Phenotypic analyses of animals with spontaneous mutations in DMT1 indicate that it functions at two distinct sites, transporting dietary iron across the apical membrane of intestinal absorptive cells, and transporting endosomal iron released from transferrin into the cytoplasm of erythroid precursors. DMT1 also acts as a proton-dependent transporter for other heavy metal ions including Mn2+, Co2+, and Cu2, but not for Mg2+ or Ca2+. A unique mutation in DMT1, G185R, has occurred spontaneously on two occasions in microcytic (mk) mice and once in Belgrade (b) rats. This mutation severely impairs the iron transport capability of DMT1, leading to systemic iron deficiency and anemia. The repeated occurrence of the G185R mutation cannot readily be explained by hypermutability of the gene. Here we show that G185R mutant DMT1 exhibits a new, constitutive Ca2+ permeability, suggesting a gain of function that contributes to remutation and the mk and b phenotypes.Divalent metal transporter-1 (DMT1/DCT1/Nramp2) is the major Fe David Clapham and colleagues offer new evidence that blurs the line between ion transporters and channels Based ondosomes . It is i2+, Mn2+, Cu2+, Zn2+, and Co2+ are required cofactors for many essential cellular enzymes. They cannot cross the plasma membrane through simple diffusion, and active uptake requires specific transporters. DMT1 is the only molecule known to mediate cellular iron uptake in higher eukaryotes. It is structurally unrelated to known Zn2+ and Cu2+ transporters, but DMT1 can transport those and other divalent metal ions  induced large inward currents in DMT1-expressing cells (42– to replace most of the Cl– ([Cl–]o = 5 mM) in low-pH bath solutions. With elimination of the background Cl– current, the proton-evoked current was inwardly rectifying (hyperbolic)  E.2+ (data not shown) or Mn2+ induced an additional current with less pronounced slow inactivation , and Fe3+ is not transported by DMT1  was recorded . In the presence of 100 μM Mn2+ (pH 4.2), the additional Mn2+-induced current was typically half the amplitude of the proton-induced current. Addition of Mn2+ alone (100 μM) at pH 7.4 did not induce any additional current. Since H+ or H+/Mn2+ induced two currents with distinct kinetics in DMT1-expressing cells, the underlying charge-carrying ion species and their relative contributions to the macroscopic currents were investigated. We monitored the reversal potential (Erev) and the current amplitude in ion-substitution experiments. Replacement of Na+ with N-methyl-D-glucamine (NMDG+) did not significantly change the Erev of H+ or H+/Mn2+-induced currents, although the net current amplitude was slightly increased  (Mn/PH can be estimated from the slope of Erev versus pH based on an extended Goldman–Hodgkin–Katz equation (+ and Mn2+). At pH 4.2, the slope of Erev versus pH did not differ significantly with or without Mn2+(see + to IDMT1 is much larger than that of Fe2+/Mn2+ (PMn/PH < 0.01), in contrast to the 1:1 stoichiometry proposed previously  Ca2+ solution  . PMn/PH equation  with two Mn2+see F. Therefeviously . Importation see C.2+ and 140 mM Na+) at pH 7.4 (G185R) was stable over minutes with no slow inactivation (see G185R in more than 85% of enhanced green fluorescent protein (EGFP)-positive cells transfected with the pTracer-G185R construct and in stable, doxycycline-induced G185R-HEK-On cells, but never in cells transfected with wild-type DMT1 . The inwardly rectifying current was cationic, since Ca2+ and Na+ substitution by NMDG+ completely abrogated the current  compared to wild-type DMT1 at similar protein expression levels , IG185R was 64 ± 7 pA/pF at −140 mV .Io 105 mM E, strengove 0 mV D, whereaG185R. The amplitude of IG185R was strongly dependent on [Ca2+]o  increased the current, indicating that Ca2+ permeated the plasma membrane of G185R-transfected cells much more readily than Na+. As shown in 2+]o not only augmented the current amplitude but also shifted Erev toward depolarized potentials. The slope of this shift (25 mV per decade) was close to the slope of 29 mV per decade predicted by the Nernst equation for a Ca2+-selective electrode . We determined G185R-specific currents by measuring the reversal potentials of the currents subtracted from two holding potentials  (G185R was less Ca2+-selective (PCa/PNa of approximately 10) than VGCCs . Single iG185R channels were not observed in cell-attached patches. Analysis of membrane current noise at −100 mV predicted a single-channel chord conductance of 0.4 ± 0.1 pS , too small to be observed under most patch–clamp conditions.We next investigated the cation selectivity of I [Ca2+]o F. With 1lectrode C. The reials see A and 4B lculated  and illuand Ba2+ D. While ransient D, the siransient , yieldinK+ > Cs+ E. Under sequence E of IG18 (VGCCs) , but IG12+ indicator dye Fura-2, we demonstrated G185R-mediated Ca2+ influx by monitoring intracellular Ca2+ levels in response to an elevation of [Ca2+]o  were −33 mV and −10 mV for control and isotonic Ca2+ solutions, respectively , nor was it blocked by antagonists of known Ca2+-permeant channels. In particular, the current was not blocked by ruthenium red (RR), Ca2+-release activated Ca2+ channel (CRAC) blockers SKF96365 and 2-APB , inhibited IG185R. La3+  blocked IG185R in a similar voltage-dependent manner , and CRAC currents, based on its current–voltage (I-V) relation, kinetics, permeation properties, and pharmacological sensitivity.Ihibition A and 4B hibition . The halectively B. The vopendence B. IG185Rnd 2-APB   and in mk enterocytes. A putative associated protein, if present in these different cell types, would have to be activated in a G185R-dependent manner. We did not find evidence of an associated protein when we immunoprecipitated wild-type or G185R DMT1 from transfected CHO-K1 cells (unpublished data). Furthermore, a distinct DMT1 mutant, G185K, also displayed Ca2+ permeability, but this mutant was less selective for Ca2+ over Na+ (unpublished data).We conclude that expression of G185R in transfected cells and in vivo in DMT1 mutants with impaired iron transport or more likely to be noticed by those managing the animal colonies. In parallel with these studies, we have generated knockout mice homozygous for a null DMT1 allele . Although detailed phenotypic characterization has not yet been completed, we have noted that –/–Dmt1 mice invariably die by the end of the first week of life, in contrast to mk/mk mice, which are poorly viable but can survive for more than a year . This suggests that the small amount of residual function of G185R DMT1, perhaps in combination with its gain-of-function Ca2+ conductance, contributes to viability.G185R mutations have occurred at least three times in rodents, which suggests that G185R not only inactivates DMT1, but may confer an unknown selective advantage. Because it has arisen in inbred colonies, the postulated selective advantage must either make the animals more viable than other 2+] has been reported to increase nontransferrin-bound iron uptake through an undefined transport system that has characteristics distinct from DMT1  in a 12-TM transporter protein conferred new Calocation . The “ch2+ entry gain of function helps to account for this remarkable pattern of remutation. Further investigation of this hypothesis will require direct and detailed comparison of DMT1-null and mk mice.We initiated these studies to investigate why a unique DMT1 mutation, G185R, has occurred independently at least twice in mice and once in rats ,1998. ThDMT1 cDNA used in this study was derived from one of four alternatively-spliced DMT1 gene transcripts. The G185R mutation was generated by using M13 phage and the oligonucleotide GTCCCCCTGTGGGGCCGAGTCCTCATCACCA. Wild-type DMT1 and the G185R mutant were tagged with a C-terminal FLAG epitope and subcloned into pTracer-CMV2 . CHO-K1 or HEK293T cells transiently transfected with DMT1 and G185R were used for the 55Fe uptake assay and Western blot analysis. To obtain a stable G185R-expressing cell line, the G185R-encoding DMT1 gene was subcloned into pRevTRE , a retroviral vector that drives expression from a Tet-responsive element. All constructs were confirmed by sequencing. DMT1 Western blot analyses were performed with an anti-FLAG M2 monoclonal antibody  and, in some cases, with a goat polyclonal antibody raised against human DMT1 .The TM, or HEK-On) was generated, and expression was induced by adding 1–10 μg/ml doxycycline into the culture medium. Unless otherwise stated, the pipette solution contained 147 mM cesium, 120 mM methane-sulfonate, 8 mM NaCl, 10 mM EGTA, 2 mM Mg-ATP, 20 mM HEPES (pH 7.4). Bath solution contained 140 mM NaCl, 10 mM CaCl2, 10 mM HEPES, 10 mM MES, 10 mM glucose (pH 7.4). Unless otherwise stated, the low pH solutions contained only nominal free Ca2+ (1–10 μM). Data were collected using an Axopatch 2A patch–clamp amplifier, Digidata 1320, and pClamp 8.0 software . Whole-cell currents were digitized at 10 kHz and filtered at 2 kHz.Wild-type and G185R mutant DMT1 were subcloned into an EGFP-containing vector  for transient expression in CHO-K1 and HEK293T cells. Cells were transfected using Lipofectamine 2000 (Invitrogen). Transfected cells, cultured at 37°C, were plated onto glass coverslips and recorded 24 (DMT1) or 30 (G185R) hrs after transfection. A stable cell line (HEK293 Tet-OnNa) was estimated according to rev upon replacing [Na+]o in nominally Ca2+-free bath solution , where X+ was Na+, K+, Cs+, or Li+. For the permeability to divalent cations (relative to PNa), bi-ionic conditions were used; Y2+ was Ca2+, Ba2+, or Sr2+ . The external solution was 140 mM NMDG-Cl, 10 mM Y2+Cl2, 20 mM HEPES (pH 7.4 adjusted with HCl). The permeability ratios of cations were estimated from the following equations  and 50 mM nitrilotriacetic acid. About 30 h after transient transfection, CHO-K1 or HEK293T cells were washed and harvested