In breast cancer it is never the primary tumour that is fatal; instead it is the development of metastatic disease which is the major cause of cancer related mortality. There is accumulating evidence that suggests that Cancer Stem Cells (CSC) may play a role in breast cancer development and progression. Breast cancer stem cell populations, including side population cells (SP), have been shown to be primitive stem cell-like populations, being long-lived, self-renewing and highly proliferative. SP cells are identified using dual wavelength flow cytometry combined with Hoechst 33342 dye efflux, this ability is due to expression of one or more members of the ABC transporter family. They have increased resistance to chemotherapeutic agents and apoptotic stimuli and have increased migratory potential above that of the bulk tumour cells making them strong candidates for the metastatic spread of breast cancer. Treatment of nearly all cancers usually involves one first-line agent known to be a substrate of an ABC transporter thereby increasing the risk of developing drug resistant tumours. At present there is no marker available to identify SP cells using immunohistochemistry on breast cancer patient samples. If SP cells do play a role in breast cancer progression/Metastatic Breast Cancer (MBC), combining chemotherapy with ABC inhibitors may be able to destroy both the cells making up the bulk tumour and the cancer stem cell population thus preventing the risk of drug resistant disease, recurrence or metastasis. In the Metastatic breast cancer (MBC) and its causes therefore remains a major concern with approximately 30% of patients with early stage breast cancer eventually progressing to metastatic disease [Cancer develops through the progressive accumulation of genetic mutations that transforms normal cells into a malignant phenotype. Carcinogenesis results in cells acquiring more primitive cell characteristics, such as the ability to self-renew and an infinite capacity to divide ,5, whichStochastic theory;Hierarchy (Cancer Stem Cell) theory.The stochastic theory predicts that every cell within a tumour can generate a new tumour when in the correct microenvironment . HoweverThe cancer stem cell (CSC) theory has been attributed to Cohnheim 1875) who postulated that stem cells misplaced during embryogenesis may be responsible for tumour formation later in life -15. Hist75 who poOver the last few years there has been a surge in the number of studies investigating the role of CSC in tumour progression, however most research has focussed on the involvement in the primary tumour. CSC are tumour cells that are capable of self-renewal and give rise to heterogeneous tumour masses ,27,28. T2.In 1889, Paget devised the “seed and soil hypothesis” of cancer metastasis in which he suggested that the seeds of cancer (the tumour cells) have an ability to survive in specific soils (organs) that are conducive to their growth . This hy3.There is accumulating evidence which suggests that CSC may play a role in metastasis . Breast Mammosphere—Forming ability -46;in vivo tumourigenicity assays;Flow cytometry—Analysis of breast cancer stem cell markers including CD44 , CD24 4,48, CD49Label retention studies—For example radioactive thymidine and BrdU -55;Quiescence—Pece and colleagues (2010) have isolated normal human mammary stem cells using the lipophilic dye, PKH26 which is retained by quiescent cells ;Functional assays—Side Population (SP) cells which have an increased ability to efflux Hoechst 33342 dye  and ALDHin vitro and in vivo functional assays. Discrepancies in marker expression may arise due to the manipulations required to prepare the samples for analysis, for example tumour dissociation into single cell suspensions and culturing of cells may alter cell behaviour and viability. Unlike normal tissue stem cells, CSC are influenced by the specific genetic aberrations of the tumour, by the stage of the disease and also by any therapeutic interventions given to the patient [The use of cell surface markers to identify CSC from solid tumours requires careful optimisation of antibodies and the gated populations must be validated using both  patient . In pati patient ,59.+CD24−/low breast CSC populations [in vivo that recapitulated the heterogeneity of the tumour from which they were isolated [+ cells have been shown to mediate invasion and metastasis in inflammatory breast cancer [At present most of what we know about the role of CSC in breast cancer metastasis has been based on ALDH-positive, CD133-positive or CD44ulations ,60-62. Bulations ,64. It iulations ,65-67, nulations , multiplulations , and malulations . In xenoisolated . ALDH+ ct cancer  and thist cancer . In factt cancer . Marcatot cancer .+ [+ [+ or CD24− cells in metastatic tissues is conflicting, Shipitsin and co-workers determined that the CD24+ cell population was increased in distant metastases regardless of the site of metastases [+ cells in primary invasive tumours and matched samples taken from metastatic sites [in vivo [−ESA+CD44+CD24−/low phenotype that were highly tumourigenic in vivo and were the only cell population capable of forming tumours in NOD/SCID mice that recapitulated the heterogeneity of the tumour from which they were isolated [+CD24−/low populations constituted a high proportion of the total cell population in basal or myoepithelial breast cancer cell lines [+CD24−/low cells [+CD24−/low cells are slow cycling, chemoresistant, capable of forming mammospheres and are enriched in drug resistant cell lines [Independently, both CD44+ ,74 and C+ [+ ,75 breasic sites . For exaic sites . Brown a[in vivo . In 2003isolated . Sheridall lines . These cow cells . Subsequll lines ,79.+CD24−/low cells in breast tumours with patient follow up have produced conflicting data. Abraham and colleagues determined that 78% of breast cancer cases assessed contained a CD44+CD24−/low population constituting less than 10% of the total cell population [+CD24−/low expressing cells and response to different therapeutic regimes was observed [+CD24−/low phenotype was significantly increased following neoadjuvant chemotherapy [+CD24−/low cells have utilised antibodies that do not discriminate between CD44 isoforms. In the future it would be beneficial to investigate expression of specific CD44 isoforms and their affect on the CD44+CD24−/low phenotype.Studies correlating the presence of CD44pulation . No signobserved . Conversotherapy . The maj+ cells have been identified in breast cancer cell lines created from Brca1-deficient murine mammary tumours and these cells were highly tumourigenic, expressed common stem cell markers and were resistant to DNA-damaging chemotherapy [+CD29hiCD133/2hi phenotype has also been identified in ERα-negative human breast tumours that was highly tumourigenic, capable of self-renewal and also expressed common stem cell markers [In breast cancer, CD133 appears to play less of a role as a stem cell marker than in other cancers . Howeverotherapy . A popul markers .4.Another putative CSC population are Side population (SP) cells. SP cells are identified using dual wavelength flow cytometry combined with Hoechst 33342 dye efflux ,83. SP c−SCA-1+), were capable of contributing to both the lymphoid and myeloid cell lineages and were highly enriched in HSC activity [− CD29hiCD24+ phenotype could function as a mammary gland stem cell reconstituting an entire functional murine mammary gland [−CD29hiCD24+ population [SP cells were first identified in murine bone marrow and were thought to be a stem cell (a cell type that has the ability to self-renew and differentiate to give rise to more restricted cell types ) populatactivity . SP cellactivity -96. Seveactivity ,97-99. Sactivity ,99-101. activity ,93-100. activity . Broadleactivity . GBM SP activity . SP cellactivity . In 2006ry gland . SP cellpulation .−/− null mice were shown to be more sensitive to mitoxantrone, a drug commonly used in chemotherapy [In haematopoietic cells, the SP phenotype has been defined by the expression of ABCG2 ,84, an Aotherapy .5.SP cells have been identified in both mouse and human mammary gland tissue ,107. Anain vitro [At present there is a debate whether mammary gland SP cells are stem cells or a more-restricted mammary gland progenitor cell population. However, regardless of their status, studies have shown that human mammary gland SP cells are able to generate luminal and myoepithelial lineages , to formin vitro ,111.6.in vitro [In vivo studies have also demonstrated that in most cancer cell lines SP are more tumourigenic [Patrawala and colleagues 2005) found that 30% of human cancer cells and xenograft tumours contain SP cells . Extensi05 found in vitro  comparedin vitro ,112,115.urigenic ,119 and urigenic ,120,121 urigenic .7.in vitro. In contrast, NSP cells could only generate NSP cells in vitro [In vivo, breast cancer SP cells have been shown to be more tumourigenic than NSP cells when transplanted into immunodeficient mice [Extensive work has been performed to characterise SP cells in breast cancer cell lines ,123-126.in vitro ,113,123.ent mice ,113,121.−CK18+EpCAM+CK19+ phenotype [+CD24−/low cells are enriched in mammosphere formation and both populations are preferentially inhibited by NF-κβ pathway inhibitors compared to their non-stem cell counterparts [The importance of ABC transporters in defining the SP phenotype in normal tissues has led to an abundance of studies investigating the role of these transporters in breast cancer SP cells. Many studies have reported increased transcriptional expression of ABCG2 in SP compared to NSP cells ,121,123 henotype  and bothterparts . These pThere is evidence to suggest that SP cells may undergo EMT and numerous studies have demonstrated an association between EMT, chemoresistance and stem cells ,127,128.Cancer SP cells are known to be chemoresistant and this has been attributed to the expression of ABC transporters ,99,131. 8.+CD24−/low [+ [+ cells. This study did not identify a prevalent breast cancer stem cell population in luminal breast tumours.SP cells have been identified in a diverse array of breast cancer cell lines that are classified within different breast cancer subtypes . HoweverD24−/low  or ALDH1−/low [+  phenotyp+PR+Her2+) breast cancer cell lines and these cell lines had increased colony forming ability in vitro compared to basal a (CK5+ and CK14+) or basal b (vimentin+) subtypes. A strong correlation was observed between Her2 expression and SP presence with the percentage of SP increasing in cell lines enforced with expression of Her2. Treatment with the Her2 signalling inhibitors, tyrophostin AG825 or trastuzumab, decreased the proportion of SP cells in breast cancer cell lines and also inhibited tumour growth in vivo [To date only Nakanishi and colleagues have identified SP cells in clinical breast tumours which were characterised as luminal cancers . The per in vivo . The aut in vivo . Unfortu9.in vitro and increased metastatic potential in vivo when compared to NSP cells using a murine liver metastasis model [As yet there is no published literature investigating the metastatic potential of SP cells in breast cancer. However, studies in other cancers have shown that SP cells may play a role in metastatic spread of disease ,120. Katis model . Nishii is model . In primis model . However10.in vitro [in vitro and inhibits breast cancer metastasis in vivo [The role of the CXCR4/CXCL12 axis in breast cancer metastasis has been well documented -137. Sevin vitro . Several in vivo -136. Pri in vivo  and in b in vivo .11.in vitro and in vivo being due to the interaction between CXCR4 and CXCL12 [in vitro [+CXCR4+ cell population is responsible for metastasis [SP cells are known to be capable of migration in response to CXCL12 ,140. Carin vitro . Perhapstastasis ), it is 12.Current treatment for MBC is limited and has little clinical success . There iin vivo and ex vivo [et al. (2010) detected higher proportions of CD44-positive cells in primary tumour samples analysed from lymph node positive supraglottic carcinoma patients compared to lymph node negative patients thus showing the importance of identifying CSC in lymph node samples [To clarify the role of SP cells in MBC, the identification of SP cells needs to be performed in all breast cancer subtypes and their presence related to the tumour characteristics and to patient follow up. Breast cancer SP cells need to be isolated from patients and extensively characterised which will involve the need to transplant these human cells into animal cancer models to elucidate the potential of these cells to contribute to invasion and metastasis. A manuscript published by Michael Clarke's group described the development of a human-in-mouse breast cancer xenograft model in which the metastatic potential of breast cancer stem cells could be determined . This gr ex vivo . It will ex vivo ,144. A d ex vivo . Lu et a samples .13.