Myelotoxicity is one of the most common treatment-related adverse events for patients receiving systemic antineoplastic therapy or radiotherapy to bone marrow–producing regions. Myeloid cytopenias, including neutropenia, thrombocytopenia, and anemia, are the most common manifestations of treatment-related myelotoxicity and one of the most common reasons for dose modifications, dose delays, or discontinuation of therapy, potentially limiting therapeutic benefit. Risk factors for myelotoxicity can be broadly categorized into three types: disease-related, host-related, and treatment- related. Familiarity with factors predictive of high-risk febrile neutropenia, bleeding due to thrombocytopenia, and cardiopulmonary compromise due to anemia will provide the advanced practitioner (AP) in oncology with critical tools for rapid identification of patients at risk, prompt implementation of established guidelines for management, and avoidance of clinical deterioration. The AP in oncology is often the primary point of contact for management of cytopenias, including administration of myeloid growth factors, transfusion of blood products, and management of acute events such as neutropenic fevers. Each of these interventions requires familiarity with the risk and benefits of treatment. This article will review the physiology of the bone marrow, risk factors for cytopenias, and current guidelines and recommendations for prevention and treatment of myeloid toxicity of cancer treatment. Myelotoxicity is one of the most common treatment-related adverse events for patients receiving systemic antineoplastic therapy or radiotherapy to bone marrow–producing regions. Myeloid cytopenias—including neutropenia, thrombocytopenia, and anemia—are the most frequently seen manifestations of treatment-related myelotoxicity and one of the most common reasons for dose modifications, dose delays, or discontinuation of therapy, potentially limiting therapeutic benefit. Lymphopenia, although less common, presents unique challenges and may place the patient at increased risk for opportunistic and often life-threatening infections. Proactive management of cytopenias can improve treatment tolerance and treatment outcomes. An understanding of the physiology of the bone marrow, normal hematopoiesis, risk factors for treatment-related cytopenias, strategies for minimizing serious adverse events (AEs), and adaptation and consistent application of these concepts for individual patient populations will limit the severity of hematologic AEs and improve treatment outcomes.The advanced practitioner (AP) in oncology is often the primary point of contact for management of cytopenias, including administration of myeloid growth factors, transfusion of blood products, and management of acute events such as neutropenic fever. The American Society of Clinical Oncology (ASCO), the National Comprehensive Cancer Network (NCCN), the American Society of Hematology (ASH), and the Multinational Association for Supportive Care in Cancer (MASCC) have published recommendations or guidelines for the management of treatment-related cytopenias. The US Food and Drug Administration (FDA) and the American Association of Blood Banks (AABB) have established guidelines for the administration of hematopoietic growth factors and blood products. Familiarity with these guidelines and recommendations, together with a working knowledge of common disease- and treatment-related risk factors, will provide a sound foundation for effective management of treatment-related myelotoxicity.JADPRO's series of articles on treatment-related adverse events .This article will focus on the clinical management of treatment-related myeloid cytopenias, including current guidelines and recommendations from the societies and associations noted above. Lymphopenia and the management of common infectious complications were previously discussed in The bone marrow is the primary source for development of the components of blood (hematopoiesis), including myeloid and lymphoid progenitor cells . Hematopoiesis occurs primarily in the axial skeleton, with the majority of production taking place in the pelvis (70%–72%), the long bones such as the femurs, the skull, the sternum, the ribs, and vertebral bodies . Extramedullary hematopoiesis, or production of the elements of blood outside the bone marrow, may occur in the spleen and other accessory sites in selected disease states such as the myeloproliferative disorders and chronic leukemia . The bone marrow comprises trabecular bone, stromal elements, hematopoietic cells, and elements of the bone marrow microenvironment (Table 1). Each of these elements plays a role in normal or abnormal hematopoiesis and represents a different percentage of the bone marrow space, in part determined by age and underlying disease.Normal myeloid hematopoietic progenitor cells arise from multipotent hematopoietic stem cells (HSCs) that are capable of self-renewal, allowing for continuous replacement of granulocytes, macrophages, and erythrocytes. The majority of HSCs remain noncycling, with only a few active at any time for release of these early progenitor cells . In the presence of more severe hematopoietic stress, such as bleeding, infection, or chemotherapy exposure, a greater number of HSCs are activated to increase progenitor production . The normal life cycle for each cell type varies. However, the continuous renewal and life cycle of the myeloid lineage is rapid compared with other tissue cell lines, playing a primary role in susceptibility to the effects of chemotherapeutic agents .The capacity for self-renewal is regulated by a number of intrinsic and extrinsic factors, including genetic and molecular mechanisms, cytokines, and signaling pathways ; see Table 1. The process of apoptosis plays a primary role in regulating the "on/off" mechanism of the self-renewal process. Defects in each cell line and in the microenvironment, including apoptosis, play a key role in the pathobiology of hematologic malignancies and contribute to susceptibility to cytopenias, the capacity for hematopoietic recovery, and the abnormal findings in the bone marrow or peripheral blood . Patients receiving antineoplastic therapies for solid tumors may develop treatment-induced abnormalities, causing similar changes and contributing to cytopenias, although the severity and duration of cytopenias are generally less severe.The concept of the stem cell niche, highly specialized bone marrow compartments with intricate regulatory processes driving stem cell development and maturation, is an evolving idea in the understanding of normal and malignant hematopoiesis . These discoveries have elucidated new areas for investigating the prognostic significance of cytopenias at diagnosis and during treatment and the potential for off-target effects of growth factor administration.Clinical evaluation of bone marrow function relies on analysis of peripheral blood (postmitotic pool) and a bone marrow biopsy and aspirate (mitotic pool/progenitor pool); see Figure 1 and Table 1. A complete blood count with differential and platelet count is the most common method for analysis of the postmitotic pool and will reflect the end results of bone marrow production with any secondary effects of the host. Bone marrow analysis is required for the diagnosis and risk analysis for hematopoietic malignancies, including cytogenetic and molecular information necessary for prognostication. The most common site for obtaining bone marrow samples is the posterior iliac crest, due to the accessibility, relative low risk, and bone marrow–producing capacity. The anterior iliac crest may also be used for sampling, although this is generally more difficult due to the architecture, accessibility, and depth of the cortical bone.Bone marrow aspirates may be obtained from the sternum using a specialized needle with a shield if there are contraindications for pelvic biopsies, such as for those patients who are intubated and are difficult to turn, for those with a fractured pelvis, or for patients who have had extensive radiotherapy to the pelvis, which may limit the diagnostic value of the specimen. Core biopsies may not be obtained from the sternum due to a high risk of sternal fracture and penetration of surrounding structures with potential for fatal hemorrhage, limiting the diagnostic evaluation. Bone marrow evaluation is performed infrequently in patients with solid tumors unless there are concerns for abnormal findings in the peripheral blood. Adequate bone marrow samples are necessary to provide a complete analysis of the mitotic pool, postmitotic pool, and disease attributes including genetic or molecular features. A summary of the key elements of the bone marrow with normal variants and life cycles for individual cell lines is included in Table 1.Risk factors for myelotoxicity can be broadly categorized into three basic types: disease-related, host-related, and treatment-related. Disease-related factors can be further divided into solid tumors vs. hematologic malignancies. The incidence, severity, and duration of myeloid cytopenias are greatest in the hematologic malignancies. Cytopenias may be present at the time of diagnosis as a result of abnormal cellular development or bone marrow infiltration. In some cases, such as chronic lymphocytic leukemia, autoimmune processes such as hemolytic anemia or idiopathic thrombocytopenia purpura may be present at diagnosis, contributing to the abrupt onset or severity of anemia or thrombocytopenia. Patients with massive splenomegaly due to acute or chronic lymphocytic leukemia or myeloproliferative disorders may present with thrombocytopenia due to splenic sequestration or peripheral destruction of platelets. Thus, evaluation of tumor burden at the time of diagnosis in patients with hematologic malignancies is a critical first step in estimating the risk for cytopenias with initiation of treatment. Furthermore, determining the extent of risk will assist in setting the expectations for both the patient and the providers as treatment is initiated.It is not uncommon to expect moderate to severe cytopenias in the early phases of treatment for hematologic malignancies, as the primary target for treatment resides in the bone marrow, and both normal and abnormal cells will be affected. Understanding the concepts of expected cytopenias, cytopenias getting worse before they get better, and sustained moderate but asymptomatic cytopenias is critical for the AP in oncology managing patients with hematologic malignancies . Treatment-related cytopenias are much less common in patients with solid tumors. However, these patients may also experience moderate to severe myeloid cytopenias as a result of bone marrow infiltration by tumor, radiation to bone marrow–producing sites, and other treatment- or host-related factors.The bone marrow’s capacity to recover is an important consideration in all patients. Patients with limited cellularity, extensive bone marrow fibrosis, underlying aplasias, or treatment-related secondary malignancies have an impaired ability to recover normal hematopoiesis. In most cases, the only option for restoration of normal hematopoiesis is an allogeneic hematopoietic stem cell transplant, which is limited primarily to patients with hematologic malignancies who meet stringent transplant eligibility criteria, including adequate organ function, a suitable donor, and the availability of a consistent caregiver.In addition to disease-related factors, selected attributes of the individual patient may increase the risk of treatment-related cytopenias (Table 2). Hematopoietic senescence is common in older patients due to the normal functional decline of the bone marrow with increasing age . The bone marrow of an older adult is generally less cellular (estimated as bone marrow cellularity % = 100 - patient age in years) with a higher fat content, increasing the susceptibility to cytopenias. Older patients more often experience compromised renal or hepatic function, which may contribute to cytopenias due to impaired metabolism of chemotherapeutic agents . Dose modification is required for selected chemotherapeutic agents in the instance of renal or hepatic impairment to limit the severity of AEs, including cytopenias. Similarly, comorbid conditions and associated medications may contribute to decreased bone marrow function or increased risk of drug interactions and treatment-related AEs .Certain drugs are known to be associated with cytopenias independent of bone marrow function, including immunosuppressive