated with the AP. The Wired Equivalent Privacy (WEP) keys were enabled for both the AP and the clients to ensure two-way authentication.The coverage of the WLAN was found to highly structure dependent. The floor of the Webb building measures about 60 by 25 meters. A single Aironet 340 AP was unable to cover the entire floor with a "good" link status. This was achieved using two (more powerful) Aironet 350 APs. Achieving the "excellent" link status on the floor required the use of the S2406P (Cushcraft) patch antenna. The wireless clients associated with the AP had a "fair" or "good" status one floor up and one floor down from the floor where the AP was located. There was a small area outside the 5-floor building in which the clients could associate with the AP with a "poor" status.In order to test the feasibility of implementing an isolated LAN around a patient's home, we chose a two-story house with the layout shown in Figure To quantify the baseline signal strength, one Aironet 350 AP was tested in the open area: "excellent", "good", "fair", and "poor" link statuses were obtained within 10, 25, 50, and 100 feet (~4, 10, 20, and 39 meters), respectively. As shown in Table The different methods and directions of file transfer might affect the rate. Methods such as FTP transfer, copying and pasting between folders, and Microsoft Briefcase synchronization were tested. Other factors that may affect transmission were also tested, including the initiation direction  and relative physical motion. One Gateway workstation and two Inspiron 4000 laptops were used. To simplify the test, a single medium-sized file (50 MB) was chosen for the purpose. We chose a 50-MB file since it corresponds to a typical high-quality EKG image. As indicated in Table We chose Ipaq Pocket PCs as PDAs due to their relatively large amount of RAM compared to other PDAs, and a cellular phone (Motorola) as alternative data transmission device when the PDA was outside the WLAN coverage (to simulate the scenario when a long-range antenna, such as a yagi antenna (Cisco), is not available). The transfer rates for single files of different sizes are summarized in Table A Cisco V3 ACS was used to improve the security of the network . As showWhen managing a WLAN with many clients, there are often insufficient public IP addresses. The solution is to use either the Dynamic Host Configuration Protocol (DHCP) or a private LAN with a Network Address Translation (NAT) server. DHCP services are not easily managed and sometimes create security challenges to network administrators in determining the user's identity. A private LAN seems to be a better solution and more applicable in most ER environments where many wireless clients may transmit at same times, thus maintaining relatively high-speed connections. Figure Health-care organizations have traditionally been slow in accepting WLAN technology in clinical practice. One of the major concerns has been its cost ,16, follAfter collecting all patient data at a patient's home, the data must ultimately be transferred to the ER. This involves two critical synchronization steps: (1) from the patient's home to a server on the ambulance, and (2) from the ambulance to the ER . Here the WLAN was employed for both of these steps, using Microsoft Briefcase and Windows Workgroups. Automatic synchronization with the destination server and batch synchronization were desired. The ultimate objective is, however, to link the two synchronization steps using a long-range antenna that reaches up to 25 miles (e.g., a yagi antenna from Cisco). This would significantly shorten the time needed to transfer data from a patient's home to the ER, since the data will reach a ER WLAN earlier. We are currently performing the associated experiments. We also tested the use of a cellular phone and other types of PDA  with network capabilities in transmitting a small (up to 50 MB) but critical file. The results suggested that cellular phones or PDAs with network cards can be effective alternatives to the use of a long-range antenna to transmit data from a patient's home to the ER.EKG data are considered very valuable in the early detection, early intervention, and possibly better outcome of heart attack patients . The useThe WLAN operates at 2.42 GHz with an output power of 100 mW, which may pose a risk of interference with medical devices using similar frequencies. Previous studies have shown that a WLAN may interfere with medical devices in close proximity  but is uThe major concern over a WLAN is its security -20, espeThe IEEE 802.11 specification addresses both the physical and MAC layers , and the OpenAir 2.4 interface standard is derived from the Wireless LAN Interoperability Forum  and needThe present study was mainly based on the Windows operating system and Cisco wireless products. IEEE 802.11a products for the next generation of WLANs are emerging quickly from various vendors. Hence the stability, compatibility, and interoperability with other vendors require further evaluation. Although currently it is relatively expensive to implement a WLAN using this new protocol, the prices and capabilities are expected to improve within the near future.Application of WLAN technology will help both paramedics and other health-care professionals in their daily acquisition of information in a localized area such as within a patient's home, an office, a small clinic, or an ER. Implementing a WLAN in a centrally managed and multiple-layer-controlled ACS is the key to ensuring its security and accessibility. Future studies should focus on product capacity, speed, compatibility, interoperability, and security management.None declared.D. Chen, the principal investigator, was most involved in conducting the experiments. H.F. Orthner participated in data collection in the simulated patient's home. H.F. Orthner was the sponsor and S.-J. Soong and G.J. Grimes were the advisors of the fellowship awarded to D. Chen from the National Library of Medicine, National Institute of Health, and they contributed significantly to the design, coordination, and performing of experiments.The pre-publication history for this paper can be accessed here:


We report that mon encodes the zebrafish ortholog of mammalian transcriptional intermediary factor 1γ (TIF1γ) (or TRIM33), a member of the TIF1 family of coactivators and corepressors. During development, hematopoietic progenitor cells in mon mutants fail to express normal levels of hematopoietic transcription factors, including gata1, and undergo apoptosis. Three different mon mutant alleles each encode premature stop codons, and enforced expression of wild-type tif1γ mRNA rescues embryonic hematopoiesis in homozygous mon mutants. Surprisingly, a high level of zygotic tif1γ mRNA expression delineates ventral mesoderm during hematopoietic stem cell and progenitor formation prior to gata1 expression. Transplantation studies reveal that tif1γ functions in a cell-autonomous manner during the differentiation of erythroid precursors. Studies in murine erythroid cell lines demonstrate that Tif1γ protein is localized within novel nuclear foci, and expression decreases during erythroid cell maturation. Our results establish a major role for this transcriptional intermediary factor in the differentiation of hematopoietic cells in vertebrates.Hematopoiesis is precisely orchestrated by lineage-specific DNA-binding proteins that regulate transcription in concert with coactivators and corepressors. Mutations in the zebrafish  A new gene acting early in red blood cell development is discovered by genetic analysis in zebrafish. The gene encodes a member of a well-known family of transcription factors Hematopoiesis involves the coordinated processes of cell proliferation and differentiation of a relatively small number of progenitor cells into billions of circulating red and white blood cells . Hematopgata1 normally in embryonic hematopoietic precursors (moonshine (mon), and another group named a noncomplementing allele vampire , a member of the TIF1 family of transcriptional coactivators and corepressors  visible in circulation (mon mutants initiate expression of gata1 in hematopoietic cells around the five-somite stage, similar to wild-type embryos (data not shown); however, based on TUNEL staining, the differentiating erythroid cells undergo programmed cell death from the 12-somite stage to 22 h postfertilization (hpf)  of all tested alleles survive to adulthood. Adult mon mutants show cardiac hypertrophy, presumably due to the severe anemia leading to a high output state /WIK strain hybrid parents carrying the tg234mon allele. The mon mutant gene was positioned on Chromosome 8 between microsatellite markers z987 and z11001 (http://www.ensembl.org/Danio_rerio/) containing a predicted zebrafish TIF1 family gene. This PAC was hybridized to a kidney cDNA library, resulting in the isolation of four clones that represented the same gene.We identified the d z11001 A . Therefore, the hypomorphic larval phenotype of the m262mon allele is likely due to partial loss of mon function or expression. The presence of mutations in each of the mon alleles indicates that defective Tif1γ function is the cause of the mon phenotype.We have identified ethyl-nitrosourea (ENU)-induced point mutations in three alleles of mon C and 3D,tif1γ is expressed in hematopoietic mesoderm, we next examined zebrafish embryos by whole-mount in situ hybridization   . This grd island . The tifse Tif1γ A and 4B.tif1γ. Using zebrafish expressed sequence tag (EST) sequences, we designed primers to RT-PCR amplify a TIF1-related cDNA from embryonic 10-hpf and 24-hpf RNA. This cDNA encodes a predicted zebrafish ortholog of human TIF1α based on predicted amino acid sequences  of tg234mon mutants show significant (greater than 200 cells in comparison to a wild-type estimate of 3,000 cells) rescue of circulating hemoglobinized RBCs in comparison to control sibling mutants (n = 75). Based on the correction of the jagged fin-fold phenotype (mon did not result in expanded blood cell numbers in wild-type embryos and was not toxic at doses that rescue the phenotype of mon mutants (unpublished data). Since