+CD24−/low breast cancer stem cell population and MBC have been shown to have aberrant MAPK components which potentially could benefit from treatment with MAPK pathway inhibitors [The emerging role of cancer stem cells in breast cancer development and progression has led to intense work to identify drugs that target the CSC population of drug resistant cells . The imphibitors .+ and CD44+CD24−/low cell populations, culminating most recently in the use of Salinomycin, a highly selective potassium ionophore, as a compound that could selectively eliminate the CD44+CD24−/low breast cancer stem cell population compared to treatment with paclitaxel alone [In vivo, Salinomycin was able to inhibit the formation of tumours in the 4T1 murine breast cancer cell model and also in the ras-transformed HMLE model. Salinomycin treatment also prevented metastasis of 4T1 cells in vivo [The majority of studies have focused on targeting the CD44el alone . In vivo in vivo ,149. The in vivo . Fuchs a in vivo . PerhapsAs the SP cell phenotype is a consequence of the expression of ABC transporters, strategies to inhibit these might be worth revisiting. Nearly all cancer chemotherapy involves a first-line agent known to be a substrate of an ABC transporter , therebyin vitro [In addition to chemoresistance, ABC transporters also play a role in cell migration, differentiation and protection against apoptosis -158. In in vitro . ABCC1 hin vitro . TherefoNakanishi and co-workers (2010) identified SP in primary cells derived from patient breast tumours  and we cThe importance of Her2 in the regulation of breast cancer SP cells has been demonstrated by Nakanishi and colleagues (2010) and SP cells have been observed to be more prevalent in Her2 overexpressing breast cancer cell lines and luminal breast tumours . The Her+ cell population [Trastuzumab and Lapatinib are targeted therapies against the Her2 receptor and the Her2 pathway and are usually only prescribed to patients with Her2-positive breast tumours  and clinpulation . Treatmepulation ,165. Per14.in vitro studies show promising results for therapeutically targeting breast CSC but they need to be translated into the clinic [Reported e clinic ,148,149.e clinic ,147. It If SP is involved in the metastatic process, therapies to target SP cells will need to be sought in order to minimise toxicity effects often experienced by treatment with systemic therapies. If ABC inhibitors/modulators are to be of use they need to be highly specific in order to reduce systemic effects as ABC transporters play a pivotal role in the blood brain barrier, many tissues and most normal stem cells , and int


Oral antiviral agents to treat influenza are challenging to administer in the intensive care unit (ICU). We describe 57 critically ill patients treated with the investigational intravenous neuraminidase inhibitor drug peramivir for influenza A (H1N1)pdm09 [pH1N1]. Most received late peramivir treatment following clinical deterioration in the ICU on enterically-administered oseltamivir therapy. The median age was 40 years (range 5 months-81 years). Common clinical complications included pneumonia or acute respiratory distress syndrome requiring mechanical ventilation , sepsis requiring vasopressor support , acute renal failure requiring hemodialysis  and secondary bacterial infection . Over half  died. When comparing the 57 peramivir-treated cases with 1627 critically ill cases who did not receive peramivir, peramivir recipients were more likely to be diagnosed with pneumonia/acute respiratory distress syndrome (p = 0.0002) or sepsis (p = <0.0001), require mechanical ventilation (p = <0.0001) or die (p = <0.0001). The high mortality could be due to the pre-existing clinical severity of cases prior to request for peramivir, but also raises questions about peramivir safety and effectiveness in hospitalized and critically ill patients. The use of peramivir merits further study in randomized controlled trials, or by use of methods such as propensity scoring and matching, to assess clinical effectiveness and safety. The influenza A(H1N1)pdm09 [pH1N1] virus emerged in April 2009 to cause a global pandemic Early in the pandemic, the only available antiviral drugs for treatment of pH1N1 were the neuraminidase inhibitors (NAI), oral oseltamivir or inhaled zanamivir. Administration of enterically- administered oseltamivir can be challenging in the ICU where patients may have difficulty tolerating oral administration of capsules or intestinal ileus may cause malabsorption In April 2009, the California Department of Public Health (CDPH) initiated mandatory reporting for critically ill and fatal cases of pH1N1. In this report, we describe the epidemiology, clinical characteristics, and outcomes of pH1N1 cases reported to CDPH who received IV peramivir during the pandemic.This study protocol was reviewed and approved by the State of California Committee for the Protection of Human Subjects (CPHS). A waiver of informed consent was granted as the data were collected as part of public health practice and were analyzed anonymously, the risk to subjects was minimal, the rights and welfare of subjects were not adversely affected, and the research could not have been practically carried out without a waiver (Common Rule 45 CFR 46.11).A case was defined as a California resident who was hospitalized in an ICU for ≥24 hours with signs and symptoms consistent with acute respiratory infection and laboratory evidence of pH1N1 virus infection by reverse-transcription polymerase chain reaction (RT-PCR). Cases were reported by providers, hospitals and county coroners to local health jurisdictions (LHJ) who subsequently reported them to CDPH. Data on demographics, clinical presentation and hospital course, co-morbid conditions, dosing and dates of antiviral medications administered were abstracted from medical records, pharmacy medication records and autopsy reports by staff at the LHJ or CDPH using the same standardized case report form. Secondary bacterial co-infection was defined by isolation of bacteria within 3 days of admission from either a sterile site  or a lower respiratory tract specimen  in conjunction with new infiltrate on chest radiograph.Comparative analysis was performed for 1) critically ill patients who received peramivir and critically ill patients who were treated with NAIs but did not receive peramivir; and 2) fatal and non-fatal peramivir-treated cases, with respect to demographics, clinical characteristics, and underlying risk factors; differences in categorical variables were analyzed using the chi-square test for comparisons of categorical variables with large numbers and the Fisher’s exact test for comparisons of categorical variables with numbers <5. All analyses were performed using SAS 9.2 .During April 3, 2009 – August 10, 2010, 1962 California residents were reported with pH1N1 virus infection that were hospitalized ≥24 hours and required intensive care. Information on whether or not antiviral treatment was given was available in 1867 (95%) cases; of these, 1684 (90%) received treatment with an NAI.Fifty-seven (3%) cases received IV peramivir . Fifty-tThe median age of peramivir recipients was 40 years (range 5 months-81 years). Fifty-six of 57 (98%) received antibiotic treatment at admission to the hospital. Almost all  had radiographic evidence of pneumonia with 54 (95%) requiring mechanical ventilation. Fifty-three cases had information on dates of antiviral treatment: 34 (64%) required vasopressor support for hypotension associated with sepsis and 19 (36%) had new onset acute renal failure requiring hemodialysis. Fourteen (25%) had microbiologic evidence of community-acquired bacterial co-infection at admission. The median length of hospital stay was 21 days (range 3–78).Twenty-nine (51%) peramivir recipients died. Fatal peramivir recipients differed from non-fatal peramivir recipients by being more likely to be diagnosed with acute renal failure requiring hemodialysis (p = 0.02), to have a shorter length of hospital stay  and to receive peramivir for a shorter duration . There was no significant difference between time from onset of symptoms and initiation of any antiviral treatment among fatal and non-fatal peramivir recipients .We also compared the 57 peramivir recipients to the 1627 cases in the ICU who were treated with other NAIs but did not receive peramivir. There was no significant difference with regard to the overall proportion with an ACIP co-morbid condition, although cases treated with peramivir had a slightly higher prevalence of obesity (p = 0.04) and morbid obesity (p = 0.02). Peramivir recipients were more likely to be diagnosed with pneumonia/ARDS (p = 0.0002) or sepsis (p = <0.0001), to require mechanical ventilation (p = <0.0001) and to die (p = <0.0001). Both peramivir and non-peramivir recipients were initiated on NAIs within similar timeframes .Our report adds to the limited existing data on use of the investigational drug peramivir in hospitalized and critically ill influenza patients. Two small case-series have described use of multi-dose peramivir during the pH1N1 pandemic; one study of 31 critically ill adults and children conducted prior to the EUA found a 56-day mortality of 41%  The case fatality proportion (51%) in our pH1N1 patient population was higher than that reported in other peramivir case series, but comparisons are impeded by the limited clinical data available. Of note, the 57 patients in our study were older (median age of 40 vs. 23 years) and had a higher prevalence of co-morbidities (72% vs. 45%) than the 31 patients who received peramivir prior to the EUA  The high case fatality proportion described in our patient population may have been due to the progressive severity of illness; the majority of requests for peramivir were for cases assessed by the clinician as not responding to several days of oral oseltamivir therapy and with signs of continued clinical deterioration. However, it also raises concerns of adverse drug events specifically due to peramivir. Most of the available safety data describes single-dose use of peramivir in adult and pediatric outpatient populations; commonly reported adverse events have included diarrhea, nausea, vomiting, and neutropenia Additionally, in our series, 37 critically ill cases received concurrent administration of oseltamivir and peramivir for more than 24 hours, with over half dying. The pH1N1 pandemic highlighted concerns that widespread use of a single antiviral agent could lead to emergence of resistant strains. Treatment with a combination of two antiviral agents could theoretically offer the advantages of additive effect, while decreasing severity of disease, duration of viral shedding and risk of developing resistance. However, because all drugs in the NAI class target the same binding pocket, drug antagonism is also a concern. Recent reports suggest that combination