Molecular markers for localized colon tumours and for prognosis following therapy are needed. Proteomics research is currently producing numerous biomarker studies with clinical potential. We investigate the protein composition of plasma and of tumour extracts with the aim of identifying biomarkers for colon cancer.By Surface Enhanced Laser Desorption/Ionisation – Time Of Flight / Mass spectrometry (SELDI-TOF/MS) we compare the protein profiles of colon cancer serum with serum from healthy individuals and the protein profiles of colon tumours with normal colon tissue. By size exclusion chromatography, we investigate the binding of HNP 1-3 to high mass plasma proteins. By microflow we investigate the effect of HNP 1-3 on mammalian cells.Human Neutrophil Peptides -1, -2 and -3 (HNP 1-3), also known as alfa-defensin-1, -2 and -3, are present in elevated concentrations in serum from colon cancer patients and in protein extracts from colon tumours. A fraction of HNP 1-3 in serum is bound to unidentified high mass plasma proteins. HNP 1-3 purified from colon tumours are lethal to mammalian cells.HNP 1-3 may serve as blood markers for colon cancer in combination with other diagnostic tools. We propose that HNP 1-3 are carried into the bloodstream by attaching to high mass plasma proteins in the tumour microenvironment. We discuss the effect of HNP 1-3 on tumour progression. The diagnostic stage of colon cancer determines survival. Patients diagnosed with localized tumours have a 75% probability of 5 year survival, whereas patients diagnosed with distant metastases only have a 5–10 % probability of 5 year survival . The samples were centrifuged at 14,000 rpm for 10 minutes and the pellet was discarded (repeated twice). The tissue protein extracts were stored at -80°C until use. Pilot studies were performed on different chips (data not shown) and the NP20  (Ciphergen) chip was chosen for the tissue screening. NP20 chips were placed in Bioprocessor (Ciphergen) and pre-treated with 50 ul tissue binding buffer (50 mM TRIS-HCl, pH 8.0) for 5 minutes on shaker (250 rpm) (repeated twice). 5 ul tissue protein extract was diluted in 50 ul tissue binding buffer and incubated in Bioprocessor on NP20 chips for 40 minutes at room temperature on shaker (250 rpm). Spots were washed twice in 250 ul tissue washing buffer (50 mM TRIS-HCl, pH 8.0) for 5 minutes. The chips were air dried for 10 minutes, followed by treatment with two times 0.6 ul 100% sinapinic (SPA) matrix solution. SPA was obtained from Ciphergen in 5 mg aliquots and dissolved (150 ul MQ water, 150 ul acetonitrile, 1.5 ul Tri-Fluoro-acetic-Acid (TFA)) immediately before the screenings.Colon cancer serum samples were obtained from patients before surgical treatment. Normal serum was obtained from a group of healthy individuals matched by age and gender to the cancer patients. All serum samples were stored at -80°C until use. Serum pilot studies were performed on different chips to monitor the presence of HNP 1-3 in serum (data not shown). The immobilised metal affinity capture (IMAC30) chip was chosen for the actual screening and was pre-treated with nickel before analysis: 5 ul 100 mM NiSO4 were added to each spot and left on shaker (250 rpm) for 5 minutes (repeated twice). The chips were placed in Bioprocessor and incubated with 100 ul MQ for 5 minutes on shaker (250 rpm). Each spot was treated with 50 ul serum binding buffer (100 mM TRIS-HCl, pH 7.5, 500 mM NaCl, 0,1% Triton X-100) and left on shaker for 5 minutes (250 rpm). Serum samples were thawed on ice and 1 ul serum was diluted in 50 ul serum binding buffer and applied to spots and left on shaker (250 rpm) at room temperature for 40 minutes. The sample solution was removed and the spots were washed twice in 200 ul serum washing buffer (100 mM PBS, pH 7.4, 700 mM NaCl), followed by one wash in 200 ul MQ water. The chips were removed from the Bioprocessor and left to air dry for 20 minutes followed by treatment with two times 0.6 ul SPA.Only freshly made matrix solutions were used and the instrument was calibrated daily. Cancer and normal samples were run side by side. The chips were analysed on a PBS II instrument (Ciphergen). All spectra in each screening were normalised based on total ion current.4HCO3, pH 8,0) was added, and the chip was left for 10 hours at 40°C in humidity chamber after which the chip was left to air dry for 20 minutes. 1 ul CHCA matrix (prepared as the SPA matrix solution) was added and the peptide map was analysed on the PBS II instrument. Peptide maps of trypsin autodiggest were used as controls. Identification was done with the PepIdent software on the Expasy server. For the reduction experiment, HNP 1-3 were first denaturation by heating (10 minutes at 80°C) followed by treatment with DTT (200 mM, 30 minutes at room temperature) and the peptides were incubated on an NP20 chip and analysed on the PBS II instrument.100 ul protein extract from colon tumour tissue in tissue lysis buffer was loaded unto a RP-HPLC column (uRPC C2/C18 ST 4.6/100, Pharmacia Biotech, Flow rate: 0.5 ml/min, Fraction size: 0.5 ml) in buffer A (0.065% TFA in MQ-water) and proteins were eluted in a gradient of 0–100% buffer B (0.05% TFA in acetonitrile (ACN)). Elution of peptides was monitored by absorbance at 280 nm. All protein-containing fractions were analysed by Matrix Assisted Laser Desorption/Ionisation-Time of flight (MALDI-TOF) on the PBS II instrument: 1.5 ul fraction was incubated with 0.6 ul SPA on a Gold array (Ciphergen) and left to crystallise, followed by an additional treatment with 0.6 ul SPA and analysed in the PBSII instrument. The HNP 1-3 containing fraction (32% buffer B) was further purified on a peptide gelfiltration column (Superdex Peptide HR 10/30, Pharmacia Biotech, Flow rate 0.9 ml/min, Fraction size: 0.5 ml, Buffer: 50% ACN, 0.1 % TFA). Elution of peptides was monitored by absorbance at 280 nm and protein-containing fractions were again analysed by MALDI-TOF. Purified HNP 1-3 were identified by on-chip trypsin digestion: 10 ul of HNP 1-3 fraction was applied to an NP20 chip and left on shaker (250 rpm) at room temperature for 40 minutes. The solution was removed and the spot was washed twice with 10 ul water. In order to denature peptides prior to digestion, the chip was left on heating block (80°C) for 5 minutes. The chip was cooled on ice for 2 minutes. 10 ul trypsin digestion solution . Elution of peptides was followed by absorbance at 280 nm. All protein containing fractions were analysed by MALDI-TOF on PBS II (Ciphergen) as described above. Maximum signal intensity of 40 individual peaks was plotted as a function of elution volume and an approximate elution curve was calculated.For micro flow experiments, canine kidney cells (MDCK cells) were plated onto poly-d-lysine coated cover slips at a concentration 3000 cells/well, grown in Dilbeccoo's Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) for five days with the result of confluent islands. Microflow was performed in an Eppendorf micromanipulator 5171 and transjector 5246 system mounted on a Leica DMIRBE inverted research microscope. Micro capillaries  were pulled to an outer diameter of 0.85 nm on a Sutter P-97 Micropipette Puller. The dye-loaded cells were visualized by excitation at 470 nm and recorded at 509-nm emission using Haupage version 3.3.18038 software and Kappa CF 15/4 MC-S camera (Leica). The MDCK cells were recorded (in CO2 independent media) on the inverted DMIRBE inverted research microscope. The capillary was placed 20 nmover the confluent cells with a constant flow (1300 hPa). The MDCK cells were exposed to peptide and calcein (20 mM) fractions for 60 minutes.-16).We performed pilot studies of colon tumour and normal colon tissue on a variety of chips with different chemical properties and with different binding and washing conditions. Based on these preliminary studies, we found that the expression of three peptides with mass/charge ratio (m/z) values of 3372, 3443 and 3486 (subsequently identified as HNP 2, 1 and 3, respectively), were upregulated in the tumour samples. The three peptides were visible on different chips and under different binding conditions (data not shown). The strongest signal of HNP 1-3 in tissue extract was obtained on the NP20  chip, whereas the strongest signal of HNP 1-3 in serum was observed on the IMAC30 (Ni) , and these conditions were chosen for the actual screenings. We emphasize that in general the protein profiles of serum and tissue were very different when using the same protein chip for serum and tissue extract. However, individual peaks were present on several types of chips, and observed in both serum and in tissue extract, for example the characteristic triplet with m/z 3372, 3443 and 3486. Protein extract from 40 colon tumour and 40 normal colon tissue samples were analysed on NP20 chips and 125 colon cancer serum samples and 100 normal serum samples were analysed on IMAC30 (Ni) chips. All spectres in each screening were pooled and normalised based on overall ion current. Each spectrum produced approximately 40 to 90 protein peaks in the range from 2 to 80 kDa FIG. . StatistCompared to other peptides in the same range, HNP 1-3 showed average signal intensity in most normal colon tissue extract, whereas the HNP 1-3 signal was extremely high in most tumour samples . In the normal serum samples the HNP 1-3 signals were weak and only slightly stronger in the cancer serum.The markers were purified by RP-HPLC, peptide gelfiltration and on-chip purification, after which they were identified by peptide mapping as HNP-2 (3372 Da), HNP-1 (3442 Da) and HNP-3 (3486 Da) Table . The mas50 ul colon tumour extract in Lysis buffer was applied to a peptide gelfiltration column. Elution of peptides was followed by absorbance at 280 nm. All fractions were analysed by MALDI-TOF on the PBS II instrument (Ciphergen). Maximum signal intensity of 40 individual peaks was plotted as a function of the elution volume and an approximate elution curve was calculated FIG. . HNP 1-3The cytotoxicity of HNP 1-3 purified from colon tumours was tested by exposing MDCK cells to different fractions purified from colon tumours. Calcein were added to the fractions and the solutions were left to flow over the cells for one hour. By fluorescence microscopy calcein was observed to accumulate in cells exposed to HNP 1-3/calcein fractions, whereas cells treated with fractions containing other (unidentified) tissue peptides did not uptake calcein FIG. . FurtherAbnormal concentration of HNP 1-3 in blood has previously been demonstrated in connection with benign conditions. Elevated concentrations of HNP 1-3 following infection  has been found in plasma, blood and other body fluids , and plaHNP expression has previously been linked to different types of tumours and cell lines. HNP-1 has been detected in lung tumours  and in tPrevious studies indicate that HNP 1-3 expression in tumours primarily originate from tumour invading eosinophils  and neutin vitro and that high concentrations of HNPs causes