there were no expanded or ectopic blood populations in the embryos, these rescue experiments suggest that mon functions as a permissive factor required for hematopoiesis.To further confirm that a mutation in the zebrafish eriments . Microinhenotype , the mestif1γ expression in erythroid cells suggest that it functions as a cell-autonomous regulator of gene expression in hematopoietic cells. In order to test this hypothesis, we transplanted wild-type adult zebrafish kidney marrow cells carrying a gata1:green fluorescent protein (GFP) transgene into 48-hpf mon mutant embryos  of these demonstrated a marked increase in erythroid cells with 100–1,000 GFP+ cells in circulation 6 d later. By day 10, these transplanted embryos showed approximately 3,000 cells in circulation, similar to the number of blood cells in normal embryos. Despite robust reconstitution of blood cells, mutant recipients did not inflate their swim bladders and thus failed to survive longer than nontransplanted sibling controls, all dying by 3 wk of age. In contrast, 13/35 (37%) heterozygous tg234mon transplants survived to early adulthood. Similar transplants of wild-type cells can fully rescue vlad tepes (gata1) mutants . In two different murine erythroleukemia cell lines (MEL and G1E), Tif1γ is also expressed in nuclear foci, and even though the overall Tif1γ protein level is reduced, this nuclear foci localization does not change with differentiation (unpublished data). This provides further support for the hypothesis that Tif1γ acts within novel nuclear foci, during erythroid differentiation.In order to examine the subcellular distribution of Tif1γ protein, we generated an affinity-purified rabbit polyclonal antiserum directed against the C-terminal 15 amino acids conserved in human TIF1γ and mouse Tif1γ. Immunofluorescence of mouse embryo fibroblast nuclei with the anti-Tif1γ antiserum demonstrates that Tif1γ is localized in small nuclear foci A. The lomoonshine  mutant embryos (mon and gata1, we tested their ability to rescue erythropoiesis. Both injection of gata1 mRNA into mon homozygous mutant embryos and injection of tif1γ mRNA into gata1 knock-down embryos failed to rescue hematopoiesis (unpublished data). We also tested for a direct interaction between Tif1γ and Gata1 proteins by coimmunoprecipitation and yeast two-hybrid assays and found no association (unpublished data). Although the mutations in each of these genes arrest cells at a similar stage of development, our results suggest that gata1 and tif1γ act independently. This does not rule out the possibility that parallel genetic pathways involving gata1 and tif1γ, operating together, regulate gene transcription within blood cells.The hematopoietic phenotype of  embryos . In an etif1γ is required as a permissive cofactor for the erythroid lineage-specific control of hematopoietic gene expression. We reasonably predict that Tif1γ protein functions as a transcriptional intermediary factor in hematopoietic progenitor cells given that both TIF1α  for GFP+ cells over a span of 12 d. On day 13 posttransplant, all surviving larvae  were placed in tanks and monitored for survival.Whole kidney marrow cells were isolated from adult Antisera against the human C-terminal TIF1γ sequence RRKRLKSDERPVHIK was generated in rabbits  and affinity purified. Mouse embryonic fibroblasts grown on coverslips were immunostained with HP1α  and Tif1γ antisera simultaneously. In brief, cells were fixed in 4% paraformaldehyde for 5 min, washed with phosphate-buffered saline, and blocked with 5% serum (PBST) for 30 min. After incubation with the primary antibodies (PBST, 60 min) cells were washed three times with PBST and incubated with secondary antibodies (Jackson Laboratory, Bar Harbor, Maine, United States) followed by three washes in PBST. Cells were embedded with Vectashield/DAPI and analyzed using an Axioplan 2 microscope (Zeiss, Jena, Germany). Digital images were processed using the Volocity 1.0 software (Improvision, Lexington, Massachusetts, United States). G1E cell differentiation experiments were performed essentially as described .gata1:GFP transgene into 2-d-old embryos reconstitutes erythropoiesis, but not viability, in montg234 homozygous mutants. Movies of live embryos at day 3 posttransplant highlight less than 100 GFP+ RBCs in circulation. Transplanted cells were observed to proliferate, resulting in thousands of donor-derived erythrocytes 7 d later. Movies present GFP-fluorescent images of live zebrafish larvae.Transplantation of wild-type zebrafish marrow cells carrying a Video S1(13.7 MB MOV).Click here for additional data file.Video S2(11.3 MB MOV).Click here for additional data file.Video S3(7.9 MB MOV).Click here for additional data file.Video S4(11.2 MB MOV)Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the genes and gene products discussed in this paper are fly bonus (AAF19646), human TIF1α (015164), human TIF1β (Q13263), human TIFγ (Q9UPN9), human TIF1γ (Q9UPN9), mon (AY59853), mouse Tif1α (Q64127), mouse Tif1β (AAH58391), and mouse Tif1γ (NP444400).The GenBank (mon/tif1γ and tif1α have been deposited in GenBank under the accession numbers AY598453 and AY598454, respectively.The cDNA sequences of zebrafish


This subunit composition is distinct from the alpha1 + beta channels found throughout the adult spinal cord. Unfortunately, the pharmacology of forebrain alpha2beta receptors are poorly defined compared to 'neonatal' alpha2 homomeric channels or 'spinal' alpha1beta heteromers. In addition, the pharmacologic properties of native alpha2beta glycine receptors have been generally distinct from receptors produced by heterologous expression. To identify subtype-specific pharmacologic tools for the forebrain alpha2beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels.Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. The β-amino acid taurine possessed 30–50% efficacy for alpha2-containing receptor isoforms when expressed in HEK 293 cells. However, taurine efficacy was dramatically reduced in L-cell fibroblasts. Similar results were obtained for β-alanine. The efficacy of these partial agonists was also strongly reduced by the beta subunit. There were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. Nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. Finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion.While exploring pharmacological properties of alphaOur results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. In the systems examine here, these effects are independent of both absolute expression level and any system-related alterations in the agonist binding site. We conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology. Because the BLA regulates a number of anxiety- or fear-related behaviors [2β compounds possess an appropriate therapeutic index, the pharmacology of these forebrain glycine receptors must be elucidated and extensively compared with the spinal isoform.It has been well established that the amygdala is important in the acquisition and maintenance of fear/anxiety-related behaviors . Strychnsitivity . This fipression . A similrneurons . It is qehaviors , it is p1 expressed in Xenopus ooctyes [1 expressed in HEK 293 cells [1, taurine efficacy is even weaker for GlyRα2 (ca. 5–10% efficacy) when expressed in Xenopus oocytes [2β receptors expressed by BLA neurons possess >50% efficacy for taurine and almost full efficacy for β-alanine [There have been conflicting reports regarding the details of glycine receptor pharmacology when expressed in heterologous systems. For example, taurine acts as a partial agonists (ca. 50% efficacy compared to glycine) for GlyRα ooctyes  whereas 93 cells . Compare oocytes . However2 and α2β receptors since these appear to be the predominate isoforms found in the embryonic and adult forebrain, respectively. Our results provide potentially important insight into the types of mechanisms that may govern brain region-to-brain region variation in glycine receptor pharmacology. Several aspects of this work have appeared in abstract form [This study utilizes whole-cell patch clamp electrophysiology to examine the influence of distinct heterologous expression systems on the β-amino acid pharmacology of glycine receptors composed of distinct subunit combinations. We have focused on the αact form ,12.2, and GluRα2/β in HEK-293 cells and in L-cell fibroblasts. Glycine-gated responses for each receptor isoform were elicited in a dose-dependent manner in both cell types  and α2β (n = 7–8), respectively. GlyR subunits expressed in L-cells displayed a similar pharmacological profile. However, the apparent glycine EC50 of both GlyRα2 (446 μM, n = 5–7) and GlyRα2β (667 μM, n = 4–8) appeared lower than apparent affinities for the same subunits when expressed in L-cells. Two-way ANOVA on the Log (EC50) values  and 570 μM (n = 7) for α2 and α2/β receptors, respectively. In L-cells, β-alanine also elicited currents in a dose-dependent manner. And, like glycine, β-alanine appeared to be less potent in these cells compared to HEK cells. EC50 values for α2 and α2/β receptors were 2.0 mM (n = 8) and 2.9 mM (n = 7), respectively. Two-way ANOVA on LogEC50 values from these studies indicate a significant effect of the expression system on β-alanine potency (F = 43.52, P < 0.0001). There was a trend for the presence of the β-subunit to influence potency but this was not significant nor was there any significant interaction between expression system and subunit composition.With the glycine pharmacological profile established, we next examined the pharmacology of the partial agonists, β-alanine and taurine. The efficacy and potency of these β-amino acids were compared to glycine by normalizing the current response at each concentration to a maximal glycine response in that same cell. In HEK cells Fig. , the ave2 and α2β isoforms had efficacies of 80 ± 6% and 55 ± 7% of the maximal glycine response, respectively. A similar trend was noted in L-cells with the α2 and α2β isoform with β-alanine efficacies being 39 ± 8% and 25 ± 5% of the maximal glycine response. Two-way ANOVA analysis of these data indicate that both expression system and subunit composition had a significant influence on β-alanine efficacy (F = 27.6, P < 0.0001 and F = 7.9, P < 0.01 respectively). There was no significant interaction between these variables. These data demonstrate that the presence of the β subunit reduced β-alanine efficacy of α2-containing receptors and that this efficacy was substantially smaller L-cells compared to HEK cells.