A major challenge of sensory systems neuroscience is to quantify brain activity underlying perceptual experiences and to explain this activity as the outcome of elemental neuronal response properties. Rats make extremely fine discriminations of texture by “whisking” their vibrissae across an object's surface, yet the neuronal coding underlying texture sensations remains unknown. Measuring whisker vibrations during active whisking across surfaces, we found that each texture results in a unique “kinetic signature” defined by the temporal profile of whisker velocity. We presented these texture-induced vibrations as stimuli while recording responses of first-order sensory neurons and neurons in the whisker area of cerebral cortex. Each texture is encoded by a distinctive, temporally precise firing pattern. To look for the neuronal coding properties that give rise to texture-specific firing patterns, we delivered horizontal and vertical whisker movements that varied randomly in time (“white noise”) and found that the response probabilities of first-order neurons and cortical neurons vary systematically according to whisker speed and direction. We applied the velocity-tuned spike probabilities derived from white noise to the sequence of velocity features in the texture to construct a simulated texture response. The close match between the simulated and real responses indicates that texture coding originates in the selectivity of neurons to elemental kinetic events. Rats move their whiskers rhythmically across surfaces to sense their environment. Textured surfaces induce distinct patterns of vibration in the whiskers that are encoded by central neural activity One goal of sensory systems neuroscience is to understand how the representations of complex, natural stimuli arise from the basic response properties of neurons. The present experiments explore the representation of textures in the rat somatosensory system. Rats have texture discrimination capacities rivaling those of humans . In ratsThere have been no reports concerning the cortical or subcortical neuronal activity generated by whisking along irregular surfaces, and the differences in activity associated with two surfaces remain unknown . HoweverTo answer these questions, the model must be challenged under conditions where the sensory input is precisely controlled and yet resembles what occurs during natural tactile behavior. Guided by this strategy, in anesthetized rats we produced whisker movements across textures while measuring vibrations of the whisker shaft. We then played back the identical vibrations to other rats, and measured the neuronal activity at two stages of the sensory pathway—the first-order neurons that innervate the sensory receptors, and the barrel cortex neurons, which are the first site of cortical integration. Texture discrimination depends on the integrity of the cortical barrels . By measn = 3), we electrically stimulated cranial nerve VII, generating 8-Hz whisking movements [The experimental strategy  was to covements ,18 that ovements . MeanwhiH) and vertical (VV) velocity. For VH, whisker protraction (forward movement) is positive and whisker retraction (backward movement) negative. For VV, upward movement is positive and downward movement negative. To better visualize the time-varying frequency content of the velocity profiles, the velocity spectrograms are also plotted  consists of six first-order neuron recordings, five cortical cluster recordings, and seven “paired” recordings—simultaneous first-order neuron and cortical cluster. The principal result is that time-varying neuronal activity in the trigeminal ganglion and cortex captured the kinetic features of the texture-induced vibrations. From the same texture library given in Two barrel cortex neuron clusters were recorded simultaneously with the first-order neuron, allowing direct comparison of different stations along the sensory pathway. The selectivity of first-order neurons and cortical clusters for the direction of whisker movement is described in more detail in Xω (referred to in previous publications as Af). The generalization of Xω to the natural, texture-induced vibration is 〈|X|ω〉Ω,Τ, a quantity known as “equivalent noise level”  and the first two P280 whisks (0 to 250 ms) of trial 50 is given in In The remarkable response locking of first-order neurons to stimulus features is further highlighted in From these observations we conclude that, under our experimental conditions, the trial-to-trial response variability of first-order neurons is caused exclusively by stimulus jitter, whereas that of cortical neurons results mainly from variations across time in sensory integration, and must emerge at some integration site between the trigeminal ganglion and cortex. A question of current interest is whether the variability in cortical responses results from noise and imprecision in neuronal integration , or elseH and VV varied randomly across time. Responses to noise stimuli allowed us to quantify velocity sensitivity and then to generate simulated spike trains based on the sequence of velocity events in the actual texture-induced vibrations. Finally, comparison between simulated and observed responses reveals the extent to which the responses to natural stimuli are explained by neuronal selectivity to velocity features: If simulated responses closely resemble real responses, we can conclude that neurons are in fact operating on natural texture stimuli according to their tuning to elemental kinetic events.We hypothesize that the firing patterns of first-order and cortical neurons during presentation of textures can be explained by their extraction of elemental features from the complex input signal, and that these elemental features are bursts of high velocity. To test this directly, we presented a “white noise” stimulus in which the two stimulus features VH and VV (white circles) across 5 ms of filtered white noise.Typically, neuronal tuning curves are mapped out using an “unbiased” stimulus—a stimulus that avoids the temporal correlations present in natural stimuli. The first step, therefore, was to map out how first-order and cortical neurons encode whisker kinetic features when these features are extracted from the context of the natural stimulus. We applied a stimulus that varied randomly in velocity—Gaussian velocity noise—and therefore was not constrained by the velocity patterns present in texture trajectories see . Figure 5,R9A . To construct the neuron's “tuning curve” in finer detail, velocity space was subdivided into 20 angular and 10 radial segments. The neuron emitted spikes with increasing probability as velocity increased, but only for restricted directions, preferring high speeds that combined retraction  and upward movement . For the simultaneously recorded cortical neuron cluster, spike probabilities in the 5–20-ms poststimulus interval are given in For the first-order neuron Zurvan, spike probabilities in the 1–2-ms postevent interval are given in Can the neurons' responses to complex, natural stimuli be explained as the outcome of these elemental tuning properties? To find out, we projected whisk velocity trajectories upon the white noise-derived tuning curves. One whisk  on P280 sandpaper is depicted on both tuning curves. The first observation is that the intersection of the velocity trajectory with the tuning curves explains why the first-order neuron was selective for whisker retraction while the cortical cluster was directionally nonselective see  and 5.P(t) and a corresponding raster plot for that trial. The 100-trial raster for one run of the simulation was summated to form a PSTH. The close match between the simulated PSTHs (black) and the real PSTHs  precise information about the kinetic features transmitted to the follicle. One kinetic feature is the “equivalent noise level”: Spike counts per whisk both for first-order neurons and for cortical neurons are proportional to the equivalent noise level of the texture-induced vibration. Thus, when texture vibrations differ in this quantity, neuronal spike counts also differ and thereby carry information that could, by itself, separate the textures. By the same token, when texture vibrations have similar equivalent noise levels, spike counts per whisk appear not to carry sufficient information. The second kinetic feature, then, is the temporal sequence of velocity events—distinctive velocity profiles induce distinctive temporal patterns in the spike trains with spike alignment of better than 0.2 ms in the first-order neurons and a few ms in the cortex.The stimulus playback method used here might not produce the identical input to the sensory receptor as occurs during active whisking . The optA classical approach to investigating sensory coding is to map the relationship between well-controlled artificial sensory stimuli and evoked neuronal activity. This can provide a complete description of neuronal feature extraction properties ,25, but In principle, one can bridge the gap between artificial and natural stimuli by (i) measuring neuronal activity during ecologically relevant stimuli, stimuli that are collected from an animal's normal interaction with the environment, (ii) constructing tuning curves under artificial stimulation , and (iii) applying the tuning curves to the natural stimuli to test whether they account for the observed response. Because artificial stimuli only partially cover dimensions of stimulus space present in natural stimuli, and because of neuronal nonlinearity, this procedure typically provides neuronal simulations that match real neuronal output with a correlation of less than 0.5 ,29,30. YAs an alternative to the temporal model outlined here, a spatial model for texture coding has recently been proposed. It begins from the observation that whisker length varies systematically across the anterior-posterior dimension of the rat's snout . When stThe resonance frequency hypothesis predicts that rats would fail to discriminate between surfaces using just a single whisker, yet they have been shown to perform texture discriminations after progressive clippings down to one or two whiskers . Our resExperiments were conducted in accordance with NIH and institutional standards for the care and use of animals in research. Subjects were ten adult male 250–350-g Wistar rats. In one set of anesthetized rats , “electrical whisking” ,18 was gH = 0), avoiding the introduction of any position or velocity discontinuity. A free whisk trial always separated two successive texture trials. A block was composed of five different texture trials (t1–5) with free whisk trials (fw) interspersed, e.g., fw-t3-fw-t5-fw-t1-fw-t2-fw-t4. Before stimulus delivery, signals were low-pass filtered at 500 Hz. All data were manipulated in MATLAB software (http://www.mathworks.com).For each texture, a 50-s continuous record was extracted and sliced into 100 trials of 500 ms, each trial composed of two-dimensional position signals across four 125-ms whisks. For free whisks, a 250-s record was sliced into 500 unique trials. The stimulus set was constructed by splicing trials together at the point of maximum retraction (Vx(t) and the whisker velocity profile asWe sought to quantify the kinetic features that characterized each texture-induced vibration. We refer to the whisker trajectory in one dimension as n)V| also corresponds to the spectrogram of the trajectory in position.Note that Xω [Xω to the texture-induced vibration. X becomes a time-varying spectrum XPrevious studies have shown that, when the stimulus set consists of sinusoidal whisker movements, the spike count per stimulus for cortical neurons is proportional to the product of the sinusoid's amplitude and frequency, Xω ,12. To tT is the entire time domain and Ω is the entire domain of frequency ω.where Xω across all time intervals and all values of ω.This quantity—known as the “equivalent noise level”—represents the average amplitude of white noise velocity that dissipates the same average power as the signal of interest. It serves to characterize the entire kinetic signature by a single quantity, equivalent to mean value of the product H and VV. nτ, the separately measured spectrograms for the two dimensions. The “velocity spectrogram” column in Our experiments measured velocity in two dimensions, Vhttp://www.fh-co.com) into the right trigeminal ganglion according to stereotaxic coordinates. Cortical recordings were obtained by inserting a 100 microelectrode array  to a depth of 700–1,000 μm in the left barrel cortex [In a second set of urethane-anesthetized rats, neuronal recordings were made simultaneously from two sites. First-order neurons were recorded by advancing a single electrode  in the first-order-only recordings were C3, E1, D6, E6, and γ (twice). The principal whiskers in the cortex-only recordings were A1, B4, E5, and E3 (twice). The principal whiskers in the paired first-order neuron-cortex recordings were δ (twice), E3 (twice), C2 (twice), and E4.Texture stimuli were delivered to a single whisker using a motor constructed from two orthogonal pairs of parallel piezoelectric wafers driven independently by horizontal and vertical signals . The whiH and VV independently from a Gaussian distribution 7,634 times per second. The stimulus was then low-pass filtered (Chebyshev type II) at 500 Hz so as to not exceed the physical capacities of the piezoelectric wafer stimulator . The noise stimulus was presented for 10 min after conclusion of the texture stimuli.We selected V1–8A) and ten radial sectors (1–10R). At each time point, angle corresponds to the direction of whisker movement, while radial distance corresponds to instantaneous speed. The positions of the radial boundaries were chosen to make each segment contain an equal number of instantaneous velocity events: Because velocity had a Gaussian distribution with a mean of zero, high-velocity events were less common, and consequently the radial boundaries were increasingly widely spaced as distance from zero increased.We adopted a method of “forward correlation” between stimulus and response where, for all occurrences of a particular stimulus event, the ensuing neuronal spike trains were averaged to construct a response probability profile for that event. This required subdividing velocity space into discrete segments. In A,R) during the whisk gave an ensuing spike probability profile. To simulate a spike train, a spike was generated in each time bin t (size 0.13 ms) with probability P(t), given by the average of the overlying spike probabilities associated with velocity events in the time window before t . After completion of the simulation, P(t) was normalized so that the overall simulated spike count matched that in the real data; this normalization did not affect the temporal profile of the simulated PSTH.To test whether the complex, texture-induced spike patterns followed directly from the tuning curves, a more elaborate analysis was necessary. Each instantaneous velocity  Trajectories of two whisks  recorded during contact with sandpaper P280.(B) To test playback, a whisker was inserted in the piezoelectric motor guide tube and whisker displacements were measured by the optical sensor placed adjacent to the insertion point of the whisker. The trace shows recordings of the playback of the same two whisks of part A.(C) Magnified view of the traces indicated in the rectangle in (A) and (B).(161 KB PDF).Click here for additional data file.Video S1http://www.redlake.com). Rat EW3, whisker C3.Each optic sensor channel consisted of a pair of light tubes . The two channels were mounted normal to each other in a metallic ring. Electrical stimulation of cranial nerve VII with 1-V pulses of 100 μs at 200 Hz for 60 ms produced whisker protraction that was followed by a passive 65-ms whisker retraction. The film shows four 125-ms free whisks in the air (8-Hz whisking), consisting of protraction (right to left) and retraction (left to right). Recorded at 500 frames per second with a Motion Scope 500 digital camera .Click here for additional data file.Video S2The motor consists of two pairs of piezoelectric wafers with axes meeting at a mobile joint. The whisker to be stimulated was placed inside the metal tube, which is orthogonal to the joint. The film shows playback of four 125-ms free whisks in the air, consisting of protraction (left to right) and retraction (right to left). Recorded at 500 frames per second.(9.0 MB MOV).Click here for additional data file.