precipitation of plasma proteins; specifically 2-macroglubulin and C1 complement [We explain the elevated concentrations of HNP 1-3 in colon cancer serum by unspecific binding between HNP 1-3 and high mass plasma proteins. We suggest that the peptides attach to plasma proteins in the tumour area and are carried into the bloodstream. The HNP 1-3 we observe in high mass fractions from size exclusion, could also be explained by multimerisation: In one study it was demonstrated that defensins form voltage dependent channels in lipid bilayer membranes and further conductance investigations suggested that the channels were formed by multimers containing 2–4 molecules  and a crmplement ,23 has bmplement  showed tmplement . TogetheCommon to beta-defensin 2 (another member of the defensin family) and HNP 1-3 is an uneven distribution of surface charges. Beta-defensin 2 has been shown to bind to a chemokine receptor . It has in vivo function of HNP 1-3. One study showed that HNP 1-3 mediates lysis of tumours in a concentration dependent manner [-4 M) are cytotoxic for human monocytes, whereas lower concentration of HNP-1 (10-8 to 10-9 M) increases TNF-alpha production by monocytes [in vivo HNP 1-3 purified from colon tumours are capable of lysing MDCK cells. Our study was based on a 60 minutes microflow study. This screening set-up did not allow us to investigate the minimum concentration of HNP 1-3 necessary for lysis.The exact concentration of HNPs in the tumour microenvironment may decide the t manner . This isonocytes . In a stonocytes  it was sThe high concentration of HNP 1-3 observed in tumours and the observation that HNP 1-3 are capable of lysing mammalian cells may lead to the conclusion that the peptides serve to the benefit of the host by killing tumour cells. However, in one study HNP 1-3 were found to bind to HLA-Class II molecules and were capable of reducing the proliferation of a HLA-DR-restricted T-cell line after stimulation  and coulOur results add to the evidence that HNP 1-3 bind to high mass plasma proteins. We suggest that the peptides are released in the inflammatory site and passively diffuse into the blood stream. HNP 1-3 have been observed in primary tumours of different tissues and the peptides are known to play a fundamental role in the innate immune system. We suggest that the peptides may serve as markers for colon cancer in combination with established diagnostic tools and as prognostic markers following therapy.This study was partly financed by Colotech ltd. and partly by Glostrup Hospital. Jakob Albrethsen, Rikke Bøgebo and Hans Raskov receive salary from Colotech A/S.Steen Gammeltoft, Jesper Olsen and Benny Winther receive salary from Glostrup Hospital.JA performed the SELDI-TOF/MS screenings, the protein identification experiments and the size exclusion study. RB did the statistical analysis, BW did the microflow study, JO obtained the biological samples. HR and SG planned the project.The pre-publication history for this paper can be accessed here:


Bacteria are particularly adept at making themselves indispensable by insinuating themselves into some fundamental aspect of an organism's biology. The endosymbiotic hypothesis proposes that this is how certain eukaryotic organelles evolved from endosymbiotic bacteria. Insights into the mechanisms governing endosymbiosis will help biologists understand how this mutually beneficial relationship evolved and provide clues to one of the fundamental questions in biology: How did the eukaryotic cell evolve?When it comes to exploiting a niche, endosymbionts take the prize. In endosymbiosis, one organism—the Buchnera aphidicola, and discover clues to the molecular foundation of their shared fate. Over 10% of insect species rely on endosymbionts for their development and survival. In this issue, David Stern and colleagues look at one of the most studied pairs, the pea aphid and Buchnera are transferred from clusters of bacteriocytes in the mother to the adjacent early-stage embryo, it has been unclear how the bacteriocytes develop. Previous studies of the bacteria's genome have failed to explain the genetic basis of Buchnera's ability to invade aphid cells. Consequently, Stern and colleagues have focused on the bacteriocytes, the specialized insect cells that house Buchnera, shedding light on the development of these cells as well as on the evolutionary adaptations in the aphid that made the bacteriocytes hospitable to Buchnera.While it is known that Buchnera's presence in the cell, and they identify three aphid transcription factors (proteins that regulate gene expression) that are expressed in three distinct stages during early-bacteriocyte development in the aphid embryo. The first protein is expressed just before Buchnera enters the embryo; a second, as the bacteria invades; and a third, after the transfer is nearly complete. A second wave of the same transcription factors occurs at a later stage in aphid embryo development and increases the population of bacteriocytes.The researchers show that bacteriocytes differentiate and proliferate independently of Buchnera-carrying aphid species, appears to be an evolutionarily conserved feature of aphids. It even occurs in an aphid species that once had a Buchnera endosymbiont and now has a yeast-like symbiont that lives outside the bacteriocytes. But this process is not observed in males of another aphid species that do not carry Buchnera. While traces of the first transcription factor activated in bacteriocytes are evident, the characteristic gene-expression pattern is not, and the aphids have no mature bacteriocytes.This two-step specification of bacteriocytes, which occurs in related Buchnera involved significant adaptations by the host.While it seems that the aphid has evolved new domains of expression in the bacteriocyte for these transcription factors—none of these transcription factors is expressed at a similar stage in other insects—the researchers cannot yet say whether these genes direct the specification of bacteriocytes. Still, these transcription factors are likely to play important roles in the bacteriocyte, suggesting that the union of aphids and


We investigated patient perspectives on switching from injection to oral B12 therapy.Although oral replacement with high doses of vitamin B12 therapy. One hundred and thirty-three patients who receive regular B12 injections were included from three family practice units  in Toronto.This study involved a quantitative arm using questionnaires and a qualitative arm using semi-structured interviews, both to assess patient views on injection and oral therapy. Patients were also offered a six-month trial of oral B12. In a multivariate analysis, patient factors associated with a "willingness to switch" to oral B12 included being able to get to the clinic in less than 30 minutes (OR 9.3, 95% CI 2.2–40.0), and believing that frequent visits to the health care provider (OR 5.4, 95% CI 1.1–26.6) or the increased costs to the health care system (OR 16.7, 95% CI 1.5–184.2) were disadvantages of injection B12. Fifty-five patients attempted oral therapy and 52 patients returned the final questionnaire. Of those who tried oral therapy, 76% (39/51) were satisfied and 71% (39/55) wished to permanently switch. Factors associated with permanently switching to oral therapy included believing that the frequent visits to the health care provider (OR 35.4, 95% CI 2.9–432.7) and travel/parking costs (OR 8.7, 95% CI 1.2–65.3) were disadvantages of injection B12. Interview participants consistently cited convenience as an advantage of oral therapy.Seventy-three percent (63/86) of respondents were willing to try oral B12 is both feasible and acceptable to patients. Oral B12 supplementation is well received largely due to increased convenience. Clinicians should offer oral B12 therapy to their patients who are currently receiving injections, and newly diagnosed B12-deficient patients who can tolerate and are compliant with oral medications should be offered oral supplementation.Switching patients from injection to oral B However, because approximately 1% of orally ingested B12 is absorbed via simple diffusion throughout the gastrointestinal tract (i.e., independently of intrinsic factor)[12 deficiency [Intramuscular injections of vitamin Bficiency -12.12 therapy would decrease physician burden, increase patient control over therapy, and avoid patient discomfort and inconvenience. Switching patients from B12 injections to oral therapy would also result in savings to the health care system [12 therapy [12.Oral Be system . While s therapy -15, litt12 are willing to switch to oral therapy and to explore the reasons for their choice.Efforts to switch patients who are well established on parenteral therapy (and who have previously been told they require lifelong injections) may fail without an understanding of what factors influence patient acceptability of oral therapy. Therefore, we combined qualitative and quantitative methods to test the hypothesis that patients offered oral B12 deficiency, offered the option of a six-month trial of oral replacement, and administered a follow-up questionnaire. Throughout the study, semi-structured interviews were conducted for the qualitative arm. We received ethics approval from the Sunnybrook and Women's College Health Sciences Centre Research Ethics Board. All patients provided informed consent.We administered a questionnaire to patients with B12 deficiency. We excluded patients if their last injection occurred more than three months before the start of patient recruitment, if they had left the practice, if their B12 therapy had been discontinued or if they had already switched to oral therapy, if they did not speak English and did not have access to a translator, or if they had been recently diagnosed with a serious illness. After applying these criteria, 133 patients were included in the study , regardless of age and etiology of B12 therapy to injections: "Studies have shown that vitamin B12 pills are just as effective and safe as B12 injections." Educational sessions on the effectiveness of oral therapy were held with the nursing staff (since they administer the injections) at all sites, and they were encouraged to pass this information on to their patients. As well, contact information for the study investigators was provided to the patients in case they had any questions.The recruitment cover letter included one sentence describing the equivalency of oral B12 injection. The initial questionnaire elicited demographic data, medication history, logistical aspects of B12 related visits, history of past B12 therapy, and attitudes about injection and oral therapy. Non-responders received up to two telephone reminders.We placed the initial questionnaire see   in pati12 tablets (one 1000 μg tablet daily), and were offered testing of serum B12 levels at baseline (one month after their last injection) and after the six-month trial. Patients had the option to withdraw from the trial and return to injections at any time for any reason. A