β-alanine efficacy was also examined in these same experiments by normalizing the maximal β-alanine response as a fraction of a maximal glycine response. In HEK cells, the α50 for taurine was 501 μM for GlyRα2 (n = 9) and 2 mM for GlyRα2β (n = 7). Because of its remarkably low efficacy in L-cells (see below), we can only provide estimates of taurine potency in this expression system. Regardless, apparent taurine affinity for both GlyRα2 and GlyRα2β expressed in L cells were ~3 mM for both isoforms (n = 4 and 7, respectively). We did not compare L cell data with that obtained from HEK cells due to the uncertainty surrounding the fits. However, there was no significant difference in apparent taurine potency between the α2 and α2β receptors expressed in HEK cells (P >> 0.05, t-test).Similar analysis of taurine pharmacology in HEK and L-cells revealed more dramatic effects of system and subunit on this partial agonist Fig. . In HEK 2 and 32 ± 4% of glycine for the GlyRα2β isoform. Efficacy for these same receptors was reduced to approximately 6 ± 1% and 5 ± 0.7% of glycine when they were expressed in L-cells in these particular studies. The system difference was significant with two-way ANOVA (F = 17.4, P < 0.001) with no substantial effects of subunit composition or interactions between these variables.Taurine efficacy was obviously quite different between the two expression systems. In HEK cells, taurine efficacy was 48 ± 12% of glycine for GlyRα2 protein using western analysis of total lysate derived from the same number of GFP+ HEK 293 or L cells from transfected cultures , where res Fig. . For thi2 and α2β receptors expressed in HEK cells compared to receptors expressed in L cells  but not subunit, nor was there a significant interaction between variables. Results from both westerns and functional experiments therefore indicate that relative expression levels of glycine receptor were different between HEK and L-cells.Maximal conductance is an independent measure of functional expression and was also larger for both αlls Fig. . Across 2 and α2β glycine receptors in a number of different heterologous systems, as well as for native receptors expressed in rat lateral/basolateral amygdala. In addition to HEK and L-cells, the heterologous systems included mouse 3T3 fibroblasts and MDCK kidney cells. α2β receptors expressed in mouse 3T3 fibroblasts had twice the current density (121 ± 34 pA/pF) of the mouse L-cells (59 ± 19 pA/pF) but had a similar taurine efficacy (13 ± 8% of glycine in 3T3 cells versus 8 ± 1% in L-cells). Similarly, α2β receptors expressed in HEK293 cells had a current density similar to GlyRs expressed in 3T3 fibroblasts (115 ± 11 pA/pF) but had a taurine efficacy compared to glycine of 48 ± 3%. This efficacy was similar to glycine receptors expressed by acutely isolated adult rat basolateral amygdala neurons (46 ± 5% of glycine) although the current density in this native system was only 57 ± 14 pA/pF. Note that the channels expressed by these neurons are composed primarily of α2+β subunits [2β current density (15 ± 5 pA/pF); yet the channels expressed by this system had the highest taurine efficacy of any cell tested (101+6%). For α2 GlyRs, the rank order of glycine receptor density was 3T3 (111 ± 21 pA/pF)> HEK cell (90 ± 11 pA/pF)> L-cell (50 ± 9 pA/pF); while the rank order of taurine efficacy for these same receptors was HEK (74 ± 9%)> 3T3 (23+7%)> L-cells (11 ± 2%). Across all subunit combinations and systems, there was no significant correlation (R2 = 0.14, P >> 0.05) between taurine efficacy and glycine current density . Indeed, no correlation between expression level and taurine efficacy was evident within any given population of cells whether the receptors were expressed in native or heterologous systems. For example, the correlation coefficients for α2β receptors between taurine efficacy and glycine current density in individual systems were 0.11, 0.13, 0.19, 0.05, and 0.31 for HEK, L-cells, 3T3 cells, MDCK cells, and amygdala neurons, respectively (P >> 0.05). Thus, while there is clearly a difference in expression level between both the systems as well as between individual cells in a given system, this particular characteristic cannot account for the apparent taurine efficacy.To further explore the interaction between expression level and partial agonist efficacy, both current density and taurine efficacy were compared for αy 121 ± 3 pA/pF of50 concentration of glycine. The strychnine KB was estimated for HEK and L-cells expressing either the GlyRα2 or GlyRα2+β subunits using the Cheng-Prusoff relationship (see Methods). This relationship takes into account the divergent Hill-slope and potencies for glycine found in these two expression systems. Receptors composed of the GlyRα2 subunit  or L-cells  in HEK cells and 38 ± 8 nM (n = 10) in L-cells. Two-way ANOVA did not reveal any significant effect of either system or subunit composition. Since strychnine is a competitive antagonist and site-directed mutation studies suggests that strychnine and glycine interact with overlapping regions of the receptor [There are a variety of possible mechanisms to account for the disparities in partial agonist pharmacology between two expression systems. One way to address this is to examine competitive antagonist binding properties in HEK and L-cells. We therefore examined the potency of the glycine receptor competitive antagonist strychnine in both systems. Following a 30 second pretreatment with the antagonist , we co-anit Fig.  had veryreceptor -16, our 50 concentration of glycine (300 μM) did not significantly affect glycine-gated currents themselves. Glycine currents were 17.3 ± 2.3 pA/pF while glycine+colchicine currents were 16.7 ± 2.2 pA/pF (p > 0.5, paired two-tail t-test, n = 7).A third possible mechanism for reduced efficacy in L-cells compared to HEK cells or neurons could be related to intracellular factors that influence channel gating . This hy2β subunits following 30 min incubation with 100 μM colchicine at 37°C, enough time to allow irreversible tubulin disruption [2β-expressing HEK cells  and β-alanine (p < 0.05, ANOVA) were significantly decreased by colchicine but not γ-lumicolchicine treatment. Taurine efficacy was 33 ± 6% of glycine in controls, 13 ± 3% following colchicine, and 28 ± 3% following γ-lumicolchicine. Similarly, β-alanine efficacy was 70 ± 7% of glycine in controls, 49 ± 6% following colchicine, and 72 ± 7% following γ-lumicolchicine. Similar treatment of α2-expressing HEK cells with colchicine  but not in GlyRα2-expressing L-cells  and 'forebrain' (α2β heteromeric) isoforms in two distinct expression systems to understand the influence of endogenous and exogenous factors on receptor partial agonist pharmacology. Although the pharmacology of the 'embryonic' GlyRα2 isoform and the 'adult spinal' isoform (GlyRα1β) have been explored more frequently in the literature, the pharmacology of GlyRα2β receptors has remained largely unexplored. Despite this, there is strong evidence that the adult 'forebrain' isoforms, specifically in the rat basolateral amygdala, is indeed α2β [2β subunits compared to their homomeric α2 counter parts.We have expressed several the 'embryonic'  replaced the β subunit when examining α2 homomeric channels.HEK 293 , mouse L-cells , NIH/3T3 fibroblasts (ATCC), and MDCK cells  were grown in Dulbecco's modified Eagle's medium (DMEM, SIGMA) with 10% fetal bovine serum (HyClone Laboratories, Logan, UT, USA) and 1X penicillin/streptomycin (Life Technologies) on Thermanox cover slips in 35 mm culture dishes. Cells were transfected during log-phase growth (30–50% confluent) using the Superfect reagent , according to manufacturers instructions. Rat glycine receptor αeviously . Briefly2 1, Mg-ATP 4, pH 7.2 with methane sulfonic acid; adjusted to 290–295 mmol kg-1 with sucrose. Whole cell capacitance and series resistance was manually compensated after opening the cell. Cells were continuously bath perfused with a HEPES-buffered saline (in mM): NaCl 150, Glucose 10, HEPES 10, KCl 2.5, CaCl2 2.5 MgCl2 1.0, pH 7.4, 305–320 mmol kg-1 Data will be analyzed off-line using pClamp software (Axon). Numerical analysis was performed using commercially available software. Independent student's t-test and two-way ANOVA were used for comparisons where appropriate; and statistical significance was based on p < 0.05. Concentration-response curves were generated from fits of data to a standard logistic equation as previously described (McCool & Botting, 2000). To derive KB from functional IC50 and EC50 data, the Cheng-Prusoff equation was used:Whole cell recordings were performed at room temperature using standard patch-clamp techniques and the axopatch-1D amplifier (Axon Instruments, Inc., Foster City CA, USA) in the voltage clamp mode. Gigaohm seals were formed using patch pipettes made from borosilicate glass (World Precision Instruments, Sarasota FL, USA). For most experiments, the internal solution contained (in mM): CsCl 100, EGTA 11, HEPES 10, CaClS = hillslope of agonist curve, A = concentration of agonist, EC50 = half-maximal agonist concentration, and IC50 = half-maximal antagonist concentration.where Stocks of glycine, taurine, β-alanine, strychnine (Tocris) and colchicine (SIGMA) were prepared fresh each day. Agonists and antagonists were applied for 4–10 sec from an array of eight HPLC-grade capillary tubes  placed within 100 μm of the cell of interest.Cells were cultured, transfected as stated above except in 10 cm Petri dishes, and harvested by scrapping with 500 μL of lysis buffer (10 mM Tris pH 7.5, 1% SDS). Proteins were quantified using the Bradford assay and loaded onto 8–10% SDS-polyacrylamide gels, separated, and transferred to a nitrocellulose membrane (Hybond C, Amersham). The membrane was blocked overnight TBS (200 mM NaCl, 10 mM Tris pH 7.5) containing 0.2% Tween-20 and 10% low-fat dry milk. Blots were then incubated in TBS/0.1% Tween-20 containing primary GlyR antibody  and monoclonal anti-gephyrin antibody (1:2000, Transduction Laboratories) for two hours at room temperature. After several washes, the HRP-coupled rabbit anti-mouse secondary antibody (SIGMA) was added for one hour (1:2000). The detection was performed by the ECL method (Amersham).GlyR – glycine receptor; BLA – lateral/basolateral amygdala; HEK – human embryonic kidney cells; MDCK – Manin-Darby canine kidney cellsJF carried out the electrophysiological recordings and participated in the western analysis. BM conceived of the study, participated in its design and coordination, performed the western analysis and some electrophysiology experiments, and drafted the manuscript. All authors read and approved the final manuscript.