with PBS . Cells were resuspended in glass test tubes at 0.5–1 million/ml in 490 μl assay buffer at 30°C. The reaction was started by adding 10 μl of 55Fe stock and stopped at 4, 8, and 16 min by quickly filtering the reaction mix on a nitrocellulose filter . Filters were washed twice with 2 ml of assay buffer, dried, and radioactivity counted by liquid scintillation spectrometry.The assay buffer contained 25 mM Tris, 25 mM MES, 140 mM NaCl, 5.4 mM KCl, 5 mM glucose, 1.8 mM CaCl2+] was constructed according to Cells were loaded with 2 μM Fura-2 AM in culture medium at 37°C for 30 min. Low levels of G185R protein were expressed in the absence of doxycycline in G185R HEK-On cells . Therefore, doxycycline-treated HEK-On cells not expressing DMT1 were used as controls in imaging experiments. We recorded Fura-2 ratios (F340/F380) on an UltraVIEW imaging system . A standard curve for Fura-2 ratio versus o = 5 mM) for all low-pH bath solutions. (C) pH-induced outwardly rectifying current in a nontransfected CHO-K1 cell. A similar current was seen also in HEK293T and HEK-On cells, with properties similar to the cloned ClC-7 channel . This cu(718 KB PDF).Click here for additional data file.Figure S2Whole-cell currents were generated by voltage steps from −140 to +80 mV in 20 mV steps, 400 ms. The interval between steps was 1,000 ms. (1 MB PDF).Click here for additional data file.Figure S3+ by NMDG+ slightly increased the proton current (approximately 20%) and this was further augmented by adding 300 μM Mn2+. The concentrations used were Na+ and NMDG+, 140 mM, (pH 4.2); Mn2+, 300 μM. (141 KB PDF).Replacement of extracellular NaClick here for additional data file.www.ncbi.nlm.nih.gov/GenBank/) accession number for DMT1 is AF029758.The GenBank (


Trigonella are important from medical and culinary aspect. Among these, Trigonella foenum-graecum is commonly grown as a vegetable. This anti-diabetic herb can lower blood glucose and cholesterol levels. Another species, Trigonella caerulea is used as food in the form of young seedlings. This herb is also used in cheese making. However, little is known about the genetic variation present in these species. In this report we describe the use of ISSR and RAPD markers to study genetic diversity in both, Trigonella foenum-graecum and Trigonella caerulea.Various species of genus Trigonella foenum-graecum and nine accessions of Trigonella caerulea representing various countries were analyzed using ISSR and RAPD markers. Genetic diversity parameters  were calculated for ISSR, RAPD and ISSR+RAPD approaches in both the species. Dendrograms were constructed using UPGMA algorithm based on the similarity index values for both Trigonella foenum-graecum and Trigonella caerulea. The UPGMA analysis showed that plants from different geographical regions were distributed in different groups in both the species. In Trigonella foenum-graecum accessions from Pakistan and Afghanistan were grouped together in one cluster but accessions from India and Nepal were grouped together in another cluster. However, in both the species accessions from Turkey did not group together and fell in different clusters.Seventeen accessions of Trigonella caerulea as compared to Trigonella foenum-graecum. The genetic similarity matrices generated by ISSR and RAPD markers in both species were highly correlated (r = 0.78 at p = 0.001 for Trigonella foenum-graecum and r = 0.98 at p = 0.001 for Trigonella caerulea) indicating congruence between these two systems. Implications of these observations in the analysis of genetic diversity and in supporting the possible Center of Origin and/or Diversity for Trigonella are discussed.Based on genetic similarity indices, higher diversity was observed in  The family Fabaceae includes many crops useful for food, forage, fiber, wood and ornamental purposes. In this family, a few legumes such as chickpea, soybean, fababean, fenugreek, lentil, pea etc. are consumed as grain legumes. The grain legumes are plants used as food in the form of unripe pods, mature seeds or immature dry seeds, directly or indirectly . The graTrigonella is one of the largest genera of the tribe Trifoliatae in the family Fabaceae and sub-family Papilionaceae . The first cluster grouped together accessions from Afghanistan (8707), Canada (1065), Pakistan , Iran (8675), and Turkey (8690). The second cluster contained accessions from India . Remaining one accession from India (8687) and one from Nepal (8706) formed the third cluster. The fourth cluster contained accessions from Egypt  and Turkey (8692). Accessions from Ethiopia (8696), Turkey (8691) and Yemen (8714) out grouped from the main clusters. Three accessions from Turkey analyzed in the present study fell into different clusters.Genetic similarity was calculated from the Nei's similarity index value for all the 17 accessions of T. foenum-graecum genotypes formed 2 main clusters [Figure not shown]. The first cluster had two subgroups, the first subgroup contained accessions from Afghanistan (8707), India , Turkey (8690) and Egypt (8698) while the second subgroup contained accessions from Pakistan , Turkey (8692), and Egypt (8679). The accession from Canada associated with these clusters. The second cluster contained accession from Iran (8685) and Nepal (8706) and the accession from India associated with this cluster. Accessions from Ethiopia (8696), Turkey (8691) and Yemen (8714) out grouped from these two clusters. In the RAPD based dendrogram also accessions from Turkey fell into different clusters.Based on RAPD markers alone, the similarity index values ranged from 0.71 to 0.91. In the RAPD based dendrogram, T. foenum-graecum accessions from Egypt  were grouped together. Accessions from Pakistan  and Afghanistan (8707) were grouped together in one cluster. Accessions from India , Nepal (8706) and Iran (8685) were grouped together. However, all the three accessions from Turkey fell in different clusters and did not group among themselves. Bootstrapping was done using the WinBoot program to estimate the relative support at clades. The robustness of the cluster was not very strong in T. foenum-graecum (50–70%).Based on both the marker systems together the similarity index values ranged from 0.65 to 0.89 Fig. . Here thT. caerulea, genetic similarity was calculated from the Nei's similarity index value considering ISSR and RAPD approaches individually as well as together. Based on ISSR marker system, the similarity index values ranged from 0.41 to 0.92 while that on the basis of RAPD markers ranged from 0.34 to 0.93. Similarity indices values based on both the marker systems together ranged from 0.38 to 0.92 indicating more diversity in case of T. caerulea. The dendrograms based on ISSR and RAPD markers showed similar clustering pattern when used individually [Figures not shown] as well as together , Alliumliaceae, ] and AsiBrassica  [Cicer [ and 15]Brassica Cajanus [ and 18]Brassica T. foenum-graecum as well as in T. caerulea the Hav and the marker index (MI) values for ISSR and RAPD markers . A dendrogram was constructed based on the matrix of distance using Unweighted Pair Group Method with Arithmetic averages. Scores entered in matrix were analyzed using TAXAN version 4.0 software based on the degree of bands sharing. Similarity matrix was generated using Dice coefficient as SI = 2Nab/Na+Nb where Na = total number of bands present in lane a, Nb = total of bands in lane b, Nab number of bands common to lanes a and b [27]. The dice values were then used to perform the UPGMA analysis. To evaluate the robustness of the grouping formed the binary data matrix was subjected to bootstrapping using WinBoot program [28]. The phenogram was reconstructed 1000 times by repeating sampling with replacement and the frequency with which the groups were formed was used to indicate the strength of the group. Correlation co-efficient for the similarity matrices generated by ISSR and RAPD data in both, T.foenum-graecum and T.caerulea, were calculated by method of Smouse et al [29].   The expected heterozygosity, Hn for a genetic marker was calculated as: Hn = 1 - pi2, where pi is the allele frequency of the ith allele [26]. The arithmetic mean heterozygosity Hav for each marker class was calculated as Hav = Hn/n, where n = number of markers or loci analyzed [18]. The average heterozygosity for polymorphic marker (Hav)p was further derived as; (Hav)p = Hn/np; where np is the no. of polymorphic markers or loci [18]. Marker index (Ml) for each marker system was also calculated as, MI = E (Hav)p; where E = effective multiplex ratio .RD carried out the PCR and molecular genetic study, participated in the design of the study and performed statistical analysis. ML participated in procuring the seeds from different sources and monitoring experiments and result. LC a taxonomist participated in procuring, growing and analyzing the seeds obtained from different sources. PR and VG conceived of the study, participated in its design and co-ordination and monitored the experiments and the results.All authors read and approved the final manuscript.


Furthermore, hypotension (systolic BP < 80 mm Hg) was present in 44% of children with severe acidosis (base deficit >15). These findings also indicate important baseline differences in two cohorts of children studied. We agree that reconsideration of guidelines for acute fluid management is warranted, particularly when current recommendations await an adequate evidence base. Nevertheless, conflicting opinions on the question of volume status in children with severe malaria can be satisfactorily resolved only through prospective randomized trials that include both fluid resuscitation and control groups. While the design and conduct of such trials will involve considerable challenges, optimal fluid management will never be resolved on the basis of theoretical consideration alone.The study by Planche et al.


