Plasmodium parasites is crucial for targeted vaccine development. While studies related to the genetic diversity of these proteins are available for Plasmodium falciparum and Plasmodium vivax, barely enough information exists regarding Plasmodium malariae. The present study aims to demonstrate the genetic variations existing among these three genes in P. malariae by analysing their diversity at nucleotide and protein levels.Genetic diversity of the three important antigenic proteins, namely thrombospondin-related anonymous protein (TRAP), apical membrane antigen 1 (AMA1), and 6-cysteine protein (P48/45), all of which are found in various developmental stages of Three surface protein genes were isolated from 45 samples collected in Thailand (N = 33), Myanmar (N = 8), and Lao PDR (N = 4), using conventional polymerase chain reaction (PCR) assay. Then, the PCR products were sequenced and analysed using BioEdit, MEGA6, and DnaSP programs.P. malariae trap, ama1, and p48/45 were 0.00169, 0.00413, and 0.00029, respectively. The haplotype diversities (Hd) of P. malariae trap, ama1, and p48/45 were 0.919, 0.946, and 0.130, respectively. Most of the nucleotide substitutions were non-synonymous, which indicated that the genetic variations of these genes were maintained by positive diversifying selection, thus, suggesting their role as a potential target of protective immune response. Amino acid substitutions of P. malariae TRAP, AMA1, and P48/45 could be categorized to 17, 20, and 2 unique amino-acid variants, respectively. For further vaccine development, carboxyl terminal of P48/45 would be a good candidate according to conserved amino acid at low genetic diversity (π = 0.2–0.3).The average pairwise nucleotide diversities (π) of P. malariae trap and ama1 as compared to p48/45 in P. malariae samples isolated from Thailand, Myanmar, and Lao PDR. Taken together, these results suggest that P48/45 might be a good vaccine candidate against P. malariae infection because of its sufficiently low genetic diversity and highly conserved amino acids especially on the carboxyl end.High mutational diversity was observed in The online version of this article (10.1186/s12936-018-2176-x) contains supplementary material, which is available to authorized users. Plasmodium malariae-related incidents are mostly observed in endemic areas [Plasmodium falciparum or Plasmodium vivax infections [P. malariae infection indicating much higher prevalence of the disease than previously expected in many regions of the world [P. malariae infection often displays low parasitaemia and can occur as mixed infection involving P. falciparum or P. vivax [P. malariae infection can remain asymptomatic for longer period of time and lead to increase in mortality by causing chronic nephrotic syndrome [P. malariae is an important species that should be included in malaria elimination programmes [msp1 gene has been previously analysed for P. malariae [ic areas  and havefections . Severalhe world –5. MoreoP. vivax . Similarsyndrome –13. Hencmalariae .P. malariae namely thrombospondin-related anonymous protein (PmTRAP), apical membrane antigen 1 (PmAMA1), and 6-cysteine protein (PmP48/45) which are known to play significant role in developmental stages of the parasite. TRAP is a sporozoite surface protein essential for sporozoite gliding, motility, and hepatocyte invasion [P. falciparum (ChAd63-MVA ME-TRAP), which could reduce burden of the liver parasite by 79–84% [P. falciparum malaria [In this context, the investigation was focused on three important antigenic proteins found in invasion –17. Thisy 79–84% . Likely,y 79–84% –24. Usin malaria . P48/45  malaria . Moreove malaria .trap, ama1, and p48/45 genes of P. falciparum and P. vivax have been analysed and reported by multiple independent studies [P. malariae has been recently reported for its evolutionary history and phylogenetic classification [P. malariae. Thus, the present study is probably the first investigation aimed at isolation and analysis of genetic diversity of genes coding for these three surface protein in P. malariae. In particular, population genetic of P. malariae was analysed based on three genes namely, P. malariae thrombospondin-related anonymous protein gene (pmtrap), P. malariae apical membrane antigen 1 gene (pmama1), and P. malariae 6-cysteine protein gene (pmp48/45). In addition, the study also includes comparative analysis of three surface proteins across six human Plasmodium species. Findings from the current study might contribute to crucial information needed for further understanding and potential vaccine development against P. malariae infection.Genetic diversity arising through antigenic polymorphisms can cause change in expression of critical epitopes resulting in reduction or even complete loss of vaccine efficacy. The success of an effective long-term vaccine development is, therefore, depended on wide-scale assessment of the degree of genetic polymorphism among parasite populations in malaria endemic areas. Genetic diversity of  studies –30. Althfication , there bP. malariae infected patients from Thailand (N = 33), Myanmar (N = 8), and Lao PDR (N = 4) in 2002–2013 (Table Plasmodium species using polymerase chain reaction (PCR) based on 18 small-subunit ribosomal RNA [P. malariae.DNA samples were extracted from total of 45 symptomatic omal RNA , 33 to cPlasmodium malariae trap, ama1 and p48/45 genes were amplified using either nested or semi-nested PCR using specifically designed primers with the optimized MgCl2 concentrations and annealing temperatures, which were validated previously , the number of haplotypes (H), and haplotype diversity (Hd) using DnaSP v5 [P. malariae trap, ama1, and p48/45 isolated from Uganda, accession numbers LT594633.1, LT594630.1, and LT594500.1, respectively. In addition, we also aligned the nucleotide sequences of P. malariae trap, ama1, and p48/45 with sequences isolated from Malaysia, Indonesia, and Guinea, accession numbers ERS1110317, ERS1110321, and ERS567899, respectively [The overall analysis procedure was divided into four major sections. Firstly, the nucleotide polymorphism of DnaSP v5 . The nucP. malariae TRAP, AMA1, and P48/45 were identified. Partial nucleotide sequences of P. malariae trap, ama1, and p48/45 were translated to deduced amino acid sequences using BioEdit and then aligned with references amino acid sequences of P. malariae TRAP, AMA1, and P48/45, accession numbers SCO93694.1, SCN12851.1, and SBT79956.1, respectively. The functional domains of these three proteins based on their reference amino acid sequences were predicted using InterPro [Secondly, the amino acid substitutions and haplotype diversity of InterPro  , AMA1 (71.40%) and P48/45 (63.34%), respectively. Additional comparison of P. malariae TRAP, AMA1, and P48/45 with P. falciparum and with P. vivax showed that the homology of P. malariae and P. vivax TRAP (45.97%), AMA1 (68.68%), and P48/45 (61.40%) were higher than the homology of P. malariae and P. falciparum TRAP (36.44%), AMA1 (56.77%), and P48/45 (58.90%), respectively  parasites obtained from previous study [P. malariae ama1 as compared to trap and p48/45. Moreover, nucleotide diversity and haplotype diversity of P. malariae trap, ama1 and p48/45 isolated during this study were compared with P. falciparum and P. vivax from previous reports [P. malariae trap, ama1 and p48/45 showed lower nucleotide diversity than P. falciparum and P. vivax (P < 0.05).Nucleotide diversity of us study . The stu reports , 30, 40 trap, ama1, and p48/45 from all P. malariae samples were translated to deduce their corresponding amino acids and analysed. The trap, ama1, and p48/45 of P. malariae were intron less genes. The translated PmTRAP was found to be located on amino acid position 58–608 of P. malariae reference strain PMUG01 accession number SCO93694.1. The tandem repeat of 12 nucleotide unit corresponded to the three tetrapeptide unit including PEDR, PENR, and PENS, located within TSP1 domain. Number of total repeat unit among those 35 samples of P. malariae isolates varied from 9 to 17 repeats. The major repeat unit was PEDR which could be found from 8 to 14 units in all isolates. All of the isolates contained only one unit of PENR, except for none in one isolate from Myanmar. The repeat unit PENS also presented for 1–2 units in most P. malariae samples, whereas only two isolates of P. malariae collected in Thailand had none. Analysis of PmTRAP in non-repeat region showed seven polymorphic sites that resulted from non-synonymous mutations. Those seven polymorphic sites were 62 (S/F), 105 (I/V), 139 (F/L), 236 , 454 , 488 , and 494 . Alignment of the 35 PmTRAP isolates revealed 17 variant types, of which the variant 9 (SILIRHD) was seen in 8 out of 35 isolates, thus, making it the most frequently observed variant type among the samples  and 203  , − 1.243 (ama1), and − 1.479 (p48/45). Besides, these three population genetic tests, the additional test for neutrality  [pmtrap, pmama1 and pmp48/45 sequences to determine whether polymorphism takes place at higher or lower frequencies than expected under a neutral model. Tajima’s D, Fu and Li D*, and Fu and Li F* tests using