agents, anti-inflammatory medications, and antibiotics. Careful review of medications that may contribute to cytopenias is necessary to limit the severity of AEs and to avoid unnecessary dose modification of antineoplastic therapies. Malnutrition affects many cellular processes, including hematopoiesis. Decreased serum albumin levels may indicate an increased risk for treatment-associated AEs and are included in the risk analysis for many hematologic malignancies .Chemotherapy-induced myelosuppression is the most common dose-limiting AE for patients receiving cancer treatment. The incidence, severity, and duration of myelosuppression vary by drug, dependent on pharmacokinetic variables of dose, frequency, route of administration, absorption, distribution, metabolism, and excretion . Each antineoplastic agent varies with respect to the onset and duration of cytopenias. In general, cytopenias are dose dependent, so dose reductions or delays may be effective in minimizing the severity of cytopenias but may also limit the therapeutic potential of treatment. Dose-intensity and combination therapies are the most common approach used in cancer treatment regimens with the intent to exploit different mechanisms of action and vulnerabilities of the tumor while balancing toxicity profiles. However, increased doses and combination therapies frequently increase the potential for cytopenias . Several guidelines and recommendations have been proposed by international oncology organizations to estimate the risk for selected cytopenias and to provide evidence-based treatment guidelines (Table 3).The combination of chemotherapy and radiation therapy is commonly used to treat esophageal, gastric, head and neck, rectal, anal, and pancreatic tumors. Each of these disease states may include areas of bone marrow–producing regions in the radiation field, increasing the potential for more severe and sustained cytopenias, complicating the delivery of systemic therapy during or following chemoradiotherapy . Administration of myeloid growth factors during radiation therapy is generally avoided in these instances, as these agents promote cell division, increasing the potential myelosuppressive effect. Patients receiving treatment over extended periods of time, such as those with metastatic disease, may develop cumulative myelotoxicity and are at risk for secondary malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). The onset of AML and MDS varies according to the agents administered, with early onset (within 3 years) for patients receiving topoisomerase II inhibitors such as etoposide, teniposide, topotecan, and doxorubicin, and late onset (5 to 10 years) for patients receiving radiation or therapeutic alkylators such as cyclophosphamide . Chromosome 5 or 7 abnormalities are most common in these patients and are associated with a poor prognosis.The late onset of cytopenias, progressive cytopenias unexplained by ongoing treatment, the presence of circulating blasts, or the onset of pancytopenia in cancer survivors will require a bone marrow biopsy and aspirate to further characterize the cause. Specimens should be sent for hematopathology, flow cytometry, and cytogenetic analysis to provide the necessary diagnostic information.Chemotherapy-induced neutropenia (CIN) is one of the most common dose-limiting toxicities associated with systemic treatment for cancer due to cytotoxic effects to the rapidly dividing neutrophils, as well as damage to elements of the stroma and cytokine milieu. Mature granulocytes, including neutrophils, have a lifespan of 1 to 3 days, thus they have a rapid mitotic rate and greater susceptibility to cytotoxic damage than other myeloid cell lines with longer lifespans . The onset and duration of neutropenia vary widely by agent, dose, frequency of dosing, and host-related factors previously discussed. Neutrophil precursors are more prevalent than erythroid or platelet cell lines, accounting for more than 50% of the hematopoietic cells in the bone marrow and postmitotic pool, with only 2% in circulation and 3% in the spleen or vasculature .The actively dividing cells in the mitotic pool and postmitotic maturation pool are the most sensitive to the effects of chemotherapy, whereas mature and fully differentiated cells in the peripheral blood are less sensitive. The degree of sensitivity for different cells in the maturation process, together with the lifespan of each cell line, helps to explain the time of onset and recovery of cytopenias and the principles of growth factor administration for treatment.The presence of neutropenia predisposes patients to infection. The severity and duration of neutropenia, together with host-related factors and secondary effects of the treatment regimen, contribute to the risk of more serious AEs, including neutropenic fevers and bacteremia. The greatest risk of severe CIN, including febrile neutropenia (FN), is in the first cycle of chemotherapy . As a result, the prophylactic use of colony-stimulating factors is recommended when the risk of National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) grades 3/4 CIN or FN is greater than 20% in the setting of potentially curable disease where dose intensity is necessary for optimal clinical outcomes ; see Table 3. Additional parameters are suggested for patients with < 20% risk of CTCAE grades 3/4 CIN or FN; see Table 4.Chan and colleagues (2011) conducted a systematic literature review to evaluate the incidence, patient characteristics, severity, supportive care strategies, and clinical impact of grades 3/4 CIN for patients enrolled in randomized phase III clinical trials evaluating emerging regimens for the treatment of common solid tumors . A total of 1,522 randomized, controlled trials for breast, lung, ovarian, colorectal, or renal cell carcinomas were evaluated for the inclusion of criteria for reporting, grading, and management of clinically significant CIN. Only 264 of the published trials reported the incidence of grades 3/4 CIN (72%) or FN (53%) overall. The incidence by tumor type for grades 3/4 CIN or FN was most common in the studies for lung , ovarian , and breast  cancer, with fewer cases reported for colorectal  and renal cell  cancers.Only 73% of the trials included descriptions of strategies for management of these events. The most commonly reported strategies for management included dose delays (36%), dose reductions (48%), and granulocyte colony-stimulating factor (G-CSF) administration. The discussion of G-CSF administration in the methods (38%) or results (19%) segment for each published study was rare. The authors note that this as a major limitation for application of the trial results to the general oncology population . Given the well-established clinical guidelines for the management and prevention of CIN and FN, inclusion of these strategies in the design and reporting for clinical trials for emerging therapies is critical to provide oncology clinicians with the data necessary to safely integrate these newer therapies into clinical practice, limit the severity of AEs, and provide patients with the most effective clinical outcomes.Patients with FN may deteriorate rapidly. Prompt management of these patients is essential to avoid more severe AEs such as sepsis syndrome, circulatory collapse, acute respiratory failure, or death. Familiarity with factors predictive of high-risk FN will provide the AP in oncology with critical tools for rapid identification of these patients, prompt implementation of established guidelines for management, and avoidance of clinical deterioration (Table 5).Patients with CIN or FN generally present with fever and/or shaking chills. Low-risk patients may be asymptomatic and found to be neutropenic on routine laboratory evaluation. The general principles of treatment for a patient with grades 3/4 CIN or FN include rapidly assessing the level of risk for deterioration and stabilizing the patient if necessary, while simultaneously obtaining additional laboratory measures, including blood cultures and urinalysis for culture and sensitivity. Prompt implementation of institutional guidelines or standing orders for the management of neutropenic fevers, including antibiotic administration, is critical to prevent more serious complications.Escherichia coli most common, 50 episodes) and gram-positive bacteria . The respiratory tract , indwelling catheters , and the gastrointestinal tract  were the most common documented sites of infection . Of the 259 episodes evaluated, 43% (107 episodes) were documented as fever of unknown origin.Park and colleagues (2010) evaluated 259 episodes of FN in patients with hematologic malignancies . The most common organisms isolated were gram-negative bacteria (p < .001).In a second trial, Klastersky and colleagues (2007) applied the MASCC risk criteria (Table 5) to a population of 2,142 cancer patients with solid tumors and hematologic malignancies receiving chemotherapy to evaluate the incidence of bacteremia in patients experiencing FN. Fifty-eight percent of the patients were considered to be at low risk for complications related to FN. A total of 499 patients  developed documented bacteremia. Gram-positive bacteremia was most common (57%), with gram-negative (23%) and polymicrobial bacteremia (10%) less common. A MASCC risk score of < 15 was associated with the poorest prognosis . In the study conducted by Park et al. (2010), the most common first choice of antibiotics administered was cefepime . The addition of a glycopeptide  for sustained fevers after 3 days of empiric antibiotics was most common, with the addition of antifungal agents based on suspected fungal etiology .p = .010) and comorbidities such as hypertension (p = .012) and liver disease (p = .029). The leading cause of death (n = 61 [24%]) was septic shock with multiorgan failure (21 cases) and respiratory failure (9 cases). Patients who developed a fever outside the hospital experienced more serious complications (p = .003), emphasizing the need to have established protocols and effective patient and caregiver communication to expedite the management of FN in patients being cared for in the outpatient setting, particularly older patients. Recovery from neutropenia was found to be the most significant factor for survival (p < .0001). Additional factors associated with poor-prognosis FN are included in Table 5.Factors associated with poor outcomes based on univariate analysis of the 259 FN episodes included age , patients with more severe episodes will require greater scrutiny. The decision to resume treatment is based on the individual patient; his or her risk profile including underlying disease; the treatment regimen including therapeutic intent; and the availability of supportive care including growth factor support, accessibility to the clinical setting, financial resources, and caregiver support. Dose modification and/or dose delays may be required in patients with metastatic or incurable disease who may require treatment over extended periods of time.The use of G-CSF as secondary prophylaxis has also been studied. Guidelines proposed by ASCO and the NCCN suggest the use of G-CSF in patients receiving mild to moderately myelotoxic chemotherapy with curative intent who have experienced an episode of grades 3/4 CIN or FN . Myeloid growth factors are not without potential AEs, the most common being bone pain (Table 6). Bone pain associated with G-CSF is generally self-limiting but may be severe . The administration of an anti-inflammatory agent such as naproxen together with an antihistamine such as loratadine at the onset of bone pain and continued for 48 to 72 hours works well to reduce the pain, with little risk of toxicity and minimal cost to the patient.Pseudomonas aeruginosa (ciprofloxacin) and Streptococcus (levofloxacin).The use of prophylactic antibiotics remains controversial owing to limited data substantiating their benefit in the general cancer population together with concerns for the development of drug-resistant bacteria. Unlike myeloid growth factors, which have proven efficacy in preventing the incidence and severity of neutropenia, including episodes of FN, prophylactic antibiotics serve to reduce the complications of neutropenic fevers . More recent trials have documented a reduction in the incidence of neutropenic fevers and bacteremia and infection-related mortality with administration of fluoroquinolones (ciprofloxacin and levofloxacin most common) in patients with hematologic or solid tumors . Fluoroquinolones provide excellent coverage against Clostridium