Once malaria occurs, deaths can be prevented by prompt treatment with relatively affordable and efficacious drugs. Yet this goal is elusive in Africa. The paradox of a continuing but easily preventable cause of high mortality raises important questions for policy makers concerning care-seeking and access to health systems. Although patterns of care-seeking during uncomplicated malaria episodes are well known, studies in cases of fatal malaria are rare. Care-seeking behaviours may differ between these groups.This study documents care-seeking events in 320 children less than five years of age with fatal malaria seen between 1999 and 2001 during over 240,000 person-years of follow-up in a stable perennial malaria transmission setting in southern Tanzania. Accounts of care-seeking recorded in verbal autopsy histories were analysed to determine providers attended and the sequence of choices made as the patients' condition deteriorated.As first resort to care, 78.7% of malaria-attributable deaths used modern biomedical care in the form of antimalarial pharmaceuticals from shops or government or non-governmental heath facilities, 9.4% used initial traditional care at home or from traditional practitioners and 11.9% sought no care of any kind. There were no differences in patterns of choice by sex of the child, sex of the head of the household, socioeconomic status of the household or presence or absence of convulsions. In malaria deaths of all ages who sought care more than once, modern care was included in the first or second resort to care in 90.0% and 99.4% with and without convulsions respectively.In this study of fatal malaria in southern Tanzania, biomedical care is the preferred choice of an overwhelming majority of suspected malaria cases, even those complicated by convulsions. Traditional care is no longer a significant delaying factor. To reduce mortality further will require greater emphasis on recognizing danger signs at home, prompter care-seeking, improved quality of care at health facilities and better adherence to treatment. Malaria continues to be the largest single component of the burden of disease in sub-Saharan Africa, even though simple, effective and affordable treatments exist. Malaria's pervasive morbidity and high mortality persist because of failed transactions between those at risk of malaria transmission and available preventive and curative health systems. The consequence is not just an intolerable burden for individuals, their families and national health systems, but is also a devastating and continuing impediment to socio-economic development on the continent. Unlike HIV and TB, the other major fatal communicable diseases in Africa, malaria deaths can be prevented by prompt treatment with relatively affordable and efficacious drugs. Yet this goal continues to be elusive. The paradox of a continuing, but easily preventable, cause of high mortality raises important questions for policy makers and health systems in Africa.). Malaria is the leading cause of out-patient and in-patient health service attendance for all the ages and the leading cause of death in both children and adults in all regions of Tanzania  you are able to recognize an episode of degedege in one day. It begins with mild fever and the next day the fever becomes more severe and results in symptoms of epilepsy. The child opens the eyes wide and the black spot cannot be seen, he begins to twist the arm and leg, and then, even if you pour cold water over the child, does not react..." .Although the local population distinguish between the illness "homa" and malaria the distinction is not always very clear to them. Analysis of case studies revealed that the illness term "malaria" has been obtained from modern health care. When mothers take their children to these health services with what had been diagnosed at home as "homa", they are told it is malaria. The following is illustrative of experiences reported:"I first thought it was normal homa (fever) and I could see the child had homa. Now, when I took the child to the hospital, they checked the child's blood and informed me the child had malaria. [...] the child was not playing. I touched the child and the body was like fire (mwili wake ulikuwa wa moto), the body was very hot". .Anaemia is not often recognized, and where recognized, is not associated with malaria.Although "homa" and especially "malaria" were seen as associated with malaria and mosquitoes, in most cases the signs and symptoms of "degedege" are not attributed to malaria. Life threatening malaria with convulsions is not only perceived as a different illness from malaria through local symptom definition but is attributed to different causes than malaria. Few households mention the mosquito as a cause of the illness described as "degedege". Popular beliefs as to the cause of "degedege" were found to include: fever, evil spirits and a change in weather/wind. The following translation is typical of quotes obtained from respondents on perceived causes of "degedege":"....Evil spirits or demons cause degedege. If it happens that evil spirits or demons pass in front of the child, then the child is likely to get degedege. This may result in paralysis of the body or leg or arm or any part of the body..." ."Homa" and "malaria" are seen as conditions that can be managed at least initially at home with modern medicine available from shops and from health facilities. But "degedege" is perceived as a serious life-threatening condition for which prompt treatment-seeking is required. People reported different sources of care they used for the treatment of "degedege". These sources encompass more than the biomedical health system and fall into three broad categories: home treatment, traditional healers and biomedical. Home treatment was reported to include the use of modern medicines, such as aspirin from local shops, in the early stages the illness. If the illness reaches a severe stage (convulsions) people claim to use traditional healers in the home or outside the home. Biomedical care ranging across government hospitals, health centres, dispensaries and equivalent private facilities was used in the later stage, when convulsions had subsided. However, some respondents perceive traditional healers as not competent to deal with such illness and claim to seek care from biomedical providers at the beginning of the illness."We use traditional remedies only to treat degedege. They (remedies) must have a very bad smell for this will chase away the evil spirit. It is just like telling you to stay in the latrine; surely you will have to find another place because of the bad smell. This is just the same case for the evil spirit attacking the child because of the bad smell it will have to find another place to stay...." ."I had gone to the dispensary for treatment; my child was suffering from homa. The first day he was given panadol tablets and chloroquine injection and was asked to return the next day for chloroquine injection. The next day while I was there at the dispensary waiting for treatment my child started convulsing. This I believed to be a sign of degedege. Immediately I left the dispensary in search of a traditional healer. Degedege is never treated in the dispensary. Child may die after being injected." ."When my child developed degedege I was at Kibiti. I had to look for transport to take the child to Songa Hospital . There you have reliable service because you find almost all kinds of investigations. I don't like going to traditional healers because they are not reliable and do not have equipment to investigate well your child. They end up telling you things related to superstition." .In the period January 1999 to December 2001 inclusive, the Rufiji DSS conducted 243,042 person years of follow-up. In this series, 3,023 deaths occurred to resident members and 2,953 (97.7%) verbal autopsies were conducted. Of these, 24.4% (722) had a cause of death suggestive of malaria as the direct or underlying cause, of which 44.3% (320) were in children less than five years of age. Among these child deaths, there was no difference in frequency between sexes, with 51.3% being male and 48.7% female. Of the child malaria-attributed deaths, 282 (88.1%) sought care at least once before death, while 38 (11.9%) did not, or could not, seek care. Convulsions  were recorded in 30 (9.4%) of these fatal cases.The verbal autopsies contained both an open-ended narrative account of the final illness and a specific chronological account of where and in what sequence care was sought. There were 13 possible sources of treatment that were collapsed for purposes of certain analyses into three sub-categories of care types  and into six sub-categories of provider types . Table Within modern care, government providers were most prominent (44.7%) followed by home care with antimalarials from private shops (20%) Table . ChildreCases with convulsions were as likely to receive initial modern care as cases without convulsions (77.8% and 78.9% respectively) Table . HoweverAmong children for whom care was actively sought, 82.4% of those with convulsions and 90.3% without convulsions sought modern care as the initial care  of malaria-attributable child deaths failed to seek modern care (9.4%) or any care (11.9%). This is considerably better than was seen in the mid-eighties, when 55% of children who died had not utilized any modern care . It is aThis study shows that most patients now include modern care early in their treatment seeking patterns for eventually severe and fatal malaria, including malaria with convulsions. So why is malaria still the largest single component in the burden of mortality? With belief systems for malaria treatment seeking now firmly on the side of modern care, there is obviously something still failing in 1) the transaction to obtain this care; 2) the quality of the care and referral once it is reached; and/or 3) patient adherence to treatment once it is obtained. This would suggest that policies, efforts and implementation research aimed at improving early recognition of symptoms and danger signs at home, prompt treatment or treatment seeking, the quality and efficacy of the antimalarial available and compliance with the full course of treatment, are now, more than ever, highly justified. When appropriate care-seeking is as high as it is in Tanzania, continuing malaria deaths should be considered as sentinel events deserving of close scrutiny and audit to identify the best remedial strategies for the health system.There are promising developments. IMCI has recently been introduced in the study area. It places heavy emphasis on training care-givers on early recognition of danger signs and the need for prompt treatment and on improving quality of assessment and care at primary health facilities ,49. ReplThis preliminary study examined what families of children who died from malaria in a holoendemic setting in Africa actually did in terms of treatment-seeking choices and sequence. It confirms that modern medicine in the form of antimalarial pharmaceuticals from shops or government or non-governmental heath facilities is now the preferred choice in an overwhelming majority of cases (78.7% and 97% as their first or second choice respectively). Traditional medicine could only be implicated in a possible delay of modern care in 9.4% of cases. 11.9% sought no care of any kind. There were no differences in these broad patterns of choice by sex of the child, sex of head of household, socioeconomic status of the household or presence or absence of convulsions. Contrary to what is concluded from much of the historical and qualitative work on this subject, modern care is now the care of first choice, even for those who seek care for children with malaria with convulsions (82.4%), although traditional medicine also played an important role in later choices. But despite high rates of modern care-seeking for all forms of malaria, and despite relatively high attendance and utilization of modern care as seen in Tanzania, malaria mortality remains high. This must, therefore, be due to excessive delay in seeking modern care, and/or poor quality of modern care (providers and/or drugs) once sought, and/or poor patient adherence to treatment regimens once obtained.Certain policy and practice implications arise: 1) public messages need to focus aggressively on improving early recognition of malaria and severe malaria at home and improving promptness of treatment seeking ; 2) quality of modern care providers and modern care must be improved in all sectors, private, NGO and Government; and 3) patient adherence with modern care at home must be simplified and reinforced.DSA Demographic Surveillance AreaDSS Demographic Surveillance SystemGIS Geographic Information SystemHBS Household Budget SurveyHH HouseholdIMCI Integrated Management of Childhood IllnessITN Insecticide-treated nettingMARA Mapping Malaria Risk in Africa CollaborationSP Sulfadoxine-pyrimethamineTM Traditional MedicineVA Verbal AutopsyDD conceived the study, participated in the design, coordination and quantitative analysis and co-wrote the article. CM conceived, conducted and analysed the qualitative studies and co-wrote the article. HM led the analysis of quantitative data. EM managed the surveillance system and participated in design and coordination. AM managed and cleaned the quantitative data. YM managed the field work. CM, HK and GR participated in the coordination and management of the study.