DnaSP v5 and MEGA6 were performed for pmtrap, pmama1 and pmp48/45. The results from Tajima’s D value was 1.83835 for pmama1 (P < 0.10), which was similar to Fu and Li’ F* test value 1.71236 (P < 0.05) . Moreover, Fu’s Fs statistic was negative for pmtrap and pmama1 which indicated population expansion of trap and ama1 of P. malariae in the study samples.Population genetics of i method  with Juk* tests) , 39 was P. malariae is more widely circulated in malaria endemic regions than previously acknowledged. Hence, this makes study of P. malariae essential to be included in malaria elimination programme [P. malariae has limited limits current understanding of the parasite.The development of modern high-throughput ultrasensitive diagnostic assays have demonstrated that rogramme –5. Effecpmp48/45 has the lowest diversity among all of the studied genes. There were only two unique amino-acid variants found in PmP48/45 compare to 17 and 20 amino-acid variants observed in PmTRAP and PmAMA1, respectively. Moreover, results from comparative analysis of six human Plasmodium species showed lowest diversity of p48/45 among the parasites. In accordance to these finding, highly conserved expression of p48/45 have been previously reported in P. falciparum [P. vivax [p48/45 nucleotide sequence coding for P48/45 protein and its relatively conserved amino acid residues may be linked to the gametocyte stage where it its mainly expressed. Interestingly, this protein has been previously investigated in other Plasmodium species for development of potential malaria vaccine targeting transmission stage of the parasite [Genetic analysis for nucleotide and haplotype diversity suggests that lciparum  and P. vP. vivax  isolatesP. vivax . Low varparasite .P. malariae ama1 suggests that P. malariae may have substantial potency to exhibit genetic variation or acquire mutation on pmama1 protein expression. In particular, most of the polymorphism were found at N-terminal position indicating possibility of directional mutation of pmama1 gene favoring N-terminal-linked functions of the protein. The genetic diversity observed in P. malariae trap was comparatively lower than pmama1. These results are in agreement with the previously published report of similar pattern of high nucleotide diversity found in P. falciparum ama1 as compared to P. falciparum trap in regions of America, Asia Pacific, and Africa [ama1 of six Plasmodium species might be related to the stage of parasite infection and functional aspect of the protein. The Plasmodium expresses AMA1 during merozoite stage of the parasite where it has to actively evade host immunity. Thus, acquiring genetic diversity in such crucial functional proteins can assist avoidance of host detection and subsequent neutralization of the parasite. Since AMA1 and TRAP both are expressed during active infection stages of Plasmodium, the evolutionary requirement level of genetic polymorphism might have been considerably higher for these genes as compared to P48/45 which is mainly expressed in gametocytes. In addition, within the P. malariae trap, the twelve nucleotides repeat was frequently observed for 9–17 times in the samples indicating possibility of this repeat to be used as genotyping marker.Noticeable levels of nucleotide polymorphism observed within d Africa . This nopmtrap and pmama1 genes analysed during this study exhibited relatively more nucleotide diversity than pmp48/45. Both of these genes and their expression proteins have been previously targeted for development of pre-erythrocytic and blood stage vaccines against P. falciparum based on their immunogenic and conserved regions [P. malariae TRAP and AMA1, which might be useful as potential vaccine target against the parasite. These regions containing largest amount of conserved amino acid sequences for PmTRAP (N = 35) and PmAMA1 (N = 43) were found to be located in positions 237–453 and 18–107, respectively. In addition, the nucleotide diversities observed in pmtrap and pmama1 during our observation were significantly lower compared to the levels previously reported in P. falciparum and P. vivax [P. malariae targeting conserved regions of PmTRAP and PmAMA1.The  regions , 25. TheP. vivax , 40. ThuP. malariae trap, ama1, and p48/45 genes suggesting positive or diversifying selection. In addition, the results from Z-test indicates that the polymorphisms in these genes were most probably achieved through positive diversifying selection. Such mechanisms have been reported to be favourable for the parasite to evade targeted host immune responses [P. malariae and their possible implications in malaria research.The ability of parasite to evolve and survive is greatly influenced by its ability to acquire genetic mutations to withstand host immunity and environmental stress , 40. Theesponses , 30, 40.P. malariae trap, ama1 and p48/45 showed lower nucleotide diversity compared to P. falciparum and P. vivax, which might have been linked to the lower transmission rate of P. malariae in these geographic regions [P. malariae samples analysed. Nonetheless, these findings were similar to previously published data that reported low level of sequence diversity of msp1 gene in P. malariae parasite as opposed to other Plasmodium species [In addition, the  regions  and the  species .trap, ama1, and p48/45 in P. malariae. Results of this study inevitability contributes to the malaria research and may potentially be a valuable resource for further vaccine development or genotyping marker.In summary, present investigation is the first reported study revealing genetic diversity of three surface protein coding genes namely Additional file 1. Specific primers and PCR conditions for isolation of 3 surface protein genes.Additional file 2. Domain prediction of P. malariae TRAP, AMA1, and P48/45 using InterPro.Additional file 3. Accession numbers of amino acid sequences and gene IDs of six Plasmodium TRAP, AMA1, and P48/45 obtained from database.Additional file 4. Amino acid identity of non-cytoplasmic region TRAP, AMA1, and P48/45 within six Plasmodium species.Additional file 5. Maximum likelihood trees of six human malaria parasites based on amino acid sequences of TRAP, AMA1, and P48/45.Additional file 6. PmTRAP1, PmAMA1, and PmP48/45 variant frequencies in Thailand, Myanmar, and Lao PDR.


Histological chorioamnionitis (HCA) is related to perinatal morbidity. However, there is no definite diagnostic method for detecting chorioamnionitis before delivery.+0 to 36+6 weeks) at Severance Hospital from January 2013 to December 2014. The women were categorized into the following two groups: (a) PPROM without HCA and (b) PPROM with HCA. The maternal white blood cell (WBC) count, neutrophil-to-lymphocyte ratio (NLR), C-reactive protein (CRP) level, and DNI were measured at admission. The DNI has been reported to reflect the fraction of circulating immature granulocytes associated with infection.We evaluated whether the delta neutrophil index (DNI) was an effective early marker of HCA in patients with preterm premature rupture of membranes (PPROM). We retrospectively evaluated 149 women diagnosed with PPROM (gestational age, 20Of the 149 patients, 87 were included in the PPROM without HCA group and 62 were included in the PPROM with HCA group. The interval between admission and delivery was significantly shorter in the PPROM with HCA group than in the PPROM without HCA group. There was no significant difference in the maternal WBC count. The serum CRP level, NLR, and DNI were significantly lower in the PPROM without HCA group than in the PPROM with HCA group, while the lymphocyte count was significantly lower in the PPROM with HCA group than in the PPROM without HCA group. A predictive equation was generated by combining the DNI, lymphocyte count, and CRP level, and the sensitivity and specificity for predicting a placental inflammatory response were 69.1% and 70.5%, respectively.The DNI could be a predictive marker for HCA in patients with PPROM. Our predictive equation involving the DNI, lymphocyte count, and CRP level may be helpful for predicting the placental inflammatory response in patients with PPROM. Preterm premature rupture of membranes (PPROM) is one of the leading causes of preterm birth , and appRecent studies have shown that the serum delta neutrophil index (DNI) is associated with diverse infectious conditions, such as bacterial peritonitis, acute appendicitis, and sepsis –11. The Therefore, we conducted this study to determine whether the DNI is an effective early marker of HCA in patients with PPROM and to develop a non-invasive, easy, and prompt predictive equation that combined maternal serum markers and the DNI.