follow-up questionnaire was placed in the charts of patients who tried the pills. This questionnaire re-assessed patients' attitudes about the different forms of B12 therapy and asked whether they would continue on oral supplementation or return to injection therapy  as the cut-off for inclusion of individual patient factors into multivariate logistic regression models. We then performed backwards stepwise regression to determine statistically significant relationships after adjustment. We generated both crude and adjusted odds ratios with 95% confidence intervals.Where variables were not already dichotomous , we dichotomized the variable of interest . To ascertain the relationship between questionnaire responses and preference for oral therapy (as "willingness to switch" and "permanently switching") we performed bivariate and multivariate analyses. We used a p-value of 0.20 . Amongst the 17 participants, four were selected from those not willing to switch, while of the 13 who were willing to try oral therapy, six were interviewed prior to, two during, and five after the trial of oral therapy.One investigator (D.C.) conducted 17 semi-structured interviews in a private meeting room at the family practice unit. A purposive sample was selected amongst those willing to be interviewed to reflect diversity in terms of sex, age, willingness to switch, and final choice of therapy after the trial of B12 therapy, his or her relationship with health care providers, perceived advantages and disadvantages of both pills and injections, and attitudes about past and current B12 therapy. Analyses of the transcripts were performed independently by two investigators (J.K. and D.T-K.), using a three-step content analysis approach to identify and collate relevant themes [The interviews were audiotaped and transcribed. They lasted 20–30 minutes, with questions about the patient's knowledge and history of their Bt themes . ThroughFor our initial questionnaire, we received responses from 86 out of the sample of 133 patients, for a response rate of 64.7%. Selected characteristics of the study population are presented in Table 12 therapy, number of non-B12 related visits per month, improvement in perceived well-being since initiating B12 injections, several perceived disadvantages of injections (risk of complications, travel/parking costs), and several perceived disadvantages of pills .Sixty-three of our 86 respondents reported a willingness to switch to oral therapy. A large number of patient factors had no clear statistical relationship to a "willingness to switch" to oral therapy (i.e., p > 0.20) and were excluded from the multivariate model: age, education, income, drug insurance coverage, study site, number of prescription medications, mode of travel to the clinic, years of past B12 injections, several perceived disadvantages of injections , and one perceived disadvantage of pills (won't see MD/nurse as often). Factors associated with willingness to switch after multivariate adjustment were: being able to get to the clinic more quickly (i.e., in less than 30 minutes) (OR 9.29, 95% CI 2.16–39.97), and believing that injection therapy is disadvantageous due to the need for frequent visits to health care provider (OR 5.41, 95% CI 1.10–26.56) and the increased costs to the health care system (OR 16.68, 95% CI 1.51–184.22)  chose pills.Fifty-two patients returned the follow-up questionnaire. Over three-quarters (39 of 51 respondents) reported being satisfied or very satisfied with oral therapy. Only 8% of patients (4/51) perceived that they felt worse with pills, while 23.5% (12/51) felt better and the rest felt the same. Self-reported compliance was good; 48 patients or 92% reported forgetting to take the pills two times or less per month. Thirty-nine patients  stated that they wished to permanently switch to B12. Patient factors that on bivariate analyses had a p-value of less than 0.20 and were consequently included in the multivariate model were: patient beliefs that frequent visits to see the doctor or nurse and the associated travel/parking costs are disadvantages of injections, and the belief oral B12 would add unnecessarily to an already large number of prescribed oral medications. Table Again, many patient factors had no association with a desire to permanently switch to oral B12 levels were obtained for 39 of the 55 patients who switched to pills . Only one patient's serum level decreased but even she remained within the normal range.Baseline and post-intervention serum BThe mean age of interview participants was 71 (range 54–92) years, with 7 men and 10 women.12 injections they had been receiving in the past were effective: "I find it gives me more energy." Patients who expressed this view were often not willing to switch to oral therapy, or if they did try the pills, they eventually went back to injections. Others felt that the injections hadn't helped much: "There is a little feeling that, 'Oh, I'm taking this. I should feel better,' but I'm not sure if I do." These patients tended to switch permanently to oral therapy.Some patients felt that the BWhen asked about the anticipated efficacy of oral therapy, some patients, particularly those who were not willing to switch, were sceptical: "Well, I don't think it will work. If the doctors suggested that I needed to go on the injections, I'm certainly pretty sure that the pills are not going to work. Otherwise he'd have put me on pills, would he not?" Many others were unsure about the efficacy of oral therapy.12 pill. Other advantages of oral therapy included savings to the health care system and ease of travel. Cited disadvantages of injections included decreased compliance due to the need for frequent visits and potential complications associated with injections. A number of patients decided to switch because they were interested in participating in a research study. Some patients acknowledged that oral therapy would benefit those who are averse to needles, however, needles did not bother most of the patients interviewed: "...needles don't bother me, because – well, I don't know. They just don't seem to bother me. I know some people are very concerned about it."Nearly every patient cited convenience as an advantage of switching to B12 and for three of the five patients interviewed afterward, all of whom permanently switched to oral therapy, there was a high level of satisfaction with oral B12. One patient even reported feeling better on oral B12: "Well, I'm more level. I don't feel at the end of the month that I'm running out of energy. I'm quite well aware of that."  and the other cited decreased energy . . see .12 is both feasible and acceptable to patients. Of those who responded to our initial questionnaire, nearly three-quarters were willing to try oral B12, and of those who did switch, most were satisfied and the majority wished to remain permanently on oral therapy. These patients believed that injections are disadvantageous because they are associated with too many visits to their health care provider and with higher patient costs (in terms of travel and parking expenses). In the qualitative arm of our study, most patients cited convenience as the reason for wanting to switch from injections to pills. We were also able to confirm findings from other studies indicating that oral B12 is biochemically equivalent to, if not better than, parenteral therapy.We found that switching patients from injection to oral BOne non-intuitive finding from the initial questionnaire assessing the factors associated with trying oral therapy Table  was that12 levels (i.e., 1 month after their last injection) and again 6 months after oral therapy, we observed that one patient was a non-responder (i.e., serum level decreased), and 4 patients had serum levels that increased but remained below 295 pmol/L (a level considered by some to be borderline B12 deficient)[12 levels and, if available, functional tests such as serum homocysteine and methlymalonic acid. The sub-optimal response in these patients may be due to patient non-compliance or sub-optimal dosing  [By monitoring trough serum Beficient). This re et al.) . FurtherThe primary strength of our study was that we achieved triangulation through the use of both quantitative and qualitative methods. By using questionnaires, we were able to provide estimates such as switch rates and levels of patient satisfaction; with the qualitative arm, we were able to more thoroughly explore patient perspectives.12. However, the sample was large enough to identify a number of patient factors associated with switching from injection to oral therapy and to determine that patient attitudes toward oral B12 therapy are greatly influenced by its convenience. A second limitation was that we did not assess the knowledge and attitudes of our patients' physicians and nurses, as they may have influenced patient's expectations and perceptions regarding the effective of oral B12 therapy. Finally, another limitation was the potential participant bias, evidenced by the differences between responders and non-responders in terms of age and sex. However, among the responders, neither age nor sex was significantly associated with switching to oral therapy, suggesting that any differences between participants and non-participants were likely irrelevant.The main limitation of our study was the relatively small sample size for the quantitative arm, which led to large confidence intervals for the estimates of the relationship between patient factors and switching to oral B12 deficiency and providing educational sessions to encourage discontinuing injections, 38% stated they would leave the practice if denied their injections [While there have been several studies examining physicians' perspectives on switching from intramuscular to oral therapy -21, we kjections . Explana12 therapy may be acceptable for most patients, it may not be appropriate for those who will not be compliant with oral medications, such as patients with significant memory impairment or cognitive dysfunction, unless they have caregivers who could ensure compliance. Oral therapy also may not be appropriate for those with swallowing difficulties; for such patients, sublingual B12 therapy, which has been shown to be equally effective, may more appropriate [While oral Bropriate .12 therapy to their patients who are currently receiving injections if they can tolerate and are compliant with oral medications. Most patients who switch from parenteral to oral therapy are satisfied and wish to stay permanently on oral therapy. They often cite the inconvenience and the travel-related expenses associated with frequent visits to the doctor as disadvantages of parenteral therapy. Therefore, oral B12 therapy is generally well-received by patients, and should be considered by clinicians as a superior alternative to the traditional injections for most patients.In summary, the results of our study suggest that clinicians should offer oral BThe author(s) declare that they have no competing interests.JK designed and coordinated the study, analyzed the qualitative and quantitative data, and drafted the manuscript. DC conducted the in-depth interviews and assisted in data collection. ID helped to develop the questionnaire and performed statistical analyses of the quantitative data. DTK analyzed the qualitative data. RU conceived the study and provided guidance on all aspects of the project. All authors participated in the development of the manuscript and gave approval to its final submission for publication.The pre-publication history for this paper can be accessed here:Initial questionnaireClick here for fileFollow-up questionnaireClick here for fileReasons for switching to oral therapyClick here for fileReasons for not switching to oral therapyClick here for file