M.I.T. offers an education to the world through its OpenCourseWare program Imagine a fledgling biology instructor at a university in the developing world. Heading into her first semester of teaching, she is armed with nothing but her college degree, some old notebooks, and—if she is lucky—a late-edition textbook. Forging a curriculum that is both current and engaging for her students could be a daunting challenge.But what if that same young instructor was given free and open access to a syllabus, complete lecture notes, and problem sets and solutions from two members of the faculty of the Massachusetts Institute of Technology (MIT)? And not just any faculty members, but David Page—the recipient of a MacArthur Foundation Prize Fellowship in 1986, a Searle Scholar's Award in 1989, and the Amory Prize for advances in reproductive biology from the American Academy of Arts and Sciences in 1997—and Chris Kaiser, who won a 1999 fellowship from MIT that recognizes his teaching excellence?This is the premise of MIT's OpenCourseWare project.Utilizing the Internet, MIT OpenCourseWare (MIT OCW) has opened MIT's curriculum and educational materials to a global audience of teachers and learners—an instructor at a new engineering university in Ghana, a precocious highschool biology student in suburban Chicago, a political scientist in Poland, a literature professor in upstate New York—who are all now able to use the same materials that MIT's professors rely on to teach their full-time students.Ten years from now, we expect that MIT OCW will have become firmly planted in MIT's educational landscape. But MIT OCW was just a leap of faith when the concept was originally proposed by a group of faculty four years ago. In the fall of 1999, Provost Robert A. Brown asked the faculty committee to provide strategic guidance on how the institute should position itself in the e-learning environment. At first, many members of the group assumed that their work would lead to an “MIT.com” venture. But after a year of analysis, market research, and development of business scenarios, the committee concluded that a revenue-generating distance-education model was not desirable for MIT.The committee went back to the drawing board and, convinced that open software and open systems were the wave of the future, came to a very simple conclusion: that MIT should use the Internet to give its teaching materials away.Brown and MIT President Charles Vest instantly recognized the simplicity and brilliance of the idea. “It seemed to me that it would be a way to advance education, by constantly widening access to our information and inspiring other institutions to do the same with theirs,” Vest said.While the 701 courses currently available represent just a third of the ultimate goal of 2,000 courses by the year 2008, MIT OCW has already had an impact on MIT's campus. We have published teaching materials from almost half of MIT's 950 faculty members, and a significant portion of the faculty have told us that they are already using materials available on MIT OCW—the lecture notes, syllabi, problem sets, and exams of their colleagues—to prepare for their classes, do research, and help their students.But the real payoff of what we hope will become the “opencourseware movement” will be its effect on educators and learners around the world. Our goal is to create a model that other universities can follow and improve upon. Ultimately, the trend toward open knowledge will help bring people of all backgrounds together and promote improved educational systems across the globe.Since April 2001, we have received more than 20,000 e-mail messages from around the world endorsing the vision and potential benefits of sharing knowledge freely. A typical message came from Andrew Wilson in the United Kingdom in October 2003: There can be no greater hope for humankind than the belief that wisdom generated through increased learning will ultimately lead to a better world. With OCW, MIT has taken an ethical stand against the belief that knowledge should only be accessible to those who can pay for it or are in proximity to it.”Just after its “official launch” in fall 2003, MIT began a rigorous data collection process to find out who is accessing MIT OCW, why and how they use it, and what difference the initiative makes. The results of this first baseline evaluation confirm what we have heard anecdotally through those e-mails: that educators, students, and self-learners around the world are using our course materials, and that, overwhelmingly, they find the materials useful in meeting their own learning and teaching goals.On average, MIT OCW clocks over 11,000 visits per day, with nearly a quarter-million unique visitors per month. About 45% of these visitors are from the United States and Canada. Outside North America, the top countries of origin are China, the United Kingdom, Germany, India, and Brazil. About 52% of visitors identify themselves as “self-learners,” 31% as “students” enrolled in a formal course of study, and 13% as “educators.” We view educators as a particularly important target audience because it is through them that MIT course materials can touch the greatest number of people and have the most profound impact on education around the world.MIT OCW asked visitors their primary purpose in using MIT course materials. Of educators who responded, about 57% answered that they use it for course or curriculum development, 33% to enhance their subject matter understanding or support research, and 7% for student advising. Elements of MIT materials have been adapted for classroom use by 47% of educators who answered our survey, while 41% report they are considering it.Among the 33 academic disciplines available are 15 courses from the MIT Department of Biology see , 63 fromEducators, students, and self-learners from a wide variety of fields will find materials they can use in their teaching and learning activities. And as the opencourseware concept spreads to other colleges and universities, we expect that access to the work of faculty from diverse disciplines and institutions will increase, by an order of magnitude, the benefits to educators and learners who  would not otherwise have access to such materials.History has proved that education and discovery are best advanced when knowledge is shared openly. Our agenda must evolve to shape the future, and to respond to new challenges and opportunities.At MIT, we believe the idea of opencourseware is one such opportunity, which we must seize during the next decade.For more information about MIT OCW, please contact Jon Paul Potts, MIT OCW Communications Manager, at E-mail:jpotts@mit.edu or 617-452-3621.


In response to the Blackburn and Rowley essay on the President's Council on Bioethics, several thought-provoking opinions on ethical challenges in biomedical research are expressed by prominent stakeholders However, it does not follow that if the benefits of embryo stem cell research had been presented more persuasively and in greater detail, then the case for ‘non-commercial, federal, peer-reviewed funding’ would be unassailable. Such a view appears to be based squarely on a utilitarian view of the moral status of embryos: that the good flowing from destructive research outweighs the evil of embryo destruction. Far from being a neutral scientific analysis, this expresses a commitment to the proposition that biomedical progress is more important than the defence of human life.If twentieth century philosophy of science has taught us anything, it is that the aspiration to pure scientific objectivity is a dangerous illusion. Research programs always embody philosophical and moral assumptions that must be openly defended. If Blackburn and Rowley want government support for embryo stem cell research, they must justify their bioethical approach and not hide behind a smokescreen of indignation over Blackburn's unwilling departure from the Council.