+0 and 36+6 gestational weeks from January 2013 to December 2014 at Severance Hospital in Seoul, Korea were enrolled in this study. This study was approved by the Institutional Review Board of the Yonsei University Health System. We excluded women with multiple pregnancies, fetal anomalies, missing placental pathologic results, and medically indicated preterm birth. Additionally, pregnant women with hematologic or any other autoimmune disease affecting hematologic parameters were excluded.A total of 149 pregnant women with PPROM between 20Ureaplasma urealyticum, and Mycoplasma hominis. The serum CRP levels were determined using an automated nephelometer  according to the manufacturer’s instructions. The detection limit of this analysis was 0.01 mg/dL, and the normal serum CRP level was considered to be <0.8 mg/dL [Clinical data included demographic variables, such as maternal age; body mass index (BMI); preterm birth history; gestational age at diagnosis; and maternal WBC count, neutrophil count, lymphocyte count, neutrophil-to-lymphocyte ratio (NLR), C-reactive protein (CRP) level, and DNI. The diagnosis of PROM was based on clinical visual examination of amniotic fluid pooling in the vaginal cavity during sterile speculum examination or a positive nitrazine test. If the nitrazine test result was inconclusive, we performed the placental alpha microglobulin-1 test (AmniSure test). Sterile cotton swabs were used to obtain vaginal secretion samples for culture in order to assess group B streptococcus (GBS), .8 mg/dL , 13. The.8 mg/dL , 14, 15.Placentas were sent to the pathologic department for detection of infection. Histopathologic chorioamnionitis was defined as microscopic evidence of infection on any part of the placenta, and deciduitis was defined as infection of the decidua. Funisitis was defined as infection of the connective tissue of the umbilical cord .All pregnant women with PPROM included in this study were divided into the following two groups according to their pathologic results: (a) patients with PPROM without HCA and (b) patients with PPROM with HCA. All patients with PPROM received prophylactic intravenous antibiotics and 12 mg intramuscular betamethasone injection twice at a 24-hour interval for lung maturation. Tocolysis was used if patients had preterm labor. All pregnant women were treated until 34 gestational weeks if they showed no signs of clinical chorioamnionitis. If clinical chorioamnionitis occurred, delivery was performed.t-test or Wilcoxon rank sum test was used for continuous variables. Multiple logistic regression analysis was performed to estimate the odds ratio of HCA prediction with adjustment for confounders. The combination of significant parameters was used to generate a predictive equation. All statistical analyses were performed using SAS version 9.2 . Statistical significance was set at a p-value <0.05.Continuous variables are expressed as means and standard deviations (SDs) or medians and ranges. The chi-squared test or Fisher’s exact test was used for categorical variables, and the two-sample +0 and 36+6 gestational weeks. Of the 149 patients, 87 were included in the PPROM without HCA group and 62 were included in the PPROM with HCA group, and we compared the clinical characteristics and perinatal outcomes between these groups .The study included 149 pregnant women who had PPROM between 20e groups . There wThe distribution of the placental pathologic findings among the study patients is shown in p = 0.001), DNI values were 0 (0–2.6) and 0.25 (0–4.7) (p = 0.006), and CRP levels were 3.3 (0.6–32.0) and 7.5 (0.6–65.2) (p = 0.008) in the PPROM with HCA group and PPROM without HCA group, respectively. The NLR, DNI, and CRP levels were significantly higher, while the Hb levels, Hct values, and lymphocyte counts were significantly lower in the PPROM with HCA group than in the PPROM without HCA group. The median neutrophil count was higher in the PPROM with HCA group than in the PPROM without HCA group; however, the difference was not statistically significant. Among the study population, the proportion of patients with positive culture results for Ureaplasma urealyticum was higher in the PPROM with HCA group than in the PPROM without HCA group . However, there was no difference in the proportion of patients with positive culture results for Mycoplasma hominis between the two groups (p = 0.102).To evaluate the differences in laboratory variables between the PPROM without HCA and PPROM with HCA groups, we compared the laboratory results, including vaginal culture findings, between the two groups . There wp = 0.009) for the CRP level, 0.348  for the lymphocyte count, and 1.818  for the DNI. Increases in the CRP level and DNI were significantly associated with HCA, while decreases in the lymphocyte count were significantly associated with HCA. The combination of the CRP level, DNI, and lymphocyte count yielded the greatest area under the receiver operating characteristic curve (0.746)  .f / (1 + ef), where f is a linear function of the selected parameters and e is the base associated with the natural logarithm. Integration of the parameters yields the following equation:A predictive equation was generated using these three parameters through logistic regression modeling. The estimated regression equation is defined as p = eWhen the predictive probability cutoff was 0.447 according to the Youden index, the sensitivity and specificity for predicting HCA were 69.1% and 70.5%, respectively.To our knowledge, this is the first study to investigate the usefulness of the DNI as a predictive marker for HCA in patients with PPROM. We found that the DNI was a useful non-invasive marker for detecting HCA in pregnant women with PPROM. Moreover, we identified a predictive equation for HCA derived from the combination of the CRP level, lymphocyte count, and DNI, which was more accurate than was the use of each factor alone. The predictive equation for HCA can be used for prenatal counseling and management planning in patients with PPROM.Intrauterine infection can be diagnosed based on amniocentesis; however, this approach is invasive and can result in complications. Recent studies have indicated that sterile intra-amniotic inflammation, which does not involve microorganisms, is highly associated with HCA , 18. RobMany researchers have suggested different combinations of biomarkers for predicting intrauterine infection. Ryu et al.  showed tp = 0.006). In addition, we evaluated the DNI value of those pregnant women without PPROM or chorioamnionitis who delivered healthy neonates after 37 gestational weeks. The DNI value in the normal pregnant group was 0 . In the logistic regression analysis, the odds ratio of the DNI was 1.818  and the odds ratio of the CRP level was 1.069 . The odds ratio was higher for the DNI than for the prevalently used CRP level. The DNI can also increase in various infectious conditions [In several studies, immature granulocytes have been investigated as predictors of sepsis because the number of immature granulocytes is known to increase in inflammatory conditions , 26. Hownditions . Therefo9/L  and that in the PPROM without HCA group increased to 8.67 × 109/L (3.42–23.41 × 109/L); however, the difference was not significant (p = 0.114). The lymphocyte count slightly decreased in the PPROM with HCA group, and this result is similar to that reported previously. The NLR is being increasingly used as a predictive marker for spontaneous preterm birth and intrauterine infection in preterm delivery [Many researchers have shown that the NLR is an independent diagnostic parameter for systemic inflammation and stress conditions, owing to the fact that leukocytes play a major role in these conditions through an increase in neutrophils and decrease in lymphocytes , 30. In delivery , 31. Thedelivery –34. In oCRP is an acute-phase protein produced by the liver, and it is widely used as an objective marker for detecting tissue damage and infection. As the CRP level increases in chorioamnionitis or neonatal sepsis conditions, it is used in the obstetrical field to detect infectious conditions. Previous studies reported that an increasing CRP level was highly associated with preterm delivery and poor perinatal outcomes in pregnant women with preterm labor or PPROM –37.The present study has some limitations. The DNI can only be calculated by measuring immature granulocytes using the ADIVA 2120 Hematology System. However, there are several hematology analyzers that can measure immature granulocytes, such as the Sysmex XE-series hematology analyzer  and Cell-Dyn series hematology analyzer . Although the measurement methods for immature granulocytes differ depending on the analyzer used, we could not identify any differences in the immature granulocyte values. Additionally, we conducted this study using only the DNI values obtained at the time when the patients with PPROM were admitted to the hospital. Therefore, changes in the DNI values are still unclear according to the course of PPROM. In addition, we were not able to prove the correlation between the severity of HCA and the predictive equation due to the unclear recording of the HCA stages in the pathologic results. Thus, a well-designed prospective study is needed in order to evaluate the usefulness of the DNI as a predictive marker of HCA.In conclusion, the serum DNI, a new inflammation parameter, could be a predictive marker for chorioamnionitis or funisitis in patients with PPROM. Our predictive equation involving maternal serum markers could be used as a non-invasive, inexpensive, and simple method for detecting the placental inflammatory response in women with PPROM.