Regulation of gene expression—deciding how much of what proteins are produced in the cell—is controlled by a myriad of different molecules. One type of naturally occurring regulatory molecule is small interfering RNA (siRNA), which selectively disrupts the production of a protein it is programmed to recognize, a process called RNA interference. These short stretches of nucleotides combine with other cellular proteins to form an RNA-induced silencing complex, called RISC, which locates and destroys a targeted messenger RNA—the molecule that carries a protein recipe from the nucleus to the site of production in the cytoplasm. While RNA interference has been widely exploited by researchers as a tool to knock out gene expression and infer the function of missing proteins, very little is known about the mechanisms behind this regulatory process.Recently, biologists have discovered hundreds of other short pieces of regulatory RNA, called microRNAs, in both plants and animals. Like siRNA, they also affect gene expression, through similar, possibly even identical RISC molecules. Animal microRNAs, however, target messenger RNA at a different stage in protein production. Though researchers have determined the sequences of these microRNAs, uncovering their function—that is, which protein they interrupt and, in turn, what the interrupted protein does—has progressed slowly and sporadically without any decisive tool to study them. Only four animal microRNAs have known biological functions, despite the intense level of work going on in this field.O-methyl oligonucleotide, whose sequence mirrors the targeted siRNA or microRNA, could bind and inhibit their function, allowing researchers an unprecedented glimpse at the regulatory roles and mechanisms behind RNA interference.In this issue, György Hutvágner and colleagues report a rapid and reliable method for knocking out both siRNAs and microRNAs and thereby exploring their functions. The authors found that a short stretch of nucleotides, called a 2′-The authors first tested their oligonucleotide design against an siRNA known to interfere with production of the firefly protein, luciferase—this luminescent protein is often used as a “reporter,” lighting up when cells successfully produce the protein. Any interference means the glow is gone. Using extracts from fruitfly embryos as the test-bed, the researchers mixed in the luciferase-associated siRNA and the sequence-specific oligonucleotide. What holds these two molecules together is complementary base-pairing, the same force that holds two molecules of DNA together. As predicted, the oligonucleotide inhibited RISC activity—it could no longer silence the production of luciferase.Because the authors could easily control the concentration of both the siRNA and the oligonucleotide inhibitor in these fly extract experiments, they were able to answer several questions about how these two molecules interact. They found that adding greater and greater concentrations of siRNA molecules did not result in equally great numbers of RISC; the process became saturated, indicating that a protein in the RISC assembly pathway limits production. Furthermore, the authors saw a marked 1:1 relationship between the concentration of the oligonucleotide and the concentration of RISC, indicating that each inhibitor binds to one RISC molecule in order to inactivate, a binding that appears to be irreversible. The results also showed that, though RISC molecules bind to the inhibitor through complementary base-pairing, a very different and more complex interaction is used by RISC molecules to find and bind their natural interference targets.The authors then went on to use the luciferase siRNA to test the function of their oligonucleotide inhibitor in cultured human cells, which had been engineered to contain the luciferase gene. This in vivo experiment, using living and metabolizing cells, showed results similar to those with fruitfly extracts. But the real test for these inhibitors was to use them in a whole animal against a previously identified microRNA where the outcome of its inactivation was already known.let-7, which blocks the production of the protein Lin-41 and is important for proper developmental timing in roundworm larvae. Larvae injected with the oligonucleotide had the exact features of a let-7 deficient worm, showing that the inhibitor did indeed block this microRNA's function. The authors also used the oligonucleotides to provide evidence that two proteins, previously suggested to be involved with let-7, were directly associated with its interfering activity.Hutvágner and colleagues constructed an oligonucleotide inhibitor based on the sequence of a microRNA called Using the technique described here, scientists could make rapid headway toward uncovering the biological functions of hundreds of microRNAs, their accessory RISC proteins, and even the proteins and genes they are programmed to interrupt. Furthermore, finding that RISC production is saturable could have significant implications for genetic studies that use RNA interference to uncover the function of sequenced, but unknown, genes; knowing the minimum required concentration of siRNA, researchers can avoid a buildup and any unwanted cell activity that goes along with it.


The current understanding of organizer formation and neural induction in vertebrate embryos is discussed In what is usually referred to as the most famous experiment in embryology, Xenopus laevis. In the past few years, however, work in other vertebrate and nonvertebrate chordate systems has come to play an important role in the field and has shed light on generalities and differences among chordates. If the present primer uses Xenopus to illustrate the process, it is because it accompanies an article in this issue of PLoS Biology dealing with neural development in this species  and Nodal factors  and also of WNT  the fibroblast growth factor (FGF) signaling pathway plays a major role in this process, and (ii) neural specification starts well before gastrulation and thus before the formation and function of the organizer. Studies on the role of FGF in early  tissues . And whi tissues , in view tissues , and the tissues  to turn FGF does not seem to behave as a classical organizer-derived neural inducer, however. Maternal FGF mRNA and protein appear to be widely distributed in the early embryo, and at least one FGF family member is expressed primarily in the animal, pre-ectodermal region during blastula stages . A detaiAn exciting recent study shows that exposure of the epiblast (ectoderm) to FGF induces, after a time delay, a transcription factor named Churchill. Churchill expression inhibits cell ingression leading to mesoderm formation; the cells remaining in the epiblast assume a neural fate . The timXenopus, it was long known that the animal region or pre-ectoderm is not uniform or naïve, in that the dorsal, organizer-proximal region is predisposed towards a neural fate  region  region . They shignaling .Xenopus, as in other vertebrates, takes place before gastrulation and does not require organizer signals; this is not to say that full differentiation and patterning of the nervous system could be achieved without organizer participation.The formation of the vertebrate nervous system thus depends on multiple signaling pathways, such as the FGF, BMP, and WNT signaling cascades, that interact in complex ways e.g., . In cont


Human T-lymphotropic virus type 1 (HTLV-1) proviral load is related to the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and has also been shown to be elevated in the peripheral blood in HTLV-1-infected patients with uveitis or alveolitis. Increased proliferation of HTLV-1-infected cells in, or migration of such cells into, the central nervous system is also seen in HAM/TSP. In the present study, we evaluated the proviral load in a cohort of HTLV-1-infected patients with arthritic conditions.HTLV-1 proviral load in the peripheral blood from 12 patients with RA and 6 patients with connective tissue disease was significantly higher than that in matched asymptomatic HTLV-1 carriers, but similar to that in matched HAM/TSP controls. HAM/TSP was seen in one-third of the HTLV-1-infected patients with RA or connective tissue disease, but did not account for the higher proviral load compared to the asymptomatic carrier group. The proviral load was increased in the synovial fluid and tissue from an HTLV-1-infected patient with RA, the values suggesting that the majority of infiltrated cells were HTLV-1-infected. In the peripheral blood from HTLV-1-infected patients with RA or connective tissue disease, HTLV-1 proviral load correlated with the percentages of memory CD4+ T cells and activated T cells, and these percentages were shown to be markedly higher in the synovial fluid than in the peripheral blood in an HTLV-1-infected patient with RA.These biological findings are consistent with a role of the retrovirus in the development of arthritis in HTLV-1-infected patients. A high level of HTLV-1-infected lymphocytes in the peripheral blood and their accumulation in situ might play a central role in the pathogenesis of HTLV-1-associated inflammatory disorders. Alternatively, the autoimmune arthritis, its etiological factors or treatments might secondarily enhance HTLV-1 proviral load. Human T-lymphotropic virus type 1 (HTLV-1) is endemic in southern Japan, intertropical Africa, Melanesia, Latin America, and the Caribbean basin  and HTLVThe possibility that HTLV-1 may cause joint disease was initially raised by reports of arthralgia and polyarthritis in patients with adult T-cell leukemia ,11. Poly+ T cells, are the main target of HTLV-1 in vivo and carry the majority of the HTLV-1 proviral load [Several findings support the hypothesis of an etiopathogenic role for HTLV-1 in HAA: ATL-like T lymphocytes have been identified in the synovial fluid and synovial tissue ,18,23; hral load . The HTLral load  and the ral load .We postulated that HTLV-1 proviral load might also influence the initiation and course of HAA, and measured this marker in PBMCs from a previously described cohort  of HTLV-6 PBMCs ranged from 14,600 to 373,000 in the patients with RA , and the difference remained significant when the analysis focused on the RA subgroup . No differences were observed between the HTLV-1-infected