Single nucleotide polymorphisms (SNPs) are extensively used in case-control studies of practically all cancer types. They are used for the identification of inherited cancer susceptibility genes and those that may interact with environmental factors. However, being genetic markers, they are applicable only on heritable conditions, which is often a neglected fact. Based on the data in the nationwide Swedish Family-Cancer Database, we review familial risks for all main cancers and discuss the evidence for a heritable component in cancer. The available evidence is not conclusive but it is consistent in pointing to a minor heritable etiology in cancer, which will hamper the success of SNP-based association studies. Empirical familial risks should be used as guidance for the planning of SNP studies. We provide calculations for the assessment of familial risks for assumed allele frequencies and gene effects (odds ratios) for different modes of inheritance. Based on these data, we discuss the gene effects that could account for the unexplained proportion of familial breast and lung cancer. As a conclusion, we are concerned about the indiscriminate use of a genetic tool to cancers, which are mainly environmental in origin. We consider the likelihood of a successful application of SNPs in gene-environment studies small, unless established environmental risk factors are tested on proven candidate genes. Genetic association studies on complex diseases have become very popular and most of them are case-control studies using single nucleotide polymorphisms (SNPs) as markers. There has been concern about the poor reproducibility of the results and the reasons for such discrepancies have been discussed -5. HowevHow feasible is it then to carry out SNP studies on cancer, particularly when the subjects are overwhelmingly unselected cases, among whom familial cases are rare. It is worrisome that genotyping is now almost a required standard component in epidemiological studies, without consideration of the expected heritable influence. Formal sample size and power calculations are irrelevant if there are no data on the assumed heritable component in the causation of a particular cancer. It is also worrisome that the fundamental differences between purely genetic and gene-environment studies regarding control populations and multiple testing problems are not appreciated. In this contribution, we first review study designs and aims of SNP studies, then we give data on familial risks for the main types of cancer, to give an idea of the upper bounds for the risks that can be expected in genotyping studies. Finally, we will calculate familial risks resulting from variants of assumed genotype relative risk and allele frequency. Such data are useful in the assessment of study designs and in the evaluation of obtained results. We use the term 'familial' to denote cancers in two or more first-degree relatives and 'heritable' when an inherited gene defect is known or inferred due to a high risk . InsteadIn the simplest form, a SNP with a known or assumed function is selected, and the genotypes are determined in cases and controls to test for association. Most studies published on cancer are of this kind, purely "genetic studies" testing the effect of the genotypes on the risk of cancer, without considering any other variables ,18. In ain vivo systems [in vivo functional test cannot be easily devised. Drug metabolism genes are a fortunate exception in this regard, because aberrant responses in humans have lead to the characterization of the underlying gene variants. One common feature of almost all the published studies is that patients have been collected without regard of a family history, thus sacrificing statistical power but attempting to compensate with a large sample size [Most studies have selected SNPs with assumed functional effects or they test the effects of haplotypes. For some genes, null or truncating alleles exist and homozygotes would then lack a functional protein. However, for missense types of SNPs the functional effects may be small or nil when tested in  systems . Unfortuple size ,25. Usinple size .5 = 3125 unique cells), and, secondly, the genes that are selected for analysis, are taken from a pool of tens or hundreds of potential candidate genes. No solutions have been found for this "two-dimensional" multiple testing problem. However, important for the present discussion, gene-environment interactions only exist if there is a heritable component in the particular cancer, and the likelihood of observing an effect is larger if the heritable component is large.A variant of the candidate gene approach is the "gene-environment study", which is founded on the assumption that in complex diseases environmental factors interact with heritable factors, and a strong effect can be detected when both are present -29. In eIn populations of random mating, it may be plausible that gene-environment interactions of epidemiologically measurable magnitude exit in the absence of a measurable familial risk. In the case of many exposures, some with harmful and others with protective effects, interacting with many genes, it may be possible that the familial risks are missed in spite of true gene-environment effects. Similarly, non-conventional dose-effect relationships, such as those suggested for blood vitamin D levels and prostate cancer , would bAccording to the complex disease paradigm, many relatively common alleles, interacting with environmental factors, cause susceptibility to common diseases ,32. SuchFamilial risk of a disease is a measure of its clustering in family members. Commonly, familial risk is defined between those who have a relative (e.g., parent or sibling) with cancer compared to those whose relatives are free from cancer, given as a familial relative risk or familial standardized incidence ratio (SIR). The SIRs shown below have been obtained from the Swedish Family-Cancer Database, the largest dataset of the kind in the world . FamiliaFamilial cases constituted 15.4% of all prostate cancers, which was the largest proportion by far. The proportion was 10.1% for colorectal adenocarcinoma and 8.5% for breast cancer, but only 0.5% for connective tissue and testicular tumors. The proportions are generally higher for common cancers. Overall familial cancers (same cancer in offspring and parents) constituted 5.5% of all cancers.We have tried to estimate the degree of environmental contribution to the familial risk by comparing cancer risks betweens spouses. Spouse concordance, which does not generally exceed an SIR of 1.3, can be noted only for cancers with known strong environmental risk factors: lung and genital cancers and early onset gastric and pancreatic cancers and melanoma ,39. SpouIf environmental causes of familial clustering have been quantified or excluded, familial SIRs and proportions give estimates on the heritable effects for cancer at the level of nuclear families (here between parents and offspring). Because of low penetrance, familial proportions underestimate true heritable effects. On the other hand, the twin model assumes that the shared environmental effects of monozygotic and dizygotic twins are identical, which may not be true. If monozygotic twins share more than dizygotic twins, the estimated heritability is exaggerated. Thus, the heritability estimates for cancer are still unreliable, and, due to possible interactions, a dichotomous classification into heritable and environmental components is conceptually inaccurate . MoreoveResults from a successful SNP study can imply that the particular variant contributes to a familial risk of the particular cancer. The resulting familial risk depends on the allele frequency of the SNP, observed OR and the mode of inheritance, i.e., on the relative risks of heterozygotes compared to homozygotes. In the dominant model, the risk of heterozygotes equals that of the variant homozygotes; in the recessive model, the risk of heterozygotes equals that of the wild type homozygotes. In the additive model, the risk of heterozygotes is the mean of the two homozygotes; in the multiplicative model, the risks between the genotypes differ by a constant multiplier.BRCA1/2, ATM, p53 and CHEK2, explain about 25% of the risk [The methods for the calculation of familial risks to offspring of affected parents . According to Table BRCA1 and BRCA2 combined. The effects of metabolic polymorphisms on various cancers have been reviewed in an IARC publication [It is of interest to examine the magnitude of familial risks which would be predicted from the published ORs for candidate genes. In a review of 34 polymorphisms in 18 different genes tested for breast cancer, a large proportion of the associations were not significant and the ORs were below 2.0 . Even malication . Among mlication ,50. SomeThe poor reproducibility of candidate gene studies has most commonly been associated to small sample size, population stratification and low prior probability, i.e., poor selection of genes or SNPs; a SNP with small functional effect would also imply a low prior probability for an effect ,32. We aIt is commendable that all available molecular and environmental data are being used in attempts to understand the mechanisms of human cancer -53. With