difficile, and the emergence of drug-resistant bacteria must be considered. However, prophylactic antibiotics in patients with hematologic malignancies or in patients at high risk for poor-prognosis FN should be considered. Combining prophylactic antibiotics with G-CSF, in particular pegfilgrastim if the schedule permits, may provide the best option for prevention in very high-risk patients .The concern for antibiotic-associated AEs, additional cost, secondary infection with Anemia is a common finding in patients with cancer, with an incidence ranging from 30% to 90% . The causes of anemia in cancer patients vary widely, including metabolic and nutritional causes, chronic disease, renal insufficiency, blood loss, inadequate production due to bone marrow disease, peripheral destruction due to autoimmune disorders, drug-induced red cell aplasia, and chemotherapy-induced anemia (CIA). Anemia is multifactorial in most cancer patients due to the generally older age of the cancer population, the existence of comorbidities with associated medications, and the effects of the malignancy and treatment . Chemotherapy-induced anemia is far less common than CIN, in part due to the difference of the life cycle of red blood cells; however, chemotherapeutic agents may contribute to anemia through disruption of normal hematopoiesis and interference of the cytokine milieu .The primary approach to the patient with anemia is as follows: (1) Establish the underlying cause(s), (2) treat the underlying cause(s), (3) evaluate symptoms of anemia with consideration of individual patient characteristics, and (4) weigh the risks and benefits of each treatment approach. The comprehensive assessment and management of anemia in the patient with cancer is beyond the scope of this article. However, supportive care for those receiving myelotoxic chemotherapy is an essential component of clinical management for these patients, including the treatment of anemia. Beyond the treatment of comorbid conditions and other contributing factors, the primary interventions for CIA include therapeutic transfusion of packed red blood cells (PRBCs) and administration of erythropoiesis-stimulating agents .The FDA, the AABB, and the NCCN have recently revised the guidelines for administration of ESAs and PRBCs based on the specific risks and benefits of each approach . Familiarity with these very recent recommendations is critical for the AP to provide informed consent to the patient, to maintain safety, and to ensure compliance with regulatory requirements, including the Risk Evaluation and Mitigation Strategy (REMS) program for erythropoiesis-stimulating agents regulated by the FDA and institutional blood bank requirements (Table 7).Consideration of the individual patient characteristics and associated risks of PRBC transfusion and ESA administration is necessary. Thresholds for transfusion in particular will vary according to patient age, comorbidities, underlying disease, expected capacity for hematologic recovery, and ongoing therapy. It must always be kept in mind that the benefits of any transfusion are temporary, and restoration of normal bone marrow function and erythropoiesis is the most desirable goal. As noted in the REMS guidelines, there are specific risks associated with the administration of ESAs, including thrombosis, inferior survival, and decreased time to progression in selected tumor types. Thus, ESAs are reserved for patients receiving chemotherapy where treatment does not have a curative intent .Thrombocytopenia and the risk of bleeding present a particular challenge in cancer patients undergoing cancer treatment. Like anemia, the cause may be multifactorial, including underlying disease, comorbidities, associated medications, disease-related destruction, or as a direct effect of treatment on the macrophages in the mitotic pool. Unlike neutropenia or anemia, the use of colony-stimulating factors for chemotherapy-induced thrombocytopenia is not yet FDA approved, in part due to concerns for off-target effects of thrombopoietic agents. The lifespan of a mature platelet in the postmitotic pool may be as little as 24 hours, thus there is a constant need for replacement. Patients with bone marrow disorders, continued peripheral destruction, or sequestration such as with splenomegaly are at particular risk for sustained thrombocytopenia. The risk of bleeding is the greatest concern for these patients. Criteria for therapeutic or prophylactic platelet transfusions, the type of platelets, and volume transfused have been reviewed .The Platelet Dose Study (PLADO), a database analysis of 3,447 hematology/oncology patients who received therapeutic platelet transfusions, found a platelet count of 10,000/µL to be the recommended trigger for prophylactic transfusion, with adaptation for patients with complicating factors, consistent with previous trials and consensus guidelines ; see Table 8. A platelet count ³ 5,000/µL is thought to be sufficient to maintain endothelial integrity, a key factor in spontaneous bleeding risk. Educating the patient and their caregivers about bleeding precautions and reportable signs and symptoms is critical to avoiding more serious adverse events.Chemotherapy-induced myelotoxicity is a common and potentially life-threatening adverse event for cancer patients. Neutropenia, febrile neutropenia, anemia, and thrombocytopenia are the result of complex processes as a result of the disease, the approach to treatment, and the characteristics of the individual patient. Assessment of individual risk using the criteria described, implementing prevention, monitoring, and treatment strategies, as well as setting expectations for the patient and family are essential to avoid more serious adverse events. Familiarity with the risks and benefits of supportive care measures for the treatment of cytopenias, recent updates to consensus guidelines, recommendations for treatment modification, and contraindications for various supportive care measures will assist the AP in oncology in effectively managing chemotherapy-induced myeloid toxicity.


Physical penetration of lipid bilayer membranes presents an alternative pathway for cellular delivery of nanoparticles (NPs) besides endocytosis. NPs delivered through this pathway could reach the cytoplasm, thereby opening the possibility of organelle-specific targeting. Herein we perform dissipative particle dynamics simulations to elucidate the transmembrane penetration mechanisms of multiple NPs. Our simulations demonstrate that NPs’ translocation proceeds in a cooperative manner, where the interplay of the quantity and surface chemistry of the NPs regulates the translocation efficiency. For NPs with hydrophilic surfaces, the increase of particle quantity facilitates penetration, while for NPs with partly or totally hydrophobic surfaces, the opposite highly possibly holds. Moreover, a set of interesting cooperative ways, such as aggregation, aggregation-dispersion, and aggregation-dispersion-reaggregation of the NPs, are observed during the penetration process. We find that the penetration behaviors of multiple NPs are mostly dominated by the changes of the NP-membrane force components in the membrane plane direction, in addition to that in the penetration direction, suggesting a different interaction mechanism between the multiple NPs and the membrane compared with the one-NP case. These results provide a fundamental understanding in the underlying mechanisms of cooperative penetration of NPs, and shed light on the NP-based drug and gene delivery. Owing to the excellent physicochemical properties and small size, nanoparticles (NPs) are considered to be one of the promising candidates as drug and gene delivery vectors, intracellular biomarkers and probes, and so on12467910111268Externally applied forces and fields may cause membrane pore opening, thereby facilitating direct penetration of NPs. For example, using the microinjection method, NPs can be mechanically delivered into living cells6986et al. observed that “striped” NPs could easily pass through the cell membrane2729303132333436et al.3839404142The surface chemical properties of NPs strongly affect the interactions between the NPs and the cell membranes18192021222425Herein, we elucidate the cooperative transmembrane penetration mechanisms of multiple NPs using the dissipative particle dynamics (DPD) simulations. We find that the NPs translocate into/through a lipid membrane in a cooperative way, which is further regulated by the interplay of the two key factors, i.e., the quantity and surface chemistry of the NPs. Importantly, the simulations demonstrate that the NP-membrane interaction mechanism in the multiple-NP case is totally different from that in the one-NP case, which fundamentally changes the penetration behavior of multiple NPs. Our findings provide a deep understanding of the collective behavior of the NPs and their interactions with cell membranes, and shed light on the rational design of NP-based diagnostic and therapeutic agents.Four types of spherical NPs with different surface chemical properties are used in our simulations : the WNP92033f the NP , as detaTo better understand the membrane penetration of multiple NPs, we first examine the translocation process of a single NP. As shown in et al.13During the translocation process, the NP interacts with the hydrophilic outer layer (lipid heads) and hydrophobic inner layer (lipid tails) of the membrane in a consecutive manner. The particle surface chemistry has a rather strong impact on the translocation. By force analysis, it is found that the resistance force acting on the WNP during the translocation is the largest, but that acting on the RNP is the smallest . For the44P’s type . TherefoWe next investigate the penetration of multiple NPs, focusing on the effect of NP quantity. Interestingly, it is found that when multiple NPs simultaneously interact with the membrane, the translocation behavior of multiple NPs may be distinctly different from that of a single NP . As show13Especially, it is worth stressing that the interesting cooperative ways of NPs in the penetration process are observed in our simulations, which can be manifested by the change in the inter-NP distance aneously , and thumembrane . On thisifferent , and conar again . Thus ththe RNPs .The surface chemistry of the NP also affects such a link. Under the similar surfaces . Thus thilar way . But forr  e.g., . As a rer  e.g., ,5. OveraSimilar to the one-NP case, the applied observed . Furtherobserved . In this process . Based o fields472049. Eve process . All the the NPs .In addition, based on the previous studies415154In summary, the cooperative penetration behavior of multiple NPs into/through a lipid membrane is investigated by computer simulations. We find that the particle quantity plays key roles in the nanoparticle penetration process. However, the increase of particle quantity does not always provide help for the NPs to penetrate a membrane due to the huge change in the NP-membrane interaction mechanism. Therefore, in experiments or biomedical applications, the concentration of the NPs should be carefully chosen according to their surface chemistry. Additionally, the coupling between the particle quantity and surface chemistry of the NPs further complicates the penetration process. Some interesting collective behaviors among NPs, such as aggregation, aggregation-dispersion, or even aggregation-dispersion-reaggregation, are observed during the particle penetration process. These behaviors, which are caused by the changes of the NP-lipid membrane forces in the membrane plane direction, strongly affect the penetration behaviors of the NPs. Our results are very helpful for the applications of NPs in drug or gene delivery system, and understanding of the interaction mechanism between NPs and cell membrane.i and j, rc is the cut-off radius of the force, and aij is the maximum possible repulsive interaction between the two beads. The simulations are performed in the NVT ensembles at the temperature Dissipative particle dynamics (DPD) is a coarse-grained modeling technique. In the simulations, the dynamics of the coarse-grains (beads) obeys Newton’s equation of motion. The inter-bead interactions include conservative force (Our simulation model consists of a tensionless lipid membrane immersed in sufficient water. The simulation box size (tion box . Accordi5 simulation steps) and three independent runs to ensure the computational convergence and consistency.Prior to the simulations, the NPs are docked above the bilayer .