Doing scientific research in post-Soviet Russia is challenging but there are solutions that could prevent the massive brain drain witnessed in recent decades A scientist living in Russia is often asked two questions: “Why haven't you left?” and “Is it still possible to work there?” The best response to the first question is, “Why should I?”—which either terminates the conversation or leads to a stimulating discussion about the fate of the world. The second question, however, deserves a serious answer. In fact, this is the question that every one of us keeps asking ourselves.There is no simple answer. The biggest problems we face are brain drain, inadequate infrastructure, and lack of money (or perhaps, lack of money, lack of money, and lack of money). In the Soviet Union, fundamental science was supported to a great extent by military expenditure. Thus, it is not surprising that Soviet physics and mathematics were more successful than other fields, such as biology. In the 1990s, military spending on science declined sharply, although the exact numbers are hard to estimate. This year, the direct funding of science constitutes only 1.78% of Russia's national budget , although the law stipulates that this figure should be at least 4%. Still, this funding amounts to 46.2 billion rubles (approximately US$1.6 billion), more than twice the amount spent in 2000. Although this figure looks negligible compared with spending on science in the United States and many European countries, it could still be sufficient to support existing actively working groups at a reasonable level.There are several mechanisms for distributing funds for research. The major share comes via Russia's Department of Science and the Russian Academy of Sciences. The Academy, unlike its Western analogs, not only acts as a consulting body of experts, but also has the authority to distribute money . The fMoney is also distributed through the Russian Foundation for Basic Research (RFBR). The decisionmaking mechanism used by RFBR is closer to Western standards, and involves anonymous refereeing followed by board discussions. Although its grants are rather small , they provide important additional support for many small and mediumsized groups that may receive several such grants for different projects. In addition, RFBR supports the publication and translation of books, travel to international conferences, the organization of conferences in Russia, and similar activities. Unfortunately, several programs (in particular, support for young scientists) have recently been transferred from RFBR to a newly established government office, and have thus become less independent.International collaboration and research grants are a major source of support for many active research groups . SeveralAnother source of financial support is direct collaboration between Western and Russian laboratories. Even after a relatively short visit, the salary of a visiting researcher abroad can be stretched for several months back home in Russia; even more importantly, experimental biologists visiting foreign labs have access to modern instruments and chemicals, which allows them to do modern research. The hosts of such visits are often  recent immigrants from Russia, and in such cases the collaborations may have roots in older days .As well as supporting Russian science directly, international collaboration plays an important indirect role because it is less influenced by local politics. In fact, one of the main positive impacts of the New York–based International Science Foundation set up by George Soros in early 1990s was that it demonstrated the possibility of open competition with clearly defined rules—something unheard of in Soviet times—and thus served as a model for the RFBR, which was organized at approximately the same time.Unfortunately, international ties, especially with the United States, have been adversely affected by recent changes in visa procedures, which have become lengthy (leading to many missed conferences) and, even worse, completely unpredictable e.g., . The grahttp://www.evrogen.com/about.shtml).The demand for fundamental and even for applied biological research from Russian industry is almost nonexistent. The pharmaceutical industry is content to produce generics, while Russian biotech companies are still exploiting old strains developed in the Soviet Union. However, some laboratories are conducting outsourced research, and there are now research outposts of Western and Japanese companies in Russia organized as standard industrial labs. On one hand, this work is a dead end for Russian scientists, because the results of such research normally cannot be published. This is a serious problem, especially for young scientists who want to establish themselves. On the other hand, royalties from patents or commercialization of the products can be used to support further research. One group that has followed this path successfully is Sergey Lukyanov's lab at the Shemyakin Institute of Bioorganic Chemistry in Moscow, Russia. They have developed the subtractive hybridization technique for enrichment of clone libraries by rare transcripts or specific genomic fragments , and areAnother major problem is the degradation of infrastructure. Only a few labs can afford modern equipment and instruments, and for many others, even standard chemicals are too expensive. This leads to a vicious circle: without equipment, a lab cannot conduct experiments at the level demanded by highimpact journals—and without such publications, it cannot compete for large grants. Smaller RFBR grants, while simpler to obtain, are insufficient to purchase large pieces of equipment, and funds from several grants or several years cannot be combined due to bureaucratic restrictions. Thus, the only hope for these labs, apart from international collaboration, is a personal connection with senior bureaucrats that might result in an (un)expected windfall.Having the funds to purchase modern equipment abroad is only the first hurdle; the many conflicting rules and restrictions, inefficiency, and corruption within the system can subsequently hold up the process. Some items, such as tissue samples or animals, are virtually impossible to import legally. The process of clearing the shipments through customs is a difficult, timeconsuming job. Grigory Kopelevich, the Howard Hughes Medical Institute's Russian representative, recalls a story of a grantee whose microscope was stopped at customs because the box contained two screwdrivers not specified in the order. Fortunately, to resolve the issue, it was sufficient to present one of the screwdrivers to a customs officer as a gift.Nature, and only two to Science. More specialized journals are available only in Moscow and perhaps St. Petersburg. This is partially offset by the proliferating open-access journals from the Public Library of Science and BioMed Central, free electronic versions of older issues provided by some journals, free subscriptions for Russian academic institutes granted by some publishers or purchased by international foundations (e.g., the e-library.ru project organized by the RFBR and supported by the Open Society Institute [the Soros Foundation, based in New York] and the Department of Education)  , reprintThese problems, along with low salaries, have naturally led to a huge brain drain. Entire generations have been decimated ; the deaDespite all this, science in Russia is very much alive. Not-yet-Nobel-prizewinner Alexei Abrikosov's repeated exhorations to the scientific community “to help all the talented scientists leave Russia and to ignore the rest” were met by universal disgust . There aWhat can be done by the international community to support what is left of Russian science? Of course, direct support in the form of competitive grants is important, especially if there are few restrictions on spending; even the most carefully considered procedure cannot foresee all possible situations. But even more useful is the creation of joint research centers, such as the one opened by the international Ludwig Institute for Cancer Research (LICR) based jointly in Zurich, New York, and London, and the Belozersky Institute of Physico-Chemical Biology of Moscow State University in Moscow, Russia. This research center began with limited support for several stronger groups, and is gradually moving toward integration of a research program in Moscow with other LICR projects, and real collaboration between Moscow groups and LICR labs elsewhere.www.oncoimmunology.ru/index_e.htm), and the participation of Western scientists in scientific conferences in Russia are important not only because they provide a fresh understanding of emerging trends, but also because they create personal contacts between Russian and Western scientists that often lead to fruitful collaboration.One of the most essential elements of successful research is access to up-to-date information. Consequently, any initiative that provides open access to scientific literature and databases is extremely useful. Seminars, lecture courses , indicating that international collaboration is the most reliable source of support for top-level research. (Text and figures in this box courtesy of Alexey Kondrashov.)