group with RA or connective tissue disease and the matched HAM/TSP controls (P > 0.05 in both Wilcoxon's test and the paired t-test). As expected, the difference between the asymptomatic carriers and the HAM/TSP group was significant .The HTLV-1 proviral load was measured in the peripheral blood of HTLV-1-infected patients with RA or connective tissue disease and in matched asymptomatic and HAM/TSP controls , even when the analysis was restricted to the subgroup with RA alone . The patient with connective tissue disease and uveitis (patient 13) had 18,420 copies per 106 PBMCs. For one of the patients with RA and HAM/TSP (patient 10), 4 consecutive frozen dry pellets of PBMCs from 1996 to 2002 were available; in these, the proviral load was relatively stable at 92,100, 73,600, 143,300, and 106,500 copies per 106 cells, respectively.Of the 12 HTLV-1-infected patients with RA, 4  had HAM/TSP . HTLV-1 proviral load did not correlate with erythrocyte sedimentation rate or C-reactive protein level . No significant difference in HTLV-1 proviral load was seen in patients positive or negative for rheumatoid factor or antinuclear antibody  or receiving or not receiving either specific treatments for RA or corticotherapy .No correlation was found between HTLV-1 proviral load and the age of the patient, the duration of illness, or the Ritchie's index score . A positive correlation was found between HTLV-1 proviral load and the percentage of CD4+ T cells expressing HLA-DR (P = 0.008), while the correlation did not reach significance for CD8+ HLA-DR T cells.Lymphocytes subsets and activation status were examined in the peripheral blood of HTLV-1-infected patients with RA or connective tissue disease. When correlations were examined between HTLV-1 proviral load and the percentage of T lymphocytes expressing CD45RO, CD45RA, or HLA-DR in the HTLV-1 infected patients with RA or connective tissue disease Figure , HTLV-1 Lymphocyte distribution and activation were compared in the peripheral blood and synovial fluid of one HTLV-1-infected patient with RA (patient 5). The percentage of CD4+ T cells expressing CD45RO was dramatically increased in the synovial fluid (98%) compared to the peripheral blood (48%), as was the percentage of CD4+ T lymphocytes expressing HLA-DR (94% compared to 18%).The etiology of autoimmune diseases, such as RA or mixed connective tissue disease, has yet to be established, but appears to result from complex interactions between host genetic and environmental factors , and ret+ cells; (3) A high HTLV-1 proviral load was found in the synovial fluid and tissue cells in a patient with RA; (4) In this patient, the percentage of memory and activated CD4+ T cells was higher in the synovial compartment than in the peripheral blood.Nevertheless, the present study provides biological data suggesting a contribution of HTLV-1 to the development of some cases of RA or mixed connective tissue disease. We found that: (1) The circulating HTLV-1 proviral load was higher in HTLV-1-seropositive patients with RA or connective tissue disease than in asymptomatic HTLV-1 carriers and similar to that in patients with HAM/TSP; (2) In the peripheral blood of patients with arthritis, HTLV-1 proviral load correlated with the percentage of memory and activated T CD4The HTLV-1 proviral load is thought to be a major determinant of HTLV-1-associated diseases. The HTLV-1 proviral load is higher in the peripheral blood from patients with HAM/TSP than in blood from asymptomatic carriers . It is aIn HAM/TSP, the HTLV-1 proviral load reaches an equilibrium set-point that is correlated with progression of motor disability, and fluctuates by no more than 2- to 4-fold over a decade . In one + T lymphocytes and the proviral load is reported to correlate with the number of memory T cells [+ T cells expressing CD45RO and negatively with that of CD4+ T cells expressing CD45RA. The HTLV-1 proviral load also correlated positively with the percentage of HLA-DR-expressing T cells. Migration of HTLV-1-infected CD4+ T cells and HTLV-1-specific CD8+ cytotoxic T lymphocytes (CTL) into the central nervous system is a critical step in the pathogenesis of HAM/TSP [+ T cells in the joint fluid of an HTLV-1 carrier with RA supports the hypothesis of a pathogenic involvement of HTLV-1-infected T lymphocytes.The unusually high proviral loads in HTLV-1 infection results mainly from the Tax-driven activation and expansion of infected cells . The HTL T cells . In our  HAM/TSP ,32. Simi HAM/TSP ,30. ThusSeveral mechanisms are potentially involved in the occurrence of rheumatological disorders during HTLV-1 infection. Firstly, HTLV-1 infection upregulates the expression of adhesion molecules potentially involved in the migration of lymphocytes into the spinal and joint compartments . SecondlIn conclusion, our data in HTLV-1-infected patients with RA or connective tissue disease are consistent with a role of the proviral load in the development of these rheumatological disorders, although the direction of causality in this interaction remains open to question. HTLV-1 might cause a systemic immune-mediated inflammatory disease potentially involving tissues other than the central nervous system, HAM/TSP being only the major syndrome. The clinical expression of this disease might be determined by the amount of HTLV-1-infected T lymphocytes, their level of activation, and their capacity to accumulate in different body compartments. Further research is needed to increase our knowledge of the molecules involved in the homing of HTLV-1-infected CD4+ T lymphocytes and of anti-HTLV-1-specific CD8+ CTL to different target tissues.The study was performed in Martinique, an island in the lesser Antilles archipelago, with a population of 400,000. Between 1988 and 2001, 280 patients with RA, defined according to the American Rheumatology Association (ARA) criteria, and 335 patients with connective tissue disease were followed on an inpatient or outpatient basis at the Rheumatology Department of the Regional Teaching Hospital. Thirteen (4.6%) of the 280 patients with RA  were found to be HTLV-1 seropositive, confirmed by Western blotting  and peripheral blood was obtained from 12 of these. In addition, 6 patients with connective tissue disease , who were seropositive for HTLV-1, were included in the study. Samples were collected between September 2001 and June 2002. For one patient, sequential samples cryopreserved since 1996 were available.The mean age at the time of sampling was 63 years for the HTLV-1-seropositive RA patients compared to 60 years for the total RA cohort, while the mean age of the HTLV-1-seropositive patients with connective tissue disease was 58 years. The mean time interval between onset of the auto-immune disease and sampling for HTLV-1 proviral load determination were 8 years and 6 years in the patients with RA and connective tissue disease, respectively. Of the 12 HTLV-1-seropositive RA patients, 4 had HAM/TSP, defined according to the WHO guidelines . Two of Each HTLV-1-infected patient with RA or connective tissue disease was matched for age (± 5 years) and sex with 2 patients with HAM/TSP and 2 asymptomatic HTLV-1 carriers.PBMCs were isolated from EDTA blood by density gradient centrifugation. Synovial fluid samples were obtained by arthrocentesis and synovial tissue was obtained during arthroscopy. The synovial tissue was minced into small pieces, washed with phosphate-buffered saline, and passed through a wire mesh to collect synovial tissue cells. Cells were cryopreserved until use.6 cells using a phenol/chloroform procedure. The HTLV-1 proviral load was quantified using a real-time TaqMan PCR method [pol gene and the dual-labeled TaqMan probe (5' FAM and 3' TAMRA) was located at 4829–4858 bp of the HTLV-1 reference sequence (HTLVATK). Albumin DNA was quantified in parallel to determine the input cell number and was used as an endogenous reference to normalize variations due to differences in the PBMC count or DNA extraction. For both HTLV-1 and the albumin gene, amplifications were performed on 10 μl of DNA extract using the TaqMan PCR Core Reagent kit, data being acquired with the ABI Prism 7700 Sequence Detector System . Standard curves were generated using ten-fold serial dilutions of a double standard plasmid (pcHTLV-ALB) containing one copy of the target regions of both the HTLV-1 pol gene and the cellular albumin gene. The HTLV-1-infected human lymphocyte line MT2 (ECACC 93121518) was used as a control for quantification, the limit for an acceptable result being taken as 2.4–3.3 copies of the HTLV-1 pol gene per cell and the variation between series being normalized on the basis of three copies per MT2 cell. All standard dilutions and control and patient samples were run in duplicate for both HTLV-1 and albumin DNA quantification. Standard curves for HTLV-1 and albumin were accepted when the slope was between -3.322 and -3.743 (corresponding to amplification efficiencies of 100% to 85%) and the correlation coefficient, r2, was >0.992. If the variation between duplicate values of HTLV-1 or albumin DNA copy numbers was greater than 30%, the analysis was repeated. The normalized value for the HTLV-1 proviral load was reported as the HTLV-1 average copy number/albumin average copy number ratio × 106 and expressed as the number of HTLV-1 copies per 106 PBMCs.DNA was extracted from 10R method . SK110/S1), allophycocyanin-conjugated anti-CD4 , peridinin chlorophyll protein-conjugated anti-CD8 , phycoerythrin (PE)-conjugated anti-CD45RO , and PE-conjugated anti-HLA-DR  , and PE-conjugated anti-CD45RA . The analyses were performed on a FACSCalibur (BD Biosciences Immunocytometry Systems).Lymphocyte subsets in PBMCs were characterized using a panel of labeled anti-human monoclonal antibodies, consisting of fluorescein isothiocyanate-conjugated anti-CD3  declare that they have no competing interests.MY carried out most of the clinical and experimental work. AL was involved in the molecular biology work and contributed to the design of the study. FD and GL performed the flow cytometry analysis. SO and GJB participated in the neurological and rheumatologic evaluations, respectively. SA supervised the design and the course of the clinical study. RC conceived the study and drafted the manuscript. All authors read and approved the final manuscript.