DNA replication forks initiated at oriC by DnaA can collapse on route to the terminus for a variety of reasons. PriA, PriB, PriC, DnaT, Rep and DnaC form multiple pathways to restart repaired replication forks. DnaC809 and dnaC809,820 are suppressors of priA2::kan mutant phenotypes. The former requires PriC and Rep while the latter is independent of them. RnhA339::cat mutations allow DnaA-independent initiation of DNA replication.DnaA is an essential protein in the regulation and initiation of DNA replication in many bacteria. It forms a protein-DNA complex at priC303::kan mutation is synthetically lethal with either a dnaA46 or dnaA508 temperature sensitive mutation at the permissive temperature of 30°C. The priC-dnaA lethality is specific for the dnaA allele. The priC303::kan mutant was viable when placed in combination with either dnaA5, dnaA167, dnaA204 or dnaA602. The priC-dnaA508 and priC-dnaA46 lethality could be suppressed by rnhA339::cat. The priC-dnaA508 lethality could be suppressed by a dnaC809,820 mutation, but not dnaC809. Neither of the dnaC mutations could suppress the priC-dnaA46 lethality.It is shown herein that a dnaA temperature sensitive mutant strains at the permissive temperature of 30°C has been documented. Models considering roles for PriC during initiation of DNA replication and roles for DnaA in replication restart were tested and found not to decisively explain the data. Other roles of dnaA in transcription and nucleoid structure are additionally considered.A hitherto unknown function for either DnaA in replication restart or PriC in initiation of DNA replication that occurs in certain  E. coli origin of DNA replication (oriC) is a highly regulated and is thought to be a key step in the assembly of the replisome. DnaB makes important contacts with the τ-subunit of DNA Polymerase III Holoenzyme and DNA primase [oriC during initiation of DNA replication is a sequence specific, cell cycle regulated event dependent on the DnaA and DnaC proteins , multiple DnaA proteins bind to four asymmetric 9 bp DnaA binding sites in the 225 bp oriC region allowing formation of a protein/DNA complex [6-DnaC6 then interacts with the DnaA/oriC complex to load DnaB at the AT rich sequence.The loading of the DnaB replicative helicase at the  primase . DnaB loiewed in -4). In v complex . This inoriC region [dnaA can affect the level of transcription from that promoter [dnaA may have global effects in gene expression and nucleoid structure as well as affecting initiation of DNA replication at oriC.It is thought that DnaA may have other roles in the cells in addition to initiation. These additional roles stem from the fact that there are many DnaA binding sites in the chromosome outside the C region ,9 and thC region . It can promoter -14. ThusE. coli, dnaA is an essential gene. Several different dnaA temperature sensitive mutant alleles have been isolated. Many of these share the property that they are double mutants : PriA, PriB, PriC, DnaT and Rep. The genes coding for these products form multiple pathways to identify the proper substrate and then help DnaC load DnaB  and is dependent on the genes in the PriA-independent pathway of replication restart, rep and priC [priA suppressor has an additional mutation (K178N) in dnaC relative to dnaC809 and makes this protein's suppression of priA mutant phenotypes independent of priC and rep. This dnaC allele is called dnaC809,820 [Bacteria since Bacillus subtilis also has a similar arrangement of PriA-dependent and PriA-independent pathways [The loading of DnaB by DnaC in  and Rep . Two typdnaC809 E6G and isdnaC809 E6G and isC809,820 . The mulpathways ,20.oriC and replication restart share several properties. The most important of these is that both strive to make protein-DNA complexes that are recognized by the DnaC protein so that DnaB can be loaded. Previous work by Kogoma and colleagues showed that oriC-DnaA-independent initiation of DNA replication could take place in an rnhA mutant strain [As mentioned above, DnaA-dependent initiation of DNA replication at t strain ,21. Thist strain  and SandpriB and priC in cSDR  requirepriB and priC are required for cSDR. During this study it was found that certain dnaAts could be introduced into a strain containing a priB mutation, but not into a strain containing a priC mutation. The standard P1 transductional cross used for the introduction of the dnaAts mutant alleles is shown in Figure tnaA300::Tn10 and dnaA508 in a wild type strain is 92% (49/53). Using a priB mutant strain as the recipient, the co-transduction frequency between tnaA300::Tn10 and dnaA508 was approximately the same as when the wild type strain was used as the recipient (data not shown). Surprisingly, when a priC mutant was used as a recipient, the co-transduction frequency was 0/72 or 0%  created by the dnaA508 mutation. To do this, the dnaA region from the 42°C R TetR transductants was amplified by standard Polymerase Chain Reaction methods using the primers, prSJS480 and prSJS481 . From these results, it is concluded that the priC303::kan mutation does not suppress the absence of dnaA508 and is synthetically lethal with it.Since it is known that the absence of other gene products  can supdnaA508-priC303::kan synthetic lethality at the permissive temperature of 30°C is allele specific or occurs with all dnaAts alleles. To distinguish between these two possibilities, several other dnaAts alleles were tested. Selection of a diverse collection of mutations to test was aided by an already large repertoire of characterized dnaAts alleles [Aquifex aeolicus [dnaA alleles that had different amino acids substitutions in different parts of the protein  using the selectable marker tnaA300::Tn10 as before. Table dnaA46, but not with dnaA5, dnaA167, dnaA204 or dnaA602. It is concluded that the synthetic lethality between priC303::kan and dnaA508 and dnaA46 at 30°C is allele specific.It is possible that either the  alleles  and the aeolicus . This alin Table . Hence, dnaA and priC genes are in operons, it is possible that the synthetic lethality seen above is due to not just the mutation in priC or dnaA, but also due to that mutation and or polar effects on downstream genes within their respective operons. While the potential polar effects of a priC303::kan insertion mutation are easily envisioned, the potential polar effects of a dnaA missense mutation are less obvious. This is tested here as well because, as introduced above and discussed below, dnaA mutations can have effects on the level of transcription of promoters in which there are DnaA binding sites. Since dnaA binds to its own promoter and autoregulates its own expression [dnaA mutation may effect transcription from its own promoter and subsequently effect dnaN and recF expression.Since both the pression ,26, it idnaA508 and priC303::kan was dependent solely on the priC gene. This was necessary to determine because priC303::kan is an insertion mutation and could be polar on the downstream gene, ybaM. This was tested by cloning priC into a plasmid  and seeing if the priC plasmid could complement the synthetically lethal phenotype. pTH1, containing just the priC promoter and gene, in the priC303::kan mutant strain (SS145), allowed the dnaA508 allele to be introduced into that strain at the wild type co-transduction frequency of 90% (data not shown).It was first tested if the synthetic lethality between the dnaA508 or dnaA46 on downstream dnaN-recF expression. This was tested in a similar way. A plasmid, pAB3 [dnaA gene in trans was introduced into the priC303::kan mutant strain (SS145). This strain was then used as a recipient in a cross with either ALO450 (dnaA46 tnaA300::Tn10) or JC12390 (dnaA508 tnaA300::Tn10). TetR transductants were selected at 30°C. In each case, several transductants were selected and screened for the presence of the dnaAts mutation by backcrosses to JC13509. The temperature sensitive phenotype associated with dnaA508 and dnaA46 was detected in each case (data not shown).It was then tested if the synthetic lethality was due to polar effects of id, pAB3 , that expriC303::kan and dnaA508 or dnaA46 is due to solely the absence of priC and the presence of the dnaAts mutation.It is concluded that the synthetic lethality seen between rnhA mutations are non-allele specific suppressors of both dnaAts and dnaA insertion mutations [priC-dnaA synthetic lethality is suppressed by a mutation in rnhA, it was attempted to introduce dnaA508 and dnaA46 into a priC303::kan rnhA339::cat (SS1531) double mutant strain. It was found that this dnaAts rnhA priC triple mutant combination was viable in each case  and priC303::kan dnaC809,820 (SS1100) strains were constructed (Table dnaA508) or AL0450 (dnaA46). Table dnaC809,820 was used as the recipient and JC12390 as the donor that 41/48 or 83% of the TetR transductants were also temperature sensitive . However, when only dnaC809 was used as the recipient, 0/63 TetR transductants inherited the temperature sensitive phenotype. It is concluded that dnaC809,820 can suppress the absence of priC in the dnaA508 mutant and dnaC809 cannot. The dnaA46 allele was additionally tested and was not suppressed by either dnaC809 or dnaC809,820  and thaRnhA339::cat, a non-allele specific suppressor of dnaA mutants role in initiation of DNA replication, could suppress the priC-dnaA synthetic lethality for both dnaAts mutant alleles. Such suppression is strong evidence that priC and dnaA may both be missing a function needed during initiation of DNA replication. It was further observed, however, that dnaC809,820 (but not dnaC809) could suppress the absence of priC in the dnaA508 mutant. Neither dnaC809 nor dnaC809,820 could suppress the dnaA46-priC synthetic lethality. DnaC809,820 is a PriC-independent suppressor of several genes required in replication restart. This suppression implicates dnaA in replication restart. Thus the inferences from the two types of suppressors seem to contradict one another.One way to answer the question of why the mutations are synthetically lethal is to see what types of mutations may suppress the lethality. dnaAts strains are missing for initiation of DNA replication that make the strain dependent on priC at 30°C? A structure-function analysis of DnaA would help to answer this question. Briefly, based on alignments of DnaA proteins, the X-ray crystal structure of the DnaA protein from Aquifex aeolicus and much research on the genetics and biochemistry of DnaA, the DnaA protein can be divided into four domains with four proposed functions: Domain I) DnaB recruitment, Domain II) Linker region, Domain III) ATP binding and Domain IV) DNA binding [priC have mutations in Domains I (DnaB recruitment) and III (ATP hydrolysis). However, several of the mutations not requiring priC also affect Domain III. An interesting aspect to dnaA genetics is that many temperature sensitive mutants have two mutations  in  binding ,35,36. TdnaAts strain? One idea is that the dnaAts protein is defective in its ability to create a region of ssDNA at oriC. Since the RRPs are also thought to help create regions of ssDNA (away from oriC) so that DnaC binds and loads DnaB, it is possible that PriC may help the DnaA mutants in this endeavor. Another type of model that is formally possible is that PriC may somehow stabilize the dnaAts protein. This seems unlikely, however, given that dnaC809,820 can rescue the synthetic lethality of priC303::kan and dnaA508. Other models may also be possible.What might PriC be doing to help initiation of DNA replication in a dnaAts allele can differentially influence the rate of initiation of transcription in some promoters with DnaA binding sites (see below). This in turn can influence replication restart in two ways. First changes in the level of gene expression of a single gene (or groups of genes) may indirectly influence the replication restart process. Second, the ability of DnaA to bind to many sites on the chromosome may influence structure of the nucleoid and the sites at which replication restart may occur.One needs to consider if DnaA may be involved in replication restart. In considering this, one needs to remember that DnaA has the ability to bind DNA at specific sites and bend it. It has been shown that different dnaA alleles (Table P+ plasmids was investigated [dnaA46, dnaA204 and dnaA508 all fail to replicate these plasmids while dnaA5, dnaA167 and dna602 can. The model proposed to explain this phenomenon suggests that DnaA is required to activate transcription at the λ PR promoter and that the dnaA46, dnaA204 and dnaA508 mutations decrease this ability[dnaA204, the inability to replicate these plasmids mirrors the ability of the dnaAts mutants to grow in the absence of priC.There are many examples where several phenotypes had been tested systematically for several stigated ,34. Tabls ability. With thoriC or is it due to a failure in replication restart based on the study of the suppressors. Since dnaA mutations can affect more than just initiation of DNA replication, it is tempting to speculate that some other dnaA function: transcription of a particular gene or set of genes or the shape of nucleoid in the priC mutant may contribute or be the cause of the synthetic lethality. Understanding the molecular mechanism underlying the dnaAts-priC synthetic lethality may require appreciation of these other aspects of dnaA biology.The results presented in this paper do not allow one to definitively know whether the synthetic lethality studied here is due to a failure in initiation of replication at dnaA temperature sensitive strains at the permissive temperature of 30°C has been documented. Models considering roles for PriC during initiation of DNA replication and roles for DnaA in replication restart were tested and found not to decisively explain the data. Other roles of dnaA in transcription and nucleoid structure are additionally considered.A hitherto unknown function for either DnaA in replication restart or PriC in initiation of DNA replication that occurs in certain E. coli K-12 and are described in Table All bacterial strains used in this work are derivatives of priC containing plasmid was called pTH1.Wildtype chromosome DNA was used as the template in a standard PCR reaction using prSJS283 and prSJS284 (Table TH carried out the initial part of the molecular genetic studies. These were completed by SJS. SJS conceived of the study and wrote the manuscript.