In sub-Saharan Africa, infectious diseases and malnutrition constitute the main health problems in children, while adolescents and adults are increasingly facing cardio-metabolic conditions. Among adolescents as the largest population group in this region, we investigated the co-occurrence of infectious diseases, malnutrition and cardio-metabolic risk factors (CRFs), and evaluated demographic, socio-economic and medical risk factors for these entities. In a cross-sectional study among 188 adolescents in rural Ghana, malarial infection, common infectious diseases and Body Mass Index were assessed. We measured ferritin, C-reactive protein, retinol, fasting glucose and blood pressure. Socio-demographic data were documented. We analyzed the proportions  and the co-occurrence of infectious diseases , malnutrition , and CRFs . In logistic regression, odds ratios (OR) and 95% CIs were calculated for the associations with socio-demographic factors. In this Ghanaian population , the proportions were for infectious diseases 45% (95% CI: 38–52%), for malnutrition 50% (43–57%) and for CRFs 16% (11–21%). Infectious diseases and malnutrition frequently co-existed . Specifically, VAD increased the odds of non-malarial infectious diseases 3-fold . Overlap of CRFs with infectious diseases  or with malnutrition  was also present. Male gender and low socio-economic status increased the odds of infectious diseases and malnutrition, respectively. Malarial infection, chronic malnutrition and VAD remain the predominant health problems among these Ghanaian adolescents. Investigating the relationships with evolving CRFs is warranted. Infectious diseases and malnutrition still constitute major public health threats in sub-Saharan Africa. In 2015, communicable diseases, protein-energy malnutrition and micronutrient deficiencies ranked among the top 10 causes of disease burden in this region. Iron defplus obesity is 3.2% among boys and 10.4% among girls according to age- and sex-specific cut-offs of Body Mass Index (BMI).[At the same time, metabolic conditions, such as overweight, type 2 diabetes and hypertension are rapidly emerging in sub-Saharan Africa. Among Ghex (BMI). Type 2 dex (BMI). Moreoverex (BMI).plus overweight, and 5% of mother-child pairs showed childhood stunting plus maternal overweight.[The epidemiologic transition from infectious diseases and malnutrition to metabolic conditions due to increased life-expectancy and lower birth rates progresses slowly in sub-Saharan Africa. As a conerweight. Today, terweight. Yet, theerweight. For insterweight. and the For this cross-sectional study, 201 adolescent boys and girls were consecutively recruited at the Presbyterian Mission Hospital in Agogo, southern Ghana between June and August 2015. Agogo Hospital is a 250-beds healthcare facility serving the Ashanti-Akim North District with a population of around 170,000. AdolesceAfter an overnight-fast, venous blood was collected into EDTA for malaria diagnosis, for biomarkers of iron status and vitamin A metabolism, and for fasting plasma glucose (FPG). Axillary body temperature (°C), blood pressure (BP) and anthropometric measures were taken by trained study personnel. Socio-demographic data and medical history were documented in questionnaire-based interviews.The study protocol was reviewed and approved by the Ethics Committee of the Kwame Nkrumah University of Science and Technology, Kumasi. Written informed consent was obtained from all caregivers and assent was given by all participants.2 in kg/m2, and BMI-for-age z-scores (BAZ) and height-for-age z-scores (HAZ) were determined using the software package AnthroPlus . According to the WHO, overweight in adolescent age was defined as 1 ≤ BAZ < 2, obesity as BAZ ≥ 2, underweight (or thinness) as BAZ < -2, and stunting as HAZ < -2.All participants underwent a routine clinical examination by the study physician, and current diagnoses were documented. We measured axillary body temperature  and anthropometric measures were taken in light clothes. Body weight was measured to the nearest 0.5 kg  and height was measured to the nearest 0.1 cm . Body Mass Index (BMI) was calculated as weight/(height)th percentile of age-, sex- and height-specific reference data.[Systolic and diastolic BP were measured in triplicates every 3 minutes with an automated device  and appropriate cuffs in a separate room after a minimum of 5 minutes resting time. Mean systolic and mean diastolic BP were calculated using the last two measurements. Hypertension was defined as having a mean systolic or a mean diastolic BP >95nce data.Trained staff conducted questionnaire-based interviews  to documLaboratory analyses were performed within 4 hours after venous blood collection. Plasma was separated by centrifugation at 8000 rpm for 10 min. Full blood and plasma aliquots were transported to Germany on dry ice and stored at -80°C.Plasmodium infection and parasite species.[Plasmodium species plus current fever (≥37.5°C) or a self-reported history of fever within the last 48h.Malaria parasites were counted microscopically on Giemsa-stained thick blood films per 200 white blood cells. Following DNA extraction , semi-nested PCR assays were performed to ascertain  species. A malariFor iron status, plasma concentrations of ferritin and C-reactive protein (CRP) were measured by immunoturbidimetry . The inter-assay coefficients of variation were 0.85–2.15% for CRP and 9% for ferritin. Iron deficiency was defined as ferritin < 15 μg/L or as ferritin < 30 μg/L, if CRP was > 0.5 mg/L.[For vitamin A metabolism, retinol concentrations were quantified by high-performance liquid chromatography (HPLC). Vitamin For FPG measurement, we used a portable device . The inter-assay coefficient of variation was 2.9–4.1%. Impaired fasting glucose was defined according to American Diabetes Association criteria as 5.6 mmol/L ≤ FPG ≤ 6.9 mmol/L.Thirteen participants with missing or implausible values for age, sex, biomarkers or covariates were excluded from the analysis, resulting in a final analytical sample of 188. Infectious diseases were defined as a malarial infection or a diagnosed infectious disease (by study physician) or self-reported symptoms compatible with another infectious disease ; malnutrition comprised underweight, stunting, iron deficiency and vitamin A deficiency; and CRFs were defined as overweight or obesity, IFG or hypertension.Given an α-level of 0.05, this study had a statistical power of 70% to detect a disease occurrence of 20% ± 7% . The significance threshold was The demographic and socio-economic characteristics of the study participants are shown in Plasmodium falciparum was the predominant parasite species . Malarial infection was more frequent in boys than in girls (p = 0.005). Symptomatic malaria was observed in 2% of the juveniles. Current diagnoses or symptoms compatible with another infectious disease were seen in 7% of adolescents with no gender difference . Iron deficiency was seen in 4% and was more frequent among girls. For vitamin A deficiency, the overall proportion was 36%, with no differences between boys and girls , and this was similar between boys and girls (nd girls .p = 0.096). FPG was normal in most teenagers (4.3 ± 0.6 mmol/L), but IFG was seen in one boy and one girl. The proportion of hypertension was 9% and this was similar between males and females.Regarding CRFs, 7% of adolescents were overweight or obese. This figure was higher in girls than in boys . Further, the combination of CRFs with either infectious diseases or with malnutrition was discernible in 6%  and 7% , respectively. The former mainly comprised malarial infection plus hypertension (n = 8/11), while the latter was largely attributable to vitamin A deficiency plus overweight or obesity (n = 7/13).In In As a sensitivity analysis, we used the same set of demographic and socio-economic variables to calculate i) associations between malarial infection or other infectious diseases and nutrient deficiencies, ii) relationships between macro- and micronutrient deficiencies, and iii) interrelations between overweight/obesity and IFG or hypertension. Neither malarial infection nor other common infectious diseases were associated with underweight, stunting or iron deficiency. The presence of vitamin A deficiency increased the odds for common infectious diseases , but not for malarial infection. Also, anthropometric markers of protein-energy-malnutrition, i.e. underweight or stunting, had no effect on micronutrient deficiencies . For IFG, the occurrence was too low (n = 2) to calculate regression models, while there was a lack of association between overweight/obesity and hypertension .In rural Ghana, we investigated the proportions and the co-occurrence of common infectious diseases, malnutrition and CRFs among 188 adolescents. Demographic, socio-economic and medical risk factors for the combined entities were assessed as well as associations of single diseases within these entities. Roughly, half of the study population had an infectious disease or was malnourished; 16% presented with a CRF. Infectious diseases and malnutrition were more common among boys, while CRFs tended to be more frequent among girls. Infectious diseases and malnutrition were strongly linked with each other (co-occurrence 28%). Particularly, vitamin A deficiency increased the risk of non-malarial infectious diseases more than 3-fold. Moreover, male gender and low household SES increased the odds of both, infectious diseases and malnutrition. The overlap of infectious diseases and malnutrition with CRFs was rather small (2 out of 25 teenagers), and no associations of demographic, socio-economic and medical factors with CRFs were observed.Plasmodium infections (41%) which were largely asymptomatic and of low or submicroscopic parasite density, arguing for a naturally acquired semi-immunity among these Ghanaian teenagers.[For malarial infection, there is a marked paucity of prevalence data from the adolescent population in Ghana. Compared to younger age groups in the country , we founeenagers. In genereenagers.The present estimates for malnutrition contribute uniquely to the scarce data of the teenage group in Ghana. Regarding macronutrient deficiencies, our findings suggest somewhat lower figures than expected for underweight (7%) , and simFor CRFs, the proportion (7%) and the female preponderance of overweight/obesity accord with previous reports from the region., 12, 26 plus at least one form of nutritional deficits. Malarial infection and vitamin A deficiency were the predominant conditions (32/53). The association between clinical malaria and malnutrition has extensively been examined [vice versa. Moreover, low occupational status of the father and low wealth score increased the odds of infectious diseases and of malnutrition, in accordance with current findings from sub-Saharan Africa, linking poverty and disease.[The vicious circle of infectious diseases and malnutrition remains a major public health challenge in sub-Saharan Africa. This seeexamined , and is examined  In our s disease. More spenditions /53. The  disease.plus hypertension. It appears unlikely that high blood pressure was an immediate consequence of malarial infection, as indicated by the lack of association in our study. Rather, malarial infection and malaria-related fever reduce systolic blood pressure [On the background of demographic and economic development, Ghana faces an epidemiologic transition from infectious diseases to metabolic conditions that appears to be delayed in rural areas and poorer social classes., 35 Conspressure , and ourplus overweight or obesity, the denominator for this constellation frequently varies. On the country level, the Double burden of malnutrition refers to considerable amounts of childhood stunting (27%) and maternal overweight (29%) in the Ghanaian population.[plus at least one overweight or obese person.[plus overweight, obesity, IFG and hypertension. A similar analysis was conducted among urban adults aged 25–60 years in Burkina Faso, and revealed that one-quarter of the study population had at least one nutritional deficiency and one CRF .[plus CRFs were present in 7% of our study population, which was mainly attributed to vitamin A deficiency plus hypertension. Hypertension rates in Ghana are projected to increase dramatically, based on population growth and aging [While the term “double burden of malnutrition” usually refers to the co-occurrence of underweight, stunting or micronutrient deficiencies pulation. On the he person. For the idaemia). Already nd aging , while vnd aging  Therefornd aging So far, data on the co-occurrence of infectious diseases, malnutrition and CRFs are scarce for the population group that forms the basis of Africa’s future—adolescents. Thus, ouIn conclusion, in this population of rural Ghanaian adolescents, asymptomatic malaria infection, chronic energy deficits and vitamin A deficiency still constitute major health threats. Already at this young age, obesity and hypertension evolve and even co-exist with infectious diseases and nutrient deficits on the individual level. Potential interrelations of malaria, malnutrition, and cardio-metabolic risk factors remain to be investigated for understanding disease trends and ultimately guide resource allocation for health care in sub-Saharan Africa.S1 Questionnaires(PDF)Click here for additional data file.