Correlation between the expression levels of genes which are located close to each other on the genome has been found in various organisms, including yeast, drosophila and humans. Since such a correlation could be explained by several biochemical, evolutionary, genetic and technological factors, there is a need for statistical models that correspond to specific biological models for the correlation structure.We modelled the pairwise correlation between the expressions of the genes in a Drosophila microarray experiment as a normal mixture under Fisher's z-transform, and fitted the model to the correlations of expressions of adjacent as well as non-adjacent genes. We also analyzed simulated data for comparison. The model provided a good fit to the data. Further, correlation between the activities of two genes could, in most cases, be attributed to either of two factors: the two genes both being active in the same age group (adult or embryo), or the two genes being in proximity of each other on the chromosome. The interaction between these two factors was weak.Correlation between the activities of adjacent genes is higher than between non-adjacent genes. In the data we analyzed, this appeared, for the most part, to be a constant effect that applied to all pairs of adjacent genes. Several studies  typically spans several genes (Roy[Gene expression is influenced by many factors (for a review, see Orphanides), many orphanides)), and aenes (Roy). Therefenes (Roy) suggestenes (Roy). And Chenes , or in  Llorente). Parisi Llorente found a  Llorente suggesteFinally, since the action of a transcription factor on a promotor gets weaker with distance, genes belonging to the same pathway should show stronger correlation if they are located close to each other (Dorsett ). Due toconsistent coregulation structure. Suppose that two genes (in this case, two adjacent genes) are co-regulated because, for example, they participate in the same pathway. They would, then, show a strong correlation because they would be co-regulated in all tissue samples. This need not necessarily be the case with the chromatin domain model: the segments of euchromatin in one tissue sample may overlap with those in another tissue sample. This latter scenario, we call an inconsistent coregulation structure. With consistent coregulation, adjacent gene pairs will show either strong positive correlation, or they will be uncorrelated. With inconsistent coregulation, all adjacent gene pairs will show a modest positive correlation.An important consequence of the evolution-based theories is that they predict a w be a fixed window size, e.g. 10. For each window of w adjacent genes, the average pairwise Pearson correlation coefficient within the window was computed. If that measure was found to be significant at, say, 1 - α = 0.999 , all the genes in the sequence were tagged. Doing this for all windows , the total number of tagged genes was counted. Then the experiment was repeated with shuffled genes , and the number of tagged genes in the shuffled experiment was subtracted from the number of tagged genes in the original experiment. This difference  grows with window size and starts plateauing for a window size of approximately 10. Spellman and Rubin interpreted this as evidence for gene interaction within regions of approximately that size.In a microarray analysis of gene expressions in 35 pools of drosophila embryos and 54 adult drosophilae  Correlation of gene expression that can be attributed to consistent coregulation, fromB) The uniform correlation expected under an hypothesis of inconsistent corregulation.We used the Drosophila data published by Spellman and Rubin. This daμ0), was 0.014 with a standard error of 0.002. . The fact that the standard deviation of the third component, σ+, is greater than that of the other components, suggests that the co-regulation of some gene pairs was stronger than that of others.The distribution of the correlation coefficient between gene pairs in general, (i.e., genes that are not necessarily adjacent) was fitted with a three-component normal mixture, based on the Arcus Tangens Hyperbolic-transform (Fisher's z) of the correlation coefficients, which was validated with a Kolmogorov-Smirnov test (p = 0.6). As shown in table μ0 and μ+ are substantially higher. This suggests that the effect of adjacency is, for the most part, a mechanism that applies to adjacent-gene pairs in general, not just to specific adjacent-gene pairs. One way to illustrate this is by means of a qq-plot (figure Table t figure , which sμ0, μ+ and the size of the third component (that is, the fraction of the genes that are positively co-regulated), for random and adjacent gene pairs in four subsets of the flies: one containing all 54 adult pools, one containing all 35 embryo pools, and two random subsets of 35 and 54 pools, respectively. One can observe that μ0  is always high for adjacent gene pairs and low for random gene pairs. On the other hand μ+ and the "+"-fraction (which can be attributed to corregulation of specific gene pairs, i.e., consistent co-regulation) are always high in heterogenous fly groups and low in homogenous one. That difference between heterogenous and homogenous groups is what we expected: In a homogenous fly group, less gene pairs will come up as co-regulated, because some pathways may either be active in all flies, or inactive in all flies. That age groups does not explain all correlation, is hardly surprising: although the adult group and the embryo group are less heterogenous than the mixed group, neither is homogenous.Table μ0 was 0.114 for the correlation between the expression of a gene and that of its direct neighbor, and 0.08 for the correlation with its second neighbor. From the fifth neighbor and until at least the tenth, a stable level of 0.03 is reached, which is still higher than for distant gene pairs. One possible interpretation of this is that two different co-regulation effects exist: a short-segment effect accounting for a correlation of approximately 0.114-0.03 = 0.081, and a long-segment effect accounting for a correlation of approximately 0.03-0.014 = 0.016. At first we had the suspicion that this stable level of 0.03 was a whole-chromosome effect, but that was not the case. We found no significant difference between the overall average correlation of random gene pairs from the same chromosome and random gene pairs from different chromosomes.To get an impression of the size of the segments involved in co-regulation, we fitted the three-component mixture to the correlation coefficient between the expressions of more distant gene pairs. μ0) for the second neighbor would be the square of that for the first neighbor. However, the unexpectedly small difference between the two values could be due to the fact that some second neighbors are physically quite close. To confirm or reject the hypothesis of a first-order process, however, the analysis must be based on physical distance rather than simple adjacency. As shown in table In a first-order autocorrelation process, tanh(μ+ is not significantly higher than for random gene pairs.Table segment effect (see Methods) was always positive in the simulated models).To validate our approach, we computed the correlation coefficients of adjacent and random gene pairs in simulated data, based on either consistent or inconsistent co-regulation. As expected, the simulated data with inconsistent co-regulation resulted in a correlation structure with only one component, whereas those with consistent coregulation showed two components. , which makes a relatively simple correlation structure plausible. Also, the data from each of their microarrays could be fit nicely with a normal mixture, which makes it possible to analyze their data on the basis of the Pearson R. As expected, most of the consistent correlation was found to be related to age group and unrelated to adjacency. Maybe more surprisingly, most of the correlation that was related to adjacency could not be accounted for by consistent co-regulation. This suggest that, at the statistical level, co-regulation of adjacent genes should be understood in terms of the mechanics of the transcription process, rather than in terms of the evolutionary origin of specific gene groups. In other words, in this particular data set, consistent correlation was