Darwin first connected pigmentation with adaptive advantage, noting that male finches with bright red plumage enjoyed greater reproductive success than their drab competitors. Explaining why coloration confers such advantages, however, has proved somewhat easier than showing how it arises. Biologists studying how neighboring regions of the vertebrate body plan develop differences in appearance and form have identified a small number of signaling pathways common to all animals. How and whether these pathways also control the developmental expression and variation of surface attributes like hair color, hair density, and hair length are unclear. By studying an old mouse mutant called Agouti gene in the ventral and dorsal regions of the developing mouse; Agouti produces a pale yellow color and thus mice with light bellies have Agouti expressed in their ventral but not dorsal region. Droopy ear was discovered more than 50 years ago by virtue of its effects on head and ear shape, but it also affects pigmentation patterns; mutant mice have expanded ventral-specific Agouti expression into the dorsal region.Many vertebrate species—be they fish, bird, or mammal—have a much lighter belly than back. Studies in mice indicate these dorsoventral pigment differences arise from differential expression of the droopy ear. When the researchers homed in on the chromosomal region known to harbor droopy ear, they found Tbx15—a member of the T-box gene family. T-box genes are found in a wide range of species and play diverse roles during embryonic development. In the droopy ear mouse, Tbx15 carries a mutation that makes the protein nonfunctional. The researchers made certain that Tbx15 really is the droopy ear gene by deleting most of the gene's coding region and showing that this “knocked-out” gene produces the typical droopy ear mouse.First, Sophie Candille, a graduate student in Barsh's laboratory, searched for the gene that underlies the defect in Tbx15 expression—determined by observing messenger RNA transcripts in developing tissues of the head, trunk, and limbs—suggests that early expression of Tbx15 in the dorsal flank sets coordinates for dorsoventral differences in hair length and pigmentation. Candille et al. demonstrate that the regional pigment differences characteristic of adults is indeed established soon after embryonic Tbx15 expression. So this boundary in pigmentation is set up very early during development. Interestingly, the early coordinates of the future pigment boundary do not correspond to any other known developmental boundary.The pattern of embryonic Tbx15 pigmentation effects seen in these lab mice, the researchers note, resembles coat variations in other mammals, including German shepherds and an endangered mouse whose lighter dorsal markings once gave it an adaptive advantage on the white sand reefs where it lives . T-box genes are also found in humans; mutations in Tbx1, Tbx4, Tbx5, and Tbx22 can cause developmental abnormalities of the heart, limbs, or of the head and neck. Mutations of human Tbx15 have not yet been identified, but could contribute to regional differences of pigmentation  or to development of the head and neck. The identification of Tbx15 adds a new player to the genes that help pattern the developing embryo—attention now turns to the controls that regulate Tbx15 and the Tbx15 targets, which set up the pattern.The


The engineering of green fluorescent protein (GFP) fusion constructs in order to visibly tag a protein of interest has become a commonly used cell biology technique. Although caveats to this approach are obvious, literature reports in which the chimeric molecule behaves differently than the native molecule are scant. This brief report describes one such case. Granulysin, a small lytic and antimicrobial protein produced by cytotoxic lymphocytes, traffics to the regulated secretory system and is subsequently released from cells upon proper stimulus. In an attempt to elucidate mechanisms by which it accumulates in and is released from cytolytic granules, GFP was fused to the C-terminus of granulysin and expressed in an NK cell line. A control construct expressing the native protein was similarly expressed. The data demonstrate that, while the fusion protein is expressed and secreted, its subcellular localization is altered in comparison to native granulysin. Thus, the addition of GFP to the C-terminus of granulysin obscures the signal(s) that cytotoxic lymphocytes use to sort it to the regulated secretory pathway despite its normal biosynthesis and secretion. This example is offered as a cautionary account for other researchers who contemplate using this technology. Aequoria victoria, termed green fluorescent protein (GFP), can be used to visualize dynamic processes in live cells in real time . Next, samples were incubated for 30 minutes with primary antibodies to granulysin (rabbit antisera) and perforin (mouse mAb) diluted in staining buffer. After washing 3X with staining buffer, samples were incubated 30 minutes with species-specific fluorescent Alexa 488 goat anti-rabbit and Alexa 568 goat anti-mouse secondary antibodies (Molecular Probes). Cells were then washed 3X with staining buffer, then twice with PBS, and glass coverslips mounted using 50% (v/v) glycerol in PBS. Images were collected using a BioRad MRC 1024 confocal imaging system mounted on a Nikon Diaphot inverted microscope.DAH designed the study, carried out all experimental procedures, and drafted the manuscript. SFZ consulted on the experimental design, provided the resources that allowed the study to be conducted and edited the manuscript. Both authors read and approved the final manuscript.


Pediculus humanus, are composed of two ancient lineages, whose origin predates modern Homo sapiens by an order of magnitude (ca. 1.18 million years). One of the two louse lineages has a worldwide distribution and appears to have undergone a population bottleneck ca. 100,000 years ago along with its modern H. sapiens host. Phylogenetic and population genetic data suggest that the other lineage, found only in the New World, has remained isolated from the worldwide lineage for the last 1.18 million years. The ancient divergence between these two lice is contemporaneous with splits among early species of Homo, and cospeciation analyses suggest that the two louse lineages codiverged with a now extinct species of Homo and the lineage leading to modern H. sapiens. If these lice indeed codiverged with their hosts ca. 1.18 million years ago, then a recent host switch from an archaic species of Homo to modern H. sapiens is required to explain the occurrence of both lineages on modern H. sapiens. Such a host switch would require direct physical contact between modern and archaic forms of Homo.Parasites can be used as unique markers to investigate host evolutionary history, independent of host data. Here we show that modern human head lice,  A phylogenetic analysis reveals that humans have two types of head lice and that one must have switched from an ancient to a modern human host, suggesting these humans had contact Homo sapiens. The debate concerns the precise manner in which anatomically modern humans arose from archaic ancestors. Empirical studies tend to support one of two prominent models of human origins, the Recent African Replacement model , which led to some degree of regional continuity. Both models can be subdivided into many variants. There are two common variants of the Multiregional Evolution model. In one variant, the transition from archaic to modern humans occurs incrementally across a large geographic region ; in the other variant, the transition from archaic to modern humans arises first in Africa then spreads through gene flow outside of Africa. This latter variant is very similar to a Diffusion Wave model recently put forth by One of the most intensely debated topics in evolutionary biology pertains to the origin of modern nt model  or the Mnt model . The Recnt model  suggestsH. neanderthalensis  data have provided additional insight into the recent evolutionary history of humans, but these data are limited mainly to extant human populations. Ancient DNA was recently sequenced from halensis , 2000 an sapiens , but eve sapiens . Only a  sapiens  or very  sapiens  fossil m(Insecta: Phthiraptera)  .(Pediculus humanus), the focus of this paper, and pubic lice(Pthirus pubis), which serve as a phylogenetic outgroup in this study. P. humanus is found in two forms (head and body lice) that are morphologically similar, but ecologically distinct. Body lice live primarily in clothing and move onto the skin to feed once or twice a day. Head lice are confined to the scalp and feed more frequently. Body lice vector the bacteria responsible for epidemic typhus, trench fever, and relapsing fever; head lice are not known to vector any agent of human disease under natural conditions  to see which model best fits the data from human lice.We reconstructed the evolutionary history of Pediculus species on chimpanzees and humans are sister taxa, which together with Pthirus form a clade that is sister to Pedicinus, the most basal member of the ingroup (p < 0.01) between the louse and primate phylogenies, thus validating the assumption of cospeciation . One of the two lineages in our data set is worldwide (WW) in distribution (p < 0.01)  . The othP. humanus (1.18 MYA) is much older than that reported by P. schaeffi, that is quite aberrant when compared to other primate lice. Phylogenetic analysis of the Kittler et al. Cytb data (downloaded from GenBank), combined with our own data, shows that the Kittler et al. sequence for P. schaeffi is 40% divergent from P. humanus and 40% divergent from our own sequence of P. schaeffi. Phylogenetic analysis places their specimen of P. schaeffi outside all other primate lice and even outside the rodent louse . The estimate of expected coalescence is based on the effective female population size (Nef), which was estimated from the sample of all P. humanus specimens to be 1.1 million female lice from the equation Θ = 2Nefμ. The estimate of Nef provides an expected coalescence time for the two divergent mitochondrial lineages of P. humanus of 1.10 million generations or ca. 0.11 MYA, which is an order of magnitude younger than the observed divergence time of 1.18 MYA. In a large randomly mating population consisting of 1.1 million female lice, one