Owing to frequent outbreaks witnessed in different parts of the country in the recent past, scrub typhus is being described as a re-emerging infectious disease in India. Differentiating scrub typhus from other endemic diseases like malaria, leptospirosis, dengue fever, typhoid, etc. is difficult due to overlapping clinical features and a lower positivity for eschars in Asian populations. Hence, the diagnosis heavily relies on laboratory tests.Costs and the need of technical expertise limit the wide use of indirect immunoperoxidase or immunofluorescence assays, ELISA and PCR. The Weil-Felix test is the most commonly used and least expensive serological test, but lacks both sensitivity and specificity. Hence, the diagnosis of scrub typhus is often delayed or overlooked. With due consideration of the cost, rapidity, single test result and simplicity of interpretation, rapid diagnostic tests have come into vogue. However, evaluation of rapid diagnostic tests for scrub typhus in the Indian population is needed to justify or discourage their use.Research studies are needed to find the most suitable test in terms of the rapidity of the result, simplicity of the procedure, ease of interpretation and cost to be used in the Indian populace.The online version of this article (doi:10.1186/s40249-016-0137-6) contains supplementary material, which is available to authorized users. Please see Additional file Orientia tsutsugamushi and is transmitted by the bite of infected larvae of the mite Leptotrombidium deliense. It is a zoonosis, with humans being accidental, dead end hosts. India is an integral component of the “tsutsugamushi triangle” which depicts a part of the globe  endemic to scrub typhus. Owing to frequent outbreaks witnessed in different parts of the country in the recent past, it is being described as a re-emerging infectious disease in India. It is one of the most under-diagnosed and under-reported febrile illnesses requiring hospitalization in the region. Eschar  is an important finding for the diagnosis of scrub typhus. However, eschar positivity in India and other Asian populations is very low [Scrub typhus is caused by Scrub typhus is essentially an occupational disease among the rural residents  in the Asia-Pacific region . Many teDifferentiating scrub typhus from other endemic diseases like malaria, leptospirosis, dengue fever, and typhoid is often difficult for a clinician. Lower positivity for an eschar in Asian populations and lack of wide availability of confirmatory tests further augments this problem. In addition, scrub typhus co-infections with other endemic diseases, due to common behavioural risk factors , increases the need for specific diagnosis –9.In such a scenario, role of rapid diagnostic tests is crucial. With due consideration of the cost, rapidity, single test result and simplicity of interpretation, rapid diagnostic tests have come into vogue. The development of rapid diagnostic tests by the use of immunochromatographic test (ICT) technologies has provided a mechanism for point-of-care serological testing . Use andO. tsutsugamushi strains [In addition, cut offs must be validated locally and local strains should be included in the antigen pool for the effective disease diagnosis owing to antigenic diversity in  strains , 12. New strains . More em strains .rickettsial diseases in India also discourage the use of rapid tests as they need further evaluation [However, such studies are yet to be reported from India. Recently published DHR-ICMR guidelines for the diagnosis and management of aluation . Hence, Research studies are needed to find the most suitable test in terms of rapidity of the result, simplicity of the procedure, ease of interpretation and cost to be used in the Indian populace.


Routine immunization (RI) is a key child survival intervention. Ensuring acceptable standards of RI service delivery is critical for optimal outcomes. Accumulated evidences suggest that ‘supportive supervision’ improves the quality of health care services in general. During 2009–2010, the Government of Odisha and UNICEF jointly piloted this strategy in four districts to improve RI program outcomes. The present study aims to assess the effect of this strategy on improvement of skills and practices at immunization session sites.A quasi-experimental ‘post-test only’ study design was adopted to compare the opinion and practices of frontline health workers and their supervisors in four intervention districts (IDs) with two control districts (CDs). Altogether, we interviewed 111 supervisor–supervisee  pairs using semi-structured interview schedules and case vignettes. We also directly observed health workers’ practices during immunization sessions at 111 sites. Data were analyzed with SPSS version 16.0.The mean knowledge score of supervisors in CDs was significantly higher than in intervention groups. Variegated responses were obtained on case vignettes. The control group performed better in solving certain hypothetically asked problems, whereas the intervention group scored better in others. Health workers in IDs gave a lower rating to their respective supervisors’ knowledge, skill, and frequency of supervision. Logistics and vaccine availability were better in CDs.Notwithstanding other limitations, supportive supervision may not have independent effects on improving the quality of immunization services. Addressing systemic issues, such as the availability of essential logistics, supply chain management, timely indenting, and financial resources, could complement the supportive supervision strategy in improving immunization service delivery. Supportive supervision (SS) as a strategy ensures that personnel carry out their activities effectively through direct, personal contact on a regular basis; this would guide and support peripheral functionaries to develop professional competence . CorrectDifferent supervision strategies influence performance. Effective and regular supervision could potentially help meet the challenges unique to health workers, especially in the context of task-shifting initiatives that transfer tasks from health supervisors to health workers–6. OtheSS usually involves record reviews, observations, performance monitoring, constructive feedback, provider participation, and problem solving. In practice, SS strategies vary greatly in approach, content, and tools . DespiteThe national universal immunization program (UIP) review 2004) and vaccine management assessment tool (VMAT) study (2007) identified inadequate SS and insufficient training  by the government as foremost gaps underlying poor coverage and quality of immunization 04 and va. ConsequFull-scale implementation of this pilot initiative was reported since May 2010. The subsequent 12 months involved extensive monitoring and on-the-job training of immunization managers and supervisors to improve supervision practices and to help providers solve immunization-related problems. Immunization managers from four intervention districts (IDs) were encouraged to apply SS guidelines in practice. Guidelines and tools for supervision included instructions for conducting supervision, namely rules of conducting supervision meetings, checklists for supervisory visits, work planning action sheets, do's and don'ts of supervision, self-assessments of supervisors’ competencies, tips on delegation, a feedback mechanism, and conflict resolution. It was mandatory for every immunization manager from the intervention group to visit sessions at least once a week during the fixed outreach session. The category of participants were medical officers (MO), AYUSH medical officers (AYUSH MO), block extension educators (BEE), block program organizers (BPO), lady health visitors (LHV), integrated child development scheme(ICDS) supervisors, and supervisors at the block level of the women and child development (WCD) department.The overall aim of this study was to assess the effect of a ‘supportive supervision’ strategy on the quality of immunization services. It is a comparative study between four IDs and two non-intervention (control) districts. The specific objectives were 1) to assess and compare the attitudes and practices of supervisee–supervisor pairs toward each other, acceptance of the SS strategy, problem-solving skills, management capacity, the communication process, and on-site correction abilities and 2) to recommend to the state government and to UNICEF the usefulness of the SS strategy for improvement of quality of services.A quasi-experimental ‘post-test only’ study design was used , 23. TheFour pre-existing IDs and two comparable control districts (CDs) were taken for data collection. Selection of CDs was performed on the basis of comparable baseline UIP indicators as of December 31, 2009, through desk review: we considered complete immunization coverage, adverse events following immunization (AEFI) cases reported, and dropout rates for such comparison. On the basis of geographic, sociopolitical, and programmatic similarities, Kandhamal and Kalahandi were taken as CDs for data collection.Link workers, also known as auxiliary nurse and midwives (ANMs), constituted the supervisees in this study. They are also termed as ‘end-users’ or ‘multipurpose health worker – female’ (MPHWF). Supervisory staff who had undergone the SS training constituted the universe of supervisors. All supervisors who had undergone the SS training but had been transferred or retired during the study were excluded from the study.Quality is synonymous with innate excellence  , 25. TheSS is defined as a process that promotes quality at all levels of the health system by strengthening relationships within the system, focusing on the identification and resolution of problems, and helping to optimize the allocation of resources promoting high standards, teamwork, and better two-way communication . We consThe primary outcome considered for calculating sample size was a composite of practices (score>2) of supervisors with regard to immunization session site supervision. With 95% confidence level and 80% power, expected frequency of 70% and worst acceptable of 50%, we calculated 16 RI sessions in each district to be sufficient for data collection. A buffer of 20% was added to the total sample to compensate for possible non-responses. Therefore, a minimum of 102 sessions and a maximum of 122 sessions were planned for directly observing sessions and interviewing supervisees as well as supervisors. We observed 111 immunization sessions  and interviewed an equal number of supervisors and supervisees. The session sites were randomly selected using a lottery method. It is important to mention here that in Odisha, RI sessions are conducted as outreach services only once a week on Wednesday. To ensure representation of each category of supervisors, a quota sampling method was adopted. In consultation with the district administration, our field investigators accompanied the selected sample supervisors at least once to the session site during data collection.Data were collected from April to December, 2011. Semi-structured interview schedules of supervisors and supervisees (each) were first developed in English, translated into the local language, and field-tested in three non-sample sessions and with six supervisor–supervisee pairs. The supervisors’ interview schedule contained both structured multiple-choice responses and open-ended hypothetical situations, whereas the supervisees’ interview schedule focused on the frequency of supervisory visits and the body language, communication style, knowledge level, attitude, and problem-solving skills of supervisors. We used a direct observation checklist recommended by the Government of India for on-site observation of session sites. It