very widespread, applying to 40% of all gene pairs table , half ofμ0 in table Spellman and Rubin concluded that the adjacent genes with correlated expressions were confined to certain domains, which spanned 20% of all genes. Our findings are different, in that we distinguished between two components of the correlation structure. We found a baseline correlation of 0.1  in tissue differentiation. Parisi et al.  found clμ0 was slightly positive. It is hard to imagine a biological effect that could cause μ0 to be significantly greater than zero, but it could be an artifact of the normalization: if gene g is expressed at the same level in all tissue samples, and gene h as well, they should have a correlation coefficient of zero, but since they will both yield elevated measurements in flies in which the normalization bias is positive, the observed correlation will be positive. The fact that μ0 is almost zero suggests that normalization bias is not a major problem with this data set. The fact that μ0 for random gene pairs is not generally higher in mixed age groups than in pure adult or embryo groups  gene pairs, ps table  is consi, Fukuoka found thFirst, it is not clear with respect to which anchor the physical distance should be defined. We have chosen to use the minimal distance (using the end nearest to the neighbor as an anchor), because it is not confounded by the length of the gene. However, depending on which co-regulation mechanism one has in mind, other distance measures might be more natural. This question becomes crucial if one wants to make subgroup analysis based on convergent, divergent and tandem gene pairs.Second, physical distance is a continuous variable, which means that the model must be augmented with an assumption regarding the relationship between distance and correlation.The question remains to what biological phenomena the baseline correlation between adjacent genes should be attributed. Several of the theories that have been proposed could account for it, and it is likely that several mechanisms play a role.In addition to the baseline correlation, we also found more consistent correlation between adjacent genes and inconsistent co-regulation, but this does not necessarily imply a combination of two mechanisms: one could imagine a kind of quasi-consistent mechanism, in which groups of adjacent, co-regulated genes could have boundaries everywhere, but with some boundary locations being more likely than others. Finally, it has been suggested that the correlation between the expressions of adjacent genes should be understood in terms of enhancer-promotor interaction, which weakens with distance(Dorsett). It is It is possible to analyze correlation structures in gene expression data on the basis of simple, parametric models with known mathematical properties and parameters with biological interpretations, or at least some candidate interpretations. When applied to the data from Spellman and Rubin, it appeared that the expressions of two genes can show positive correlation for either of two largely distinct reasons: because they share some confounder unrelated to adjacency , or because they are located close to each other on the chromosome. The underlying biological mechanisms remain unknown, but there appears to be a component of the correlation that depends on distance only and not of the biological function of the genes. If this is true, gene clustering algorithms might benefit from making distinction between the adjacency-related and not-adjacency-related co-regulation, for example by subtracting the effect of adjacency from the correlation of adjacent genes. Doing that would, according to our findings, lead to more reliable identifications of gene pairs with related biological functions.Ygi be the log-scale measured activity of gene g in fly i, fly i having development stage s (either "adult" or "embryo"). A natural model for Ygi would be a two-component normal mixture:First, we constructed a model for those co-regulation effects that were unrelated to the relative location of the two genes. Suppose that a gene can be active either in adults, in embryos, in both, or in neither. Let g in the other development stage should be taken into account. We therefore assumed a three-component normal mixture:Since the data were normalized so that the average logscale activity across all 89 flies was zero, the activity of gene Actually, of the 89 flies, the gene expressions in 87 of them could be nicely fitted with a three-component, normal mixture, while in two of the adult flies we identified only two components (the component of genes that were passive in adults while active in embryos vanished in those two cases).Rgh for two genes, g and h, isIf the three-component model is correct, Fisher's z-transform, which is the hyperbolic arcus tangens of the Pearson correlation coefficient μ0 to be close to zero. This model was validated by implementing it on the neighbor correlations in a shuffled data set, that is, for each gene its Pearson R with a random gene was computed. This procedure was repeated with 10 shuffled data sets, each generated by shuffling the genes in the original data set and subsequently computing the Pearson R for each pair of adjacent genes. For each shuffled data set, the parameters in the model were estimated with the EM algorithm .Second, the distribution of the correlation coefficients of the pairs of adjacent genes in the unshuffied data set were compared to the same distribution for the shuffled data sets. Doing that, we could distinguish two different contributions to the correlation; A) co-regulation effects that are unrelated to the fact that two genes are adjacent, and B) co-regulation effects related to the fact that two genes are adjacent. We also analyzed each gene's correlation with its second, third etc. (up to the tenth) neighbor, in order to get an idea of the lengths of the chromatin domains in question.Third, we applied the above two procedures to two subsets of the data, namely the data for adult flies and the data for embryos. If our assumption, that all (or at least most) correlation could be attributed to the development stage, one would expect much less correlation in those subsets. However, those subsets differ from the entire data set by the mere fact that they are smaller. Therefore, we also applied the same procedure to two random subsets of 35 and 54 flies, respectively, stratified by development stage.For those gene pairs where the start and the end of the open reading frame (ORF) was available, we defined the physical distance as the distance between the end of the ORF of the first gene and the start of the ORF of the second gene. We chose that definition because other distance definitions (defined on the basis of the direction of transcription, or on the basis of mid-points) would be confounded by the length of the ORFs.consistent co-regulation, in which the same segments were used for all 100 tissue samples, and one data set with inconsistent co-regulation, in which a separate segmentation was sampled for each tissue sample. If gene g belongs to segment s in sample i, the expression Ygi was given byFinally, in order to validate our method, we applied it to 6 simulated data sets, which we simulated on the basis of the two alternative hypothesis of consistent and inconsistent co-regulation. The simulated data sets contained 100 tissue samples and 1000 genes. The genes were randomly divided into either 67, 200 or 500 segments, corresponding to average segment sizes of 15, 5 and 2. This means that each gene belonged to exactly one segment. For each average segment size, we simulated one data set with Ygi = xsi + εgi     (4)xsi + and εgi were both sampled from the standard normal distribution. Notice that this model does not account for different tissue classes, so it should be compared to the results from the adults-only and embryos-only subsets. The error term εgi should be interpreted as a combination of measurement errors and biological effects that are unrelated to the segmentation.where HT did the explorative data analysis and suggested the concept of consistent co-regulation and using three-components mixtures for the correlation structure. AZ suggested using Fisher's z-transform for the correlations. HT did the literature review. Both authors contributed to the manuscript.