would expect to maintain two distinct haplotypes for only ca. 0.11 million years (MY). This suggests that we can reject panmixia if we assume that Nef prior to the bottleneck was roughly similar to what we see today. If estimates of Nef were drastically higher  prior to the bottleneck, then expected time to coalescence could be much longer. The Fst value, a measure of genetic population differentiation, calculated for the WW and NW clades was 0.96, indicating substantial population structure, which also supports the rejection of panmixia.We can calculate an expected date of mitochondrial coalescence for P. humanus shows evidence of a recent population expansion (Fu and Li's D* = −2.80 [p < 0.02). We estimated the date of this population expansion from the mismatch distribution. The estimate was calculated by comparing the average pairwise difference within the WW clade of P. humanus (4.21 mutations) to the pairwise difference between P. humanus and P. schaeffi (220 mutations), which diverged 5.6 MYA. The population expansion of the WW clade is estimated to be 0.11 MYA, similar to the estimated date of population expansion of modern humans out of Africa ca. 0.10 MYA (P. humanus does not exhibit the signature of a recent population expansion (Fu and Li's D* = 0.17), but instead shows a more stable population size.The WW clade of  = −2.80 ; p < 0.00.10 MYA . In contP. humanus dates to 1.18 MYA (for mtDNA), which is roughly midway between the estimated ages of H. neanderthalensis (0.60 MY) and H. erectus (1.8 MY). We used a maximum likelihood (ML) analysis to test whether our two divergent lineages of lice could have diverged in tandem with H. sapiens and H. neanderthalensis . H. neanderthalensis is the only other species of Homo for which DNA sequence data are available  the H. sapiens/H. neanderthalensis split as a node of cospeciation with the two clades of P. humanus because the branch length between H. sapiens and H. neanderthalensis is far too short to explain the louse DNA sequence data. In other words, the split between H. sapiens and H. neanderthalensis is too recent to have been contemporaneous with the divergence of the two lineages of lice. If one artificially lengthens the branch between H. sapiens and H. neanderthalensis to approximate the split between H. sapiens and H. erectus (anywhere from 1.2 to 1.8 MYA), the LRT fails to reject this hypothesis of cospeciation.The age of the MRCA of vailable . The tesnce data . In cospnce data . A likelP. humanus could conceivably be the result of sequencing a nuclear copy of a mitochondrial gene. However, several lines of evidence strongly suggest otherwise. Because we amplified two different mitochondrial genes (COI and Cytb) that show the same divergent lineages and similar percent sequence divergences, copies of both mitochondrial genes would have had to enter the nucleus simultaneously, which is unlikely. In addition, we amplified each gene with a nested set of overlapping primers, and we never amplified more than one gene copy, even during bouts of cloning. Nucleotide base composition for our COI and Cytb data do not deviate from the mean values for all louse COI and Cytb sequences in GenBank (unpublished data), which would not be the case for a nuclear copy of a mitochondrial gene. Finally, the deep divergences seen in our mitochondrial genes are confirmed by preliminary analyses of nuclear data . Therefore, we are confident that the DNA sequences used in this study are mitochondrial in origin, and we must attempt to explain the occurrence of such ancient mitochondrial haplotypes in human lice.Morphological and molecular data agree that primates and their lice have been cospeciating for over 20 MY. Indeed, it is this cospeciation that permits us to use host fossil evidence to calibrate portions of the louse phylogenetic tree. This has resulted in the discovery of two extant lineages of human lice that diverged 1.18 MYA. This ancient divergence is surprising because humans, and presumably their lice, are thought to have passed through a population bottleneck ca. 0.05–0.10 MYA . Such boP. humanus . It is useful to determine an expected time to coalescence from the estimated Nef of 1.1 million female lice, even though this estimate seems high for a parasite of humans, who themselves have had very small effective population sizes  and recently went through a population bottleneck ; therefore, we must consider the alternative explanation of extensive population subdivision.Gene trees  can be considerably older than species trees, and therefore our louse mitochondrial lineages could predate the actual origin of the species ttleneck . AlthougH. sapiens could disrupt gene flow and allow the retention of very old lineages, making the age of P. humanus seem much older than it actually is. However, there is no evidence of such pervasive geographic isolation in the modern human hosts of these lice. Other species of lice have been shown to have substantial geographic substructure  even when hosts show no geographic isolation  would show signs of the recent population expansion of humans during the last ca. 0.10 MY. However, only the WW clade shows evidence of this event, which very closely matches the timing of human population expansion.Substantial isolation among populations of lice on modern solation . If popuH. sapiens, we conclude that this lineage has a common evolutionary history with modern H. sapiens. In contrast, the NW clade appears to have diverged from the WW lineage 1.18 MYA, and has had a distinctly different evolutionary history. We are left unable to explain the retention of two ancient louse lineages, each with a different evolutionary history, within the confines of a single host, modern H. sapiens. Given the history of cospeciation between primate lice and their hosts, it is necessary to look beyond modern H. sapiens to determine whether the two divergent lineages of P. humanus are legacies of a more ancient divergence.Because the WW clade is commonly found worldwide, and shows a population expansion concurrent with that of modern H. neanderthalensis as having diverged from H. sapiens contemporaneously with the two divergent lineages of lice. The mitochondrial MRCA of Neandertals and humans is 0.60 MYA . We refer to them as “species” mostly for convenience of writing. Whereas the WW lineage has population genetic characteristics that are similar to those of modern H. sapiens, the geographically restricted NW lineage does not. It likely evolved on a now extinct species of Homo only to switch to modern H. sapiens very recently. For example, H. erectus and switched to modern H. sapiens. Interestingly, Taenia asiatica, is entirely restricted to Asia. This is consistent with the depiction in T. asiatica evolved on H. erectus. Although divergence dates are not available, it is intriguing that some Native American strains of HTLV have closer affinities to Asian primate strains than to other human strains of HTLV, suggesting an independent Asian origin of this virus in humans. One must still explain how these parasites came to be on modern H. sapiens, but taken together, the parasitological evidence  suggests that they might have evolved on H. erectus and switched recently to H. sapiens. If true, this implies that H. erectus was contemporaneous with modern H. sapiens in eastern Asia, as suggested by There is much debate regarding the past 2 MY of hominid evolution. However, one area of broad agreement is that, prior to 2 MYA, our ancestors were confined to Africa, then left the continent ca. 1.8 MYA. This first migration out of Africa resulted in archaic species of  sapiens . The 1.1H. sapiens arose as the result of a speciation event ca. 0.13 MYA, and then replaced without introgression  non-African archaic humans . In contrast, the sensu stricto Multiregional Evolution model assumes that modern H. sapiens evolved from early African descendants (up to 2 MYA). Characteristics of modernity were spread geographically through intercontinental gene flow, but local regional characteristics were maintained through admixture between modern and archaic forms (H. sapiens left Africa (ca. 0.13 MYA) and replaced archaic humans through a process of introgression, natural selection, and gradual demic expansion.Explanations of modern human origins are dominated by two competing models, Recent African Replacement and Multiregional Evolution. The Recent African Replacement model assumes that anatomically modern ic forms . Most deP. humanus is somewhat consistent with all three models of modern human origins mentioned above; however, the number of ad hoc assumptions required to reconcile host and parasite evolutionary histories varies among the three views of human origins. Each model can account for the deep divergence between the two clades of P. humanus because each recognizes divergences between archaic species of Homo ca. 2 MYA. However, the model that best fits the louse data must account not only for the 1 MY of isolation between archaic and modern forms of lice, but also for a recent population expansion in just one louse lineage, the WW clade. The model must also explain how archaic louse DNA might have been incorporated into the lice of modern H. sapiens.The evolutionary history of H. sapiens. This scenario does not provide the louse populations with the degree of isolation necessary (ca. 1.18 MY) to maintain the two divergent louse lineages, unless we assume that gene flow between human populations was considerably greater than gene flow among their populations of lice. There is no reason to assume such a disparity in gene flow between hosts and parasites. The Multiregional Evolution model also predicts that we should detect the same genetic fingerprint of recent population expansion in both clades of P. humanus, which we do not.The sensu stricto model of Multiregional Evolution  predictsH. sapiens left Africa ca. 0.10 MYA, more than a million years after archaic species of Homo left Africa, and that the modern and archaic humans remained distinct . Furthermore, it explicitly assumes a recent population expansion in modern H. sapiens, which would account for the population expansion seen in our WW clade. However, in the strict sense, this model also predicts that modern H. sapiens replaced archaic forms of humans without introgression , which leaves no obvious mechanism for archaic louse DNA to reach the lice of