In complex graphical representations, the relevant information for a specific task is often distributed across multiple spatial locations. In such situations, understanding the representation requires internal transformation processes in order to extract the relevant information. However, digital technology enables observers to alter the spatial arrangement of depicted information and therefore to offload the transformation processes. The objective of this study was to investigate the use of such a representation control (i.e. the users' option to decide how information should be displayed) in order to accomplish an information extraction task in terms of solution time and accuracy. In the representation control condition, the participants were allowed to reorganize the graphical representation and reduce information density. In the control condition, no interactive features were offered. We observed that participants in the representation control condition solved tasks that required reorganization of the maps faster and more accurate than participants without representation control. The present findings demonstrate how processes of cognitive offloading, spatial contiguity, and information coherence interact in knowledge media intended for broad and diverse groups of recipients. Complex graphical representations such as depictions of the results of elections frequently occur in traditional as well as online media ,2. WhileFor printed media, designers of graphical representations face a conflict between ease of use and usefulness: When the viewers' information task is known in advance, the graphical representation can be tailored accordingly by leaving out task-irrelevant information and focusing on task-relevant information ,4. The gDigital graphical representations  offer a solution to mediate this conflict between ease of use and usefulness; designers may include information suitable for a wide range of tasks and allow the viewers themselves to interactively adapt a given graphical representation to their individual needs and task requirements. While such interactive graphical representations seem to combine both a high degree of ease of use and usefulness, adapting graphical representations requires metacognitive knowledge as well as task-specific knowledge about the structure of the current task. Furthermore, a new tradeoff arises between the motoric and cognitive costs of altering the depicted information and the reduced cognitive effort that is necessary to subsequently comprehend the altered information .Tradeoffs between costs of motoric actions and cognitive effort are not unique for graphical representations but provide a rather broad principle of cognitive psychology. In fact, even actions such as writing notes or calendar entries can be considered to be cognitive offloading in order to relieve internal processing capacities. This externalization of internal processes is referred to as cognitive offloading ,7 that iWith regard to information complexity, infographics clearly exceed the materials of previous studies on cognitive offloading. These studies used Tetris blocks  or colorThe change of spatial organization affects the spatial contiguity  of the depicted information –24,26. SThe second transformation (selection) is based on the distinction between relevant and irrelevant information. According to the coherence principle of the cognitive theory of multimedia learning , simplerIn the present study, we investigated how representation control contributes to cognitive offloading as well as task performance, using complex graphical representations as stimuli. We asked participants to perform an information extraction task with political maps on which the relevant information was either spatially aligned (match) or misaligned (mismatch) with the task demands. In our experiment, we compared offloading as well as performance between two groups that were either allowed to alter the graphical representation (i.e. representation control) or not (static condition). We hypothesized that when available, representation control will be used in a task-appropriate manner to increase task effectiveness. More specifically, we predicted:(a)Users provided with representation control will adjust the complex graphical representations in cases in which the overall spatial organization does not correspond to the task requirements.(b)Users with representation control will reduce the information density of the complex graphical representations in order to increase coherence of the depicted information.(c)Users with representation control will be faster in the information extraction tasks with  mismatching graphical representations than users in the static condition.(d)Users with representation control will be more accurate than users in the static condition.Leibniz-Institut für Wissensmedien, Tübingen. They were randomly assigned to the two experimental conditions. The experimental procedure was approved by the ethics committee of the Leibniz-Institut für Wissensmedien, Tübingen. Most of the participants were university students (90%) with a relatively high self-reported experience in the use of computers —to 4 : M = 1.06; SD = 0.27) but low self-reported experience in the use of tablets . All participants provided written informed consent prior to testing. They received monetary compensation (10 €). One participant was excluded due to self-reported color blindness. An additional participant was excluded due to no correct responses  in the mismatch trials. Both participants were part of the static group.The final sample consisted of 88 participants  who were recruited from the participant pool of the The experiment was conducted on Microsoft Surface Pro2 tablets with a 10.6-inch screen (1920 x 1080 pixels). All user input was given through the touch-sensitive display.The graphical representations included two maps that were arranged side by side and depicted fictitious election results of two consecutive election periods and two types of votes. The two types of votes, called first vote and second vote, were based on the German election system, in which the seats of the parliament are distributed according to the combined result of the two types of votes. The exact layout and the components of the display are depicted in The depicted election results were either organized by type of vote  or electThe questions in the question/answer field either focused on the two types of votes within one election period or on one type of vote across the two election periods. For instance, the question ‘In how many election districts did the party SVP gain the majority of the first votes, in both 2009 and 2013?’ required the participants to compare the first votes of the two election periods, whereas the question ‘In how many election districts did the party SVP gain the majority of the first and the second votes in 2009?’ required the participants to compare the first and the second votes within one election period. All questions were of the described format, only varying in the parties, the election periods, and the type of votes. Participants indicated their responses by marking one of the 19 answer options below the infographic.The combination of the two types of map organization and the two types of questions resulted in match and mismatch trials. In match trials, all information required for solving a task was visible within one map. Therefore, participants only had to identify the color of the party in question as well as the relevant map in match trials in order to subsequently count the states. In mismatch trails, however, the information required to solve a task was distributed between the two maps. Therefore, the participants also had to identify the color of the party as well as the relevant information; however, in these trials the relevant information was displayed as either the right bar or the left bar in both maps. Thus, the participants had to compare the bars in the two maps in order to count the states with consistent votes. Importantly, within the representation control condition, the participants were able to change the organization criterion of the maps. In this condition, the terms match and mismatch refer to the initial state of the maps.After providing informed consent, participants received instructions explaining the information extraction task. Participants in the representation control condition watched an additional instruction video that explained the condition-specific interactive functions. We instructed the participants only how the representation control features could be used in principle; however, we did not encourage them to use these features whenever applicable. Next, the participants completed four practice trials , of which three had to be answered correctly. In case of more than one error, the practice trials were repeated until the criterion was met. Following these practice trials, the participants completed 24 experimental trials. One half of the trials consisted of match trials, the other half of mismatch trails. Match and mismatch trials alternated, the order being consistent among the participants. At the beginning of each trial, all information layers (types of votes and election periods) were visible.In the representation control condition, participants could reorganize the maps by switching the organization criterion in an interaction menu on the left side of the screen see . ParticiWe analyzed response times (RTs) and error rates (ERs) as dependent variables. In the representation control group, we additionally analyzed changes to the map organization and the information density. The trials with a response time larger than four standard deviations from the mean (0.90%) were excluded from the analysis. Errors were dichotomously coded and the trials with inaccurate responses were ignored in all subsequent analyses. We used linear mixed effect models (LME) to analyze the log-transformed RTs (due to their leftward inclination). In order to analyze the ERs and layout changes, we used generalized linear mixed effect models (GLME) with the logit as link function.Fixed effects in the RT and the ER analyses were condition (representation control vs. static), matching (match vs. mismatch), and the interaction between condition and matching. In the analysis of the changes of the layout, only the fixed effect matching was included in the model because layout changes were possible in the representation control condition only. All predictor variables were effect coded prior to analysis. In the models, we included a random intercept for each participant, as well as a random intercept for each task. The regression models were conducted using R  and the 2(1) = 81.71, p < .001. Planned t-tests with Tukey adjustments for multiple comparisons confirmed that the RTs were faster in the representation control condition than in the static condition in mismatch trials only, t(102.90) = -4.57, p < .001, whereas there was no difference between the two conditions in the match trials, t(100.06) = 0.19, p = 0.851. Furthermore, the LME revealed a significant main effect for matching, χ2(1) = 31.27, p < .001 as well as a main effect for condition, χ2(1) = 5.06, p = .025.The mean RTs for the representation control and the static condition across match and mismatch trials are depicted in 2(1) = 8.15, p = .004. The representation control condition and the static condition differed in mismatch trials, z = 6.70, p < .001, and also in match trials, z = 3.55, p < .001. Additionally, there was a main effect of condition, χ2(1) = 33.78, p < .001, whereas the main effect for matching did not reach significance, χ2(1) = 0.91, p = .340.As depicted in Beyond RTs and ERs, we analyzed how frequently participants adjusted the spatial organization and information density in the representation control condition. Particularly, we were interested in whether these interactions differ for match and mismatch trials. For this purpose, we dichotomously coded whether the participants had adjusted the spatial organization of the graphical representation and/or the information density. The results are depicted in 2(1) = 66.57, p < .001. As depicted in 2(1) = 0.76, p = .383. The information density of the graphical representations was reduced equally often in match trials (73%) and in mismatch trials (74%; Regarding the spatial reorganization, a GLME analysis revealed a significant effect of match vs. mismatch trials, χals 74%; .The present experiment investigated whether task performance benefits from representation control in complex graphical representations. In line with our hypothesis, participants who were able to alter the spatial organization of the presented information as well as to reduce information density of the graphical representation outperformed participants in the static group with respect to response time and accuracy. The beneficial effect of representation control was particularly pronounced in mismatch trials in which the relevant information for the question at hand was distributed across both maps. This is in line with our hypothesis that representation control is more beneficial in mismatch trials than in match trials as the option to reorganize the infographic is only useful in mismatch trials. Therefore, in mismatch trials, the participants could benefit from both transformations: the option to reduce information density and to reorganize the information, the former being in accordance with the coherence principle  and the The differentiated pattern of usage of the two transformation functions of representation control–reducing information density in most match and mismatch trials and reorganizing information almost only in mismatch trials–represents a strategic, purposeful action that is executed with the aim to reduce task complexity. Consequently, participants of the representation control condition answered the questions in the mismatch trials faster and more accurately than participants in the static condition.meta-representational competence [Our study provides evidence for the beneficial effects of the cognitive offloading of transformation processes . Whereasmpetence ,46,47. TOverall, the findings of the study show that digital graphical representations offer a solution to the conflict between ease of use and usefulness. Designers may include information suitable for a wide range of tasks in a graphical representation, thus increasing its usefulness. At the same time, representation control allows the viewers themselves to interactively adapt a given graphical representation to task requirements, thus increasing its ease of use. Consequently, our participants spontaneously used representation control options in order to solve the information extraction task in an efficient manner.Note that the present study was conducted with student participants. Almost all of them reported daily use of computers. Apart from their general knowledge of digital media, they were prepared to use the representation control by an instructional video and practice trials. In Germany and German speaking countries between 75 and 80% of the population above 14 years of age are using the Internet at least sometimes ,49. HoweOther inter-individual differences that we did not address in the present work are ability and metacognitive knowledge. As the aim of our study was to investigate whether representation control is used in infographics in order to offload cognitive processes and to test the effects thereof, we did not measure metacognition nor the abilities of our participants. Instead, we used a rather large and homogeneous sample to minimize individual influences. However, former studies found an influence of ability and metacognitive evaluations on offloading behavior ,16. RiskIn the present experiment, we allowed our participants to change the spatial organization and to reduce the information density. These two transformations were selected based on theoretical assumptions and allowed us to investigate the effect of representation control in a controlled experimental setup. A closer look at recent interactive infographics and maps makes clear that the options for how representation control could be implemented are manifold . For exaThe main part of the interactive infographics that we used in the present study consisted of pairs of maps. In a recently published research agenda, Roth and his colleagues  identifiThe present findings demonstrate how cognitive offloading, spatial contiguity, and information coherence may inform the design of interactive knowledge media that is intended for a broad and diverse group of recipients. Particularly in online media as well as in informal educational settings in which the information goals vary across individuals, well-designed interactive features may contribute to resolve the conflict between ease of use and usefulness of complex graphical representations such as infographics or maps.


It is widely perceived that there is a problem in giving a naturalistic account of mental representation that deals adequately with the issue of meaning, interpretation, or significance (semantic content). It is suggested here that this problem may arise partly from the conflation of two vernacular senses of representation: representation-as-origin and representation-as-input. The flash of a neon sign may in one sense represent a popular drink, but to function as a representation it must provide an input to a ‘consumer’ in the street. The arguments presented draw on two principles – the neuron doctrine and the need for a venue for ‘presentation’ or ‘reception’ of a representation at a specified site, consistent with the locality principle. It is also argued that domains of representation cannot be defined by signal traffic, since they can be expected to include ‘null’ elements based on non-firing cells. In this analysis, mental representations-as-origin are distributed patterns of cell firing. Each firing cell is given semantic value in its own right – some form of atomic propositional significance – since different axonal branches may contribute to integration with different populations of signals at different downstream sites. Representations-as-input are patterns of local co-arrival of signals in the form of synaptic potentials in dendrites. Meaning then draws on the relationships between active and null inputs, forming ‘scenarios’ comprising a molecular combination of ‘premises’ from which a new output with atomic propositional significance is generated. In both types of representation, meaning, interpretation or significance pivots on events in an individual cell.  The concept of representations-as-input emphasizes the need for an internal ‘consumer’ of a representation and the dependence of meaning on the co-relationships involved in an input interaction between signals and consumer. The acceptance of this necessity provides a basis for resolving the problem that representations appear both as distributed (representation-as-origin) and as local (representation-as-input). The key implications are that representations in the brain are massively multiple both in series and in parallel, and that individual cells play specific semantic roles. These roles are discussed in relation to traditional concepts of ‘gnostic’ cell types. Concepts of mental representation are widely invoked in neurobiology, linguistics, artificial intelligence, and philosophy. Yet, as From the outset I wish to emphasize that the problem I address relates only to what may be called ‘occurrent’ or ‘active’ representations in which signals are sent and received on specific occasions. There is another use of the term that might be called a ‘dispositional representation’ – an acquired pattern of cellular connectivity underlying memory, knowledge, or concept acquisition, that disposes the brain to generate occurrent representations in response to stimuli . I will type, or level, of biophysical location that could support a fitting causal role, and with appropriate information capacity (‘bandwidth’). There are those who would argue that we have a rough answer: that representations can be equated with patterns of neural activity, or firing. However, as discussed below, this fails to address key problems, justifiably of concern to philosophers of mind. Meaning is not to be solved so easily.The naturalization problem is not so much about whether a representation is to the right, left, front or back of the brain, or what connection tracts are involved. The more basic problem is defining the type of spatial pattern and location is likely to be central to a theory of meaning. Secondly, recognizing that reductive analysis of mechanism is only part of the story does not mean that fruitful progress in neural mechanisms should be abandoned half-finished and replaced by hand-waving. Rather than, as It might be argued that searching for a detailed substrate type for mental representation is overly reductionist or, in theoretical modeling terms, simply premature. It might even be considered immaterial to understanding of how a representation can have a meaning, either in terms of external referents or internal ‘meaning to the subject.’ However, I think the search is justified on the following grounds. Firstly, spatial pattern is about the only way meaning can be encoded in a brain at any point in time, as far as we know, so at least Moreover, the ability to suggest at least one plausible physical example for any theoretical model is a requirement that is arguably never premature. A search for such examples can render explicit contradictions in popular concepts. The key proposal here is that neuropsychology may benefit from a greater focus on the input aspect of mental representation. The author’s background is in immunology. It was not until we insisted on a grounding in a dynamics of integration of signals into individual cells that we began to understand leucocyte behavior in immune recognition and memory . HypotheRepresentation is a term used in a variety of ways that are not always transparent. It is not simply ‘re-presentation,’ and not just because ‘presentation’ might be a better label. It can also imply ‘proxy’ or ‘symbol.’ In the mental case, where representations do not resemble their referents in any simple way, the meaning of the term will be preconditioned not only by presumptions about how brains work but also metaphysical standpoint. A materialist may think in terms of brain states representing external ‘things’ whereas someone taking a dynamist or structural realist approach (as I do) may think in terms of internal dynamic relations representing external dynamic relations . There wMental representation may be invoked simply as part of an account of the human brain as a machine that generates outputs from inputs. A mental representation can be seen as the equivalent of local currents or magnetizations in a computer. As long as we accept that brain cells send messages around in a way vaguely similar to computer components, we can consider the nature of mental representations in this context as just a technical issue, like the difference between Microsoft Windows and Mac OS-X, without raising too many philosophical questions. ‘Representation’ is being used here purely to imply some internal dynamics that co-vary usefully with external world dynamics.There is, nevertheless, even here, a need to define a representation more precisely than just that total pattern of brain activity that arises in a specific context, whether the presence of a red square or blue circle, or when thinking ‘I suspect the recession will double-dip.’ A representation is not just a pattern of events; it is a pattern with a causal role. A red square will trigger patterns in the retinae, geniculate bodies, primary and secondary visual cortices, temporal, parietal and frontal lobes, all with different causal roles. To function, the content of any individual representation must be available to some functional component at a causal nexus: what Millikan calls a ‘consumer’ . Thus weThe second motivation for talking about mental representations is in the context of questions about first person experience, as conceived from positions on the nature of ‘mentality’ ranging from Cartesian to eliminitivist . Thus ‘mone instance of this ‘percept’ type of representation in a brain at a time, and there has been extended debate over whether this is local or distributed  can be explained by the interactions of separate neuronal units  as well as their inputs. This is self-contradictory for any computational system that obeys standard concepts of causality – what something has access to is its input – and neuroscience consistently indicates that these concepts of causality hold good.This might seem self-evident. However, this second premise is worth emphasizing because literature on consciousness often appears to take a different view. Representations may be seen as associated with computational or ‘information processing’ operations, which involve not inputs but input–output relations, or ‘roles in the world’ – the essence of ‘functionalism’ . The ‘coI must emphasize that this is a low-level analysis dealing with individual neuro-computational steps. Events within feedback systems taken as whole, as in anticipatory models of perception  can, in Both in neuroscience and philosophy, representations are often considered in terms of patterns of cell activity, with no specific reference to input or output. The problem here is that to consider a pattern as an operant representation implies that the total activity pattern is accessible to something. A pattern of activity of 73,456 out of a bank of 1,000,000 right occipital cells might seem to represent a scene. However, each of these cells may have 10,000 branches to its axonal output, some feeding forward, some back. Only 6,228 cells may send branches to each of a bank of temporal cells, and 18,992 to a bank of prefrontal cells (through any one direct or indirect route) and, moreover, there will be variation (and plasticity) in this between individual sending and receiving cells in each bank. Although the activity of the 73,456 cells is a representation in a certain legitimate vernacular sense, there seems to be another important sense in which it underpins, together with whatever other ‘null cells’ whose non-firing may contribute critically to the content being conveyed, not one, but many, representations-as-inputs, diverse in content and function.to something specified. It is sometimes implied that there are no ‘inner receiving entities’ for representations in a brain, but, again, this is inconsistent with our understanding of causality. To be part of a causal chain, and thus reportable, the information encoded in a representation must be made available to something that generates a response. A word of text in a forgotten language embedded in an opaque medium that cannot be removed without destroying the text cannot function as a representation. Similarly, a pattern of lines of cellular activity in my visual cortex that bears a homotopic relation to a pattern of tree trunks I am viewing is not acting as a spatial representation by dint of homotopy, since no part of me, including the cells themselves, is informed of the spatial relations of active and inactive cells. The cells provide a representation in the form of presenting sensory data to other parts of my brain through patterns of downstream synaptic transmission, but the homotopic spatial relation of their cell bodies is itself of no consequence. Representation must be linked to a causal path.In other words, an act of representation must ultimately imply an input non sequitur. If the problem is the same as for the whole brain then that must surely also suffer from the regress. The reverse conclusion applies: if the problem has any solution for the brain it may also have a solution for a homuncular subdomain and it may only have a solution there  with novel  non-local properties, at any structural level. Neurobiology has shown that we can push the concept of ‘input’ as far in as interpretable empirical observation will allow, and well within the confines of the human body or brain. Pressure from an intervertebral disk on a lumbar nerve root gives pain in the foot. Cochlear implants give deaf people an experience of sound. Stimulation of cerebral cortex in the awake individual can evoke sensations and memories. The evidence indicates that sensory pathways, at all points up to that where a percept is experienced, are simply providing an input to the next stage, which often can be mimicked artefactually.The work of In summary, despite speculations in other directions in some fields of study, the two assumptions of the neuron doctrine and the doctrine of percepts as based on inputs to perceiving entities appear to be worth retaining.Armed with this basic causal standpoint, it is possible to ask general questions about the location of the representations as percepts and the nature of the entities to which these are available. The starting premise is that at least one domain exists in a waking brain that supports an experience correlated with input from sense organs, contextualized by anticipations derived from kinesthetic monitoring, etc. We want to describe such a domain in dynamic physical terms. The prima facie case is that it will be a dynamic domain comprising part or all of one or more neurons, receiving inputs derived from all sensory modalities, and other internally generated signals, like names and concepts retrieved from memory , and capable of sending a sequence of outputs that can connect to all, or most, motor pathways. Conventional neuroscience indicates that the input will be of signals leading to patterns of depolarization of cell membrane. Since we are considering input this ought to be a pattern within dendrites .because it determines synchronized arrival at some site of input.It might be questioned that any single domain has inputs of all perceptual modalities and also concepts. However, our ability to mix raw sensory data and concepts in use of language indicates that somewhere in the brain signals with these disparate types of meaning are integrated – i.e., are co-inputs to some computational unit. Moreover, introspection indicates that human perceiving subjects experience them concurrently in a meaningful relationship and do so alongside the use of relevant language. Synchronization of signals may be important for optimal computation but as I agree with Absence of a mechanism for this sense of interpretation may seem puzzling. However, immediate local correspondence between physical dynamics and meaningful experience seems to be something that, like Descartes, we have to take as brute fact. Ascribing it to processes prior to the point of input to the perceiving entity makes things no easier. There is no means by which to carry interpretation forward from previous events, since we have no evidence for anything other than the physical input itself being available to the receiving unit. Moreover, the idea of ‘carrying meaning forward’ generates an absurdity. Since the history of past events contributing to any causal interaction is immeasurably complex an immeasurably large number of ‘interpretations’ from earlier events should be carried forward in causal chains and that is not what we experience. Both the existence and the richness of the meanings inputs have to human perceiving subjects may be things for us to wonder at, but trying to delegate richness elsewhere is no solution.It seems that representations as meaningful percepts ought to occur in neural dendrites.A further consideration is helpful in narrowing down options for the domain of a percept. The content of a percept almost certainly has to be an interpretation of both signals associated with membrane excitation and ‘null signals’ corresponding to where membrane might have been excited but was not. Unless signals are interpreted in the context of all possible signals in a domain we lose what appears to be essential for a complex percept: encoding of information in patterns of inter-relation. A summation of all and only the black spots of a set of printed words can have only one meaning: black. (Or if black is coded null the sum of white areas just means white.) Moreover, it is indeterminate whether the spots included are on one page, or in a whole library. Only if both active and null signals and their relations are included do we have diverse meaning and bounded domains of meaning. In visual cortex, a ‘line’ of uniform color within a block of the same color is not interpreted as a line. The interpretation of ‘a line’ implies the absence of signals encoding similar color on either side of the line.currently carrying signal traffic.This means that the domain that supports a representation with meaning cannot be defined by a pattern of active signal traffic; it cannot be defined in terms of where signals are occurring. It must include null signals, so there must be some intrinsically defined structural domain within which signals and null signals are co-interpreted. The domain receiving signals interpreted as a percept cannot be an ‘active circuit’ in the sense of a set of pathways There is a distinction here between the processing units in a brain and in a computer. In a computer there are ‘gates’ in which electrical signals ‘open’ or ‘close’ connections between units, forming and breaking electrical circuits. The brain does not have gates in this sense. Connections remain unchanged, at least over periods of hours, regardless of traffic. The processing units are integrators, but not gates. Something akin to gating will occur during refractory periods and if input signals show differential synchronization in relation to refractory periods there may be triage, so that some active signals are ‘let through’ and others not. However, these signals will still operate in the context of null signals within the non-refractory time window.single and local domain. That leaves options of one very extended domain or multiple local domains.Representations-as-percepts, if only in a degraded form, survive damage to large areas of cerebral cortex. Damage to certain areas produces predictable defects, but does not appear to remove the capacity for some sort of perceptual experience, even if there is agnosia in the sense of not being aware that the percept is defective. The inference is that if the type of domain receiving representations as percepts is indeed cortical then there is no The idea