There is considerable interest in identifying target genes and chromatin binding sites for transcription factors in a genome-wide manner. Such information may become useful in diagnosis and treatment of disease, drug target identification, and for prognostication. In cancer diagnosis, patterns of transcription factor binding to specific regulatory chromatin elements are expected to complement and enhance current diagnostic predictions of tumor behavior based on protein and mRNA analyses. Signal transducer and activator of transcription-5 (Stat5) is a cytokine-activated transcription factor implicated in growth and progression of many malignancies, including hematopoietic, prostate, and breast cancer. We have explored immunoaffinity purification of Stat5-bound chromatin from breast cancer cells to identify Stat5 target sites in an unbiased, genome-wide manner.in vitro and in vivo as tested by electrophoretic mobility shift assay and chromatin immunoprecipitation, respectively. While there was no binding to fragments lacking a Stat5 consensus binding sequence, presence of a Stat5 binding sequence did not assure binding.In this report, we evaluate the efficacy of a Stat5-bound chromatin library to identify valid Stat5 chromatin binding sites within the oncogenome of T-47D human breast cancer cells. A general problem with cloning of immunocaptured, transcription factor-bound chromatin fragments is contamination with non-specific chromatin. However, using an optimized strategy, five out of ten randomly selected clones could be experimentally verified to bind Stat5 both in vivo Stat5 chromatin binding sites in cancer, including abnormal regulatory sites in tumor-specific neochromatin.A chromatin library coupled with experimental validation may productively identify novel  Transcription factors function uniquely at the interface of the genome and the proteome. A significant portion of transcription factors serve not only as executors of gene transcription programs, but also as biochemical sensors of extracellular stimuli. For instance, members of the nuclear receptor family are directly activated by lipophilic extracellular ligands, and transcription factors of the Smad and Stat families are activated by phosphorylation in response to cytokine stimulation of cell surface receptors. Chromatin-bound transcription factors that act both as sensors of extracellular cues and as transcriptional effectors carry exceptional instructive value about the biological state of individual cells. Their high biological information value makes such factors particularly attractive for use as markers to predict disease activity and outcome, as well as predictive markers of disease-responsiveness to drugs.ENCyclopedia Of DNA Elements), has been initiated with the ambitious goal of identifying all regulatory elements of the human genome, including chromatin binding sites for individual transcription factors [Based on related and broader rationale, the second phase of the human genome project, ENCODE  and chromatin immunoprecipitation (ChIP).In contrast to transcription factors that bind to chromatin in a constitutive manner, Stat5 is a latent cytoplasmic transcription factor that is activated by tyrosine phosphorylation and binds tightly to DNA in response to extracellular cytokines, such as prolactin . We usedMethods. Before subcloning of the captured chromatin fragments, the specificity of immunocapture was verified by analysis of binding to known human Stat5 target chromatin. In particular, we took advantage of earlier work that has identified a group of Stat5 regulated genes that have been shown to contain the Stat5 consensus sequence, TTCNNNGAA, in the traditional promoter element [Cytokine-Inducible SH2 Protein (CISH), β-Casein, and α2-Macroglobulin. Due to the average chromatin fragment size of 400 bp, primers were designed to yield shorter PCR products of 200 – 300 bp.Sonication is necessary to shear genomic DNA into fragments that can be easily manipulated for PCR amplification, cloning, and sequencing. Fragments of approximately 400 base pairs (bp) allow for a complete sequencing read-through and are of sufficient size to localize the fragment within the human genome with a high degree of statistical certainty. Optimal shearing of chromatin from formaldehyde-fixed T-47D cells into approximately 400 bp fragments was established empirically Figure , lane 5. element ,15. AliqCISH gene response element in T-47D cells  insert-containing PCR products. Figure The enriched, Stat5-bound chromatin pool was then cloned into a bacterial vector to generate a chromatin library. Because sonication generates random overhangs in double stranded DNA , T4 DNA in vitro by EMSA. Stat5 binds to the consensus sequence TT(N5)AA and to relatively conservative variations, with TTC(N3)GAA considered to be optimal [in vitro was detected in seven of the ten chromatin fragments, as evidenced by prolactin-inducible DNA-binding complexes that could be supershifted by a specific anti-Stat5 antibody, but not by non-specific IgG  prolactin and (+) prolactin samples. In all cases there was no detectable product from the replicate samples that had been immunoprecipitated with a non-specific IgG antibody (data not shown), indicating a specific Stat5-mediated capture of genomic elements. All PCR amplifications were performed at least twice on at least two separate pools of immunoprecipitated genomic elements.As a second and independent means to validate the quality of the Stat5 chromatin library, the same randomly selected fragments were analyzed for inducible Stat5 binding in vitro Stat5 binding by EMSA, five (CCFs #5, #18, #23, #28, and #29) were also consistently positive for in vivo Stat5 binding by ChIP assay  was purchased from Sigma Chemical Co. (St. Louis, MO).The human breast cancer cell line T-47D was grown to confluence in RPMI-1640 medium (Biofluids, Rockville, MD) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin-streptomycin (50 IU/ml and 50 μg/ml, respectively) at 37°C with 5% CO7 cells/T175 cm2 flask), T-47D cells were serum starved for 24 h and then treated with or without 10 nM hPRL for 30 min. For prodifferentiation experiments with glucocorticoid pretreatment, confluent cultures were maintained in serum-free RPMI-1640 with 1 μM Dex dissolved in DMSO or in DMSO alone for 96 hours, then stimulated with or without PRL for 30 min. Proteins were then crosslinked to the chromatin by the addition of formaldehyde (Fisher Scientific, Fairlawn, NJ) to a final concentration of 1% and incubated for 30 min at 37°C. The cells were rinsed, scraped, and pelleted in ice-cold PBS with 1 mM PMSF, 2 μg/ml aprotinin, and 2 μg/ml pepstatin A. Cell pellets were resuspended in 400 μl lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0, 1 mM PMSF, 2 μg/ml aprotinin, and 2 μg/ml pepstatin A) and the lysates were sonicated using a Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA), fitted with a tapered microtip set to an amplitude of 50% and were pulsed twice for 30 s. Debris was pelleted by centrifugation for 10 min at 13,200 RPM at 4°C. The supernatants were then diluted 10-fold in IP buffer (0.1% SDS, 1.1% Triton-X, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 16.7 mM NaCl, 1 mM PMSF, 2 μg/ml aprotinin, and 2 μg/ml pepstatin A) and 1% was set aside for Pre-IP or input sample. The solution was then pre-cleared with beads (50% protein A-sepharose, 1 mg/ml poly dI-dC, 0.1% BSA, in TE, pH 7.4) for 30 min at 4°C. Next, the samples were incubated overnight with Stat5 antibody (N-20, Santa Cruz Biotechnology) or IgG IP-control followed by immunoprecipitation with pre-incubated beads. The samples (beads) were then washed once with each of these buffers in the following order: Wash Buffer 1 (0.1% SDS, 1% Triton-X, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl), Wash Buffer 2 (0.1% SDS, 1% Triton-X, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 500 mM NaCl), Wash Buffer 3 (0.25 M LiCl, 1% NP-40, 1% Na-deoxycholate, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA), then twice with Wash Buffer 4 (TE, pH 8.0). The samples were eluted from the beads in 1% SDS and 0.1 M NaHCO3. Next, the cross-links were reversed with 0.2 M NaCl overnight at 65°C followed by protein digestion with proteinase K for 1 hour at 45°C. The DNA was recovered by phenol:chloroform extraction and ethanol precipitation. This pool was then used as PCR template or could be blunted with T4 DNA polymerase (New England Biolabs, Beverly, MA) at 37°C for 5 minutes under standard conditions for uniform cloning. Taq DNA polymerase (New England Biolabs) was then used to add a 3' A overhang for cloning into a TOPO TA cloning kit (Invitrogen, Carlsbad, CA). The ligated vectors were transformed into electrocompetent bacteria, DH-5αE (Invitrogen), using the manufacturer's recommendations for transformation in a Bio-Rad Gene-Pulser (Hercules, CA), and plated on S-Gal/Ampicillin plates . Positive clones by blue-white screening were then analyzed directly by colony PCR in a standard reaction with vector-specific M13 and T7 primers that flank the cloning site and were incubated at 94°C for 4 min and cycled at 94°C for 30 s, 55°C for 45 s, and 72°C for 30 s. Colonies with quantifiable inserts were then purified , sequenced, and localized using BLAST (NCBI, NIH, Bethesda, MD).After grown to confluence  = 6.67, as described previously . Paralleα32P dATP . Initially the samples were incubated at 94°C for 1 min, then cycled 36 times at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The reaction was then held at 72°C for 5 min following the cycling to allow for product fill-in and addition of a 3' terminal "A". After completion of the cycling, the PCR products were purified using the Qiagen PCR purification kit, according to manufacturer's instructions. The final products were eluted in 50 μl and stored at -20°C until use.Radiolabeled products were generated by PCR in a 10 μl reaction under standard conditions with the addition of 0.25 μl 32P-labeled PCR probe and incubated for 20 min at room temperature. The samples were then resolved in a 3% non-denaturing polyacrylamide gel, as previously described [The DNA-protein binding reactions were performed in a 10 μl mixture containing 3 μl of nuclear extract from the respective sample, and 1 μg of double-stranded poly dI:dC (Boehringer Mannheim, Indianapolis, IN), as previously described . After 1escribed .CCF #5 forward 5' TGA CAT CAG TGA GAG TGG AGG 3', reverse 5' TGA GGC TGT AAT GTC ACT CAG AA 3'; CCF #11 forward 5' GGA CAC ATC CAC TAC TGC CA, reverse 5' AAA AGA AGT GCA GTT CAG GAT AA 3'; CCF #14 forward 5' GCC TGT GTG ATA TCA TAT GGA AAG 3', reverse 5' TTT CCA AGA ACT TAT CAG AAT GAC TT 3'; CCF #18 forward 5' TCA GTC TTC CTC TTT CCC CA 3', reverse 5' CGG CTT ACA TTC TGT GTC GC 3'; CCF #23 forward 5' CAT ATC TGT CTA CAA ATA ACA GTT CCC 3', reverse 5' AAT AGA GGG CAG AGT TAA GAA TCA AA 3'; CCF #28 forward 5' AAG CCT GAA AAG AAA AAT CTC A 3', reverse 5' CCT GAC ACA TCC AGA CTT GG 3'; CCF #29 forward 5' CAG ACG TTT GCT GGA AGA TAT G 3', reverse 5' TTG TTC CAT GAA ACC AGG G 3'; CCF #30 forward 5' AAG GCC ATT GAA GTG AGG TG 3', reverse 5' CCA TCA GCC AGT CAT TGA AG 3'. The PCR was performed with standard conditions using template immunoprecipitated from cells treated with or without prolactin to indicate Stat5 inducibility and was executed at least twice on each of 2 separate pools.As an independent validation technique, we designed primers specific to each cloned chromatin fragment (CCF) then performed PCR on a separately-generated enriched pool of immunoprecipitated Stat5-chromatin interaction sites. The following primers were used for PCR: MJL carried out the experimental studies, participated in the design of the study, and drafted the manuscript. JX participated in the design of the study and scientific discussion of the results. HR conceived and participated in the design of the study, contributed in the evaluation of the results, and in the preparation of the manuscript. All authors read and approved the final manuscript.