NAI therapy may be less effective than monotherapy; in a randomized controlled trial of over 500 outpatients with pH1N1 influenza, treatment with concurrent oral oseltamivir and inhaled zanamavir was associated with more side effects (nausea and vomiting) and shedding of higher viral titers for longer periods compared to those receiving either drug alone Some limitations are important to note. There was likely underreporting of cases during the pH1N1 pandemic, including those who received peramivir. While 57 cases were identified by search of the CDPH surveillance database, during the same timeframe the CDC received 130 requests for peramivir from California , suggesting that treatment was not administered to many cases, due to clinical improvement, death, or other reasons. Serial testing for influenza by PCR, which might inform decisions on when to modify or cease antiviral therapy, was not performed in our 57 peramivir recipients. It is possible that some patients may have had poor outcomes following peramivir treatment related to the presence of the H275Y mutation in viral neuraminidase, which confers resistance to oseltamivir and reduced in-vitro susceptibility to peramivir http://clinicaltrials.gov) and IV zanamivir is also available on a compassionate use basis . However, while the high mortality (>50%) we observed could be due to the pre-existing clinical severity of cases prior to request for peramivir, it also raises questions about peramivir safety and effectiveness in hospitalized and critically ill patients. Although ours is the largest case-series describing the use of peramivir in criticaly ill patients published to date, the nature of our surveillance and the criteria for requesting peramivir under EUA precludes any comparison of effectiveness between the oral and IV NAIs. Large randomized clinical trials are required to assess these questions appropriately among hospitalized influenza patients, or, in the absence of data from randomized controlled trials by use of methods such as propensity scoring and matching to assess the clinical effectiveness and safety of new investigational agents for influenza treatment.In conclusion, current guidelines strongly recommend treatment with oral oseltamivir as soon as possible in influenza patients who are hospitalized, have ACIP-defined high risk conditions, or have severe or progressive clinical presentation


Plasmodium falciparum-infected erythrocytes (IEs) in the microvasculature of vital host organs. Identification of P. falciparum cytoadherence phenotypes that are associated with severe malaria may lead to the development of novel strategies against life-threatening malaria.Severe malaria has been attributed partly to the sequestration of P. falciparum isolates from Mozambican children under 5 years of age with severe malaria (cases) were examined and compared to 46 isolates from sex and age matched Mozambican children with uncomplicated malaria (controls). Cytoadherence properties such as platelet-mediated clumping, rosetting and adhesion to purified receptors , were compared between these matched pairs by non-parametric tests. The most common clinical presentation associated with severe malaria was prostration. Compared to matched controls, prevalence of platelet-mediated clumping was higher in cases (P = .019), in children presenting with prostration (P = .049) and in children with severe anaemia (P = .025). Prevalence of rosetting and gC1qR adhesion were also higher in isolates from cases with severe anemia and multiple seizures, respectively (P = .045 in both cases), than in controls.Forty-six These data indicate a role for platelet-mediated clumping, rosetting and adhesion to gC1qR in the pathogenesis of severe malaria. Inhibition of these cytoadherence phenotypes may reduce the occurrence or improve the prognosis of severe malaria outcomes. Plasmodium falciparum causes approximately 500 million clinical episodes of malaria per year P. falciparum-infected erythrocytes (IEs) in the microvasculature of vital host organs leading to mechanical obstruction of blood flow, hypoxia, tissue damage and, ultimately, organ failure P. falciparum IEs to host receptors expressed on the endothelium lining host capillaries, on uninfected erythrocytes to form ‘rosettes’ P. falciparum, which is responsible for most of the malaria related deaths, is known to sequester P. falciparum variant types suggests that IEs can exhibit preferential adhesion in the body The following two theories are proposed to explain the patho-physiological mechanisms leading to severe malaria. The cytokine-based hypothesis suggests that inflammatory mediators such as cytokines and nitric oxide released by host cells following exposure to malaria ‘toxins’ have a major role in the onset of the pathology of severe malaria In vitro analysis of the cytoadherence phenotypes of P. falciparum isolates derived from infected individuals has shown a wide range of binding affinities to numerous host receptors such as intercellular adhesion molecule ICAM-1 P. falciparum isolates, namely, adhesion to ICAM1, CD36 and gC1qR, rosetting and platelet-mediated clumping, and disease severity in children less than 5 years of age residing in a malaria-endemic area of Mozambique.Here we have examined the relationship between specific adhesion phenotypes of Among the 142 parasite isolates included in the study, 111 (78%) matured to trophozoite stages. The proportion of isolates that did not recover from cryopreservation was similar for parasite isolates collected from children with severe  or uncomplicated malaria . Parasitemia at thawing was higher for isolates from children with severe malaria  than in children with uncomplicated malaria . Of the 111 isolates that matured to trophozoite stage, 46 matched case-control pairs of parasites could be used for analysis. The characteristics of the 92 patients that provided the parasite isolates used are summarized in 2; standard deviation 8.78), we established the cut-off for positive binding to endothelial host receptors as 19.5 per mm2 (mean+ 2 standard deviations).Isolates were considered positive for rosetting if frequency of rosettes was higher than 2% P. falciparum isolates tested (65%) formed clumps in presence of platelets. Among those isolates showing platelet-mediated clumping, patient's platelet count decreased with increasing frequency of clumps . No correlation was found between frequency of clumps and parasitemia at thawing . Results from platelet-mediated clumping assays performed without adjusting parasitemia correlated well with results from the same assay performed with parasitemias adjusted to 1% . Both assays gave similar results in terms of prevalence of clumping , although medians for frequency of clumping were slightly higher in assays with unadjusted parasitemias  compared to assays performed at 1% parasitemia .I was doing it and I will do my best to finish Sixty out of 92 P. falciparum isolates , followed by platelet-mediated clumping , adhesion to ICAM1 , to gC1qR  and rosetting . Among the isolates showing cytoadherence, median adhesion (parasites per mm2) was highest for CD36 , followed by gC1qR  and ICAM1 . Median frequency of rosetting and platelet-mediated clumping was 3.0% (IQR [1.9–5.3]) and 7.2% (IQR [1.9–23.3]), respectively.Adhesion to CD36 was the most frequent cytoadherence phenotype among Cytoadherence phenotypes were compared between isolates from children presenting with severe malaria or specific severe malaria syndromes and their matched controls. For the rest of the cytoadhesion phenotypes, a uniform trend towards higher levels of cytoadherence among parasites isolated from children with severe malaria was observed, although these differences were not statistically significant. Platelet-mediated clumping assessed at unadjusted parasitemias was also higher for those parasites isolated from children with prostration and severe anemia, as compared to their matched controls  and 3. SThe results of this case-control study suggest that platelet-mediated clumping is associated with severe malaria. Moreover, to our knowledge, this study reports for the first time that platelet-mediated clumping is associated with prostration, and that adhesion to the recently identified cytoadherence receptor gC1qR The 46 patients with severe falciparum malaria participating in this study are representative of this condition in Manhiça (Mozambique) P. falciparum isolates infecting Mozambican children were characterized by their predominant adhesion to CD36 (87%), followed by platelet-mediated clumping (65%), adhesion to ICAM1 (45%), to gC1qR (42%) and rosetting (36%). It could be argued that cryopreservation of parasites may reduce the adhesive ability of IEs, although it has been reported that it does not alter the amount of parasite proteins on the surface of IEs as assessed by flow cytometry In accordance with previous studies from other sites Since pathogenesis may be quite different among the syndromes used to define severe malaria, we have also analyzed cytoadherence separately for each syndrome. Prostration, the most prevalent severe malaria presentation in this study population, was found to be associated with platelet-mediated clumping. Multiple seizures were observed to be associated with adhesion to gC1qR. Seizures are frequent among severe malaria cases. They may occur as part of CM or independently Severe anemia was associated with higher platelet-mediated clumping and rosetting, suggesting a role for these cytoadherence phenotypes in the etiology of malaria-related anemia. Destruction of uninfected erythrocytes attached to IEs in rosettes or down regulation of erythrocyte production by abnormal erythrocyte aggregates might also contribute to severe anemia. The role of platelet-mediated clumping in severe anemia is unclear and requires further investigation. If platelet-mediated clumping is found to have a causative role in severe anemia, novel anti-adhesion interventions that prevent platelet-mediated clumping might have an impact on reducing severe anemia in malaria endemic areas. Such interventions might be of public health relevance, as severe anemia is an important contributor to malaria-related morbidity and mortality in children residing in sub-Saharan Africa Role of adhesion to CD36 in the physiopathology of severe malaria is still a subject of debate, as some studies have found higher binding in isolates infecting children with severe malaria P. falciparum may be an important virulence factor. The potential for inhibiting or reversing platelet-mediated clumping and adhesion to gC1qR with antibodies or receptor analogs could improve severe malaria outcomes and should be actively explored.In summary, the results of this case-control study support the hypothesis that cytoadherence properties of The study protocol was approved by the National Ethics Review Committee of Mozambique and the Ethics Review Committee of the Hospital Clinic of Barcelona.P. falciparum infections with an average entomological inoculation rate of 38 infective bites per person per year in 2002 The study took place in Manhiça District, southern Mozambique. The study area, which is characterized by perennial malaria transmission with some seasonality, has been extensively described elsewhere P. falciparum malaria were recruited into the study after written informed consent was given by their parents or guardians. Blood smears were stained with Giemsa and examined by microscopy according to quality-controlled procedures P. falciparum≥500/µLon thin blood film examination. This definition has both a sensitivity and specificity of greater than 90% in children from Manhiça Between April and November 2006, a sex and age (+/−3 months) matched case-control study was conducted, the cases being children under five years of age with severe malaria (n = 71) and controls being outpatient children with non-severe malaria (n = 71). Children attending the Manhiça District Hospital with a primary clinical diagnosis of Before treatment, 10 mL of peripheral blood was collected by venipuncture into a tube containing lithium heparin. All blood samples were collected by trained persons in presence of a paediatrician. The amount of blood collected was less than the limit of 3 ml per kg of bodyweight as recommended by ethical guidelines msp-1 ormsp-2 alleles detected in the sample by PCR DNA was extracted from blood onto filter papers using the QIAGEN QIAamp DNA Mini Kit. The number of concurrent infections  was estimated as the highest number of P. falciparum isolates were thawed by serial dilutions in a gradient of salt solutions following standard methods. Erythrocytes were washed in RPMI 1640 medium containing 2 mM L-glutamine, 25 mM HEPES, 25 mM sodium bicarbonate and 10 µg/mL gentamicin (incomplete RPMI) and cultured at 37°C in complete RPMI (incomplete RPMI supplemented with 0.5% Albumax I and 50 mM hypoxanthine) as previously described Cryopreserved 2 was counted by observation of 10 fields (using 100× objective) under light microscopy. Adhesion to BSA was subtracted to yield specific binding to each receptor.Measurement of parasite adhesion to purified receptors was done as previously described An aliquot of culture suspension at pigmented trophozoite stage was stained with 20 µg/mL of ethidium bromide in rosetting media (incomplete RPMI supplemented with 10% AB+ human sera). Twenty µL of the suspension at 2% hematocrit was examined by fluorescence and direct light microscopy, and the proportion of IEs in rosettes was measured for each sample in triplicate. Two hundred IEs were scored in each experiment, with the adhesion of two or more uninfected erythrocytes to an IE constituting a rosette. The level of rosetting is reported as the percentage of IEs forming rosettes.g for 5 min. Platelet rich plasma was transferred to a separate tube, equal volume of fresh CCDAT buffer  was added drop wise and incubated for 15 min. Platelets were collected by centrifugation at 1000 g for 20 min and resuspended in CCDAT buffer. Homogenous platelet suspension was transferred to new tubes and centrifuged at 1000 g for 20 min. Platelets were finally resuspended in Hanks buffer at 108 platelets per mL, stored in sterile condition at room temperature and used within 4 days of collection. All steps were performed at room temperature. P-selectin (CD62) was not detected on the surface of platelets by flow cytometry Blood was collected from donors by venipuncture in citrate-phosphate dextrose solution (Sigma Aldrich), and centrifuged at 250 Parasites were thawed and used for clumping assays as described below. The assays were performed at thawing parasitemias and 2% hematocrit, as well as at 1% adjusted parasitemias  and was analysed using the Stata version 9.0 (Stata Corporation). A Table S1Platelet-mediated clumping results in assays conducted at 1% parasitemia and 5% hematocrit.(DOCX)Click here for additional data file.


To better understand the underlying mechanisms, we aimed to map the physiological stretch-induced molecular changes related to cytoskeletal (CSK) structure and contractile force generation through integrin receptors. Using ultrasound, we measured airway constriction in isolated intact airways during 90 minutes of static transmural pressure (Ptm) of 7.5 cmH2O or dynamic variations between Ptm of 5 and 10 cmH20 mimicking breathing. Integrin and focal adhesion kinase activity increased during Ptm oscillations which was further amplified during constriction. While Ptm oscillations reduced β-actin and F-actin formation implying lower CSK stiffness, it did not affect tubulin. However, constriction was amplified when the microtubule structure was disassembled. Without constriction, α-smooth muscle actin (ASMA) level was higher and smooth muscle myosin heavy chain 2 was lower during Ptm oscillations. Alternatively, during constriction, overall molecular motor activity was enhanced by Ptm oscillations, but ASMA level became lower. Thus, ASMA and motor protein levels change in opposite directions due to stretch and contraction maintaining similar airway constriction levels during static and dynamic Ptm. We conclude that physiological Ptm variations affect cellular processes in intact airways with constriction determined by the balance among contractile and CSK molecules and structure.Recent reports suggest that tidal stretches do not cause significant and sustainable dilation of constricted intact airways  While AHR is often considered a consequence of inflammation Recently, LaPrad et al., however, challenged the notion that tidal stretches mitigate airway constriction in intact whole airways The aforementioned studies present compelling data showing a lack of influence of tidal stretch on intact airway function. However, it is unclear if and how tidal stretches affect the effector proteins and their interactions to regulate bronchial ASM contraction in the intact airway wall. The mechanical factors that determine airway constriction include the contractile force of the ASM, all passive loads against which contraction occurs, and the applied forces due to tidal breathing Since the above studies suggested that tidal stretches minimally influence the responsiveness of whole bronchial airways All chemicals were from Sigma-Aldrich  or otherwise stated.2O ranging from 2.1 to 4 mm) was freed from parenchyma, and the side branches were closed off. The airways were cannulated at each end and mounted horizontally in a tissue bath containing gassed (95% O2–5% CO2) and heated (37°C) Krebs solution . The airways were stretched longitudinally (∼110% of its resting length) and held fixed at its extended length for the entire experiment to ensure that only radial dilation occurs when the airway is pressurized. This amount of axial stretch mimics airway lengthening during tidal breathing −5 M) challenge, as previously described Bovine lungs were obtained from a local slaughterhouse  immediately after euthanasia and kept chilled. A bronchus of the right lung  or a static Ptm level  for 90 minutes with (n = 10) or without (n = 9) a single dose ACh (10−5 M) induced constriction. Ultrasound images were taken in real time. Images at the start (T0) and end (T90) of the protocol were used for normalization and for comparison. The airway lumen and the two airway walls were digitally segmented from the images and airway diameter was determined in the middle of the airway segment.The details of the experimental system are described elsewhere 2O, 0.2 Hz) was applied for 1.5 hours with a single dose of ACh (10−5 M).To block polymerization of microtubules or actin filaments nocodazole  or cytochalasin D  was used respectively. The length and diameter of each airway was measured and the inner volumes were calculated. The airways were mounted in the system horizontally as described above and both cannulated ends were closed off to insure constant concentration of the inhibitors. Noc  or CD  was injected into the lumen of the airways. After a 30 minute incubation period the closed ends were opened and the dynamic loading condition  for histological processing and the other ∼10 mm of the tissue for biochemistry was dropped into T-PER Tissue Protein Extraction Reagent to ensure membrane bound protein solubilization  containing Halt Protease Inhibitor Cocktail and Halt Phosphatase Inhibitor Cocktail  , both at 1.5X final concentration to avoid any protein degradation. The latter was immediately homogenized using PowerGen 125  and frozen overnight at −80°C and re-homogenized the next day . To ensure the complete solubilization of membrane bound proteins by the T-PER Tissue Protein Extraction Reagent sodium dodecyl sulfate (SDS) was added. The amount of protein in the supernatant samples was measured using BCA-Reducing Agent Compatible protein assay reagent kit .Equal amounts of total protein (7.8 µg/sample) were separated using 4–20% SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes . Western blot analysis was carried out to assess the levels of β-actin, α-tubulin, MLC, the activated form phosho-MLC (ph-MLC), MLCK, MYPT1, SMMHC2, ASMA, integrin-β1 (Int), its phophorylated form (ph-Int), FAK and phospho-FAK (ph-FAK). GAPDH was used as loading control. All primary and secondary antibody incubation as well as the bovine serum albumin blocking step was done for one hour. All antibodies were from Abcam Inc.  exept MYPT1 . Quantitative densitometry was performed after chemiluminescence detection using Pierce ECL substrates or SuperSignal West Pico Chemiluminescent Substrate  and corrections were made for background and loading control.2O2 and sections were washed in 10 mM sodium phosphate buffer, 150 mM NaCl (PBS), pH 7.5. Protein blocking step was performed with horse serum and sections were incubated for 1 hr with anti-β-actin or anti-α-tubulin or anti-ASMA or anti-F-actin antibodies. Mouse or rabbit IgG (20 ng/ml) as well as omitting the primary or secondary antibodies were used as technical controls. All antibodies were from Abcam Inc. . After PBS washes, the mouse or rabbit HRP conjugated secondary antibodies  were applied for 1 hr. Sections were washed in PBS and incubated for 30 min in VECTASTAIN ABC reagent (Vector Lab). Enzyme substrates (Vector Lab) were applied until the right colors developed: DAB (brown), Vector SG (blue/gray). After this step, either counter staining  and dehydration-clearing-mounting or, in the case of ASMA, a second antigen labeling with F- actin antibody was applied following the protocol as above on a second set of sections. All conditions were processed simultaneously for each antibody (n = 30/condition). Images were captured by a Nikon Eclipse 50i microscope and SPOT camera  and histological evaluation was performed.Formalin  fixed, paraffin-embedded sections of airways were deparaffinized in xylene and rehydrated in decreasing alcohol series. Endogenous peroxidase activity was quenched by 1% HAll data were expressed as means ±SE. One-way repeated measures ANOVA was used to determine the effect of the Ptm loading conditions on luminal radii. One-way ANOVA and unpaired t-tests were used on the cytoskeletal, adhesion and contractile molecules of intact airways for no Ach and Ach conditions, respectively. When the normality test failed, the corresponding non-parametric statistical procedures were used. Post hoc comparisons included Holm-Sidak and Tukey tests for parametric and non-parametric one-way ANOVA, respectively. Statistical significance was accepted at p<0.05.2O) or dynamic  loading conditions. Applying dynamic or static mechanical stresses resulted in similar diameters of intact airways without constriction. The application of Ach reduced airway diameter to about 55% of its value before constriction (p<0.05) independent of the mechanical loading condition consistent with our earlier findings 2O divided by the radius at Ptm = 5 cmH2O at baseline, showed a decreasing trend with time without Ach and without inhibitors  but it did not change with Ach, CD or Noc. On average, the strain was 2.7±0.8% in agreement with previous findings We measured the changes in airway diameter of intact bovine bronchi during static Ptm and dynamic Ptm applying physiologically realistic pressures to mimic breathing. To evaluate the involvement of the cytoskeleton in airway responsiveness, we characterized the levels of the tensile element β-actin and the compressive element α-tubulin, the two major load bearing components of the cytoskeleton Next, we assessed how the two major contractile cross-bridge forming proteins, ASMA and SMMHC2, respond to stretch and stimulation. Both protein levels were significantly influenced by stretch pattern and Ach stimulation . WithoutWe also determined the level and activation of MLCK, MYPT1 and MLC. While MLCK activates the regulatory 20 kDa MLC through phosphorylation allowing