Recent technological advances in high-throughput data collection allow for experimental study of increasingly complex systems on the scale of the whole cellular genome and proteome. Gene network models are needed to interpret the resulting large and complex data sets. Rationally designed perturbations  can be used to iteratively refine hypothetical models, suggesting an approach for high-throughput biological system analysis. We introduce an approach to gene network modeling based on a scalable linear variant of fuzzy logic: a framework with greater resolution than Boolean logic models, but which, while still semi-quantitative, does not require the precise parameter measurement needed for chemical kinetics-based modeling.We demonstrated our approach with exhaustive search for fuzzy gene interaction models that best fit transcription measurements by microarray of twelve selected genes regulating the yeast cell cycle. Applying an efficient, universally applicable data normalization and fuzzification scheme, the search converged to a small number of models that individually predict experimental data within an error tolerance. Because only gene transcription levels are used to develop the models, they include both direct and indirect regulation of genes.Biological relationships in the best-fitting fuzzy gene network models successfully recover direct and indirect interactions predicted from previous knowledge to result in transcriptional correlation. Fuzzy models fit on one yeast cell cycle data set robustly predict another experimental data set for the same system. Linear fuzzy gene networks and exhaustive rule search are the first steps towards a framework for an integrated modeling and experiment approach to high-throughput "reverse engineering" of complex biological systems. In partFuzzy logic  providesThe problem of rule set combinatorial explosion is addressed by the union rule configuration (URC) developed by Combs and Andrew , which alac operon of E. coli  in sets 1 (Low), 2 (Med), and 3 (High) respectively, the estimated numerical result of the evaluation, , is given by the centroid for the points located at -1, 0, and +1 for each set respectively, or method" , with pof(x) = x and f(x) = -x are quantitatively equal to monotonic fuzzy rules, which can be written using notation from the following section as [1 2 3] and [3 2 1] respectively) so it generally will not introduce systematic error in the model.The fuzzy set definition and centroid defuzzification of Equations 1 and 2 were selected to maximize computational efficiency during exhaustive search: all 27 rules can be represented by easily implemented algebraic functions and it is possible to design the implementation to avoid as many costly if/then comparisons as possible. In addition, the scheme perfectly reproduces monotonic linear positive and negative interactions .To apply this scheme for defuzzification and fuzzification scheme, experimental data must be projected on to the interval -1.0 through +1.0. Thus, log base 2 expression ratios are normalized by taking the arctangent of each ratio and dividing by π/2, yielding a symmetric transformation covering the desired interval. Previous work normalized expression ratios by the maximum value found in the experiment  or used r. For example, the rule r = [3 2 1] corresponds to the linguistic rules:The fuzzy rule relating the input of a single gene to an ouptut node gene can be expressed as a rule vector Input is Low (1) then Output is High (3)If Input is Med (2) then Output is Med (2)If Input is High (3) then Output is Low (1)If y = [y1 y2 y3] obtained using Equation 1 and the general fuzzy rule r = [r1 r2 r3], the resulting fuzzified expression of the output gene z will be:Given the fuzzified expression of an input gene N input genes acting on the output gene simultaneously. In the linear fuzzy logic scheme, the rule for each input gene is evaluated separately, leading to intermediate outputs zi:In general, node behavior is the result of These intermediate fuzzy values are summed algebraically to obtain the final resulting fuzzy value for node gene expression:3 or 27 possible rules describing the effect of a single gene on another gene. Thus, if there are N input genes for a node, there are 27N total possible rule combinations describing the behavior of the node gene.This result is defuzzified using Equation 2 to evaluate the output of the node. For three fuzzy sets, there are 3M data of the output gene x = {x1,x2,...,xM} asIn general, no rule combination will be an exact fit to real experimental data. Given some tolerance to fitting error, there will be multiple possible rule combinations, representing plausible hypothetical gene network models. In our present work, we define the error of the fit for the  is the set of defuzzified numerical predictions  and  is the mean of the experimental data set x. A perfect fit results in a maximum E of 1.0. This error score was chosen because while it is quantitative, it emphasizes the correlation in qualitative behavior between the fit and prediction instead of the absolute numerical fit, which can be difficult to model with the limited resolution of three fuzzy sets. We can use the fitting error to rank these models, and use rule patterns consistent throughout all plausible models as a basis for constructing the template of a final network model that can be tested experimentally.where G1, G2, G3) with log base 2 expression ratios measured at three different times:As an example to illustrate fuzzy gene networks using a simple rule combination, we consider three genes , Medium (2), and High (3). Consider the following rules for G1 and G2 as input genes to G3:with vectors for each time point containing set membership in Low (G1:G3 = [3 2 1]G2:G3 = [1 2 3]where the rules can be written in English asG1 is Low (1) then G3 is High (3)If G1 is Med (2) then G3 is Med (2)If G1 is High (3) then G3 is Low (1)If G2 is Low (1) then G3 is Low (1)If G2 is Med (2) then G3 is Med (2)If G2 is High (3) then G3 is High (3)If Now, the evaluations of the rules taken individually areG1:G3 = {[0 0.205 0.795] [0 1.0 0] [0.795 0.205 0]}G2:G3 = {[0 0.814 0.186] [0 1.0 0] [0.186 0.814 0]}G3 for the three time points, which can be defuzzified using the point-centroid method (Equation 2) and transformed back to real numbers on :The sum of two intermediate outputs (Equation 3) is the predicted fuzzy behavior of G3 = {[0 1.019 0.981] [0 2.0 0] [0.981 1.019 0]} = {0.491 0 -0.491}These numbers can be transformed back to a Log2 expression ratio by inverting the normalization (multiplying by π/2 and taking the tangent):G3 = {0.97 0 -0.97}G3 to calculate the fit error for this rule combination:Finally, we use Equation 4 and the experimental data for E = 1.0 for a perfect fit.which compares to a maximum 16 possible rule combinations for each of the 12 genes, making the exhaustive search method practically impossible. Thus, the number of possible inputs to a node must have a maximum constraint to make exhaustive search tractable.In general, a possible model for a node can include any combination of the genes available to act as inputs. In the work described here, we consider potential interactions of 12 genes. Thus, a rule for any one gene can include as inputs any combination of any number of up to all 11 other genes. Since each input gene can influence the node by any one of the 27 possible fuzzy rules, there are approximately 10all possible combinations of 1 through 4 of the genes are searched sequentially. For example, in our fitting of rules to CLN3, we considered the following potential input combinations: SIC1 alone, SIC1 and CLN1 together, SIC1-CLN1-CLN2, SIC1-CLN1-CLN2-CLN3, CLN1, CLN1-CLN2, CLN1-CLN2-CLN3, CLN1-CLN2-CLN3-SWI4, CLN2, CLN2-CLN3, etc. If we include all combinations from 1 through 4 of the genes taken from the 11 total possible inputs, then the total search space for each of the 12 genes consists of approximately 108 rules (taking about 10 minutes on a PowerMac G4 using a single 450 MHz processor). Simulation files used to generate all the data presented here are available from the authors upon request.Studies of network topology through the experimentally observed association of proteins suggest that in many cases only few regulatory proteins are observed to directly influence the expression of a gene ,30-32. FBAS originated the concept of applying scalable (URC) fuzzy logic to modeling biological systems, implemented the scheme described within on the data set, and was the primary author. JPF conceived of the approach to use exhaustive search for biological network reconstruction. JNQ and AAQ developed and initially implemented the method of combinatorial input selection for the exhaustive network search, and AAQ contributed to the text. All authors read and approved the final manuscript.Microsoft Excel spreadsheets of simulation results. See descriptive text in the workbook.Click here for file


Methylococcus capsulatus (Bath), obtained by the shotgun sequencing approach. Analysis revealed a 3.3-Mb genome highly specialized for a methanotrophic lifestyle, including redundant pathways predicted to be involved in methanotrophy and duplicated genes for essential enzymes such as the methane monooxygenases. We used phylogenomic analysis, gene order information, and comparative analysis with the partially sequenced methylotroph Methylobacterium extorquens to detect genes of unknown function likely to be involved in methanotrophy and methylotrophy. Genome analysis suggests the ability of M. capsulatus to scavenge copper  and to use copper in regulation of methanotrophy, but the exact regulatory mechanisms remain unclear. One of the most surprising outcomes of the project is evidence suggesting the existence of previously unsuspected metabolic flexibility in M. capsulatus, including an ability to grow on sugars, oxidize chemolithotrophic hydrogen and sulfur, and live under reduced oxygen tension, all of which have implications for methanotroph ecology. The availability of the complete genome of M. capsulatus (Bath) deepens our understanding of methanotroph biology and its relationship to global carbon cycles. We have gained evidence for greater metabolic flexibility than was previously known, and for genetic components that may have biotechnological potential.Methanotrophs are ubiquitous bacteria that can use the greenhouse gas methane as a sole carbon and energy source for growth, thus playing major roles in global carbon cycles, and in particular, substantially reducing emissions of biologically generated methane to the atmosphere. Despite their importance, and in contrast to organisms that play roles in other major parts of the carbon cycle such as photosynthesis, no genome-level studies have been published on the biology of methanotrophs. We report the first complete genome sequence to our knowledge from an obligate methanotroph,  Methylococcus capsulatus deepens our understanding of methanotroph biology and its relationship to global carbon cyclesMethanotrophs are bacteria that use methane as a sole carbon source. The genome sequence of  Methylococcus capsulatus are responsible for the oxidation of biologically generated methane , which are all members of the Gammaproteobacteria, utilize ribulose monophosphate (RuMP) as the primary pathway for formaldehyde assimilation, whereas those of type II, which are all Alphaproteobacteria, use the serine pathway  (M. capsulatus (Bath) to obtain a better understanding of the genomic basis of methanotrophy, a globally important microbial process.The powerful tool of whole-genome sequencing has been applied to microorganisms that carry out other important components of the carbon cycle, such as photosynthesis  and methns (AM1) , 2004, bM. capsulatus (Bath) comprises a single circular molecule of 3,304,697 bp. General features of the genome and its 3,120 predicted coding sequences (CDSs) are summarized in (gidA) gene(MCA0001), adjacent to the chromosome-partitioning proteins encoded by gidB, parA, and parB and the operon that encodes F1F0 ATP synthase.The genome of M. capsulatus (Bath) genome contains 51 identifiable insertion sequence elements from various families (M. capsulatus (Bath) genome, repeated cycles of duplication and subsequent deletion, or gene conversion. Twenty elements belonging to the IS4 family of insertion sequences . Inteins are rare, but when present are often found in genes associated with nucleotide metabolism, such as ribonucleotide reductases.Two putative prophages  were identified in the genome. The Mu-like prophage is unusual in encoding an intein within F (Mu gp30), a cofactor in head assembly. This intein region is most similar to inteins present in several archaeal translation initiation factor IF-2 sequences from the genera M. capsulatus (Bath). The M. capsulatus (Bath) Mu-like prophage could be engineered to resemble the Mu derivatives, which have been excellent tools for random mutagenesis in other species (M. capsulatus proteins) or for manipulating M. capsulatus live vaccine vectors.Bacteriophages have been important tools for genetic manipulation of bacterial genomes, and such tools are currently lacking for  species . Conditi species . The putM. capsulatus (Bath), including the methane oxidation pathway, mechanisms for carbon fixation, glycolytic and gluconeogenic conversions, and the tricarboxylic acid (TCA) cycle, from analysis of the genome data. These pathways, together with those known from previous studies, are depicted in M. capsulatus (Bath) that is based on available genome data.We have attempted to predict central metabolic pathways in M. capsulatus is known to possess both a particulate membrane-bound form, pMMO (detected by centrifugation studies and encoded by pmo), and a soluble form, sMMO (encoded by mmo), and these enzymes have been extensively studied (pmoCAB (pmoCAB and a third copy of pmoC (pmoC3) were previously identified , which catalyze the first step of methane oxidation . M. caps studied . The pMM . The transposase (MCA1197) is oriented in the same direction as the mmo genes and, thus, may be transcribed under sMMO-promoting growth conditions. The functions of these sMMO components have been previously determined  ((homologs of MxaJGRACKLD)  contains a second methanol dehydrogenase–like cluster, mxaFJ, (MCA0299–0300) lacking a homolog of mxaI that normally encodes the small subunit of methanol dehydrogenase. The function of mxaJ is unknown, and it is not clear whether this mxaFJ cluster encodes components active in methanol oxidation. There is a third cluster of genes required for methanol dehydrogenase synthesis, mxaACKL (MCA1527–1530).Methanol is available to adation) , and itsadation) . We haveGRACKLD) . There ilotrophs , 1997b. M. capsulatus +-linked formaldehyde dehydrogenase is active when copper concentrations are low rogenase  best matrogenase , could nrogenase  helps reM. capsulatus, as was recently found alongside the THMPT pathway in M. extorquens -linked and THMPT-linked pathways of formaldehyde oxidation . In additorquens . In M. e pathway . M. capsM. extorquens, all three are expendable, indicating that this last step in methane oxidation plays a minor role and that formate can be dissimilated in other ways . Multiple formate dehydrogenases occur in other bacteria ; in M. eher ways . The impM. capsulatus exploits different systems under variable environmental conditions . It is plausible that M. capsulatus balances its requirement for formaldehyde-derived carbon and reducing power with the toxicity of formaldehyde by taking advantage of three enzymes for formate oxidation and multiple pathways for formaldehyde oxidation under different environmental conditions. This redundancy has implications for future attempts to manipulate the genes of this pathway; simple knockouts may not be achievable.The genomic redundancy in the set of methane oxidation pathways suggests that M. capsulatus is known to assimilate formaldehyde through the RuMP pathway ; this dual function may have been facilitated by the redundancy resulting from this tandem duplication. The M. capsulatus (Bath) genome appears to contain some parts of the alternative serine pathway of formaldehyde assimilation , reversibly converting glyceraldehyde-3-phosphate to xylulose-5-phosphate, bypassing the typical ribose-5-phosphate to fructose-6-phosphate segment ; cell suactivity , and a gM. capsulatus metabolism, and genomic evidence suggests three potential pathways for serine synthesis not previously described in M. capsulatus: a phosphorylated pathway from glycerate-3-phosphate, a nonphosphorylated pathway from glycerate, and a nonphosphorylated pathway from glycolate-2-phosphate  were not identified in the M. capsulatus (Bath) genome, and phosphoserine phosphatase may perform this function as described in Pseudomonas aeruginosa  are present, but the latter two may have alternate functions in vitamin B6 biosynthesis  or as horuginosa .M. capsulatus (Bath) genome also encodes a putative phosphoglycerate mutase (MCA0753), to interconvert 3- and 2-phosphoglycerate , uses a complete TCA cycle to grow on glucose as its sole carbon and energy source , one complete (MCA1941–1944) and one partial (MCA1924) ATP-binding casette (ABC) family transporter with predicted specificity for sugar uptake were identified. Additionally, components of transporters for peptides (MCA1264 and MCA1268), carboxylates (MCA1872), and a variety of amino acids  are present.An incomplete TCA cycle lacking 2-oxoglutarate dehydrogenase activity has been found in nearly all type I methanotrophs, including psulatus . Howeverpression  may playy source ; M. capsM. capsulatus (Bath) is able to fix atmospheric nitrogen  were previously shown to be contiguous ; this organization has been found in Chlorobium tepidum and some nitrogen-fixing methanogenic Archaea. Two 2Fe-2S ferredoxins (MCA0232 and MCA0238) and two genes identified as conserved hypotheticals (MCA0236–0237) are interspersed with the nif genes in the same orientation. The conserved hypothetical genes share the highest sequence similarity with genes from other organisms capable of nitrogen fixation, suggesting that they also have a role in this process.nitrogen , conferrntiguous , as theyM. capsulatus exhibits considerable versatility in its combined nitrogen conversions, including nitrification and denitrification. Ammonia is oxidized to nitrite by both pMMO and sMMO because of their lack of substrate specificity , suggesting that ammonium is an important nitrogen source for M. capsulatus. Methane oxidation is inhibited by the presence of ammonia and ammonia oxidation is inhibited by methane  genome has a relatively large complement of putative c-type cytochromes; 57 proteins containing a heme-binding motif were identified, and 23 of these contain two or more heme-binding motifs. Analysis of the genome reveals electron transport components previously known to be associated with the methane oxidation pathway, such as cytochrome CL (MCA0781), a specific electron acceptor for methanol dehydrogenase. Other novel electron transport components are encoded in several physical locations on the chromosome; there is genomic evidence for chemolithotrophy and the ability to live at a variety of oxygen tensions.The +; and (c) a membrane-bound Ni-Fe hydrogenase (MCA0163–0165). Activity of two hydrogenases (one soluble and one membrane-bound), and the role of molecular hydrogen in driving MMOs, was previously reported  and could supply exogenous hydrogen for chemolithotrophic oxidation. The removal of this hydrogen by M. capsulatus metabolism may aid in driving these reactions forward and hence constitute a syntrophic partnership.The genome encodes three predicted hydrogenases: (a) a multisubunit formate hydrogenlyase (MCA1137–1142), most likely involved in the conversion of formate to dihydrogen and carbon dioxide; (b) a soluble cytoplasmic NAD-reducing hydrogenase (MCA2724–2726), which transfers electrons to NADreported . The memreported . The preM. capsulatus (Bath) genome; however, two ferredoxins (MCA0238 and MCA0232) physically located within a cluster of genes encoding proteins involved in nitrogen fixation (see Nitrogen Metabolism section above) more likely serve in this capacity. In aerobes, these carriers are usually reduced by NADH/NADPH, although reverse electron transport could be involved in this organism.Nitrogen fixation requires reducing power, which in aerobes can be supplied by reduced flavodoxin or ferredoxins. There is one candidate flavodoxin present (MCA1697) in the M. capsulatus (Bath) genome encodes homologs of a two-subunit high oxygen-affinity cytochrome d (MCA1105 and MCA1106), which suggests the ability to live under microaerophilic conditions. This evidence for life at low oxygen tensions is supported by the presence of enzymes indicative of fermentative activity  genome includes a region of approximately 25 kb (MCA0421–0443) encoding novel proteins related to energy metabolism  and hypothetical proteins. The same 25-kb region contains six multiheme c-type cytochromes that are members of the cytochrome c553o family. This previously described family is unique to M. capsulatus (Bath); genome analysis has widened our knowledge of this family from three  and the second a signature for fumarate lyase. The genome also encodes homologs to the putidaredoxin family of ferredoxins.A novel monoheme c-type cytochrome (MCA1187) has eight transmembrane-spanning regions and is located near another c-type cytochrome and a proton-translocating pyrophosphatase . This configuration suggests that the monoheme protein has a role in ATP production via chemiosmosis. Two other monoheme CDSs (MCA2188 and MCA2196) are members of a paralogous family similar to a cytochrome found in cis and trans configuration  appears to catalyze an alternative dehydratase reaction at the C10 level of fatty acid synthesis, followed by a synthase reaction carried out by 3-oxoacyl-acyl carrier protein (ACP) synthase (MCA2879), resulting in cis-vaccenate . Trans-unsaturated acids are obtained by isomerization of preformed cis-unsaturated fatty acids, to control membrane fluidity; putative fatty acid cis/trans-isomerases were identified in the genome (MCA1585 and MCA1806).The membrane phospholipids of methanotrophs are unique, with mono-unsaturated fatty acids consisting of a series of even- and odd-numbered positional isomers of both the guration . Our anaM. capsulatus is one of only a few prokaryotes known to synthesize sterols de novo (M. capsulatus (Bath) are methylated; genome analysis indicated four putative proteins involved in the conversion of squalene to 4,4-dimethylcholest-8(14)-en-3β-ol . de novo , and it  de novo . The maitrans-transferase, the mechanism for converting acetyl-CoA to squalene (the first major intermediate) is not apparent from genome analysis. Instead, M. capsulatus (Bath) contains genes for the alternative mevalonate-independent pathway from glyceraldehyde-3-phosphate and pyruvate , so it is possible that the organism employs the same squalene synthesis pathway found in plants and many Gram-negative bacteria  genome reveals several elements that may relate to processing of copper.Copper is known to be important in the regulation of MMO activity; high copper concentrations are essential for the formation of extensive intracytoplasmic membranes and pMMO activity, and copper is thought to play an active role in both the catalytic site and the electron transport chain . In cont(Methylosinus) is known to excrete copper-binding compounds  that may scavenge copper; another methylotrophic bacterium ompounds . The NRP+/heavy metal cation antiporters. The two-module and six-domain organization of this PKS is atypical; it contains domains with unknown functions, and its role is difficult to predict. A cation membrane transport system  is located near the NRPS, and the 4′-phosphopantetheinyl transferase needed for activation of both PKS and NRPS is also present (MCA1522), indicating that these multimodular enzymes may be active.A single polyketide synthase gene (PKS) (MCA1238) is found adjacent to a gene encoding a sensor protein with diguanylate cyclase and diguanylate phosphodiesterase activities (MCA1237), often found in environmental sensing proteins and HM. capsulatus (Bath) has a large repertoire of 12 P-type cation ATPases, including multiple predicted copper ion pumps, which correlates with the role of copper in regulation of methane oxidation in this organism. There are also 18 resistance/nodulation/cell division–type metal ion and drug efflux pumps; the large number of these pumps, together with a variety of other metal cation uptake and efflux systems, highlight the significance of metal ion homeostasis in M. capsulatus.Escherichia coli, CopA is regulated together with CueO, a multicopper oxidase, by CueR, a member of the MerR-family transcription regulators. No evidence for a CueR homolog was found, indicating a different mechanism of regulation of the copA and cueO genes in M. capsulatus. The genome encodes one potential cusCBA gene cluster (MCA2262–2264), with the cusA candidate  having the same copper-binding and transport motif found in the E. coli gene. No indication of homologs of the CusF periplasmic copper chaperone was found. However, it is interesting to note that the cusB candidate (which encodes the CusB outer membrane protein) carries the metal-binding motif typically found in CusF, suggesting that the putative CusB may have a dual function as CusF. No evidence for the CusRS two-component response system regulating the E. coli cusCFBA operon was found in M. capsulatus (Bath).The genome encodes three homologs  of P-type ATPases with the characteristic copper-binding P-type ATPase motif , which mM. capsulatus (Bath); the lack of the same full complement of genes and identifiable regulators raises questions about the exact operation of copper regulation and suggests future experiments to resolve this central mechanism.In summary, there are elements of previously studied copper transport and regulation systems in the genome of M. capsulatus possesses an insoluble polysaccharide capsule  is somewhat desiccation resistant  genome lacks genes for asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase. However, a heterotrimeric glutamyl-tRNA amidotransferase (MCA0097–0099) is present, suggesting that a single amidotransferase forms asparaginyl-tRNA and glutaminyl-tRNA by transamidation of mischarged aspartyl-tRNA or glutamyl-tRNA, as found previously in many Gram-positive and some Gram-negative bacteria, archaea, and eukaryal organelles  exhibits redundancy in many pathways, as described in more detail in the relevant sections above. The redundant genes fall into two categories. The first category comprises those that appear to be lineage-specific duplications (see M. capsulatus (Bath) than to all other complete genomes. In some cases, these genes are found adjacent to each other in the genome, implying that they may have been generated by a tandem duplication, and that they may be transient (tandem arrays are prone to deletion). Other lineage-specific duplications, including those of many genes encoding hypothetical and conserved hypothetical proteins, may not simply be transient mutations and may instead have been maintained because they confer an evolutionary advantage on the organism. The second category contains redundant genes that do not appear to be recently duplicated, and are evolutionarily divergent, suggesting ancient duplications or exogenous acquisition. The divergent phylogeny of these genes is inconsistent with ancient duplication and subsequent vertical transmission, but we cannot determine the origin, direction, and exact path of a possible lateral transfer. Past exchange of genetic material between a methanogen and a methanotroph ancestral to M. capsulatus, whether direct or indirect, is certainly plausible, given their biochemical dependency. The presence of both categories of redundancy for a given enzyme or pathway makes it more plausible that the enzyme or pathway is functionally important and that its redundancy is advantageous to the organism.The genome of ions see , identifM. capsulatus, like some other bacteria , one of which is located at the putative origin of replication. Only one of these genes, that encoding the ATP synthase F1 epsilon subunit, appears to be recently duplicated . Phylogenetic analysis suggests that the other ATP synthase genes are not recently duplicated. Genes of the operon located at the origin of replication (MCA0006–0013) are of a type found only in Gammaproteobacteria or Betaproteobacteria, whereas the nonorigin genes (MCA2699–2708 and MCA1556) are divergent and related to the methanogenic Archaea and C. tepidum. The cooccurrence of the divergent genes in another organism able to fix nitrogen (C. tepidum) suggests that they may be involved in generation of extra ATP required to fix nitrogen.The genome also contains two sets of ATP synthase genes, as has been found in four other completed genomes , which is involved in methionine and selenoamino acid metabolism and has a role in activation of formate dehydrogenase; the GlpG glycogen phosphorylase (MCA0067 and MCA2540), which has a role in starch and sucrose metabolism; and the cell division protein FtsH (MCA0851 and MCA1848), which is a proteolytic regulator of cell division under stress. One of each duplicated pair is most closely related to genes from other Gammaproteobacteria, whereas its partner is either most closely related to genes from cyanobacteria, or occupies a deep-branching position.M. capsulatus suggests that this enzyme  is instead functioning in reverse, in THMPT-linked formaldehyde oxidation , an enzyme central to methanogenesis in Archaea. The fact that methanogenesis has not been previously reported in xidation   with the incomplete genome data from the methylotroph M. extorquens were used to identify additional novel genes. Phylogenetic profiling identified four genes not previously known to have a role in methane oxidation pathways in M. capsulatus. Two of them (MCA0180 and MCA3022) clustered with the gene that ecodes methylene THF dehydrogenase (transfer of C1 compounds) together with a gene from Pirellula, and the others (MCA0346 and MCA2963) grouped with pmoC3 (oxidation of methane to methanol) and a gene from Nitrosomonas.Phylogenetic profiling  and compM. extorquens, which possesses a much larger genome (7.6 Mb) than that of M. capsulatus (Bath) (M. capsulatus (Bath) and M. extorquens (best hits) yielded a total of 572 genes in 88 role categories. The majority of these shared genes are of unknown function. Putative orthologs detected included 24 conserved hypothetical genes. Phylogenetic profiling showed that ten of the 24 occur in a species distribution similar to proteins of the methane oxidation pathway, suggesting that they may also have a role in methane oxidation. Three of the ten  were found within methanotrophy gene “islands” (see below), and three had the highest levels of similarity to M. extorquens  supporting a putative role in the methane oxidation pathway.Specific comparisons with s (Bath) , revealeM. extorquens, we found orthologs of 69, mostly in the categories of energy and carbon metabolism; the remaining 20 genes found in M. extorquens but not M. capsulatus (Bath) are involved in the metabolism of other C1 compounds not used by M. capsulatus. Many (41 of 69) of these shared methylotrophy genes are clustered on the chromosome into 13 groups, seven of which contain more than three genes, and the largest of which contains nine. Three hypothetical proteins were identified in these clusters, suggesting a role in C1 metabolism.Of the 89 genes putatively involved in methylotrophy in M. capsulatus (Bath) genome has illuminated the genomic basis for the highly specialized methanotrophic lifestyle, including redundant pathways involved in methanotrophy and duplicated genes for essential enzymes such as the MMOs. We used phylogenomic analysis, gene order information, and comparative analysis with a partially sequenced methylotroph to detect genes of unknown function likely to be involved in methanotrophy and methylotrophy. Many methylotrophy genes were found to be clustered in gene islands in both organisms. We found genomic evidence for the organism's ability to acquire copper (including a previously unknown NRPS) and to use copper in regulation of methanotrophy, but the exact regulatory mechanisms remain unclear.Our analysis of the The genome sequence suggests previously unexpected metabolic flexibility, including the ability to oxidize chemolithotrophic hydrogen and sulfur and to live under reduced oxygen tension, both of which have implications for methanotroph ecology. There is a clear need for experimental validation of these genome-based hypotheses.M. capsulatus (Bath) deepens our understanding of methanotroph biology, its relationship to global carbon cycles, and its potential for biotechnological applications, and it provides a set of hypotheses of gene function that can now be experimentally tested. In addition, the annotated genome provides a source of gene probes for detection and differentiation of methanotrophs in environmental samples.The availability of the complete genome of M. capsulatus (Bath) was purchased from National Collection of Industrial and Marine Bacteria  as strain NCIMB 11132, and its DNA was isolated as previously described  proteins were analyzed using the Automated Phylogenetic Inference System . This system automates the process of sequence similarity, alignment, and phylogenetic inference for each protein in a genome. Sequence alignments and phylogenetic trees were refined using the methods described previously (Identification of putative protein-encoding genes and annotation of the genome were performed as previously described . An initeviously .M. capsulatus [Bath]), and (c) to determine the presence of homologs in different species. Orthologs between the M. capsulatus (Bath) genome and that of M. extorquens were identified by requiring mutual best-hit relationships  among all possible pairwise BLASTP comparisons, with some manual corrections. A total of 89 genes involved in methylotrophy in M. extorquens (M. capsulatus (Bath) and M. extorquens. Comparative genome analyses were also performed using the Comprehensive Microbial Resource (Proteins were searched by BLASTP  against torquens  were obtResource .core of the putative prophage (identified using MUMmer [mom DNA methyltransferase) demarking the 5′ and 3′ boundaries of the region  per protein sequence query.Putative prophage regions were defined as containing genes that encode proteins bearing sequence similarity to known phage or prophage proteins. We are using “prophage” to refer to sequences with similarity to lysogenic bacteriophages that have not been experimentally demonstrated to form infectious particles. Additional supporting information included the presence of direct repeats representing the attg MUMmer ), the coe region . Best mae region  was imple region , reportihttp://www.ncbi.nlm.nih.gov/Genbank/) accession number for the Methylococcus capsulatus genome discussed in this paper is AE017282.The GenBank (