had questions related to the micro-plan, the availability of logistics and supplies, and practices directly related to the quality of immunization, such as needle touching, waste management, vaccine returning practices, use of cards, delivery of key messages, and so on. The researchers and supervisors  separately collected the data from the same session, using the same tool. To avoid bias in data collection, the supervisor was not informed beforehand about the need to collect such information. We found all supervisors carried the supervisory checklist when visited the session along with the researcher; after the session visit, the researcher collected a photocopy of the checklist from the supervisor. To ensure uniformity, six field investigators were hired and trained on the data collection tools for data collection. A team led by the principal researcher monitored the quality of data collection through regular field visits and cross-checking.t-tests, were used to infer the significance of associations and differences.Quantitative data were entered into Microsoft Excel and then exported to SPSS version 16.0 for analysis. Questions on attitude scales were analyzed through derivation of means; statistical techniques, such as chi-square and We obtained ethical approval from the institutional ethical committee of the Indian Institute of Public Health – Bhubaneswar (IIPHB). Confidentiality and anonymity were maintained throughout the data collection process. Informed consent of all respondents was taken before instituting the interview schedules. Written consent of all respondents was obtained, and they were briefed about the study objectives. The respondents were free not to respond to any question and were free to leave the study at any stage.The main limitation of the study is non-availability of baseline data, which makes it difficult to attribute the results to the intervention. There is the possibility that a modest role of intervention in improving service outcomes could be due to a type II error, resulting from an inadequate number of districts (we chose two CDs against four IDs). Finally, the short duration of the intervention may have restricted its potential to bring the expected outcomes.Supervisors’ interviews revealed that there are significant differences in their understanding of the basic principles behind SS. The interview schedule of supervisors had both positive and negative questions. On questions related to the importance of supervisors motivating supervisees, the role of punitive measures in SS, and the importance of supervision in the improvement of supervisees’ performance, the mean score of CDs was significantly higher than that of intervention groups . On otheWhen asked about hypothetical situations to assess the problem-solving abilities of supervisors in a series of open-ended questions, we received variegated responses. A joint meeting of UNICEF representatives, IIPHB faculty, and senior officials of the directorate of family welfare, Government of Odisha, decided to rank the responses on a scale of 0–3, wherein 0 represents a totally wrong answer, 1 is a partially correct but unacceptable answer, 2 is an incomplete but correct answer, and 3 means a fully correct answer. Analysis of the findings indicated that on issues related to waste disposal, correction of site of a DPT  injection, reconstitution of diluents, correction of needle touching, improvement of skills among health staff, and rectification of doses of hepatitis B vaccine, the supervisors of IDs had significantly higher levels of correct responses as compared to those of CDs.In contrast, when asked about the ways of dealing with melted icepacks, return of unused vials, key messages at immunization sites, use of red bags for waste disposal, late arrivals of ANMs for sessions, and photocopying the passbook, CDs had better responses . Levene'Through interviewing the supervisees, we assessed the number of supervisory visits made in the past 6 months, and the communication styles and on-site correction practices of supervisors. We found that 40.5% of supervisors in IDs and 35.9% of supervisors in CDs had more than three visits to the session in the past 6 months. Two-way communication was higher in CDs (76.9%) as compared to the IDs (69.4%), although this was not statistically significant. With respect to the availability of session-monitoring checklists, we found the CDs had better results than IDs. Furthermore, as many as 15 out of 72 and 18 out of 39 sample supervisees reported that their supervisors had visited the session site only once during the past 6 months in intervention and CDs, respectively .In the second category of questions, assessments of the knowledge, attitude, and problem-solving skills of supervisors were ranked by the supervisees on a scale of 0–4 . This question was asked only to those supervisees whose supervisors visited the session sites more than once in the past 6 months. An analysis of results revealed that supervisees of CDs ranked their supervisors better than by the IDs, although this was statistically not significant . One posThe researchers ranked the body language, communication process, and correction process of the supervisors at the session site on the basis of the response given by the supervisees. Through direct observation of immunization session sites, we assessed the regularity in holding sessions, availability of logistics, and practices of supervisees. Availability of antigens and other logistics in CDs was better than in IDs, although such differences were statistically not significant. One of the probable explanations behind such unexpected results is that decentralization as a policy directive has not yet percolated down to the district and sub-district levels. Furthermore, the availability of supplies and logistics is a function of sufficient availability of stock at central and block stores, which is dependent upon timely indenting and receipt of items. Consequently, we infer that SS training had not improved the skills of supervisors with respect to these management functions. We wanted to cross-verify these findings with data triangulation. Thus, direct observation results of the supervisor was analyzed, and it was found that similar, rather significant differences existed in the reports of supervisors with regard to the availability of supplies and logistics in immunization sessions. Significant differences were found with regard to their reports on the practice of alternate vaccine delivery (AVD); the availability of measles vaccines, oral rehydration solutions (ORSs), blood pressure apparatus, black and red bags, DPT vaccines, and iron folic acid (IFA) tablets; and their compliance with the micro-plan. In the next step, we analyzed the difference in observation of findings on key practices in IDs and CDs. Results indicated a mixed response. For instance, with regard to the use of hub cutter, delivery of four key message, such as next immunisation date, possible side effects, how to address the side effects and the importance of mother and child protection card, and proper handling of syringes by the ANMs, IDs performed better than CDs. However, such differences were statistically not significant.We analyzed the differences in the findings of the researcher and the supervisor to better understand the accuracy of reporting by the supervisors, who are also called ‘internal monitors’. To ensure neutrality in reporting, the researchers were specifically trained to objectively record the findings from the session. Results revealed that there were notable differences with respect to reporting of each and every item, although both researcher and supervisor were using the same checklist. Therefore, objectivity in session assessment by the supervisors was not maintained, and individual differences in perspectives of reporting existed. The difference was significant with respect to reporting on the availability of black and red bags, the weighing machine, BP apparatus, the use of hub cutter, and four key message delivery. All of these items were over-reported by the supervisors as compared to their actual status in the sessions.The results of the study suggest that the intervention package, which included SS guidelines, district-level trainings, continuous supervision and support during a 12-month period, monitoring of provider performance, and resource allocation for travel and communication of supervisors. The intervention independently contributed to improved knowledge of SS among supervisees, and it helped in removing self-perceived barriers such as availability of resources, a lack of formats for field visits, and a lack of recognition among providers regarding the importance of SS. Discussion with district officials indicated that they attached high importance to the utility of supervision as a key strategy to improve immunization quality. Given that most of the important public health program implementation is being performed by health workers, stationed in peripheral health posts and health centers, faulty supervision by higher officials could pose daunting management challenges at the block and district levels. One possible explanation of the results shown in Over the past 10 years, we have noticed the establishment of new peripheral facilities; usually, the facilities are remote, have poor communication with the rest of the health care system, lack even basic supplies and equipment, and are staffed by under-trained and poorly paid personnel who typically work by themselves. Motivation is hard to maintain in such an atmosphere. Regular supervision of sessions, ILR points, and vaccine stores is an important step to ensuring quality immunization services. While supervision can be a very participatory process, traditional supervisory visits focus more on inspection and fault finding rather than on problem solving to improve performance. If health workers don't receive sufficient guidance or mentoring on how to improve their performance, they will remain undirected.Other similar studies have pointed out the necessity of improving supervisory support to these facilitiesTraditional approaches to performing supervisory visits are effective to an extent, but with several limitations: the supervisors tend toward facility inspection rather than human capital development. Often, supervisors themselves lack skills, tools, and resources and are over-burdened with other administrative duties on a daily basis. This needs to be addressed at the district level with utmost sincerity. There is very limited evidence on the subject in India. However, the benefits of supervision, as reflected in some of the published literature, include: helping service providers to achieve work objectives by improving their performance, ensuring uniformity to set standards, identifying problems and solving them in a timely manner, making a follow-up on decisions reached during a previous supervision visit, identifying staff needs and providing opportunities for personal development, and reinforcing administrative and technical links between higher and lower levels , 35.There are institutions that provide training on performance improvement, but governments have not seriously dwelled on this option mainly for two reasons: first, investing in long-term capacity-building measures pre-necessitates the deputation of peripheral health staff for a longer period of time; and, second, although there are many examples and case studies where SS has been used to improve health worker performance and immunization coverage, long-term and sustainable results have not been thoroughly documented.The large-scale health care reforms of the past decade included the need to ensure that immunization managers carry out