the formation of actomyosin and subsequently initiating ASM contraction via the sliding filament process, MLCP attenuates this process Following Ach challenge , both loSince inhibition of actin and microtubules resulted in substantial physiological responses , we nextAll the above molecular responses and the subcellular structures are influenced by cell-ECM interactions through integrins. We thus examined these interactions by assessing the effects of stretch and Ach challenge on integrin-β1 and one of its signaling kinase FAK as well as their activation through phosphorylation, summarized in In this study, we characterized the cellular and molecular determinants of airway responsiveness to stretch and constriction in intact bovine bronchi having radii between 2 and 4 mm corresponding to human airway generations of 4 to 9 Our results showed that the cytoskeletal β-actin was not different at the beginning and at the end of the protocol without constriction . HoweverThe contractile machinery was significantly influenced by the nature of stretch including the cross-bridge forming ASMA and SMMHC2  as well Interestingly, the changes in integrin- β1 and FAK also appeared to follow an inverse pattern of the β-actin suggesting that while muscle stiffness may have decreased during dynamic loading, the cells tended to interact more strongly with the ECM potentially also canceling each other's effect.2O) with and without intermittent DIs, at least at two mean Ptm levels (5 and 7.5 cmH2O) and a wide range of Ach doses (from 10−7 to 10−3 M) since little difference was found in airway responsiveness during static and dynamic loading for these conditions in two independent studies These inverse effects during static and dynamic loading can thus maintain the airway at the same luminal diameter following Ach-induced stimulation. A proposed mechanism is summarized in The internal structure of ASM visualized in What determines the final level of constriction for a given Ach dose as seen in What are the possible implications of our results to asthma? The airway wall structure in asthmatics is certainly different from that of normal subjects and varies with asthma severity in vivo the airways can also shorten axially. Such axial shortening is not likely to be considerable during constriction because the pitch angle of the muscle spiral around the airway is only about 13 degrees ex vivo preparation does not have innervation. We used Ach as a stimulant which is the neurotransmitter released from the parasympathetic nerve endings that regulate airway tone in situ. Our results are likely not species specific because Noble et al. found similar lack of sustained influence of stretch on constriction in human airways There are several limitations of the study. The experimental setup has fixed boundaries at the two ends of the airway whereas in vitro is related to an inverse regulation of contractile proteins by stretch pattern and contractile stimulation while the level of constriction is determined by the availability of ASMA/phMLC and the structure of both actin and tubulin. In order to be able to exploit the mechanisms revealed in this study for the treatment of asthma, experiments should validate the findings in animal models of asthma and ultimately in asthmatic patients.In summary, we found that the lack of airway responsiveness to tidal stretching in intact airways stimulated


Human presence and activity in tropical forest is thought to exert top-down regulation over the various ‘green-world’ pathways of plant-based foodwebs. However, these effects have never been explored for the ‘brown-world’ pathways of fecal-detritus webs. The strong effects of humans on tropical game mammals are likely to indirectly influence fecal detritivores (including Scarabaeine dung beetles), with subsequent indirect impacts on detrivore-mediated and plant-facilitating detrital processes. Across a 380-km gradient of human influence in the western Brazilian Amazon, we conducted the first landscape-level assessment of human-induced cascade effects on the fecal detritus pathway, by coupling data on human impact, game mammal and detritivore community structure, and rate measurements of a key detritus process . We found evidence that human impact indirectly influences both the diversity and biomass of fecal detritivores, but not detritivore-mediated processes. Cascade strength varied across detritivore groups defined by species' traits. We found smaller-bodied dung beetles were at higher risk of local decline in areas of human presence, and that body size was a better predictor of cascade structure than fecal resource manipulation strategy. Cascade strength was also stronger in upland, unflooded forests, than in seasonally flooded forests. Our results suggest that the impact of human activity in tropical forest on fecal-detritus food web structure is mediated by both species' traits and habitat type. Further research will be required to determine the conditions under which these cascade effects influence fecal-detritus web function. Abundant evidence now supports a role for vertebrate regulation of the structure and function of foodwebs Alternatively, predator-mediated reduction in herbivore fecal resource availability may instigate four-level trophic cascades along the fecal detritus web. For example, mammal overhunting in tropical forests is predicted to negatively impact fecal detritivore communities by reducing the diversity and availability of fecal detritus inputs from the game mammals targeted by rural hunters Along a 380-km gradient of human impact along the Juruá River in the western Brazilian Amazon , we quanFirst, does human activity in tropical forest influence the community structure of fecal detritivores by decreasing the availability and diversity of mammal fecal inputs? The ability of trophic cascades to substantially alter the biomass or diversity of entire trophic levels (i.e. community-level cascades) remains controversial Alternatively, the strength of trophic cascades may be driven by variation in values of species' trait important for trophic interaction Second, can human impact-mediated trophic cascades influence a key detrital process - the secondary dispersal by dung beetles of seeds excreted in mammal feces? We used a field experiment to quantify the proportion of seeds buried by detrivores across a gradient of human impact. We expected that (i) heavier human impact would be correlated with reduced seed burial rates due to the indirect influence of hunting on community-level dung beetle biomass and diversity, (ii) the negative effects of human impact would be disproportionately strong for the largest sized seeds terra firme) represent the dominant forest type across the Amazon, and are associated with heavily leached and oligotrophic soils. Seasonally flooded forests (várzea) along white-water rivers account cover roughly 180,000 km2 of the 7 million km2 (2.6%) of the Amazon basin To better understand the generality of top-down trophic processes in fecal detritus webs, we explored these questions across two common Amazonian forest types. Upland forests  and the Uacari Sustainable Development Reserve . These reserves are contiguous and bisected by the Juruá River, a large white-water tributary of the Amazon (Solimões) River . The twoWithin the reserve system, approximately 4,100 local residents are distributed across 74 variable-sized human settlements. Adjacent to the reserve system lies the municipal town of Carauari and the satellite community of Riozinho, with a total estimated population of 25,200 2) 25 = −0.59, p = 0.001).Human impact was represented as the size of each permanent human settlement (i.e. number of households) nearest each census transect terra firme forest N = 15, várzea forest N = 11) distributed across the study region Between January 2008 and December 2010, medium and large-bodied (≥1 kg) terrestrial mammal assemblages were characterized using standardized line-transect surveys across a total of 26 sites (Dung beetles (Coleoptera: Scarabaeidae: Scarabaeinae) were sampled using standardized baited pitfall traps  buried flush with the ground and baited with 20 g of fresh human dung. Human feces are routinely used as standardized collection protocol in Neotropical dung beetle biodiversity studies terra firme and 11 várzea forest sites, a total of 15 traps were placed every 50 m along linear transects, beginning at the 400 m mark of the same transect used for surveying mammals. These trail segments were those nearest to local communities along each transect, thereby maximizing any spatial effects of human activities on the fecal detritus system. Trapping was conducted twice at each site, coinciding with the late-dry (August–September 2009) and early-wet seasons (December–January 2010). Fewer sites were sampled in the wet season due to accessibility issues . Traps were operated for one 24-h period at each site. Captured specimens were separated to species paracoprid (i.e. tunneler) species locate their nests underneath the fecal deposit; telocoprid (i.e. roller) species locate their nests at great horizontal distances from the fecal deposit; and endocoprid (i.e. dweller) species nest directly within fecal deposits In each of 15 terra firme forest (n = 15) transects, and within the dry season. The border of each mesocosm arena was delimited by a mesh fence , and at the center of each mesocosm, we placed a single 150 g experimental fecal deposit of fresh human feces. Each fecal deposit was mixed with 70 plastic seed mimics in three size classes . Seed mimics  are an ideal proxy for real seeds in tropical forest, as they are not subject to rodent or ant seed predation or removal and have similar burial rates by beetles We set up a seed burial experiment the day before dung beetles were sampled. The sampling protocol consisted of establishing four circular, 1 m diameter mesocosm plots, spaced 100 m apart, and located within the first 400 m of transects used to survey mammal and beetles. Due to logistical constraints, we only measured seed burial within All necessary permits were obtained for this work from the Brazilian Council Scientific and Technological Development (CNPq) and the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA) under SISBIO permit 16620-1. Collections took place in lands within sustainable development forest reserves, under the jurisdiction of State of Amazonas Secretariat for the Environment and Sustainable Development (SDS-CEUC) and the Chico Mendes Institute of Biodiversity Conservation (ICMBio). No protected species were captured, sampling of all mammal species were restricted to non-invasive line-transect censuses, and no primates, rodents, ungulates  were handled during the study.terra firme and várzea). We explored all models with both multiple generalized linear mixed models (GLMM) and generalized multilevel path analysis (GMPA) to examine the causal support for overall cascade structure with statistical controls We examined the evidence for three distinct cascade structures, represented as either community-level  or traitTo examine community-level cascades with generalized multiple linear mixed models, we modeled (i) game mammal abundance as a function of human impact (i.e. settlement size), (ii) dung beetle community species richness or biomass as a function of human impact and game mammal abundance, and (iii) the probability of seed burial as a function of human impact, game mammal abundance and dung beetle species richness or biomass. To discern which dung beetle community attribute (i.e. species richness or biomass) best explained seed burial probability, we compared two separate models including either beetle species richness or biomass with AIC-based model comparison.terra firme models that included seed burial, we separated seeds into size classes . To discern which property of dung beetle community structure (i.e. species