Plasmodium-infected mosquito bites a human, it injects the parasite—sequestered in the mosquito's salivary glands in its sporozoite stage—into the victim's bloodstream. Within hours, the sporozoites invade the liver—a critical stage for establishing infection—and spend the next few weeks asexually dividing inside liver cells, eventually releasing thousands of merozoites into the bloodstream. Merozoites quickly invade red blood cells and begin a second round of asexual proliferation. The infected cells rupture and die, releasing more parasites and toxins. The toxins cause malaria's characteristic fever and chills, and the liberated merozoites initiate another cycle of red blood cell attacks.Plasmodium, the microscopic parasite that causes malaria, passes through two hosts, two reproductive modes, four habitats, and over half-a-dozen distinct developmental stages in one lifecycle. When a An unresolved question has been how the circulating sporozoites reach the liver cells in the first place, since liver cells are separated from the bloodstream by a layer of endothelial and Kupffer cells, which form the walls of the liver capillaries. (Kupffer cells project into the bloodstream and remove contaminants.) Having identified a protein required for sporozoite migration through the capillary lining, Tomoko Ishino, Masao Yuda, and their colleagues at Mie University School of Medicine in Japan may have found an answer.Plasmodium species affect humans. P. falciparum, the most pathogenic of the human-infecting species, is closely related to avian and rodent species. One rodent species,—P. berghei—shares fundamental aspects of structure, physiology, and lifecycle with P. falciparum and so serves as a model for the human parasites. Since sporozoites must infect mosquito salivary glands before they can infect the mammalian liver, Yuda's team searched for sporozoite genes that are predicted to encode secretory or membrane proteins and are expressed only in mosquito salivary glands. Their search revealed a coding region conserved in several species of Plasmodium.Only four of the roughly 150 vertebrate-infecting Plasmodium motility and invasion, the researchers predicted this protein would also be important in migrating to or invading liver cells. They named the protein SPECT, for sporozoite microneme protein essential for cell traversal.Tracing the gene's activity through the parasite's life cycle, Yuda's team confirmed that it was expressed only in sporozoites in the mosquito salivary gland—not in the mosquito midgut, where sporozoites are produced after mosquitoes feed on the blood of an infected person. The corresponding protein was localized to micronemes, specialized secretory organelles found at the front end of malaria parasites. Because micronemes are known to play a central role in spect-disrupted mutants and observing how the altered parasites affected their hosts. spect disruption did not affect parasite proliferation in rat red blood cells or interfere with parasite development in the mosquito midgut or salivary glands, but it did have an effect on the parasite's ability to infect the liver. Rats injected with spect-disrupted parasites had significantly lower levels of liver infection than rats injected with nonmutant parasites. Since it was unclear whether the spect-disrupted mutants lost their infectivity or simply could not pass through the cell layer, the researchers inoculated human liver cells with the mutants and found that they infected the cells normally.Yuda's team tested SPECT's function by generating spect-disrupted parasites completely lost their ability to pass through cells. Since traversal of the cellular barrier between liver cells and the circulatory system is a crucial step in malarial infection, the authors conclude, SPECT and other proteins involved in shuttling sporozoites into liver cells could be effective targets for malaria treatment and prevention.Yuda's team also tested SPECT's impact on sporozoite cell-passage ability; if the mutants couldn't reach the liver cells, they couldn't infect them.


Recent technological advances in integrated circuits, wireless communications, and physiological sensing allow miniature, lightweight, ultra-low power, intelligent monitoring devices. A number of these devices can be integrated into a Wireless Body Area Network (WBAN), a new enabling technology for health monitoring.Using off-the-shelf wireless sensors we designed a prototype WBAN which features a standard ZigBee compliant radio and a common set of physiological, kinetic, and environmental sensors.We introduce a multi-tier telemedicine system and describe how we optimized our prototype WBAN implementation for computer-assisted physical rehabilitation applications and ambulatory monitoring. The system performs real-time analysis of sensors' data, provides guidance and feedback to the user, and can generate warnings based on the user's state, level of activity, and environmental conditions. In addition, all recorded information can be transferred to medical servers via the Internet and seamlessly integrated into the user's electronic medical record and research databases.WBANs promise inexpensive, unobtrusive, and unsupervised ambulatory monitoring during normal daily activities for prolonged periods of time. To make this technology ubiquitous and affordable, a number of challenging issues should be resolved, such as system design, configuration and customization, seamless integration, standardization, further utilization of common off-the-shelf components, security and privacy, and social issues. Wearable health monitoring systems integrated into a telemedicine system are novel information technology that will be able to support early detection of abnormal conditions and prevention of its serious consequences ,2. Many Important limitations for wider acceptance of the existing systems for continuous monitoring are: a) unwieldy wires between sensors and a processing unit, b) lack of system integration of individual sensors, c) interference on a wireless communication channel shared by multiple devices, and d) nonexistent support for massive data collection and knowledge discovery. Traditionally, personal medical monitoring systems, such as Holter monitors, have been used only to collect data for off-line processing. Systems with multiple sensors for physical rehabilitation feature unwieldy wires between electrodes and the monitoring system. These wires may limit the patient's activity and level of comfort and thus negatively influence the measured results. A wearable health-monitoring device using a Personal Area Network (PAN) or Body Area Network (BAN) can be integrated into a user's clothing . This syIncreased system processing power allows sophisticated real-time data processing within the confines of the wearable system. As a result, such wearable system can support biofeedback and generation of warnings. The use of biofeedback techniques has gained increased attention among researchers in the field of physical medicine and tele-rehabilitation . IntensiTypical examples of possible applications include stroke rehabilitation, physical rehabilitation after hip or knee surgeries, myocardial infarction rehabilitation, and traumatic brain injury rehabilitation. The assessment of the effectiveness of rehabilitation procedures has been limited to the laboratory setting; relatively little is known about rehabilitation in real-life situations. Miniature, wireless, wearable technology offers a tremendous opportunity to address this issue.We propose a wireless BAN composed of off-the-shelf sensor platforms with application-specific signal conditioning modules . In thisContinuous technological advances in integrated circuits, wireless communication, and sensors enable development of miniature, non-invasive physiological sensors that communicate wirelessly with a personal server and subsequently through the Internet with a remote emergency, weather forecast or medical database server; using baseline , sensor (WBAN) and environmental (emergency or weather forecast) information, algorithms may result in patient-specific recommendations. The personal server, running on a PDA or a 3 G cell phone, provides the human-computer interface and communicates with the remote server(s). Figure A WBAN can include a number of physiological sensors depending on the end-user application. Information of several sensors can be combined to generate new information such as total energy expenditure. An extensive set of physiological sensors may include the following:• an ECG (electrocardiogram) sensor for monitoring heart activity• an EMG (electromyography) sensor for monitoring muscle activity• an EEG  sensor for monitoring brain electrical activity• a blood pressure sensor• a tilt sensor for monitoring trunk position• a breathing sensor for monitoring respiration• movement sensors used to estimate user's activity• a "smart sock" sensor or a sensor equipped shoe insole used to delineate phases of individual stepsThese physiological sensors typically generate analog signals that are interfaced to standard wireless network platforms that provide computational, storage, and communication capabilities. Multiple physiological sensors can share a single wireless network node. In addition, physiological sensors can be interfaced with an intelligent sensor board that provides on-sensor processing capability and communicates with a standard wireless network platform through serial interfaces.The wireless sensor nodes should satisfy the following requirements: minimal weight, miniature form-factor, low-power operation to permit prolonged continuous monitoring, seamless integration into a WBAN, standard-based interface protocols, and patient-specific calibration, tuning, and customization. These requirements represent a challenging task, but we believe a crucial one if we want to move beyond 'stovepipe' systems in healthcare where one vendor creates all components. Only hybrid systems implemented by combining off-the-shelf, commodity hardware and software components, manufactured by different vendors promise proliferation and dramatic cost reduction.The wireless network nodes can be implemented as tiny patches or incorporated into clothes or shoes. The network nodes continuously collect and process raw information, store them locally, and send them to the personal server. Type and nature of a healthcare application will determine the frequency of relevant events (sampling, processing, storing, and communicating). Ideally, sensors periodically transmit their status and events, therefore significantly reducing power consumption and extending battery life. When local analysis of data is inconclusive or indicates an emergency situation, the upper level in the hierarchy can issue a request to transfer raw signals to the upper levels where advanced processing and storage is available.The personal server performs the following tasks:• Initialization, configuration, and synchronization of WBAN nodes• Control and monitor operation of WBAN nodes• Collection of sensor readings from physiological sensors• Processing and integration of data from various physiological sensors providing better insight into the users state• Providing an audio and graphical user-interface that can be used to relay early warnings or guidance (e.g., during rehabilitation)• Secure communication with remote healthcare provider servers in the upper level using Internet servicesThe personal server can be implemented on an off-the-shelf Internet-enabled PDA  or 3 G cell phone, or on a home personal computer. Multiple configurations are possible depending on the type of wireless network employed. For example, the personal server can communicate with individual