their activities effectively. Through regular and personal contact, supervisors were mandated to improve the competence of peripheral health workers. However, recent reports are non-conclusive regarding the effects of such reforms . With thIn Odisha, peripheral health staff are responsible for providing primary health care services, where supervision continues to be a problematic issue. Lack of experienced and quality human resources can easily jeopardize the success of any health program, including immunization . RI is aReporting by supervisors was not objective, and there were significant variations with regard to reporting on the availability of logistics and instances of undesirable activities. However, whether or not such distortions in reporting by supervisors were deliberate needs further scrutiny. There was a general improvement in the coverage of immunization services in both groups of districts. This can be attributed to improved knowledge of supervisees and increased acceptance of on-site corrections made by supervisors. The trend in improvement of service outcomes in both IDs and CDs can be attributed to other factors, such as an overall improvement in health care financing after the advent of NHMs and targeted service provision to the ‘hard-to-reach’ areas. Allocating resources to supervision is likely to result in improved performance of health workers.SS can have independent positive effects on improvement of the knowledge and practices of supervisors and supervisees related to conducting immunization sessions. However, whether or not it has any conclusive effect on improvement of the overall program outcomes, because of the existence of many intermediate factors in the chain of events, needs further investigation. Thus, SS as a strategy may be conditionally envisaged for quality improvement within the overall framework of a national immunization program. Better health system preparedness is a necessity for the success of this strategy . As reflThe old paradigm that most performance problems can be solved by training alone needs to be reconsidered. Further delineation of roles, mandatory provision for supervisory visits, the allocation of resources for field trips, and monthly reviews at the district and sub-district levels are key district-level interventions for SS to work. SS would require staff time and travel and per diem costs for supervisors. In the Odisha context, the health budgets frequently either do not allocate sufficient funds to conduct SS or do not transfer funds to peripheral facilities; this makes the job of supervisors difficult to finance and coordinate his or her visit: this may be addressed at district review meetings. Furthermore, supervisors need support and authority from the district to implement supervision or recommend context-specific changes to improve services in the periphery. Since the ranking score of supervisors with regard to communication skills, problem-solving skills, and frequency of supervisory visits was suboptimal, it is recommended to have modular training programs. An RI program also requires the active involvement of accredited social health activists (ASHAs) and anganwadi workers (AWWs). Therefore, establishing a platform of intersectoral convergence at the block and district levels could be crucial to discuss and address the systematic bottlenecks.BP conceptualized the study design, wrote the manuscripts, supervised the data collection, and conducted data analyses. SP contributed to the finalization of study tools, data analysis, and manuscript writing. SN contributed to development of study tools, data analysis, and manuscript writing. ASC undertook the literature review and writing of the data analysis. AA finalized the study design, data collection tools, and manuscript editing. MS helped in data collection, analysis, and writing. SZ provided technical support to finalize study design, data analysis, and manuscript editing.


Systems biology, a holistic approach describing a system emerging from the interactions of its molecular components, critically depends on accurate qualitative determination and quantitative measurements of these components. Development and improvement of large-scale profiling methods (“omics”) now facilitates comprehensive measurements of many relevant molecules. For multicellular organisms, such as animals, fungi, algae, and plants, the complexity of the system is augmented by the presence of specialized cell types and organs, and a complex interplay within and between them. Cell type-specific analyses are therefore crucial for the understanding of developmental processes and environmental responses. This review first gives an overview of current methods used for large-scale profiling of specific cell types exemplified by recent advances in plant biology. The focus then lies on suitable model systems to study plant development and cell type specification. We introduce the female gametophyte of flowering plants as an ideal model to study fundamental developmental processes. Moreover, the female reproductive lineage is of importance for the emergence of evolutionary novelties such as an unequal parental contribution to the tissue nurturing the embryo or the clonal production of seeds by asexual reproduction (apomixis). Understanding these processes is not only interesting from a developmental or evolutionary perspective, but bears great potential for further crop improvement and the simplification of breeding efforts. We finally highlight novel methods, which are already available or which will likely soon facilitate large-scale profiling of the specific cell types of the female gametophyte in both model and non-model species. We conclude that it may take only few years until an evolutionary systems biology approach toward female gametogenesis may decipher some of its biologically most interesting and economically most valuable processes. Since the foundation of the Institute for Systems Biology in the year 2000 and the formal definition of systems biology at the beginning of the twenty-first century , such as transcriptomics (RNA profiling), proteomics , and metabolomics (profiling of metabolites). However, also epigenetic regulatory processes based on the modification of chromatin components or DNA (epigenomics), the translation of mRNAs to proteins (translatomics), complex formation of proteins with proteins or nucleic acids (interactomics), the investigation of protein modifications, e.g., phosphorylation important for the regulation of their activity (phospho-proteomics), and the transport of ions or metabolites (fluxomics) need to be taken into account to achieve a full picture of the dynamic processes of a cell or organism  of a system, as exemplified for yeast  is a widely used model system appreciated as the currently best understood cell  can be used to sort fluorescent cells based on their light scattering characteristics and fluorescence , the most popular microarray for Arabidopsis has for example been used to profile a large variety of different tissue types . However, due to the frequent use of oligo-dT priming during cDNA synthesis or the hybridization to microarrays covering only coding regions of the genome, many studies are restricted to mRNAs or a subset of mRNAs. Several types of microarrays were produced and extensively used for the analyses of gene expression in different plant species, including the model plant de novo assembly of the short reads into transcripts or by the use of a reference transcriptome either produced separately or taken from a public database . Examples for such an approach are the central cells of Arabidopsis thaliana, and cells of the female reproductive lineage in Hieracium praealtum and Boechera gunnisoniana  have been developed over the last years and are now routinely used for transcriptional profiling  add additional levels of complexity. From a systems biology perspective the aim is the combination of cell type-specific proteomics with transcriptomics and metabolomics to elucidate and model regulatory networks  to activate the expression of a down-stream gene encoding a selectable marker. Apart from a high false-positive rate, the use of yeast itself is a major drawback of the method. While cell type-specific cDNA libraries can be used to profile pairwise protein interactions, the system does not truly reflect the in vivo state of a specific plant cell . Several systems similar to Y2H assays have been established to specifically study membrane proteins  is split into two non-fluorescent halves that are reconstituted to a fluorescent protein upon interaction of the bait and target proteins they are fused to  screens, Affinity Purification Mass Spectrometry (AP-MS), or Bimolecular Fluorescence Complementation (BiFC)  and the MASSBANK Database (www.massbank.jp).Due to the high complexity of plant metabolites coming from both primary and secondary metabolism, the plant metabolome is highly complex. Although by far not comprehensively elucidated to date, about 200,000 different metabolites are estimated to be represented in plants . WGBS is therefore a very promising method for the profiling of specific cell types in plants.DNA (cytosine) methylation is a heritable epigenetic modification of the genome and is involved in various cellular and developmental processes in a wide range of species, including animals, fungi, and plants. Several methods for genome-wide profiling of the DNA cytosine methylation status have been established. These include the hybridization onto whole-genome DNA microarrays after digestion of genomic DNA with methylation-sensitive restriction enzymes, or the precipitation of methylated DNA with antibodies targeting methylated cytosines , followed by either microarray hybridization (MeDIP-chip) or NGS , and the hypocotyl and cotyledons with their various tissue types  and the embryo sac . They are typically formed from one spore (meiotic product) and, at maturity, they consist of only a few cells and cell types, including the male and female gametes, the sperm cells and the egg and central cells, respectively , Zea mays (maize), Lilium longiflorum (lily), and Plumbago zeylanica (white leadwort) (Table Lilium davidii var. unicolor (Lanzhou lily), and tomato . Nonetheless, several cell type-specific transcriptomes  Table , and sevto Table . Due to to Table . Howeverto Table , and intto Table . In contes Table  as well es Table  are currPodostemaceae, where the central cell seems to degenerate before pollen tube arrival, resulting in a single fertilization event , bisporic (two spores), and tetrasporic . Further variation includes the location of the degenerating spores and the positioning of the spores in the tetrasporic types. Likewise, megagametogenesis can vary in the number of mitotic divisions, the arrangement of the nuclei/cells, and late divisions of individual cells after cellularization  is the formation of a syncytium during the divisions of the nuclei prior to cellularization. In angiosperms, gametogenesis and early stages of endosperm development are the two major examples for the formation of a syncytium. In contrast, the plasmodial tapetum, for example, is formed by degeneration of the cell walls and the fusion of the resulting protoplasts  mutant, which produces supernumerary nuclei differentiating according to their position within the FG . This may be overcome by either increased sensitivity of the profiling method, or through a simplified collection of a large number of cells. However, most high-throughput isolation methods  rely on the existence of a specific marker  and the possibility to generate transgenic plants. In addition, typically a certain abundance of the cell type of interest in the sample is required for efficient