We strategically selected the GrB ELISPOT assay because GrB is a hallmark effector molecule of cell-mediated destruction of target cells.This study assessed the Granzyme B (GrB) ELISPOT as a viable alternative to the 51Cr-release assays were made using human LAK cells.We optimized the GrB ELISPOT assay using the human-derived TALL-104 cytotoxic cell line as effectors against K562 target cells. Titration studies were performed to assess whether the ELISPOT assay could accurately enumerate the number of GrB-secreting effector cells. TALL-104 were treated with various secretion inhibitors and utilized in the GrB ELISPOT to determine if GrB measured in the ELISPOT was due to degranulation of effector cells. Additionally, CD107a expression on effector cells after effector-target interaction was utilized to further confirm the mechanism of GrB release by TALL-104 and lymphokine-activated killer (LAK) cells. Direct comparisons between the GrB ELISPOT, the IFN-γ ELISPOT and the standard 2+, thereby preventing degranulation. The number of effector cells expressing the degranulation associated glycoprotein CD107a increased after interaction with target cells and correlated with the stimulated release of GrB measured in the ELISPOT assay.Titration studies demonstrated a strong correlation between the number of TALL-104 and LAK effector cells and the number of GrB spots per well. GrB secretion was detectable within 10 min of effector-target contact with optimal secretion observed at 3–4 h; in contrast, optimal IFN-γ secretion was not observed until 24 h. The protein secretion inhibitor, brefeldin A, did not inhibit the release of GrB but did abrogate IFN-γ production by TALL-104 cells. GrB secretion was abrogated by BAPTA-AM ethane-N,N,N', N'-tetraacetic acid tetra(acetoxymethyl) ester), which sequesters intracellular Ca51Cr-release assay to measure MHC non-restricted cytotoxic activity of innate immune cells. Compared to the IFN-γ ELISPOT assay, the GrB ELISPOT may be a more direct measure of cytotoxic cell activity. Because GrB is one of the primary effector molecules in natural killer (NK) cell-mediated killing, detection and enumeration of GrB secreting effector cells can provide valuable insight with regards to innate immunological responses.Because of its high sensitivity and ability to estimate cytotoxic effector cell frequency, the GrB ELISPOT assay is a viable alternative to the  TALL-104 cells were resuspended at 2.5 × 105 cells/ml and 2.5 × 104 cells/ml in PBS without divalent cations for BAPTA-AM and brefeldin A treatment, respectively. Cells were pretreated with the indicated concentrations of BAPTA-AM for 45 min or with 5 μg/ml brefeldin A for 1 h at 37°C, washed twice, assessed for viability by trypan blue exclusion, and resuspended at 2.5 × 104 cells/ml in assay media.TALL-104 cells were treated with inhibitors of cellular secretion prior to use in the GrB and IFN-γ ELISPOT assays. Inhibitors used included 1,2-bis-(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid tetra(acetoxymethyl) ester , a cell permeant chelator of intracellular Ca51Cr-release assay. Briefly, one million target cells were labeled at 37°C for 1 h with 100 μCi Na251CrO4 . Target cells were washed and resuspended in TCM at 5 × 104 cells/ml. Five thousand target cells per well (100 μl) were added to a 96 well plate  following the appropriate number of effector cells (100 μl/well). The defined effector:target (E:T) ratios were plated in triplicate. Cytotoxicity assays were performed at 37°C for 4 h. Percent specific lysis was calculated using the following equation:Cytotoxicity of TALL-104 and LAK cells was assessed using the standard (ER - SR)/(MR - SR) × 100,where ER = experimental release, SR = spontaneous release and MR = maximum release.4 target cells per well (100 μl). After the specified effector-target cell incubation, the plates were washed and 100 μl/well of biotinylated anti-human GrB detecting antibody  was added. Plates were incubated for 3 h and 50 μl of Streptavidin-Alkaline Phosphatase  was added for 1 h. Spots were visualized with 100 μl/well of BCIP-NBT phosphatase substrate  and subjected to automated evaluation using the ImmunoSpot Imaging Analyzer system .Granzyme B secretion was measured using the GrB ELISPOT assay as previously described . BrieflyFor assessing IFN-γ secretion, MultiScreen-IP plates  were coated overnight at room temperature with 50 μl/well of anti-human IFN-γ antibody  as previously described . After eDegranulation of TALL-104 and LAK cells in response to target cell recognition was assessed by monitoring surface antigen expression of CD107a , a surface antigen transiently present on the cell surface after release of cytolytic granules. Expression of CD107a has been used as a marker to measure degranulation by flow cytometry ,29.5) were resuspended in 400 μl of phenol red-free TCM in polystyrene tubes . PKH67-labeled K562 target cells (1 × 105 cells in 50 μl) were then added to appropriate tubes, the tubes were spun for 30 sec at 500 rpm and incubated for the specified periods in a 37°C water bath. Reactions were quenched with cold PBS and then 20 μl of CD107a-PE-Cy5 (PharMingen) was added. Mouse IgG1-biotin  and streptavidin-PE-Cy5  were utilized for controls. Tubes were incubated with antibody for 20 min at room temperature, washed with buffer and analyzed using a FACScan instrument  equipped with a 15 mW argon-ion laser. The percent of effector cells expressing CD107a was determined by gating the PKH67 negative (effector cells) population. Within the PKH67 negative gate, CD107a expression was determined from histograms based on forward and side scatter analysis. Effector cells run in the absence of target cells were evaluated for baseline expression of CD107a. The percentage of effector cells induced by target cells to express CD107a was calculated by subtracting background (spontaneous) expression from experimental samples.To distinguish target cells from effector cells, K562 cells were first labeled with PKH67 dye (Sigma) according to the manufacturer's instructions and washed extensively prior to use in the assay. Effector cells (2 × 10t test and Pearson correlation coefficient (R2).Statistical analysis was performed using Student's 2 = 0.98. Therefore, the GrB ELISPOT assay is capable of measuring the frequency of effector cells stimulated to secrete GrB.GrB ELISPOT assays were performed using a constant number of K562 targets and decreasing numbers of TALL-104 effector cells per well. As the number of TALL-104 cells decreased, the resulting number of GrB spots decreased linearly. Limited GrB spots were detected in wells containing TALL-104 alone demonstrating that the ELISPOT assay measured primarily stimulated release of GrB  inhibit IFN-γ secretion  therefore, the optimal ratios for the ELISPOT assays are below the level of sensitivity of the 51Cr-release assay . EGTA, which sequesters extracellular Ca2+, could also abrogate GrB secretion measured in the ELISPOT assay. However, significantly higher concentrations of EGTA (4–40 mM) than BAPTA-AM were needed to block GrB secretion (data not shown). These data are in good agreement with recently published findings [2+, but not Ca2+ [To confirm that GrB measured in the ELISPOT assay was released via degranulation, effector cells were treated with inhibitors of cellular secretion: brefeldin A, BAPTA-AM and EGTA. Brefeldin A reversibly disrupts protein translocation from the endoplasmic reticulum to the Golgi apparatus, blocking the production and subsequent secretion of newly synthesized proteins . BAPTA-Afindings  which denot Ca2+ . Thus, t2 = 0.89). Both the release of GrB and the expression of CD107a was observed as early as 30 min after LAK cells were stimulated with relevant target cells, and increased with extended effector-target cell interaction. These data confirm that the GrB ELISPOT assay is an excellent measure of cytotoxic capacity mediated by effector cell degranulation.Recently, a flow cytometric assay based on CD107a  mobilization was developed to measure degranulation of cytolytic cells ,29. CD1051Cr-release assay, they demonstrated higher sensitivity with both TALL-104 and LAK cells as effector cells, data consistent with previous studies [51Cr-release assay measures cytotoxic ability regardless of the mechanisms of the killing. Therefore, unlike the 51Cr-release assay, the ELISPOT assays are both qualitative and quantitative.When the ELISPOT assays were directly compared to the  studies ,25,36,3751Cr-release and the GrB ELISPOT assay measure different aspects of cell-mediated killing – target cell death and effector cell function, respectively. A limitation of the GrB ELISPOT assay is that it measures degranulation, not direct target cell lysis. As such, degranulation may not always equate to cell death if target cells contain serpin proteinase inhibitor 9 (PI9), a protein that inhibits the proteolytic activity of GrB, [51Cr-release assay, the GrB ELISPOT provides the precusory frequency of cells with the potential to kill targets. Although a number of flow cytometric assays have been developed to assess target cell cytotoxicity as well as identify the phenotype of the effector cells mediating the immune response, these assays are not as sensitive as the ELISPOT assay.It is important to emphasize that  of GrB,  or if ef51Cr-release assay [The use of the IFN-γ ELISPOT assay as a surrogate measure for CTL and NK responses has recently gained increased application as an alternative to the se assay ,39-43. Hse assay -48. The se assay ,50. Ther51Cr-release assay to measure granule-mediated cytotoxicity and could be easily adapted to reliably and accurately measure MHC non-restricted cytotoxicity indicative of NK and LAK activity. The GrB ELISPOT assay measured GrB release due to degranulation of stimulated effector cells. Additionally, GrB is a more specific candidate marker than IFN-γ to measure the cytotoxic capacity of innate immune effector cells such as NK cells.Our data demonstrate that the GrB ELISPOT assay is a viable alternative to the standard None declared.KSW and AM designed the study, analyzed the data and drafted the manuscript. KSW carried and prepared the cell lines and performed the majority of the immunogical assays. MWB performed and analyzed the flow cytometric CD107a assay data. SS participated in the design of the study and preparation of the manuscript. TS, DK and MB participated in the design of the study and served as expert advisors. All authors read and approved the final manuscript.