WBAN nodes using the Zigbee wireless protocol that provides low-power network operation and supports virtually an unlimited number of network nodes. A network coordinator, attached to the personal server, can perform some of the pre-processing and synchronization tasks. Other communication scenarios are also possible. For example, the personal server running on a Bluetooth or WLAN enabled PDA can communicate with remote upper-level services through a home computer; the computer then serves as a gateway Figure .Relying on off-the-shelf mobile computing platforms is crucial, as these platforms will continue to grow in their capabilities and quality of services. The challenging tasks are to develop robust applications that provide simple and intuitive services . Total information integration will allow patients to receive directions from their healthcare providers based on their current conditions.We envision various medical services in the top level of the tiered hierarchy. A healthcare provider runs a service that automatically collects data from individual patients, integrates the data into a patient's medical record, processes them, and issues recommendations, if necessary. These recommendations are also documented in the electronic medical record. If the received data are out of range or indicate an imminent medical condition, an emergency service can be notified . The exact location of the patient can be determined based on the Internet access entry point or directly if the personal server is equipped with a GPS sensor. Medical professionals can monitor the activity of the patient and issue altered guidance based on the new information, other prior known and relevant patient data, and the patient's environment (e.g., location and weather conditions).The large amount of data collected through such services will allow quantitative analysis of various conditions and patterns. For example, suggested targets for stride and forces of hip replacement patients could be suggested according to the previous history, external temperature, time of the day, gender, and current physiological parameters (e.g., heart rate). Moreover, the results could be stored in research databases that will allow researchers to quantify the contribution of each parameter to a given condition if adequate numbers of patients are studied in this manner. Again, it is important to emphasize that the proposed approach requires seamless integration of large amounts of data into a research database in order to be able to perform meaningful statistical analyses.The ActiS sensor was developed specifically for WBAN-based, wearable computer-assisted, rehabilitation applications. With this concept in mind, we integrated a one-channel bio-amplifier and three accelerometer channels with a low power microcontroller into an intelligent signal processing board that can be used as an extension of a standard wireless sensor platform. ActiS consists of a standard sensor platform, Telos, from Moteiv and a custom Intelligent Signal Processing Module – ISPM Figure . A blockThe Telos platform is an ideal fit for this application due to small footprint and open source system software support. A second generation of the Telos platform features an 8 MHz MSP430F1611 microcontroller with integrated 10 KB of RAM and 48 KB of flash memory, a USB  interface for programming and communication, and an integrated wireless ZigBee compliant radio with on-board antenna [The ISPM extends the capabilities of Telos by adding two perpendicular dual axis accelerometers  and a bio-amplifier with a signal conditioning circuit. The ISPM has its own MSP430F1232 processor for sampling and low-level data processing. This microcontroller was selected primarily for its compact size and ultra low power operation. Other features that were desirable for this design were the 10-bit ADC and the timer capture/compare registers that are used for acquisition of data from the accelerometers. The F1232 has hardware UART that is used for communications with Telos.The ISPM's two ADXL202 accelerometers cover all three axes of motion. One ADXL202 is mounted directly on the ISPM board and collects data for the X and Y axes in the same plane. The second ADXL202 is mounted on a daughter card that extends vertically from the ISPM.The user's physiological state is monitored using an on-board bio-amplifier implemented using an instrumentation amplifier with a signal conditioning circuit. The bio-amplifier could be used for electromyogram (EMG) or electrocardiogram (ECG) monitoring. The output of the signal conditioning circuit is connected to the local microcontroller as well as to the microcontroller on the Telos board via the expansion connector. The AD converter on the Telos board has a higher resolution (12 bit) than the F1232 on the ISPM (10 bit). This configuration gives flexibility of utilizing either microcontroller to process physiological signals.x and Ay. Rotations of the sensor in the vertical plane (Θ) can be estimated as Θ = arctan(Ax / Ay). A compensation for non-ideal vertical placement can be achieved using the second accelerometer (not mounted in this photo) at 90-degree angle. Instead of calculating the angular position, many systems use off-the-shelf gyroscopes to measure angular velocity for the detection of gait phases [An example application of the ActiS sensor as motion sensor on an ankle is given in Figure t phases . A typicsystem design and social issues should be resolved. If resolved successfully, WBAN systems will open a whole range of possible new applications that can significantly influence our lives.WBAN systems can capitalize on recent technological advances that have enabled new methods for studying human activity and motion, making extended activity analysis more feasible. However, before WBAN becomes a widely accepted concept, a number of challenging The development of pedometers and Micro-ElectroMechanical Systems (MEMS) accelerometers and gyroscopes show great promise in the design of wearable sensors. The main system design issues include:• types of sensors• power source• size and weight of sensors• wireless communication range and transmission characteristics of wearable sensors• sensor location and mounting• seamless system configuration• automatic uploads to the patient's electronic medical record• intuitive and simple user interfaceet al [As for sensors, accelerometers and gyroscopes offer greater sensitivity and are more applicable for monitoring of motion since they generate continuous output. Bouten et al  found thet al ,35. As eet al  systems et al . Both Laet al . Lancastet al .The majority of foot-contact pedometers are designed to count steps only. Although they have been studied for use in complex energy estimation and have even shown a high degree of accuracy for walking / running activities  they areTo be unobtrusive, the sensors must be lightweight with small form factor. The size and weight of sensors is predominantly determined by the size and weight of batteries. Requirements for extended battery life directly oppose the requirement for small form factor and low weight. This implies that sensors have to be extremely power efficient, as frequent battery changes for multiple WBAN sensors would likely hamper users' acceptance and increase the cost. In addition, low power consumption is very important as we move toward future generations of implantable sensors that would ideally be self-powered, using energy extracted from the environment.The radio communication poses the most significant energy consumption problem. Intelligent on-sensor signal processing has the potential to save power by transmitting the processed data rather than raw signals, and consequently to extend battery life. A careful trade-off between communication and computation is crucial for an optimal design. It appears that the most promising wireless standard for WBAN applications is ZigBee, as it represents an emerging wireless technology for the low-power, short-range, wireless sensors.et al use two accelerometers on the SenseWear armband [et al [et al developed a loose fitting lab coat and trousers [et al [Although the purpose of the measurement does influence sensor location, researchers seem to disagree on the ideal body location for sensors. A motion sensor attached to an ankle is the most discriminative single position for state recognition, while a combination of hip and ankle sensors discriminates the states even more . In a st armband . Lee et d [et al  placed atrousers  consistis [et al developedThe intelligent WBAN sensors should allow users to easily assemble a robust ad-hoc WBAN, depending on the user's state of health. We can imagine standard off-the-shelf sensors, manufactured by different vendors, and sold "over-the-counter" . Each seApplication-specific algorithms mostly use digital signal pre-processing combined with a variety of artificial intelligence techniques to model user's states and activity in each state. Digital signal processing include filters to resolve high and low frequency components of a signal, wavelet transform algorithms to correlate heel-strike and toe-off (steps) to angular velocity measured via gyroscopes , power sMost of the algorithms in the open literature are not executed in real-time, or require powerful computing platforms such as laptops for real-time analysis.Social issues of WBAN systems include privacy/security and legal issues. Due to communication of health-related information between sensors and servers, all communication over WBAN and Internet should be encrypted to protect user's privacy. Legal regulation will be necessary to regulate access to patient-identifiable information.The WBAN technology can be used for computer-assisted physical rehabilitation in ambulatory settings and monitoring of trends during recovery. An integrated system can synergize the information from multiple sensors, warn the user in the case of emergencies, and provide feedback during supervised recovery or normal activity. Candidate applications include post-stroke rehabilitation, orthopaedic rehabilitation (e.g. hip/knee replacement rehabilitation), and supervised recovery of cardiac patients . In the In addition, WBAN systems can be used for gait phase detection during programmable, functional electrical stimulation , analysiA wearable Wireless Body Area Network (WBAN) of physiological sensors integrated into a telemedical system holds the promise to become a key infrastructure element in remotely supervised, home-based patient rehabilitation. It has the potential to provide a better and less expensive alternative for rehabilitation healthcare and may provide benefit to patients, physicians, and society through continuous monitoring in the ambulatory setting, early detection of abnormal conditions, supervised rehabilitation, and potential knowledge discovery through data mining of all gathered information.Continuous monitoring with early detection likely has the potential to provide patients with an increased level of confidence, which in turn may improve quality of life. In addition, ambulatory monitoring will allow patients to engage in normal activities of daily life, rather than staying at home or close to specialized medical services. Last but not least, inclusion of continuous monitoring data into medical databases will allow integrated analysis of all data to optimize individualized care and provide knowledge discovery through integrated data mining. Indeed, with the current technological trend toward integration of processors and wireless interfaces, we will soon have coin-sized intelligent sensors. They will be applied as skin patches, seamlessly integrated into a personal monitoring system, and worn for extended periods of time.