that a percept is an interpretation of the inputs to cells over a wide area of brain generates a range of problems, quite apart from the basic problem noted by These and related concerns may have motivated the proposal by sense of multiplicity (of the perception of being one of many subjects) since multiplicity would not itself be encoded, represented or, therefore, perceived by anything, being a fact about parallel reception events, not a property of the receiving unit, or the content of its input.In summary, although the discussion so far might suggest that the question of what domain supports a perceptual representation is just what it must always have been – which cell or cells – it may need a subtler formulation. How many of which sort of neuron have a perceptual representation encoded in their input(s) and do they constitute one domain of one representation of this type at any one time or are there multiple domains, with multiple representations based on the same sensory data? It is important to note that the latter should not be expected to evoke a At this point the reader may sense that the concept of representation is too confusing to be useful, and there is a case for that position! I would argue, however, that if some historic confusions in the literature are unpacked it is possible to restore the usefulness of the idea, with some riders that add significant explanatory power.seems ‘single’ is immaterial, since there would be no reason for there to be representation (and thus perception) of multiplicity, or a sense of ‘other copies,’ within each of multiple representations.The simplest hypothesis for the domain of a percept, now universally taken as a null hypothesis, is that of a single pontifical cell, as discussed by quite different sort of ’pontifical’ cell to explain recognition. Sensory data relating to an object such as a dog enters through many 1000s of receptors. Recognition would appear to require sequential stages of discrimination, each leading to a reduced number of possible interpretations. This might be expected to form of a ‘pyramid’ with fewer cells at each stage until the input finally converged on one cell responsible of recognizing dogs. There would be a pontifical cell for a dog, another for a cat and another for grandmother.It is useful to raise here a potential confusion in terminology between sites of representation and sites of recognition. A key point here is that we have no reason to think that only the cell with the job of recognizing dogs will receive input signals encoding doggy features. If 100 cells each recognized a different mammal we would not expect the presence of a dog to lead to input to only one of these. We would expect all the cells to receive signals encoding doggy features but only one (or some) to fire. It could be argued that synapses receiving signals encoding long snouts will atrophy on koala-recognizing cells but at least to be able to learn to recognize new animals we have to assume that cells with catholic inputs exist.Figure 1). It highlights the fact that a representation is always a step in a causal chain and is thus always a representation to a domain at a particular point in that chain. Thus a pattern of data, perhaps encoding legs, fur and muzzle, would represent a dog to a ‘dog-pontifical cell’ as well as to a lot of other cells, untuned, or tuned to other creatures. In turn the firing of the dog-pontifical cell and not its neighbors would denote ‘dog’ to the rest of the brain. The two types of representation would be quite different. Moreover, intuition tells us that whatever domain has a percept of a dog of the sort normally discussed it must have an input encoding both the key features of a dog – legs, fur, etc. – and the sense of these being part of a dog, apparently putting the relevant domain downstream of the site of dog-recognition with additional parallel input encoding the original upstream context-dependent sensory data.Thus if a representation is based on an input pattern we do not expect sites of representation and recognition to be commensurate. This emphasizes the need to consider a causal chain as potentially involving many levels of representation with multiplicity at each level  expect to support a ‘percept’ of granny in the sense of manifestation of all of grandmother’s features, whether or not they fired. And it would be the pattern of firing and non-firing of these latter cells that would ‘represent’ (in the denoting sense) to domains in the rest of the brain the presence, but not the pattern of features, of this individual. Whether or not within this latter group of cells there are cells with 100% sensitivity and specificity for grandmother is a different issue that need not bear on the search for the domains supporting the representations known as percepts.The odd thing here is that Barlow appears to be describing the activity of cells upstream of a site of recognition of grandmother. If each cell is responding to signals which together encode a feature not entirely specific and sensitive for grandmother then grandmother can only be recognized, and social responses activated, by a downstream group of cells receiving inputs from these thousand cells, More recently, The above discussion emphasizes a number of issues relating to this crucial question. It seems that representations (in the broadest sense) of a given referent in the brain must be multiple and diverse. At each level many cells will be involved in representing. Representation and recognition are not likely to be commensurate. So far the discussion has been in terms of individual cells despite the general assumption in the literature that representations in brains each involve many cells. The grounds for such an assumption need be revisited in the light of the preceding arguments.to anything within the machine beyond the four trivial input options for an electronic gate of on/on, on/off, off/on and off/off. Moreover, we do not require that anything in a computer interprets, or attributes meaning to, input signals. It might be argued that a sequence of signals passing through a gate might constitute a representation. However, since each signal contributes to a separate computation this is problematic. The sequence of incoming signals is not subjected as a whole to a computation, other than as arbitrarily defined by a programmer. Within the machine any temporal ‘chunking’ of serial signals into ‘representations’ adds nothing to the causal account and at the gate in question no chunking should be apparent.As already noted, to be useful, the concept of representation-as-percept, has to imply a step in a causal chain with content encoded in the input to some domain. It is also difficult to see how a representation can have a meaning, or interpretation, to a domain, unless its content is encoded in the co-temporal input of a pattern of active signals and null signals to the domain. Representations like this do not occur in computers. Stored data in a computer can represent something meaningful to a human user accessing it via a screen but no representation based on a pattern of co-temporal input occurs are units that receive complex patterns co-temporally: neurons. Moreover, they are the only units that receive patterns relevant to percepts as far as we know. Barlow’s 1000 cardinal cells are not a unit receiving a pattern of features of grandmother. Each has a separate input encoding one feature. For all 1000 features to contribute co-temporally to a representation 1000 cardinal cells must send all 1000 active or null signals to converge on at least one downstream neuron, which is within the range of neuronal inputs. The neuron doctrine, as was probably evident to Leibniz, entails the simple but surprising conclusion that representations qua percepts in brains can only be in individual neurons  rather than structure. The conclusion might be branded over-reductive but one of its key features is that it makes explicit the boundary between reductive analysis and the non-reductive relation of ‘interpretation,’ rather than invoking an ill-defined internal no-man’s-land where both are claimed to apply at different ‘levels.’to the single computational (rule based input–output) units that are neurons is that it implies that the brain does not perform single operations on ‘atomic’ (structureless) symbols, but rather it performs operations on ‘molecular’ representations. That is to say that the basic data units that the brain operates on are irreducibly complex, with many degrees of freedom. This begins to address the puzzle of how the manipulation of symbols can be associated with an experience of complex patterns that reflect the complexity of their referents. It also provides a reason why, as appears to be increasingly recognized, syntax and semantics cannot be totally dissociated when considering meaning  is an instance of a pattern, as in a picture or map, that acts as origin for a representation in the other sense (r) of an instance of representing to something via its input. At every stage of neural computation we can expect a representation-as-input to lead to an output that can act as representation-as-origin for the next stage. At every stage banks of cells will be involved but whereas such a bank of cells will hold a single representation-as-origin it will hold as many representations-as-inputs as there are cells in the bank. Perhaps surprisingly, although building a representation-as-origin is likely to be labor-intensive, much larger numbers of representations-as-inputs, which we could expect to correspond to percepts, would appear to come free of charge.Figure 1, hierarchies of representation-as-origin give the opportunity for representations-as-input downstream to ‘pick-‘n’-mix’ inputs from more than one level of this hierarchy. Thus there is nothing very surprising about the idea that the representations that guide our rapid actions appear to overlap in content in most but not all situations with those that form the basis of our percepts.We are used to the idea that the nervous system generates motor output from sensory input at several levels of complexity. There are spinal reflexes, brainstem reflexes, automatic but co-ordinated responses involving cerebellum, routine purposive actions and deliberated actions. All of these can be expected to be associated with different levels of representation-as-origin and representations-as-input so we should not be surprised by the idea of multiple spatial representations even in terms of representations-as-origin. Perhaps more interestingly, as indicated on the right side of Mainstream neuroscience prides itself in being rigorously physicalist, in the sense of adhering to the basic precepts of natural science and general principles of causality. A consideration of representations in such a rigorous causal framework leads to the conclusion that all representations in the brain, including those that may form the basis of percepts, must ultimately be considered in terms of how they are cashed out in the inputs to individual neurons. These representations as inputs will occur at multiple levels of sensory processing and will be multiple at all levels, including levels associated with pattern recognition, denotation and reportable percepts. Such a model is counterintuitive but resolves certain important problems relating to the distributed nature of representation and may provide clues to the basis of meaning and language.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.