sorting and purification. Given that these preconditions are generally not met by low abundant cell types of of non-model organisms, it is likely that plant systems biology will profit the most from an increase in sensitivity and the development of novel profiling methods. In the following sections, we will therefore focus on a subset of omics approaches, which are readily available or which bear great future potential for routine large-scale Arabidopsis , in which stably cross-linked cDNA amplicons are sequenced directly within a biological sample, thereby not only quantifying gene expression, but also detecting the subcellular localization of the transcripts  sequencing projects, reviewed in Wuest et al. ], cell tTorenia, Alstroemeria, and Arabidopsis  plays an important role in the epigenetic regulation of plant genomes. While WGBS has not yet been reported for isolated cells of the female gametophyte, bisulfite sequencing of specific sequences has already been applied for To date, cell type-specific systems biology in plants is frequently constrained by the difficulties associated with the isolation of the cell type of interest in large enough amounts. Robust and simple isolation methods exist only for a few cell types. Consequently, the comprehensive profiling of all cell types of an organism with different large-scale profiling methods, allowing the detailed understanding of all biological processes ongoing in the biological system, is still an unreached goal. While the in-depth understanding of complex organisms over their lifespan is a major aim for systems biology, the use of simple model organisms bears advantages, given the persisting technical limitations. We introduce the female gametophyte of angiosperms as an attractive model system for future systems biology approaches in plant development. Apart from its relatively simple organization, it is of great biological and agronomical importance, for example with respect to seed production and plant breeding.Arabidopsis thaliana, Oryza sativa). However, a biological system may be best understood in the context of evolution. In addition, a detailed understanding of the cellular processes in major agriculturally important species including wheat, where an additional challenge is the genome size and its hexaploid nature, are a precondition for targeted crop improvement. Such studies would thus not only be of potential applied value, but would also help to understand the common concepts and divergent mechanisms active in different species. Therefore, methods facilitating large-scale profiling of specific cell types in model as well as non-model organism are of crucial importance. Parallel high-throughput profiling of several organisms covering a phenotypic gradient, or including gain, loss, and alternative usage of modular building blocks along the phylogeny, will enable evolutionary systems biology. This approach may ultimately help to reconstruct the emergence of evolutionary novelties and to find the underlying genetic and molecular networks. Such an understanding would in turn allow the control of the underlying processes with an unprecedented resolution. In perspective, this can be an important precondition for targeted improvement of crop species, including the engineering of apomixis into crop plants.Currently, most high-throughput isolation methods with broader application  are limited to model organisms (e.g., Even though the isolation of individual cell types is currently still very challenging, the rapid technical advances observed over the past few years in, for example, transcriptional profiling, are clear indications for the tremendous improvement of large-scale profiling technologies. In this light, we emphasize methods for transcriptomics, proteomics, metabolomics, and methylomics, in which we see great future potential. However, cell type-specificity and single-cell resolution are just one step towards a more comprehensive view on developmental processes and environmental responses. Clearly, monitoring subcellular localization of molecules and their interactions will be essential to understand certain patterning processes and specific cellular functions. In analogy to the hypothesized distribution of mRNA within the syncytial female gametophyte, subcellular localization of mRNA may also occur within the cell types of the mature female gametophyte to, for example, target the proteins they encode to a specific subcellular region. In this respect, technologies based on high resolution imaging, allowing large-scale profiling without prior cell isolation, for example MSI or FISSEQ, are very promising for future applications. The growing amount of data and data types also points to the need for novel computational solutions addressing the problems of data storage, integration, and analysis (see Ahrens et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The transition to lactation period in dairy cattle is characterized by metabolic challenges, negative energy balance, and adipose tissue mobilization. Metabolism of mobilized adipose tissue is part of the adaptive response to negative energy balance in dairy cattle; however, the capacity of the liver to completely oxidize nonesterified fatty acids may be limited and is reflective of oxaloacetate pool, the carbon carrier of the tricarboxylic acid cycle. Alternative metabolic fates of acetyl-CoA from nonesterified fatty acids include esterification to triacylglycerides and ketogenesis, and when excessive, these pathways lead to fatty liver and ketosis. Examination of the anaplerotic and cataplerotic pull of oxaloacetate by the tricarboxylic acid cycle and gluconeogenesis may provide insight into the balance of oxidation and esterification of acetyl-CoA within the liver of periparturient dairy cows. The transition to lactation period is the most metabolically challenging period for dairy cattle and is characterized by homeorhetic changes, negative energy balance (NEB), and increased risk for metabolic and reproductive disorders. Over 60% of all dairy cows develop fatty liver during the transition to lactation period, which is a direct response to an energy imbalance, and may decrease hepatic efficiency, animal health, and milk production. Recent research suggests that regulation of key hepatic genes may play an important role in liver lipid accumulation and ketone accumulation during this period.Voluntary intake reduction around the time of calving, coupled with increases in energy requirements to meet the needs of lactation, results in cows entering a state of NEB. During periods of NEB, triglycerides (TAG) are mobilized from adipose stores ,4,5,6,7.Once liberated, NEFA can be metabolized by many maternal and fetal tissues for energy production and fat synthesis ,11,12. COnce taken up by the liver, fatty acids are β-oxidized to acetyl-CoA units with four possible fates: complete oxidation through the TCA cycle, incomplete oxidation through ketogenesis, TAG synthesis and packaging as very-low density lipoprotein for export from the liver , or TAG synthesis for storage as liver lipids . When avOften developing in parallel, accumulation of liver lipids during early lactation can be as high as 500 g/d, and it is predicted that 60% of dairy cows have severe or clinical fatty liver, defined as a liver lipid content greater than 10% on wet weight basis at one day postpartum ,19. AccuHepatic uptake of NEFA is reflective of both the circulating NEFA concentration and blood flow to the liver, resulting in an increase in hepatic NEFA uptake during negative energy balance. During the periparturient period, both blood flow to the liver and the circulating NEFA concentration are increased and hepatic NEFA uptake can increase by thirteen times . The degThe progression of ketosis and fatty liver is the response to poor adaptation to the challenges associated with the transition to lactation period. While the hepatic mechanism of bovine fatty liver and ketosis is not completely understood, it is clear that regulation of TAG uptake, lipolysis, oxidation, and storage play a key role in the onset and progression of the disorders.E. coli [Maintaining a balance between anaplerosis, the synthesis of metabolic intermediates, and cataplerosis, the extraction of metabolic intermediates for breakdown, is critical in preserving hepatic carbon homeostasis. Anaplerosis was originally used to describe the flux of carbons into the tricarboxylic acid cycle (TCA) in E. coli . AlthougE. coli  and may In vertebrates, the TCA cycle functions to oxidize acetyl-CoA for energy production and to supply intermediates to other biological pathways such as gluconeogenesis and fatty acid synthesis . In rumiThe oxidative capacity of the TCA cycle is dependent on availability of OAA, which serves as the carbon carrier for the cycle. Supply of oxaloacetate originates from either the carboxylation of pyruvate by pyruvate carboxylase (PC) in mitochondria, or from net maintenance of propionate carbon within the TCA cycle . OxaloacAnaplerotic supply of OAA into the TCA cycle through increased PC activity serves to replenish or offset carbon loss due to cataplerosis. In the absence of cataplerotic pull, increased PC generation of OAA can increase the capacity of the TCA cycle, potentiating increased oxidation of acetyl-CoA. Therefore the role of PC in the formation of OAA, a pivotal molecule in metabolism, is critical to anaplerosis and cataplerosis in many tissues and impacts several biological pathways ,26,27.Cataplerosis is only possible if anaplerosis is adequate and the pool of TCA cycle intermediates is maintained ,24,28. EDuring periods of feed restriction and adipose tissue mobilization, or when circulating NEFA and TAG are elevated due to diet or disease, the capacity of the TCA cycle to completely oxidize acetyl-CoA can be exceeded. As shown in Examination of the balance of PC and PEPCK during different physiological states or during nutritional challenges highlights the importance of anaplerotic balance. Increased hepatic expression of PC, but not PEPCK, is observed at calving ,35,36,37The balance of PC and PEPCK can also serve to fuel cataplerosis and gluconeogenesis. When propionate supply from the rumen is increased post-calving, with rumensin, or during glycerol feeding, increased expression of PEPCK-C allows for cataplerosis of OAA carbons into gluconeogenesis for glucose production ,42,43. TIncreased oxidative stress and inflammation during the peripurient period has been documented in dairy cows ,45,46,47Pyruvate carboxylase catalyzes the generation of OAA from pyruvate, a critical anaplerotic and cataplerotic reaction that impacts TCA cycle capacity, gluconeogenesis, and fatty acid synthesis. Given that liver uptake of fatty acids and TAG is proportional to the concentration of fatty acids and TAG in circulation, understanding the limitations to hepatic fatty acid metabolism is critical to developing potential prevention or treatment strategies. Understanding the balance of anaplerotic and cataplerotic and the resulting shifts in the TCA cycle oxidation of acetyl-CoA, can provide biochemical insight into hepatic oxidative capacity and lipid accumulation. The balance of PC to PEPCK serves as a control point to shift net carbon flux towards glucose production when precursors are adequately available, or to favor TCA cycle oxidation and prevent OAA pool depletion when maximal oxidative capacity is required. Taken together, characterization of individual gene regulation and recent whole genome data, suggests that differential regulation of PC and PEPCK, and the resulting ratio, are a part of the coordinated response to optimize hepatic metabolism of the dynamic substrates available during the periparturient period.