Functional magnetic resonance imaging offers the promise of peeking into the human mind. What signals in the human brain can we really detect and how should the technology be used? Functional magnetic resonance imaging—fMRI—opens a window onto the brain at work. By tracking changes in cerebral blood flow as a subject performs a mental task, fMRI shows which brain regions “light up” when making a movement, thinking of a loved one, or telling a lie. Its ability to reveal function, not merely structure, distinguishes fMRI from static neuroimaging techniques such as CT scanning, and its capacity to highlight the neural substrates of decisions, emotions, and deceptions has propelled fMRI into the popular consciousness. Discussions of the future of fMRI have conjured visions of mind-reading devices used everywhere from the front door at the airport terminal to the back room of the corporate personnel office. At least one “neuromarketing” research firm is already trying to use fMRI to probe what consumers “really” think about their clients' products.But will fMRI's utility in the real world ever match the power we currently imagine for it? Is fMRI likely to leave the clinic for widespread use in the courtroom or the boardroom? Are there neuroethical nightmares just around the corner? Or are all these vivid specters really just idle speculations that will never come to pass?To understand the potential, and the limitations, of fMRI, it's helpful to know how the technique works. The heart of the apparatus is a large donut-shaped magnet that senses changes in the electromagnetic field of any material placed in its center, in particular—when a head is scanned—the blood as it flows through the brain. When a region of the brain is activated, it receives an increased flow of oxygenated blood . This influx of oxygenated blood alters the strength of the local magnetic field in proportion to the increase in flow, which is detected and recorded by the imaging machinery.The resolution of the best fMRI machines—the smallest “volume picture element,” or voxel, they can distinguish and make an image of—is currently about 1.5 mm ×1.5 mm × 4 mm, the size of a grain of rice. There are approximately 150,000 of these little volumes in the typical brain, and the immense computers hooked up to the scanners record and integrate signals from all of them. In a typical experiment, a subject, lying still with his head surrounded by the magnet, does nothing for thirty seconds, then performs some task for thirty seconds, then lies still for thirty seconds. For each voxel, the signal during the task is compared to the signal at rest; those areas of the brain with stronger signals during the task are presumed to be processing the information that underlies the performance of the task . AccordiThe first caveat about fMRI's imaging power, though, and one that every neuroimager stresses, is that a voxel is a long way from a neuron. There are an estimated 100 billion neurons, so at best, an fMRI is signaling blood flow changes associated with the increased activity of tens of thousands of neurons. As a result, says Hirsch, fMRI “falls short when we want to ask about more detailed brain processes. We're not learning that much about how neurons are doing local computing.” While resolution will improve over time, it seems unlikely that fMRI will ever detect the activity of individual neurons, and so its ability to dissect the “fine structure” of thought is inherently limited. These limitations have not prevented fMRI researchers from making some major discoveries about brain function, however. Hirsch, for instance, showed in one study that minimally conscious individuals still process human speech, and in another, that those who become bilingual as young children employ overlapping language areas in the cerebral cortex, while those who learn a second language later in life use a different area for the second language. The key strength of fMRI, she says, is that it provides the ability to test these kinds of hypotheses about structure–function relationships in the normal brain.But the hypotheses that can be tested and the conclusions that can be drawn are still largely about group averages, not about the functionings of individual brains, and therein lies a second major caveat about the use of fMRI beyond the clinic. John Gabrieli, Associate Professor of Psychology at Stanford University, has shown that distinct activation patterns in the brains of dyslexic children normalize as they improve their reading skills . It seemthis scan in this individual on this day does or doesn't reflect his underlying racial attitudes.There are similar limitations to most other applications of fMRI—while conclusions can be made about aggregated data, individual scans are for the most part too hard to interpret. There is not yet any real understanding of how brain patterns change over time in an individual, or how interindividual differences should be interpreted in relation to the conclusions that are valid for groups. This makes fMRI an unlikely tool for job screening, for instance. While one study has shown a brain signature in a group of white people that is associated with racial bias, denying a particular individual a job on the basis of such a scan would likely lead straight to a lawsuit, with experts debating whether On the other hand, Hirsch has used individual scans to help locate a patient's language structures that must be spared during neurosurgery. “If you are a neurosurgeon planning a resection, you don't want an average brain at all. Millimeters matter.” But her success is precisely because she is not using group data to make inferences about the individual—she is not leaping over the chasm, but instead is toiling entirely on the other side of it. “The goal is personalized medicine,” she says.Even this kind of personalized approach with fMRI is fraught with problems when researchers attempt to apply it outside the clinic, because of limitations in the technology itself. One researcher with firsthand knowledge of these problems is Daniel Langleben, Assistant Professor of Psychiatry at the University of Pennsylvania School of Medicine. In 2002, Langleben showed that when subjects were hiding information in an attempt to deceive , they had intense activity in five distinct brain areas not seen when they were telling the truth. In effect, Langleben used the fMRI as a lie detector. It is potentially even more powerful than a standard polygraph test, he says, because there are thousands of brain regions which can be scanned for deception-triggered variation, versus only three variables—skin conductance, respiration, and blood pressure—used in the standard polygraph. Not surprisingly, Langleben got a lot of press after he announced his results, and his experiment led directly to speculation that we might eventually see fMRIs installed at airports, scanning the brains of would-be terrorists trying to deceive security screeners, or in courtrooms, catching perjurers red-handed (or perhaps red–anterior-cingulate-gyrused?).Langleben is enthusiastic about the potential for an fMRI-based lie detector, and has even applied to the Department of Justice for a grant to develop the technology (they turned him down, saying it was too expensive). But he is also clear about how difficult it will be to get one that really works outside the highly structured confines of the research lab. “We are a long way from making a working polygraph,” he says. Even with a “Manhattan Project” type effort, he speculates it would take at least ten years. “There are still essential discoveries to make along the way,” he says, “and there's a good chance it would end in total failure.” It's not just a matter of developing the imaging technology, he stresses—“we'll need fundamental developments in semantics, too.” This is because “a lot still depends on how you ask the question”—the subtlest of differences can dramatically shift which areas of the brain respond. Given the sensitivity of the fMRI result to such seemingly minor perturbations, it's hard to imagine it could be reliably adapted to the hurly-burly of an airport security checkpoint.Even well-performed scans done in topnotch clinics may not easily find their way into the courtroom. Perhaps the least likely use of fMRI is in determining if a defendant is telling the truth, according to Hank Greely, Professor of Law at Stanford Law School, since compelling someone on trial to submit to an fMRI could be seen as a violation of the Fifth Amendment right against self incrimination, just as giving spoken testimony against oneself is. On the other hand, says Greely, DNA samples and fingerprints can be compelled—whether a brain scan is more like testifying or more like submitting to a blood test is an open question. Still, for the moment, scanning under duress simply isn't feasible, since all you have to do to ruin a good scan is move your head. Motion-correcting algorithms can be used, but they are nowhere near advanced enough to correct for large-scale movements by an unwilling subject. It's much more likely that an fMRI of a willing defendant would be introduced to convince the jury he is telling the truth, or performed before trial to rule out an innocent suspect. While to Greely's knowledge fMRI evidence hasn't yet been used in court, “it's certain to be tried,” and the barrier to its admission will fall as both the reliability and the ease of administration increase. “The easier, the cheaper, the more pleasant a technique is, the more likely it is to be used in the legal system.”Other forensic uses of fMRI are likely to arrive sooner rather than later. Could scans showing diminished impulse control—a function controlled by several regions of the brain, including the striatum and the ventromedial prefrontal cortex—be used to support more lenient sentencing, or even acquit a defendant, because he couldn't control his violent impulses? Or alternatively, will those same scans be used to argue for harsher sentences, since the defendant is clearly “hardwired” to commit similar crimes again? Courts already consider other factors, such as a history of child abuse, in an attempt to more fully understand the psychological state of the defendant. Will brain scans be seen as the ultimate “objective” look into the mind of the person on trial?Deciding all these issues of admissibility will be judges who will need to weigh competing claims from lawyers with competing interests, backed up by expert witnesses with competing theories. Here, the desire to apply the science may rush ahead of its demonstrated validity.Langleben, for one, doesn't think fMRI will be legitimately ready for the courtroom for a long time. On the other hand, he says, “if you want to abuse this technique and claim that it works, you can create tests that will produce results—I can see how it could be done. We know enough to rig it.” But still, he says, “we have all the tools we need to prevent this—there are enough people who are sufficiently honest [who would counter the premature use of fMRI in these contexts].”For now, at least, given the problems inherent in current fMRI technology, the neuroethical nightmare scenarios of widespread brain scanning seem unlikely to come to pass, at least until radical advances make it far cheaper, much less invasive, far less sensitive to subtle perturbations, and with a much more robust ability to legitimately extrapolate from a finding about a group to a prediction about an individual. Where fMRI is concerned, “a penny for your thoughts” is currently more like “a million pennies for a group-averaged hemodynamic response to highly constrained stimuli under entirely artificial conditions.”In light of this, bioethical concerns about fMRI applications should perhaps be viewed not as predictions of a certain future but rather as worstcase scenarios, a reminder of what we want to avoid. “It's a funny thing about the bioethics field,” says Greely. “The general approach is to look for bad news.”While many of these “worst cases” seem highly unlikely to come to pass, Judy Illes, of the Stanford Center for Biomedical Ethics, thinks some action is warranted now, if only to generate a better understanding of the ethical dimensions of fMRI research. She notes that “bioethicists are often viewed as the ethics police,” but she doesn't see regulations as the right path to shape the future uses of fMRI. Instead, she thinks a coalition of involved parties—scientists, lawyers, ethicists, politicians—should work together to develop guidelines that all will find acceptable. “I'm not in the business of stopping anything.”What everyone apparently already agrees on is the need for carefully designed experiments and cautious interpretation of the data. “A huge message in imaging is that you really have to look at the experimental setup at the common-sense level,” says Gabrieli, and avoid the tendency to “pick the most dramatic interpretation.” “The public needs to be reminded of the limitations of these findings,” agrees Hirsch. And as Langleben puts it, expressing his skepticism that there will ever be a one-size-fits-all, foolproof fMRI mind reader: “I don't think we'll ever be able to be stupid about it.”