Human resources (HR) constraints have been reported as one of the main barriers to achieving the 2005 global tuberculosis (TB) control targets in 18 of the 22 TB high-burden countries (HBCs); consequently we try to assess the current HR available for TB control in HBCs.A standard questionnaire designed to collect information on staff numbers, skills, training activities and current staff shortages at different health service levels was sent to national TB control programme managers in all HBCs.Nineteen HBCs (86%) replied, and 17 (77%) followed the questionnaire format to provide data. Complete information on staff numbers at all service levels was available from nine countries and data on skill levels and training were complete in six countries. Data showed considerable variations in staff numbers, proportions of trained staff, length of courses and quality of training activities. Eleven HBCs had developed training materials, many used implementation guidelines for training and only three used participatory educational methods. Two countries reported shortages of staff at district health facility level, whereas 14 reported shortages at central level. There was no apparent association between reported staff numbers (and skills) and the country's TB burden or current case detection rates (CDR).There were few readily available data on HR for TB control in HBCs, particularly in the larger ones. The great variations in staff numbers and the poor association between information on workforce, proportion of trained staff, and length and quality of courses suggested a lack of valid information and/or poor data reliability. There is urgent need to support HBCs to develop a comprehensive HR strategy involving short-term and long-term HR development plans and strengthening their HR planning and management capabilities. The performance of health care systems is closely related to the numbers, distribution, knowledge, skills and motivation of its workforce, particularly of those individuals delivering the services . ImproveTuberculosis (TB) constitutes the third most important cause of death and disability  among inThe World Health Assembly (WHA), in 1991, pledged countries to achieve detection of at least 70% of estimated infectious TB cases (SS+) and to cure 85% of them by the year 2000 . LikewisLow-income and middle-income countries (LMIC) urgently need a sufficiently large health care staff with appropriate expertise, experience and motivation, working at the right places. Lack of HR strategies, inadequate HR planning and management, poor deployment practices, inflexible contracting arrangements and inability to create new posts or increase salaries resulting from international regulations capping social sector spending have contributed to the global HRH crisis . In addiIn 2003, national TB programme (NTP) managers from 18 of the 22 TB high-burden countries , ranked inadequate HR first within the top five constraints to reaching the WHA global TB control targets [There is very limited published literature on HRH issues, particularly in LMIC and there are both scanty information on methods to assess HR capacity and lack of evidence on how best to evaluate interventions to strengthen and/or build HR capacity . The deaCurrently there is increasing awareness that HRH concerns must be addressed in order to reach the MDGs, to expand access to priority interventions, to promote health systems development and to achieve global health equity ,15. HoweThis paper reports the results of a questionnaire sent to NTP staff in the 22 HBCs to assess the workforce available for TB control , as well as the estimated HR requirements for appropriate TB control in HBCs. It aims to inform the development of more reliable methods of gathering qualitative and quantitative information on HR and to stimulate a long-overdue discussion on HR for TB control issues, so that governments of endemic countries and technical and financial partners can finally begin to address them jointly.NTP managers and country-based WHO staff in the 22 HBCs: Afghanistan, Bangladesh, Brazil, Cambodia, China, Democratic Republic of the Congo, Ethiopia, India, Indonesia, Kenya, Mozambique, Myanmar, Nigeria, Pakistan, Philippines, the Russian Federation, South Africa, Tanzania, Thailand, Uganda, Viet Nam and Zimbabwe. These countries account for more than half of the world's population and approximately 80% of the global TB burdenDiscussions with NTP managers and a literature review on HRH informed the development of a questionnaire to ascertain current staff provision; quality and intensity of training; time and type of personnel involved in performing different TB control activities; and estimated staffing needs at different health service levels. The questionnaire was pilot-tested internally within the Stop TB Department (STB), WHO headquarters, Geneva; and externally with staff from the NTP in Indonesia.The first section of the questionnaire assessed staff numbers and skills. It included open questions about absolute numbers of staff involved in delivering TB control activities at each service level . Skills were assessed using a composite of the proportion of staff receiving training in the previous three years (at each level) and the quality of the training provided  – materials) as proxy measures.Section two of the questionnaire addressed estimated HR gap. Since this paper specifically refers to TB control, we estimated the workload involved in adequate TB control in HBCs. Workloads were assessed by means of open questions on the different processes involved in the management of new SS+ patients , the estimated duration of each task and the type of staff involved in delivering them. A series of worksheets containing the different tasks required for TB case management were designed to assist respondents completing the questionnaire and to improve standardization. Capacity of health services at two different CDRs were assessed (current CDR and at the target 70% CDR). Workloads at current CDRs were calculated by multiplying the current numbers of new SS+ patients by the estimated time needed to treat a new SS+ patient; workloads at the 70% CDR were calculated by multiplying the figure corresponding to 70% of the estimated SS+ TB cases for each country by the time needed to treat a new SS+ patient. Eleven hours was used as the time needed to treat a new SS+ patient; this had been estimated previously by a TB experts' consensus .In March 2003, the questionnaire, together with detailed instructions in English, was e-mailed to NTP managers and country-based WHO staff in the 22 HBCs. Three e-mail reminders were sent monthly after the return deadline; follow-up with several countries included e-mail and telephone communications to clarify responses. Data were entered into spreadsheet software for the analysis; qualitative answers were discussed by the authors.Nineteen of the 22 HBCs returned the questionnaire (86% response rate). Two countries provided information on their HR for TB control but did not use the questionnaire format and were thus excluded from the analysis; one country reported that it would take a substantial amount of time to get reliable information from such a large country and provided other readily available HR information; another reported that given the country's current staffing deficits and competing priorities they were unable to complete the survey. Further discussions revealed that some HR information was available in this country, although it was scattered in different sources and lengthy to compile. Both countries requested technical support to assess staffing needs at different levels and to assist their government's HR development programmes. Despite e-mail and telephone remainders, three countries failed to respond.Information on estimated numbers of staff was available from all 17 countries but complete in nine (53%) of them. Staff numbers within the same service level varied considerably between countries; i.e. numbers of staff at provincial level varied from 8704 in one country to 6 staff in two countries (Table Complete information on numbers of staff trained in the previous three years was available in only six countries (35%). There was also great variation in the numbers (or proportions) of trained staff Table . IndonesInformation on length of training courses and development of training materials was also incomplete. Length of courses for staff at the same service level varied greatly between countries; for example, courses for TB coordinators at provincial level (data from 11 countries) varied from 4 to up to 60 days, whereas at district level (information from 10 countries) courses varied from 4 days to 4 months Table . SimilarThe estimated time needed to treat a new SS+ patient is shown in Figure Sixteen countries Table  reportedAll countries provided information on their estimated HR gap at district level; nine 53%) countries reported existing staff needs at district level ; for other health care staff, adequate competence is ensured through regular training and supervision. Apart from Indonesia and Viet Nam, there was no correlation between information on numbers of staff, attendance of training courses, length of courses and development of training materials. In general NTP managers in HBCs had limited information on staff attendance of training courses and on the characteristics, duration and intensity of training activities. HBCs need to develop needs-based comprehensive training policies and training strategies for health care staff at all service levels, incorporating pre-service training, re-training, in-service support and continuous professional/career development. Similarly, it is important to emphasize the role of periodic monitoring and supervisory visits as part of the staff continuous education and support processes. Appropriately designed strategies fostering career paths could increase staff motivation and performance; improve staff recruitment, retention and distribution; and even have a positive effect on enlisting of students into training programmes  and expaThe quality of training materials was also of concern: many countries used inadequate training tools, few had developed specific modules and even fewer used problem-based learning or participatory methodology to facilitate adequate skill development. The length of courses also varied greatly: while very short courses do not allow the development of skills and competences needed to improve performance, lengthy courses have economic and logistic implications. Differences in the quality and intensity of training could translate into service or performance variations with detrimental effects to programmes and ultimately to patients. There is a need to standardize training in terms of contents, competences, methodologies, course duration and quality of training materials. Improvement and standardization of training curricula and methodologies will facilitate the adoption of a universal standard of care for TB patients in HBCs, leading to a more rational use of the health care workforce, improved staff motivation and productivity and better outcomes for TB patients.Although the majority of countries described deficiencies of staff at central level, few reported shortages in the actual numbers of posts. However, there were concerns about the distribution and/or the skill mix (competences and efficiency) of staff. There was no clear relation between reported data on staff needs and either the TB burden in the country or the actual performance of the NTP. It is of concern that only Uganda and Myanmar reported shortages of staff at current CDRs, whereas countries with very low current CDRs did not. Only five countries projected shortages of staff at the 70% CDR; four of them estimated large numbers of staff required.If the assessment of the HR needs was based on the actual workload, it is possible no substantial gap was determined because most of the current diagnostic and treatment tasks are carried out satisfactorily by existing staff, but an unrecognized HR gap could hamper improvement in case detection rates. The inability of some countries to increase the number of posts because of recruitment ceilings imposed under structural adjustment programmes could lead to countries underreporting their HR gap. Further analysis and recommendations on staff needs at different levels is hindered by the lack of accurate information on numbers, quality and distribution of existing staff.This survey is, to our knowledge, the first attempt to ascertain HR for TB control in HBCs. The response rate was high and responses were analysed in the context of the countries' estimated TB burden and current CDRs.Limitations of the study are evidenced by the great variation in reported staff numbers and the lack of correlation between information on numbers of trained staff, length of courses, development of materials and performance, suggesting a shortage of valid information, poor standardization and/or data reliability.The fact that the questionnaire was in English could have generated some inaccurate answers. Indonesia, for example, reported 100% training completion based on its 2002 training goals rather than in absolute numbers , but countries were re-contacted (by e-mail or telephone) to clarify inconsistent replies. Responses could have been biased towards greater HR needs if countries perceived this as an opportunity to request increased support; however, this did not seem to happen, since few countries reported staff shortages.The performance assessment component of the questionnaire assumed NTP managers could assess the HR gap by comparing existing staff numbers (at different service levels) with the product of the additional SS+ cases by the time spent in diagnostic/treatment activities. Although all the tasks were listed in the questionnaire, the interpretation of what constituted achieving each task was left to NTP managers; this could have affected their estimation of the time required. We are currently developing a more detailed task analysis tool that includes the breakdown of each TB control activity into specific single tasks and a description of what constitutes each task; this will facilitate future studies. Furthermore, this method accounted only for new SS+ cases: in some countries, health facility staff will spend more time dealing with relapses, treatment failures or other TB patients: On the other hand, in some HBCs some tasks are performed by NGOs or the private sector and this was not discounted.Constraints to HR development such as poor HR data quality, lack of a comprehensive national HR plan/strategy and little attention to continuing education programmes  were comThe 2nd ad hoc committee on the TB epidemic recommended addressing the health-workforce crisis by collaboratively developing policies to reduce barriers to creating and filling posts in HBCs; increasing staff recruitment and retention by improving working conditions in the health sector; promoting task analysis and HR needs assessments, HR planning and training at country level; and working together with other stakeholders to develop strategies to further mobilize HR for TB control . In gene declare that they have no competing interests.