richness or biomass) and trait-defined pathway (i.e. body size or nesting strategy) best explained seed burial probability, we compared four separate models for each seed size class with AIC-based model comparison. All analyses used beetle data taken from the 10 traps farthest (≥300 m) from the nearest mesocosm used to measure function to avoid potential bias in beetle capture rates. Ungulates and rodents were excluded from várzea forest models due to their rarity in this dataset. All predictors were centered on their means to facilitate interpretation. For all models, we used appropriate error structures  and incorporated both season (i.e. wet or dry) and transect identity as random effects in an unreplicated, crossed design To examine trait-defined cascades with a GLMM approach, we re-ran these same models after separating both the dependent and independent variables into groups defined by species traits relevant to trophic interactions. We separated game mammals into broad taxonomic groups  that both encounter differential selective hunting pressure (e.g. Jerozolimski and Peres 2003) and present distinct feces morphologies and deposition locations  U of independence claims implied by a DAG that express the full set of dependence and independence claims implied by the causal graph, when taken as a set  in a causal graph that lack an arrow between them and then conditioning each of those k pairs by the set of variables Z that are either a direct cause of Xi or of Xj . The overall hypothesized causal structure implied by the path diagram can then be tested by combining values of ip using Fisher's test statistic C as:P value associated with the C statistic is smaller than the specified α-level  after comparison to a chi-square (χ2) distribution with 2k degrees of freedom. A significant P value supports a rejection of the proposed DAG, as it implies that the data depart significantly from what would be expected under such a causal model To additionally assess the causal influence of human impact on cascade structure, we used generalized multilevel path models (GMPA) to examine the support for overall cascade structure set e.g. . BU is oor of Xj . The prok independence claims implied by each proposed causal path models (see TS6). These GMPAs used identical model structures as the GLMMs described above. Values of ip were taken from the P value associated with the t statistic for the regression coefficient of the composite variable. All analyses were conducted using the ‘nlme’ We represented each of our three overall hypotheses about community and trait-defined trophic cascade structure with a DAG  and testterra firme or várzea forest sites were similar in both size  and distance to censused transects .Across the 26 forest transects sampled for both mammals and dung beetles, we conducted a total of 8,430 km of mammal census walks . These transects were associated with 15 neighboring human settlements, varying in size from five to 500 households . Human communities adjacent to terra firme and várzea sites , while terra firme sites supported significantly higher mammal species richness .Mammal surveys resulted in observations of 38 species see . Primateterra firme: N = 5,887, S = 83; várzea: N = 5,513, S = 57; see várzea forest , while biomass and species richness were higher in terra firme . There were no significant differences in mean individual beetle body mass between forest types . Dung beetle biomass and species richness per trap were significantly higher in the dry season .A total of 10,819 dung beetle individuals in 90 species were captured  and used a tunneling nesting strategy (58%). For our analyses, a total of 68 species were classified as ‘small’  and 31 species as ‘large’ . The distribution of nesting strategies across small and large species was similar, with the majority classified as ‘tunnelers’ , while ‘rollers’ represented 30% of small species and 20% of large species. The proportion of seed mimics  removed by detritivores per forest site was greatest for large seeds, lowest for medium seeds and intermediate for small seeds .terra firme and várzea forests  and ungulate abundance . Seed dispersal was further unrelated to the biomass of large-bodied dung beetles and rodents  and negatively associated with primate abundance .The probability of community-level seed burial was equally well explained by models that included beetle biomass or species richness . In neitterra firme forest showed a significant negative relationship with human impact  and biomass , suggesting the absence of density or biomass compensation.Across trait-defined mammal groups, only hunted primates in terra firme and várzea forest . The biomass of species with a ‘roller’ strategy was negatively associated with game rodent abundance , while the species richness of rollers was negatively correlated with ungulate abundance in terra firme forest . In várzea forest, the abundance of hunted primates was unrelated to all measures of nesting strategy , as well as ungulate abundance . The likelihood of small seed burial was negatively associated with primate abundance . Despite this strong overall model support, individual path coefficients from both forest types were weak . In contrast, species with a ‘rolling’ nesting strategy were negatively associated with the abundance of both caviomorph rodents  and ungulates . While the predation of adult dung beetles by caviomorph rodents and ungulates is a possible explanation for these results We found no consistent patterns of association between dung beetle nesting strategy and responses to human presence or game mammal abundance. Beetle species with a ‘dwelling’ strategy were positively associated with primate abundance in We found stronger support for trophic effects defined by dung beetle body-size than those defined by nesting strategy. Contrary to our initial expectations First, the high vagility of large dung beetles may enable the detection and pursuit of fecal resources across wider spatial extents Alternatively, the neutral abundance response of larger-bodied beetles in areas of human settled areas may arise from a positive relationship between local resource scarcity and capture rates terra firme forest, we observed a decoupled response to human activity  and the abundance of large-bodied ateline primates (positive) that were themselves strongly impacted by human activity. This decoupled response may reflect the high demographic consequences of dispersal through the faunal depletion zone for large-bodied primates For large-bodied dung beetles in Any of these three of these size-biased processes may translate into a degree of functional ‘spatial insurance’ We found no relationship between human activity in tropical forest and detritivore-mediated rates of secondary seed dispersal. Strong attenuation between top-down forces and processes related to plant growth and demography are not uncommon in trophic cascade studies, particularly those that focus on density-mediated indirect interactions between predators and plants We posit that part of this variability may arise from a widespread sampling artifact. In studies that collect data on ecological process rates and biodiversity in separate steps , differences in the size of the baits used to measure function (150 g) or attract beetle diversity in pitfalls (20 g) may introduce a large-beetle bias in ecological process measurements, as larger-bodied beetles can respond from greater distances to the larger scent plumes emitted by functional mesocosms. Horgan (2005) reported marked differences in observed beetle-function relationships when mesocosm data were compared with biodiversity data collected either directly within the mesocosms, or in neighboring pitfall traps (as used here). This suggests that size-biased sampling artifacts can indeed influence the observed correlations between dung beetle community attributes and function. This artifact can be overcome with simple modifications to the collection of either paired functional or biodiversity data.terra firme, rather than várzea forests. These differences were strong, and are likely associated with the biological and socio-economic features unique to each forest types, despite their naturally close spatial proximity. Biomass of all vertebrates terra firme forests, and location of manioc fields within terra firme sites may support the exploitation of large-bodied primates in upland forest, beyond the threshold at which active pursuit is predicted on the basis of hunting effort alone terra firme and várzea forests were specifically present across our study region, where the two forest types differed in mean basal area We also found that forest type strongly mediated trophic cascade strength, with clearer evidence for human impact-induced cascade effects in Finally, we found that our use of two analytical approaches (i.e. GLMM and GMPA) resulted in strikingly different assessments of both cascade structure and strength. Path analysis suggested that a community-level model of cascade structure was most appropriate for both forest types, a result potentially linked to the higher number of parameters in trait-defined models see . Despiteweaken detrital cascades strengthen them relative to plant cascades, by tightly coupling consumption to resource depletion. A quantitative exploration of these questions will demand new information on the topology of ecological interaction networks formed by fecal detritus producers and consumers, as well as an improved understanding of the spatial dynamics of feces producer and consumer co-decline.Our study also raises an interesting and currently underexplored issue, related to the net effects on ecological function of cascade dynamics in donor-controlled systems. When the indirect interactions that lead to detritivore decline are mediated by a reduction in the availability of detrital resources, and the ecological processes of interest result from the consumption of those detrital resources, does biodiversity loss beget a true net loss of ecological functioning? This question stems from the donor-controlled nature of the detritus pathway, and is generalizable to any detritus based system. While feedbacks between plants and plant consumers in green-world pathways largely determine overall cascade structure and strength Our landscape-level study allowed us to detect evidence for a cascading impact of human activity on detritivores, but not detritivore-mediated processes Figure S1Relationship between human settlement size and the abundance of large and medium diurnal tropical mammals.(TIFF)Click here for additional data file.Table S1Summary of mammals encountered through 8,430 km of line transect sampling in both várzea and terra firme forest sites .(DOCX)Click here for additional data file.Table S2Total number of captures per habitat type and values of three species traits: mean body mass, activity period and food relocation strategy. Abundances represent total captures from terra firme (n = 369 trap nights) and várzea forests (n = 277 trap nights).(DOCX)Click here for additional data file.Table S3Generalized linear regression results of medium and large-bodied diurnal game mammals as a function of human impact in terra firme and várzea forest.(DOCX)Click here for additional data file.Table S4Generalized linear regression results of detritivorous dung beetles as a function of human impact, game mammal abundance and sampling season in terra firme (A) and várzea forest (B).(DOCX)Click here for additional data file.Table S5AIC model selection results (A) and generalized linear regression results (B) for AIC top models of dung beetle-mediated secondary seed burial.(DOCX)Click here for additional data file.Table S6The Bu basis set of d-separation for three alternative causal path models of the direct and indirect impacts of human activity in terra firme and várzea forests.(DOCX)Click here for additional data file.