Plasmodium falciparum multiple epitope (ME)– thrombospondin-related adhesion protein (TRAP) candidate vaccines are designed to potently induce effector T cells and so are a departure from earlier malaria vaccines evaluated in the field in terms of their mechanism of action. ME-TRAP vaccines encode a polyepitope string and the TRAP sporozoite antigen. Two vaccine vectors encoding ME-TRAP, plasmid DNA and modified vaccinia virus Ankara (MVA), when used sequentially in a prime-boost immunisation regime, induce high frequencies of effector T cells and partial protection, manifest as delay in time to parasitaemia, in a clinical challenge model.Many malaria vaccines are currently in development, although very few have been evaluated for efficacy in the field. P. falciparum. Analysis was per protocol.A total of 372 Gambian men aged 15–45 y were randomised to receive either DNA ME-TRAP followed by MVA ME-TRAP or rabies vaccine (control). Of these men, 296 received three doses of vaccine timed to coincide with the beginning of the transmission season (141 in the DNA/MVA group and 155 in the rabies group) and were followed up. Volunteers were given sulphadoxine/pyrimethamine 2 wk before the final vaccination. Blood smears were collected weekly for 11 wk and whenever a volunteer developed symptoms compatible with malaria during the transmission season. The primary endpoint was time to first infection with asexual p = 0.49). Incidence of malaria infection decreased with increasing age and was associated with ethnicity.DNA ME-TRAP and MVA ME-TRAP were safe and well-tolerated. Effector T cell responses to a non-vaccine strain of TRAP were 50-fold higher postvaccination in the malaria vaccine group than in the rabies vaccine group. Vaccine efficacy, adjusted for confounding factors, was 10.3% (95% confidence interval, −22% to +34%; DNA/MVA heterologous prime-boost vaccination is safe and highly immunogenic for effector T cell induction in a malaria-endemic area. But despite having produced a substantial reduction in liver-stage parasites in challenge studies of non-immune volunteers, this first generation T cell–inducing vaccine was ineffective at reducing the natural infection rate in semi-immune African adults. Inducing an immune response is just one part of what a vaccine needs to do before it will protect against episodes of malaria. Plasmodium falciparum parasites [Anopheline spp. vectors [P. falciparum [P. falciparum malaria, were manufactured to explore this approach [The disease burden of malaria has increased in recent years partly because of the rise of drug-resistant arasites  and inse vectors . There ilciparum ,6,7,8,9.lciparum ,11. DNA approach . γ-interapproach .DNA and modified vaccinia virus Ankara (MVA)'s excellent safety profiles in malaria-naïve and semi-immune volunteers have been discussed previously . In seve8 plaque-forming units, produced very strong vaccine-induced CD4+ and CD8+ T cell responses in previous phase I studies in the United Kingdom [Priming with three 2-mg intramuscular DNA ME-TRAP vaccinations at 3-wk intervals, followed by boosting with one intradermal MVA ME-TRAP vaccination of 1.5 × 10 Kingdom . The imm Kingdom . DNA ME- Kingdom .P. falciparum infection. We chose a two-DNA prime, one-MVA boost regimen with 3-wk between doses because this is a three-dose regimen that would be amenable to integration with the World Health Organization/United Nations Children's Fund Expanded Program on Immunization, with the necessary supporting safety and immunogenicity data both from adults in the United Kingdom and Gambia. We used 3-wk intervals because 4-wk intervals had not been evaluated in phase I trials previously, hence bridging studies would be necessary to bridge to the three-dose, 4-wk interval Expanded Program on Immunization schedule.To follow up these encouraging findings in volunteers, we have conducted a randomised, controlled trial of DNA ME-TRAP/MVA ME-TRAP in a rural part of Gambia to explore whether this vaccine combination could provide protection against natural P. falciparum antigens. It also contains two non-malarial CD4+ T cell epitopes [P. falciparum TRAP [P. falciparum used to produce the vaccine construct is T9/96. The candidate malaria vaccines were manufactured to Good Manufacturing Practice by contract manufacturers . DNA ME-TRAP was supplied as a single dose of2 mg in 2-ml vials. MVA ME-TRAP was supplied as two-dose vials, each containing 3 × 108 plaque-forming units in 0.8 ml. The rabies vaccine (Chiron Behring, Marburg, Germany) was supplied as a lyophilised single-dose vial with accompanying diluent and syringe. This vaccine was chosen because of its public health benefit in Gambia.The malarial DNA sequence is known as ME-TRAP. The ME string contains 14 CD8+ T cell epitopes, one CD4+ T cell epitope, and two B cell epitopes from six pre-erythrocytic epitopes . The ME rum TRAP ,24,25. Trum TRAP . The strApproval was obtained from the Joint Gambian Government/Medical Research Council Ethics Committee, the Oxford Tropical Research Ethics Committee, and the London School of Hygiene and Tropical Medicine Ethics Committee. An independent Data and Safety Monitoring Board provided oversight for the trial. In addition, independent clinical trial monitors monitored the trial for adherence to International Committee on Harmonisation Good Clinical Practice guidelines.Malaria incidence is highly seasonal in Gambia. The climate is typical of sub-Sahelian Africa, with a long dry season followed by a relatively short rainy season from July to October. Rainfall averages about 600 mm per year. Morbidity and mortality from malaria both occur more frequently during the rainy season. However, in 2002, Gambia experienced a drought, and the malaria season was delayed, with few disease episodes before October. The entomological inoculation rate varies between less than 1 and greater than 100 in Gambia . Based oBefore recruitment, meetings were held with village heads and elders, followed by general village meetings at which the study was explained. Volunteers received information sheets and consent forms translated into the three local languages in Arabic script, as well as in English. After written informed consent was obtained by a study physician, age and identity were checked, pre-test HIV counselling occurred, and potential volunteers underwent clinical evaluation, including a full medical history and clinical examination. They were screened for haematological (full blood count), renal (plasma creatinine), and hepatic  dysfunction, and duplicate malaria smears were made. Exclusion criteria included any chronic illness detected by clinical evaluation, ALT greater than 42 , creatinine greater than 130 (micromoles/litre), packed cell volume less than 30%, positive antibody ELISA to HIV-1 or HIV-2, simultaneous participation in another clinical trial, blood transfusion in the month prior to vaccination, previous experimental malaria vaccination, administration of another vaccine within 2 wk of vaccination, previous rabies vaccination, allergy to any previous vaccine or to sulphadoxine/pyrimethamine, history of splenectomy, and any treatment with immunosuppressive drugs. Eligible volunteers were given a unique study number and a photographic identity card. Parental written informed consent was obtained for volunteers aged 15–17 y.A member of the Data and Safety Monitoring Board generated and held the randomisation code that associated each study number with a specific vaccine. A blocking procedure was used, and whole villages were enrolled with sequential study numbers to ensure balanced numbers in each group. During the course of the study investigators and volunteers did not know the size of the blocks, nor were they aware of which vaccine preparation was administered to a particular volunteer. Opaque sealed envelopes were used for vaccine allocation. Study numbers were not preprinted on vials but were written on vials at vaccination. Used vials were checked for correct allocation off-site. Vaccination was performed by nurses who played no other part in the trial.8-plaque-forming-units dose of MVA ME-TRAP or (2) three doses of rabies vaccine; injections were given on days 0, 21, and 42, timed to coincide with the start of the rainy season. The first two doses of vaccine consisted of two intramuscular injections, one into each deltoid muscle. DNA ME-TRAP was given as 1 ml and rabies vaccine as 0.5 ml into each arm. The third dose of vaccine was given as four intradermal injections into the skin overlying the deltoid muscle, with two injections into each arm. The malaria vaccine group received MVA ME-TRAP as four 0.1-ml injections, whereas the control group received four 0.05-ml injections of rabies vaccine. Two weeks before administration of the third dose, all volunteers received three tablets of sulphadoxine/pyrimethamine to clear blood-stage P. falciparum infections [Volunteers were randomly assigned to receive either (1) two 2-mg doses of DNA ME-TRAP followed by a single 1.5 × 10fections .After each vaccination volunteers were observed for 1 h and visited at home on the first, second, and seventh day postvaccination for assessment of local adverse events (discolouration, induration, blister formation, pain, or limitation of arm motion), systemic adverse events , and unsolicited adverse events. One week and 13 wk after the third vaccination, venous blood was collected for repeated measurement of full blood count, ALT, and creatinine.P. falciparum parasites at any parasitaemia with either an axillary temperature of 37.5 °C or more or one or more of the following symptoms: headache, myalgia, arthralgia, malaise, nausea, dizziness, or abdominal pain.Since vaccination with MVA causes a characteristic local reactogenicity in some subjects, specific steps were taken to ensure that the participants were evaluated in a double-blinded manner. Field workers who assessed reactogenicity after the third dose were different from those who undertook surveillance during the parasitological follow-up period. During the surveillance period, starting 2 wk after the third dose of vaccine, volunteers were visited twice weekly and asked whether they had attended a health centre. At weekly visits blood smears and axillary temperatures were taken. At midweek visits, blood smears and temperature were taken if symptoms compatible with malaria were present. Investigators and field supervisors made random visits to ensure accurate data collection. This active case detection was supplemented by passive case detection by study nurses to whom volunteers had 24-h access at three of the study villages and by a clinic at Medical Research Council Farafenni (20 km from the study villages). Symptomatic malaria was defined as the presence of asexual P. falciparum parasites was confirmed by a supervisor before a slide was declared positive. The arithmetic mean of the A and B slides was used to determine parasite density. If parasite densities for A and B slides were markedly discrepant, a third read was performed by the supervisor and this read was used for analysis. Parasite density was expressed per microlitre . Full blood counts were performed and packed cell volumes were measured in a CA620 cell analyser (Medonic, Stockholm, Sweden). ALT  and creatinine (micromoles/litre) were measured in a Visual analyser (bioMérieux, Craponne, France).When blood smears were obtained, two sets of duplicate blood smears  were made. A Field's stain was performed on films obtained from subjects with possible clinical malaria and the films read immediately. Two further smears (“A” and “B” slides) were stained with Giemsa after overnight drying; 100 high-power fields were read by two slide readers before a film was declared negative. The presence of 5 peripheral blood mononuclear cells (PBMCs) were assayed as described [P. falciparum.Effector T cell responses were assessed in ex vivo γ-interferon enzyme-linked immunospot (ELISPOT) assays for 98 volunteers randomly selected from a substudy list containing a 3:1 ratio of participants receiving malaria vaccines to participants receiving rabies (control) vaccines. For this assay, 4 × 10escribed , using MP. falciparum, defined as the number of days from the start of the surveillance period to the date of the first positive slide. Vaccine efficacy was calculated from the hazard ratio estimated by Cox's regression, adjusting for the effects of prognostic variables. Volunteers who received fewer than three doses or who were parasitaemic both prevaccination and at the beginning of surveillance without an intervening negative blood smear were excluded from the primary analysis but included in a secondary analysis. Observations on individuals who were lost to follow-up or were missing from trial data for 3 wk were censored. Analyses were done with Stata version 7 (Stata Corporation, College Station, Texas, United States). ELISPOT responses were analysed as follows. After subtraction of medium-alone values from each pooled peptide response, responses were summed across T9/96 and 3D7 pools. Geometric means were calculated for T9/96 TRAP, 3D7 TRAP, and ME string responses. Responses in the two groups were compared using the Mann-Whitney test.An analysis plan, written before unblinding, specified exclusion criteria, statistical methods, and important covariates (age, village of residence, and bednet use [defined as sleeping nightly under an intact or impregnated bednet]). Ethnic group, though not specified as a