modern H. sapiens. This lack of host introgression implies, but does not require, a lack of direct physical contact between modern and archaic humans. It is conceivable that direct contact between modern and archaic humans was sufficient to allow the lice to switch hosts without making the assertion that the hosts were interbreeding. Therefore, the Recent African Replacement model is fairly consistent with the louse data, so long as one assumes some level of direct contact  between modern and archaic humans.The Recent African Replacement model provides the isolation necessary between archaic and modern forms, because it assumes that modern H. sapiens leaving Africa (ca. 0.13 MYA), modern and archaic humans had distinct types of lice owing to ≥1 MY of isolation. As modern humans began to replace archaic forms, direct contact between hosts during introgression allowed archaic lice to switch to modern H. sapiens hosts.Eswaran's Diffusion Wave model (2002) is similar to the Multiregional Evolution model in that it permits some level of introgression between modern and archaic humans. However, it is also similar to the Recent African Replacement model in that it assumes the same recent population expansion of modern humans out of Africa (ca. 0.10 MYA), thus providing both the isolation and population expansion necessary to accommodate our louse data. The additional assumption of introgression between modern and archaic forms of humans, which is proposed to have occurred only in the last 0.10 MY, provides a ready vehicle that would have transported archaic louse DNA into the modern louse population. Eswaran's model applied to lice suggests that at the beginning of the diffusion wave of modern As previously stated, the recent literature addressing human origins boils down to models that do not permit introgression (strict-sense replacement models) and those of many types that do . All things being equal, our parasite data are most consistent with a limited amount of admixture between modern and archaic humans, because this process presents the opportunity for host switching. However, introgression between modern and archaic humans over a protracted period of time would erode the isolation required to maintain the two louse lineages that we have observed. For example, some variants of the Multiregional Evolution model reject a single origin of modernity in Africa ca. 0.13 MYA in favor of a piecemeal acquisition of modern traits over a long period of time. This long-term admixture is precisely what would disrupt the isolation required to maintain the two louse lineages. Eswaran's Diffusion Wave model, on the other hand, confines admixture to the last ca. 0.10 MY.Pthirus pubis, the human pubic louse, might shed light on the subject of human admixture because unlike head and body lice, pubic lice are primarily transmitted during intercourse.Our data cannot directly address whether host introgression occurred, because nonsexual, direct contact between hosts is sufficient for parasite transmission. We are confident that “direct contact” would be required for a host switch because these obligate parasitic lice cannot move between individuals without direct physical contact  and furtH. erectus to H. sapiens were true, then the host switch would have brought together two long-separated taxa of lice. It is impossible to know whether this long separation affected the reproductive compatibility of the two louse taxa once reunited. Discriminant function analysis shows no morphological differences between members of the two divergent molecular haplotypes of head lice  compared to the WW clade (0.54 MYA). Presumably, the archaic form  of louse either was extirpated along with its host or was assimilated into modern P. humanus. Regardless of the mechanism, ancient louse lineages can be found among the lice of modern H. sapiens.If our scenario involving lice switching from lice see . There alice see  whose pofive such pairs caused Ashford to ask, were humans once two distinct populations that rejoined after a long separation? The ancient divergences seen in mitochondrial data from P. humanus are clearly consistent with some level of long-term host isolation, and preliminary evidence from nuclear markers  reveals similarly ancient divergences (unpublished data). Furthermore, the two tapeworm species from humans showed amazingly concordant divergences  from many localities, ranging from remote areas such as the Papua New Guinea highlands to metropolitan areas like Boston (P. schaeffi (from chimpanzees), Pthirus pubis (from humans), Pedicinus hamadryas (from baboons), and Fahrenholzia pinnata (from a rodent) to use as outgroup taxa in phylogenetic analyses. All lice were preserved in 95% EtOH and stored at −80 °C. DNA was extracted from lice by separating the thorax and abdomen and placing both in digestion buffer . Digestion proceeded for 48 h at 55 °C, then followed the manufacturer's protocol. After digestion, each louse was reassembled on a microscope slide as a voucher specimen corresponding to each DNA sequence. Voucher specimens were deposited in the Price Institute of Phthirapteran Research (PIPeR) collection at the University of Utah.We collected human head and body lice e Boston . We alsoP. humanus (from humans), P. schaeffi (from chimpanzees), Pthirus pubis (from humans), Pthirus gorillae (from gorillas), Pedicinus hamadryas (from baboons), and F. pinnata (from a rodent). Morphological data were scored in the software package MacClade v. 4.05 , and heuristic searches consisting of random stepwise addition  and tree bisection/reconnection branch swapping were performed in PAUP* v. 4.0b10 . Branch support was estimated with bootstrapping . The complete data matrix is available from TreeBASE (http://www.treebase.org/) as study accession number SN1969.Considerable morphological variation exists among different species of primate lice. We examined 155 unordered morphological characters for 113 specimens of The morphological data set  was compared to the host phylogeny , gorilla), baboon), rodent) using reconciliation analysis in Treemap v. 2.0 with default parameters. Treemap determines whether the two phylogenies are more congruent than expected by chance based on randomizations of both the host and parasite phylogeny. Significant congruence between host and parasite phylogenies is interpreted as being the result of a shared evolutionary history .P. humanus lice in detail to test for morphological correlates of the differences detected at the molecular level. P. humanus. The predictor variables were used to build a set of discriminant functions that maximized variation among groups while minimizing within-group variation. The first two canonical discriminant functions explained 100% of the variation within the data.We examined the morphology of These discriminant functions, which were built using existing museum specimens, were then applied to our newly collected specimens in a blind test to determine whether the specimens could be identified as head or body lice from morphology alone. We were able to classify our samples as head or body lice with a probability of ≥0.95. Indeed, the assignment of adult specimens proved to be 100% accurate when checked against microhabitat data for the new specimens.P. humanus, P. schaeffi, Pthirus pubis, Pedicinus hamadryas, and F. pinnata. PCR primers were as follows: (5′−3′) COI, C1-J-1718 GGAGGTTTTGCTAATTGATTAG and H7005 CCGGATCCACNACRTARTANGTRTCRTG; Cytb, L11122 GAAATTTTGGGTCWTTRCTNGG and H11823 GGCATATGCGAATARGAARTATCA. PCR parameters included 94 °C for 30 s, 48 °C for 30 s, and 72 °C for 1.5 min (five cycles), then 30 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 1.5 min. Amplified fragments were sequenced in both directions, assembled using Sequencher v. 4.1 , and deposited in the NCBI database  of the mitochondrial (mtDNA) genes COI (854 bp) and Cytb (671 bp) from 69 individuals of base see . To ensuThe computer program modelTest  was usedPedicinus and Pediculus + Pthirus. The date is based on fossil evidence . Each of these bootstrapped datasets was calibrated with the same 22.5 ± 2.5 MYA divergence.We used the significant cospeciation shown between the primate and louse phylogenies see  as a basevidence  of the sediculus . All recediculus . Using tHomo. We calculated the branch length between H. sapiens and H. neanderthalensis (the only species of Homo for which DNA sequence data are available) based on the human mitochondrial hypervariable region II of the D loop (see P. humanus to conform to a prescribed relative branch length. The resulting likelihood score was compared with the unconstrained tree score using an LRT (d.f. = taxa − 2). If the constrained and unconstrained tree scores are not significantly different, then the host tree topology and branch lengths describe the parasite DNA sequence data as well as the parasite tree itself . The computer software package DnaSP . Ten additional haplotypes from P. humanus, we used estimates of theta  to calculate louse Nef from the equation Θ = 2Nefμ. The mutation rate (μ = 9.0 × 10−9 substitutions per site per generation) was calculated by determining the expected number of substitutions per site between P. humanus and P. schaeffi under the Tamura-Nei + Γ model of nucleotide substitution. This mutation rate for P. humanus is roughly five to six times faster than that of human mtDNA, excluding the D-loop  given the formula 2Nef (1 − 1/n), where n is the number of haplotypes detected in the population. One can also ask the similar question, what is the probability that two lineages, which are expected to differ by ke substitutions, actually differ by k* or more substitutions? The expression used, (ke /(1+ ke) ^ k*), is derived from the geometric distribution .Population genetic analyses were performed on a pruned dataset, which contained only specimens of ge DnaSP  was usedge DnaSP ) and mute D-loop , when bohttp://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for items discussed in the text are as follows: the EF1-alpha sequences from P. humanus from P. schaeffi, AY695939; the Cytb sequence from P. schaeffi, AY316793; the human mitochondrial hypervariable region II of the D loop, M76311, AY195756, AY217615, AF282972, AF142095, X97709, X98472, X93336, X93337, X93347, and X93348; the ten haplotypes from Dennyus hirundinus shown in GenBank (The amplified PCR fragments discussed in