We found that salt-loaded pregnant rat dams were hypernatraemic at day 20 gestation (147±5 vs. 128±5 mmoles/L). Increased extracellular salt impeded murine kidney development in vitro, but had little effect in vivo. Kidneys of the adult offspring had few structural or functional abnormalities, but male and female offspring were hypernatraemic (166±4 vs. 149±2 mmoles/L), with a marked increase in plasma corticosterone . Furthermore, adult male, but not female, offspring had higher mean arterial blood pressure , member 3 (SLC9A3) together with altered faecal characteristics and electrolyte handling, relative to control offspring. On the basis of these data we suggest that excess salt exposure, via maternal diet, at a vulnerable period of brain and gut development in the rat neonate lays the foundation for sustained increases in blood pressure later in life. Hence, our evidence further supports the argument that excess dietary salt should be avoided per se, particularly in the range of foods consumed by physiologically immature young.Hypertension is common and contributes, via cardiovascular disease, towards a large proportion of adult deaths in the Western World. High salt intake leads to high blood pressure, even when occurring prior to birth – a mechanism purported to reside in altered kidney development and later function. Using a combination of  Between a quarter and a third of the world's adult population suffer from hypertension and as a result are significantly predisposed towards diseases of the cardiovascular system (CVD) including stroke, coronary and/or ischemic heart disease The regulation of blood pressure is a classic example of a complex, multi-factorial and polygenic trait; hence, the aetiology of essential hypertension – a sustained above-average increase in blood pressure with no known cause – has occupied the hypertension field for many decades Whilst increased salt consumption has demonstrable, direct pressor effects in adult individuals Nevertheless, the ability to excrete salt and participate in optimal fluid homeostasis is dependent upon the anatomical integrity and physiological capacity of the kidneys. Maternal malnutrition may impact upon both of these aspects of kidney function in the offspring, although a programmed deficit in nephron number has received most attention (for review see in vitro and in vivo approaches (using multiple cohorts) to examine the effect of high extracellular salt on kidney development and function. After characterisation of our phenotype partially supported the null hypothesis  a further cohort was established in which we repeated our analyses of renal function in the offspring (at 8 weeks of age) but also conducted a preliminary analysis of gastrointestinal electrolyte handling and faecal characteristics in the male and female offspring at this time.Hence, in this study we chose to use the paradigm of maternal salt-loading to examine the programming of renal function and blood pressure in the adult (12 week old) male and female offspring. We hypothesised that maternal salt-loading would lead to hypernatraemia in both dam and fetus which blunts development of the fetal kidney and compromises adult renal function; an effect characterised by renal sodium retention, hypernatraemia and hypertension in both male and female offspring. We used a combination of All procedures involving animals were carried out under license and in accordance with the Home Office Animals (Scientific Procedures) Act 1986 and were approved by the local ethical review committee of the University of Nottingham. The study was designed to test the hypothesis that maternal intake of excess salt impedes normal development of the fetal kidney leading to poorer control of salt balance later in life, an effect mediated at the level of the kidney.ad libitum standard laboratory chow  for 1 week prior to being randomly assigned to 1) Control diet  fed purified standard chow  and tap water or 2) Salt diet  fed purified standard chow with 4% NaCl added  and tap water. Rats were habituated to the diets for 4 weeks and remained on the diets through mating, conception (plugging designated as d0), gestation and lactation (offspring weaned at 3 weeks of age). Weight gain and other descriptive parameters in dams were not influenced by diet (data not shown). Proportions of dams were euthanized  at different stages of gestation  for blood collection (into Li-heparin tubes) and plasma. At day 20 gestation, maternal and fetal organs were recovered and either snap frozen in LN2 (stored at −80°C) or fixed  and plasma obtained (stored at −20°C). Remaining dams  proceeded to term with litters standardized to eight pups at birth . At weaning, dams were euthanized and the remaining pups group housed according to sex and fed standard chow diet thereafter, unless otherwise indicated. Due to occasional experimental difficulties not all measurements were available for all variables in dams and the appropriate experimental n is indicated in individual Figures and Tables.59 Sprague Dawley female rats  were housed in a temperature (20–22°C) and humidity (55–65%) controlled environment and subjected to a 12 hour light/dark cycle (0700–1900 h). Dams were fed After weaning and between 8–12 weeks of age, two siblings from each litter  were entered into one of four protocols:Baseline renal function at 8 and 12 weeks of age. Baseline renal function was established in two cohorts of offspring at 8 and 12 weeks of age  by 24 h urine collection in a metabolic crate (after 24 h acclimatisation to the environment) with a paired blood sample collected at 24 h.Salt-stimulated renal function at 12 weeks of age. In a separate cohort, salt-stimulated renal function was established in 12 week old offspring . In brief, renal function was assessed as described above but after rats were fed salt-diet for 4-days (including 24 h acclimatisation to the met crate).Blood pressure assessment by telemetry. A proportion of offspring  were surgically implanted with a radiotelemetric probe at 9 weeks of age, as previously described −1 of each i.p.), for probe implantation  as described previously −1) and analgesia administered  together with a long-acting antibiotic . After 5–7 days, blood pressure was measured continuously for a 48 h period at a sampling rate of 2×15 sec periods per min .Tissue collection at 8 and 12 weeks of age. Any remaining offspring from each cohort  were euthanised at 8 or 12 weeks of age without exposure to any experimental procedure for collection of control (unstimulated) plasma and organs. At 8 weeks of age, faecal matter (the first fully formed stool in the colon) and gastrointestinal tissue  were specifically collected for fixation (4% PFA) or snap-frozen in LN2 and stored at −80°C for later analysis.3) using a microwave  followed by addition of 4 ml H20. Physiological measurements are presented adjusted to body weight (kg BW) to allow informed assessment of between-sex differences . Urine flow rate (Vurine) was measured in ml per 24 h and is presented as ml/min/kg BW. Creatinine (Ccr), albumin  or osmolal (Cosm) clearance (ml/min/kg BW) were calculated as /Crplasma). Free water clearance (CH2O) was calculated as urine flow rate – COsm.All dams and/or offspring were euthanised between 09.00–11.00 hrs. Plasma or urinary osmolality was determined by freezing point depression  with intra-assay variability being <1%. Tissue dry weights were determined by freeze-drying. Biofluid and faecal electrolytes  were determined by inductively-coupled plasma mass spectrometry  with intra-assay variability being <2%. Faecal matter was first acid-digested . Plates were read at 450 nm  or 405 nm (vasopressin) on an ELISA MRX plate reader. Values were interpolated from a 4-parameter logistic curve and reported cross reactivity for vasopressin was <0.001% . The reported minimum detection limit for the hormones was: corticosterone, 6.6 pg/ml; aldosterone, 3.4 pg/ml; vasopressin, 3.39 pg/ml.Plasma and urinary corticosterone and aldosterone were measured by ELISA as described by Rat glomeruli were counted at day 20 gestation and at 8 weeks postnatal age as described previously 2. Sodium chloride, mannitol or urea was added to the media to achieve an increase in ECF osmolality of 0 mM, 25 mM, 50 mM, 100 mM, 200 mM; 25 mM NaCl being reflective of the differences in plasma osmolality observed in vivo after salt-loading, whereas the higher concentrations were used to illustrate the toxic effect of high-salt on organ development. Organs were imaged using light microscopy  and surface area determined with Image Pro Plus (Media Cybernetics Inc. USA).Female mice (outbred ICR strain) were time mated between 09.00 hrs and 13.00 hrs and at embryonic day 12 dams were euthanized and the fetal kidneys/lungs were removed and cultured on polyethylene terephthalate tissue culture plate inserts . Cultured kidneys were maintained in DMEM-F12  supplemented with insulin (10 mg/L), transferrin (5.5 mg/L), sodium selenite (5 µg/L), penicillin and streptomycin. Cultured lungs were maintained in BGjB medium Fitton-Jackson Modification  containing 25 mg/dl ascorbic acid and 1% heat inactivated fetal bovine serum . All organs were maintained at 37°C in 5% CO3VO4, 10 mM NaF) containing a protease inhibitor cocktail . Approximately 50 µg protein was probed with an antibody raised against 11β hydroxysteroid dehydrogenase type II . Bands were visualised using ECL Advance reagent . Standard PCR was used to visualise the presence or absence of SLC9A3 in proximal or distal colon (×30-cycles) according to the manufacturers protocol (Sigma-taq) using the primer sequence: forward, TATCTTCGCCTTCCTGCTGT; reverse, GCTCTGAGATGTTGGCCTTC. 18S was used as the internal control.Western blotting was carried out as previously described 10-transformed before analysis, as required. Data are presented as estimated marginal means from the model with ± standard error of the mean (SEM) or of the difference between means (s.e.d.) or 95% confidence interval, as appropriate to represent the error for each comparison. Where male and female siblings were included in the statistical model then the dam was added as a random effect (to account for reduced intra-litter variance) and data were analysed by a General Linear Mixed Model . For cardiovascular circadian analyses all continuously recorded cardiovascular data /w) to derive four parameters α, set-point; β, amplitude; w, wavelength and ε, offset, which were analysed by 2-way ANOVA.The study was designed and analysed as a 2 ×2  factorial ANOVA. Data and residual distributions were first checked and logvs. 