covariate in the analysis plan, was found on analysis to be associated with the risk of infection, and was included as a covariate. The primary endpoint was time to first infection with asexual In total, 489 volunteers were screened , of whomNo clinically significant differences in packed cell volume, ALT, or creatinine were seen in either vaccine group. One volunteer who received rabies vaccine had a history of breathlessness and chest pain several years prior to enrolment, experienced a relapse of symptoms, and deteriorated and died, probably from heart disease, 3 mo after he received the last dose of vaccine. This event was regarded as unrelated to vaccination. There were no other serious adverse events. Adverse events were rare after first and second doses and were not increased in the DNA ME-TRAP group compared to the rabies vaccine group (data not shown). Injection site pain, limited arm motion, headache, and malaise in the first 24 h after vaccination  were more common after MVA ME-TRAP vaccination than rabies vaccination . Some vop <0.001, range 6.25–2148.75). A large cross-reactive T cell response to 3D7 TRAP, a strain with 6% amino acid variance to T9/96, and a weaker response to the ME string were also seen at this timepoint  per million PBMCs for T9/96 and 3D7 strains of TRAP and the ME string, respectively. In the malaria vaccine group, the effector T cell frequency to the vaccine strain of the TRAP antigen, T9/96, was geometric mean 251.1 SFCs per million PBMCs (80-fold increase above control group, imepoint .p = 0.49). Similar results were obtained when all participants who received at least one dose of vaccine were included in the analysis .By the end of the study, 171 participants developed parasitaemia, 80/141 (57%) in the malaria vaccine group and 91/155 (59%) in the rabies vaccine group. The distribution of time to first infection was similar in the two groups . VaccineP. falciparum densities in first infections were similar in the two groups .Geometric mean During surveillance, there were ten episodes of symptomatic malaria in the malaria vaccine group and 13 in the rabies group. The risk of malaria-related symptoms during an episode of parasitaemia was similar in both vaccine groups. Percentage mean packed cell volume at the end of the trial was similar in both groups .Within the ELISPOT substudy group, the risk of developing parasitaemia was not associated with effector T cell response to the 3D7 strain of TRAP. The 80 men from the substudy group who received three doses of either malaria vaccine (55 men) or rabies vaccine (25 men), completed 11 wk of surveillance, and had complete ELISPOT data after dose three were divided into four quartiles. Men with the highest effector T cell responses had hazard ratios for infection similar to those with the lowest after adjustment for age, bednet use, and village of residence.The incidence of parasitaemia decreased with increasing age, and was decreased in those of Fula ethnicity compared to those in the Mandinka and Wollof ethnic groups . The useP. falciparum infection rate in a semi-immune adult African population. This provides a second comparison between protection in malaria-naïve and malaria-experienced adults. RTS,S/AS02, a circumsporozoite protein malaria vaccine based on a hepatitis B surface antigen virus-like particle formulated in a proprietary adjuvant, provided about 40% sterile protection in the artificial challenge model [This trial demonstrated that vaccination with two doses of DNA ME-TRAP followed by a single dose of MVA ME-TRAP is safe and highly immunogenic for effector T cell induction but that it did not reduce the ge model  and 71% ge model . The lacge model , whereasge model . The preAnother possible reason for the observed low efficacy is that the frequency of the effector T cell response declines from 7 d after boost, and so efficacy would be prevented if very high frequencies of circulating effectors are needed for protective efficacy. Alternatively, a suboptimal regional memory T cell pool in the liver may be responsible , or, lesIn previous studies in East and West Africa, summed T cell responses to TRAP in unvaccinated semi-immune adults by ex vivo γ-interferon ELISPOT were geometric mean less than 20 SFCs per million PBMCs. The candidate regimen represents a new method for induction of unprecedented effector T cell frequencies, which are about 50-fold higher than those induced by lifelong natural exposure. Estimates of the reduction in liver-stage parasite burden induced by these vaccines in the human challenge model are of the order of 80%–90% of infected hepatocytes ,33. It iSecond-generation prime-boost vaccine strategies for malaria currently in or near to clinical evaluation include the following: use of a different viral vector as the priming agent that may lead to proportionately greater CD8+ rather than CD4+ T cell induction , as is the case with fowlpox-MVA immunisation; the use of a different antigen, the circumsporozoite protein, or polyprotein constructs  to addreWe were unable to obtain a useful estimate of the likely efficacy of the DNA ME-TRAP/MVA ME-TRAP vaccination regime against clinical disease. Even for an adult population, the incidence of clinical disease was lower than expected. Sulphadoxine/pyrimethamine was administered 4 wk before the start of surveillance in this study and in an RTS,S field efficacy study . There iThis study highlights North Bank Division in Gambia as an excellent malaria vaccine field trial site both for adults and, by extrapolation, for children. In a low-transmission year, cumulative incidence overall in men aged 15–45 y was 72% over 11 wk, which was higher than expected. Also, compliance was good despite a demanding study design, and migration from the study area was acceptably low.This paper adds to the body of data detailing the very gradual acquisition of anti-infection immunity in adults resident in sub-Saharan Africa . While sImmunological analysis of the high level of protection inducible by immunisation of humans and animals with irradiated sporozoites has encouraged attempts to generate protective immunity by subunit vaccines that induce strong cellular immune responses. To date the induction of high-level protective T cell responses against malaria and some other infectious pathogens has generally required two-component prime-boost vaccination approaches . We repoMalaria kills 1–2 million people a year, mostly children under the age of five who live in sub-Saharan Africa. Scientists are trying to develop cheap, safe, and effective vaccines that could be given to people living in regions where malaria is very common to prevent them from developing the disease.The researchers enrolled 372 Gambian men aged 15–45 years into the study. They injected half the men with two malaria vaccines, one after the other, and half the men with a rabies vaccine that does not protect against malaria  just before the rainy season, when malaria is especially prevalent. The scientists took blood smears from the men once a week and checked to see if they had been infected with the parasite that causes malaria. They found that the men who had been vaccinated became infected just as quickly as those who had not. Although the two malaria vaccines in concert did not work, neither did they cause any serious side effects. The men given the malaria vaccines did produce an immune response to the vaccines, though not one that was clinically useful.Plasmodium falciparum, the parasite that causes malaria. However, there are other vaccines in development that have not been tested yet.It looks as though the combination of these two vaccines is not effective at preventing infection with http://www.lshtm.ac.uk/gmp/Gates Malaria Partnership (which co-funded the study): http://www.malariavaccine.org/Malaria Vaccine Initiative: http://www.mmv.org/pages/page_main.htmMedicines for Malaria Venture: http://rbm.who.int/partnershipRoll Back Malaria Partnership: http://www.wellcome.ac.uk/en/malaria/The Wellcome Trust (which co-funded the study): http://www.controlled-trials.com/isrctn/trial/1/0/05221133.html.This trial has been submitted for registration in the International Standard Randomised Controlled Trial Number (ISRCTN) Register. The ISRCTN is ISRCTN05221133; Web site Table S1(55 KB DOC).Click here for additional data file.


Polyketides, natural products from microorganisms, have been a main source of antibiotics. Understanding the 'programming' of the enzymes that produce these complex molecules has opened a new field of drug discovery For half a century, natural products from microorganisms have been the main source of medicines for treating infectious disease. The most important chemical class of these antibiotics, apart from the penicillins, is the polyketides. They are made by the stepwise building of long carbon chains, two atoms at a time, by multifunctional enzymes that determine the chain length, oxidation state, and pattern of branching, cyclisation, and stereochemistry of the molecules in a combinatorial fashion to produce an enormous variety of structures. Recent elucidation of the genetic ‘programming’ of the enzymes has opened a new field of drug discovery based on rationally engineering the enzymes to produce ‘unnatural natural products’ with novel properties.Streptomyces is the best-known genus. These organisms make an amazing array of so-called secondary metabolites that have evolved to give their producers a competitive advantage in the complex soil environment, where they are exposed to stresses of all kinds  is simply the most discussed of a series of threats , which are related to the fatty acid synthases that make the lipids essential for the integrity of cell membranes, but they carry out much more complex biosynthetic routines. Repeated rounds of carbon chain building and modification use a series of independently variable reactions selected according to a ‘program’ characteristic of each PKS . Recent  threats . How do The heart of PKS function is the synthesis of long chains of carbon atoms by joining (condensing) together small organic acids, such as acetic and malonic acid, by a so-called ketosynthase function. This uses the building units in the form of activated derivatives, called coenzyme A (CoA) esters, so we speak of acetyl-CoA and malonyl-CoA, for example. The special form of condensation that joins them is driven by loss of carbon dioxide. Thus, when acetyl-CoA, with two carbon atoms, joins with malonyl-CoA, with three carbons, one of the latter is lost and a chain of four carbon atoms results . FurtherChoices of the number and type of the building units are variables in determining polyketide structure. Another concerns the keto groups (C=O) that appear at every alternate carbon atom in the growing chain as a result of the condensation process (accounting for the name polyketide). They may remain intact. Alternatively, some may be modified or removed by a series of three steps , any of Streptomyces coelicolor (S. coelicolor itself) and tetracenomycin. The main variable in their structure is carbon chain length, with few choices of different building units or keto group modification, so the programming would (in principle) be simple. The DNA sequences responsible for such PKSs revealed sets of genes encoding proteins, including ketosynthases, ketoreductases, and acyl carrier proteins (ACPs)  and filled the need for spare parts for the engineering of countless new polyketides . SeveralMeanwhile, the programming of the aromatic PKSs was harder to understand. They had been found to contain only a single ketosynthase, which has to operate a specific number of times to build a carbon chain of the correct length, so how is this determined? How does a single reductive enzyme know which keto groups to modify? And how is the starter unit for building the carbon chain selected ? Considerable progress had been made in constructing novel compounds by mixing and matching PKS subunits, but this was largely based on empirical knowledge about which components to put together . A speciIn the first paper , the autWhat about the choice of starter unit? Most aromatic polyketides start with acetyl-CoA. An important earlier publication by Leadlay and colleagues  had showPloS Biology, is that the PKSs for these compounds consist of two modules of active sites. The components of each module are not activities carried on the same protein, as in the PKSs for the complex polyketides, but are all separate proteins. They form functional modules nevertheless. The newly recognized modules in the producers of compounds that start with nonacetate units have a dedicated ACP and a special ketosynthase that carries out a first condensation, joining the unusual starter unit to the first malonyl-CoA extender unit. The starter module then hands the resulting ‘diketide’ on to the second module (first reducing it, if appropriate, using reductive enzymes ‘borrowed’ from fatty acid biosynthesis) for typical extension by successive condensation with malonyl-CoA units to complete the chain. If the starter module is not present, the second module defaults to its typical habit of decarboxylating malonyl-CoA to acetyl-CoA and starts the chain with that.What The excitement of the work for biotechnology is that it offers the prospect of engineering promising drug candidates by making novel combinations of starter and extender modules and perhaps of feeding the starter modules with a whole range of unnatural substrates . It is e