278±2 mosmoles/kg H2O for SD vs. CD dams, respectively). Continued dietary salt-loading maintained this difference in maternal plasma osmolality, for example, when measured at day 20 gestation . With no difference in plasma glucose, albumin or urea between diet groups (data not shown) the diet-induced difference in osmolality was likely due to increased extracellular fluid (ECF) sodium, an effect confirmed when measured at day 20 gestation  is 143±8 mmoles/L. At day 20 gestation, salt-loaded pregnant rat dams had renal hypertrophy  accompanied by polydipsia and polyuria with significantly increased free water clearance (+) excretion  the culture media tended to impede in vitro lung growth  or not cross (e.g. mannitol) plasma membranes. When grown in NaCl for two days, growth of murine fetal kidneys was reduced at 25 mM NaCl  but markogenesis . To deteg growth , but belg growth . Thus, evs. 298±1 mosmoles/kg H2O for SD vs. CD fetuses, respectively). In addition, glomerular number, a marker for the degree of branching renal morphogenesis at this time was not different between treatment groups . Furthermore, fetal and placental (wet and dry) weights were also not different between treatment groups or sex  and the maternal diets are fed throughout this time (to weaning at day 21). Hence, further potential effects of maternal salt diet on renal structure and function of the subsequent adult offspring were investigated.Fetal plasma osmolality was similar in both male and female fetuses, and was not influenced by maternal salt diet  and thus osmolality  with no influence of offspring sex being evident . The adult male offspring of salt-fed dams had significantly higher blood pressure throughout the circadian cycle whilst female offspring tended to have chronically lower blood pressure relative to respective control offspring , the male and female prenatally salt-exposed offspring had significantly increased plasma sodium concentration . When adjusted to plasma osmolality (i.e. osmolar clearance) the treatment effect disappeared  for 5 days and repeated the renal functional analyses. Dietary salt loading did not have a significant additional effect on plasma osmolality of offspring , but as expected, resulted in polydipsia and polyuria in all animals  were similar, renal histology did not indicate any glomerular or tubular injury/hypertrophy or adverse infiltration of inflammatory cells  which is glucocorticoid-inducible vs. 67.3±1.2% water) or total measured electrolytes . However, analysis of individual electrolyte concentrations in faecal matter indicated subtle effects of maternal diet on offspring colonic electrolyte handling; faecal Ca2+ content was significantly increased .Baseline plasma corticosterone was significantly elevated  in the male offspring of prenatally salt-exposed animals , with lincreased , there wecreased  and faect effect . Faecal in utero. However, we show that their adult offspring, despite no direct exposure to salt diet, retain their hypernatraemic phenotype contributing toward plasma hypertonicity and hypertension – the latter effect being markedly sex-specific . In vitro, increased extracellular salt in the media bathing a developing kidney significantly impairs its growth – an effect not observed in the lung, which also grows by branching morphogenesis. In vivo, fetal plasma is not influenced by maternal salt diet; hence fetal kidney development under these conditions is apparently normal, resultant adult kidney function is also relatively normal with no tendency for greater salt retention to explain persistent hypernatraemia and hypertension in salt-exposed male offspring. However, our preliminary evidence suggests that maternal salt-loading at a vulnerable and transitional  period for development of the offspring gut and brain may influence functional aspects of each to underpin hypertension as an adult. In the adult gut, maternal salt loading influenced electrolyte handling. In the adult brain we hypothesise an effect on the threshold for regulation of plasma osmolality, leading to increased plasma corticosterone  which further influences gastrointestinal electrolyte handling e.g. through the major glucocorticoid-sensitive sodium-coupled transporter in the proximal colon, SLC9A3.Moderate salt-loading of rat dams before and during pregnancy leads to hypernatraemia and marked changes to their fluid balance, but few overt effects on their offspring The level of hypernatraemia and thus increased osmolality in the offspring of salt-loaded dams despite little direct exposure is considerable, interesting and important. Increased sodium retention is commonly associated with glucocorticoid excess (apparent or otherwise) and hypertension Sodium intake. In adult offspring, daily food and thus salt intake was not different between groups either under basal conditions  in which either production of AVP is lacking  or kidneys are insensitive to AVP (nephrogenic DI) Increased salt-retention or decreased salt-excretion. Plasma corticosterone was considerably elevated (approximately 10 fold) in adult offspring of salt-fed mothers  tract. It is well established that glucocorticoids regulate sodium uptake across the gut via induction of expression of a number of sodium/proton antiporters, particularly SLC9A3 at specific locations along the gastrointestinal tract including ileum and proximal colon in vivo evidence to support a marked effect of maternal salt diet on offspring kidney anatomy and physiology; nephron numbers in near-term offspring (i.e. during nephrogenesis) and in adulthood (i.e. after completion of nephrogenesis) were similar between groups. We also found no evidence of renal damage in offspring kidneys, suggesting that, at least in our animals, there had been no direct structural impact on offspring kidneys of developmental exposure to a maternal high-salt diet. This variation in outcome despite apparently similar diets may reflect different experimental protocols used to measure similar endpoints or suggests that the influence of maternal salt varies with different populations (genotypes) of animals. Nevertheless, in vitro, it is clear that growth of cultured fetal kidneys is impaired when exposed to elevated extracellular NaCl  in the incubating media. By 2 days, growth was significantly impaired even at the lowest NaCl dose  was observed and the authors were able to identify an absence of oestrogen in males as the causal factor Higher blood pressure after exposure to a maternal high salt diet is one of the few repeatable phenotypes within the paradigm of developmental programming 2O, leaves the subsequent lambs with elevated plasma osmolality (due to hypernatraemia) – which is maintained even after infusion with hypotonic saline – and hypertension leads to offspring hypernatraemia  and hypertension . It is important to put in a comparative context the likely timing of the micronutrient insult relative to the developmental phase in the two species; late gestation in the fetal sheep and the neonatal period in the rat are considered particularly ‘vulnerable’ periods of brain development when similar developmental processes in the brain are occurring per se evokes an early and marked plasma volume expansion enabled, in part, through central downward resetting of the osmotic threshold for AVP release from the posterior pituitary Finally, it is of interest that plasma osmolality was responsive to salt-loading in pregnant rats  but not in their offspring, despite differing baselines between groups, suggesting rigid control of osmolarity around the set-point in all groups of offspring. The implication from these data is that an upward shift in the osmotic threshold has become established in the male and female offspring of salt-loaded rats and is fully-functional at that level  – negligible plasma vasopressin levels, despite high plasma osmolality, suggests adequate volume control at this elevated level in utero. The prevailing scientific literature suggests programming of kidney development and function primarily underpins this phenotype. We provide an alternative hypothesis: at a developmentally vulnerable period for the brain and gut in the rat i.e. during transition from parenteral to enteral feeding and the necessary physiological adaptation required in the offspring gut-brain axis, then increased salt exposure at this time – here, passively via the dam – may detrimentally affect this axis to have longer-term effects on the osmotic and pressor balance of the adult offspring. We acknowledge increased glucocorticoid action in males may, in part, underpin the sex-specificity of our phenotype but cannot ascribe cause or effect to this response since it is likely that many other endocrine pathways such as local renin-angiotensin-aldosterone action may equally be involved. Future work can begin to tease apart these multivariate effects. In conclusion, our study adds weight to the argument that salt intake should be reduced per se but particularly in the range of foods consumed by vulnerable babies and neonates.From a nutritional perspective, one price of progress is an inevitable and inescapable dietary intake of excess salt, which increases the blood pressure of an individual consuming excess salt but also the next generation exposed Figure S1in vitro lung growth.Increased extracellular salt has no effect on A–R: representative images of lungs (n = 4–6 replicates) cultured for 3 days in media with varying osmolality, generated using NaCl, mannitol or urea, at concentrations indicated on y-axes.(JPG)Click here for additional data file.Figure S2Renal structure and hormone output is unaffected by maternal diet. A,B; representative histological sections  from a single adult offspring (8-weeks of age) from dams fed a control, low-salt, (A) or high-salt  diet. C–F; data are for offspring of dams fed control diet  or 4% salt diet with water ad libitum . Plasma and urinary steroid hormones were measured by a rodent specific ELISA in plasma and urine collected after 24 h in a metabolic crate. Data are presented with mean (±95% CI) indicated and were analysed by mixed effects models with treatment  and sex  or their interaction as fixed effects and dam as a random effect (Genstat v14). Steroids were analysed as log10 transformed to normalise the error distribution and are shown as antilogs for clarity. NS, not significant.(TIF)Click here for additional data file.