Heterochrony is an enabling concept in evolution theory that metaphorically captures the mechanism of biologic change due to mechanisms of growth and development. The spatio-temporal patterns of morphogenesis are determined by cell-to-cell signaling mediated by specific soluble growth factors and their cognate receptors on nearby cells of different germline origins. Subsequently, down-stream production of second messengers generates patterns of form and function. Environmental upheavals such as Romer’s hypothesized drying up of bodies of water globally caused the vertebrate water-land transition. That transition caused physiologic stress, modifying cell-cell signaling to generate terrestrial adaptations of the skeleton, lung, skin, kidney and brain. These tissue-specific remodeling events occurred as a result of the duplication of the Parathyroid Hormone-related Protein Receptor (PTHrPR) gene, expressed in mesodermal fibroblasts in close proximity to ubiquitously expressed endodermal PTHrP, amplifying this signaling pathway. Examples of how and why PTHrPR amplification affected the ontogeny, phylogeny, physiology and pathophysiology of the lung are used to substantiate and further our understanding through insights to the heterochronic mechanisms of evolution, such as the fish swim bladder evolving into the vertebrate lung, interrelated by such functional homologies as surfactant and mechanotransduction. Instead of the conventional description of this phenomenon, lung evolution can now be understood as adaptive changes in the cellular-molecular signaling mechanisms underlying its ontogeny and phylogeny. Heterochrony is an enabling concept in evolution theory because it captures the image of dynamic biologic change diachronically across space and time. The term heterochrony see  below waThe spatio-temporal patterns of vertebrate embryogenesis are determined by the elaboration of cell-specific growth factors signaling to their cell surface G-Protein Coupled Receptors are neighboring cells of differing germline origins to form patterns of growth and differentiation, from the zygote  to the oThe terrestrial forms of the vertebrate lung, kidney, bone, skin and brain all evolved during the water-land transition in adaptation to terrestrial life. The epitome of the mechanisms underlying these phenotypic changes is the duplication of the Parathyroid Hormone-related Protein Receptor (PTHrPR) , expressIn hindsight, it is obvious that land vertebrates had to evolve lungs in order to adapt to air breathing, yet phylogenetically the fish organ of gas exchange—the gill—is an analog of the lung, not the functionally ancestral homolog—counterintuitively, the fish swim bladder, which facilitates buoyancy, is actually the functional homolog of the lung . This exThe lung has adapted to atmospheric oxygen phylogenetically and ontogenetically by reducing the surface area of the gas exchange unit, increasing the ratio of the gas-exchange surface area to the blood volume for increased oxygenation. Concomitant ontogenetic and phylogenetic increases in the biological activity of lung surfactant secreted into the alveolar space prevented the alveolar collapse that would otherwise have been caused by the increase in surface tension resulting from the decreased surface area (surface tension being inversely related to surface area by the Law of Laplace). The stretch-regulated mechanism of alveolar surfactant production  is funct2 [The alveolar lipofibroblast (LIF) is a molecular Rosetta Stone , “transl2 , referri2 ; and (3)2  to regul2 . For oriet al. [et al. [et al. [LIFs in the alveolar wall of the rat lung were first described by Hitchcock et al. , and extet al. ,47,48 anet al. . However [et al. , who sho [et al. .The breakthrough in understanding the functional nature of these cells in lung alveolar physiology came with the co-culture of LIFs containing radiolabeled triglyceride and naive ATIIs, resulting in rapid passage of the tagged triglyceride from the LIF to the ATII, and their subsequent robust, enriched incorporation into surfactant phospholipid , termed 2 (PGE2), secreted by ATIIs [2 receptor-mediated release of TGs by LIFs, and leptin produced by the LIFs facilitates the uptake of TGs by binding to its cell surface receptors on ATIIs [As mentioned above, during the course of these studies, it was empirically discovered that ATIIs could not absorb triglycerides (TGs) , whereasby ATIIs , specifion ATIIs .in utero [The climax of these coordinated cell-cell interactions mediating and facilitating the production of lung surfactant was the discovery that Neutral Lipid Trafficking is stretch-regulated, providing key insights into both the cellular-molecular basis for the mechanism of alveolar ventilation-perfusion matching , and to in utero , and prein utero . The elug conditions using a Rotating Wall Vessel Bioreactor [g caused loss of polarity and budding. The former is a reflection of the inability to mediate calcium flux [The ancestral relationship between the effect of stretch on PTHrP expression and microgravity was subsequently shown empirically. ATIIs were subjected to 0 × oreactor . The amooreactor . Exposurium flux , and theium flux . These aium flux .These insights led to the realization that the endodermal and mesodermal components of the alveolar wall evolved over evolutionary time to generate their structural-functional properties through cell-cell interactions ,36,62. Iin vitro [Gravitational force is the oldest, constant, unidirectional force on Earth. As such, it affects biologic systems through mechanotransduction to affect cellular physiology. The fundamental nature of this effector is reflected by experiments in which yeast were exposed to microgravity, showing phenotypic effects on polarity and budding . The forin vitro , which win vitro . The mRNin vitro , consistAffecting such fundamental adaptive mechanotransductive mechanisms has produced heterochronic changes over the history of organisms.Oxygen has profoundly affected the evolution of terrestrial organisms, fueling their metabolism, causing increased growth  and diffAs added evidence for the interrelationship between physiologic stress and the co-evolution of the neuroendocrine and respiratory systems, lung surfactant has evolved to optimize surface tension reducing activity during the transition from poikilotherms to endotherms . InitialAnd since all of these properties are the net result of changes in cell-cell signaling mechanisms for structure and function, they can be characterized as heterochronies.Many chronic diseases such as emphysema and Bronchopulmonary Dysplasia are characterized by structural simplification , atavistSimilarly, modification of these cell-cell communications was responsible for the evolution of the lung from the swim bladder  phylogenPPARγ regulates peroxisome formation , and as Goodpasture’s Syndrome exhibits a similar evolutionary etiology. The disease state is due to the formation of autoantibodies against an isoform of Type IV collagen, namely Alpha 3(IV)NC1. It first appears phylogenetically in fish, and is omnipresent in amphibians, reptiles, mammals and birds. Evolutionarily, it is more hydrophobic than other Type IV collagen isoforms, preventing water loss across lung and kidney epithelial barriers in terrestrial vertebrates. Its appearance in land animals was probably due to this adaptive property.Bearing in mind that the extracellular matrix is generated by cell-cell interactions, this isoform of Type IV collagen would have been the result of a heterochronic process.The perspective expressed in this paper is that heterochrony is not a random mutational event, but instead is like Jacob’s “tinkering” mechanism , reallocBy seeing the process of evolution as communication, novel insights are gained that would otherwise remain tautologies and dogma . Kuhn 1 defined


Ritual use of ayahuasca, an amazonian Amerindian medicine turned sacrament in syncretic religions in Brazil, is rapidly growing around the world. Because of this internationalization, a comprehensive understanding of the pharmacological mechanisms of action of the brew and the neural correlates of the modified states of consciousness it induces is important. Employing a combination of electroencephalogram (EEG) recordings and quantification of ayahuasca's compounds and their metabolites in the systemic circulation we found ayahuasca to induce a biphasic effect in the brain. This effect was composed of reduced power in the alpha band (8–13 Hz) after 50 minutes from ingestion of the brew and increased slow- and fast-gamma power  between 75 and 125 minutes. Alpha power reductions were mostly located at left parieto-occipital cortex, slow-gamma power increase was observed at left centro-parieto-occipital, left fronto-temporal and right frontal cortices while fast-gamma increases were significant at left centro-parieto-occipital, left fronto-temporal, right frontal and right parieto-occipital cortices. These effects were significantly associated with circulating levels of ayahuasca’s chemical compounds, mostly N,N-dimethyltryptamine (DMT), harmine, harmaline and tetrahydroharmine and some of their metabolites. An interpretation based on a cognitive and emotional framework relevant to the ritual use of ayahuasca, as well as it's potential therapeutic effects is offered. Banisteriopsis caapi, translated as "vine of the soul" , co, coPeganB. caapi .When using the concentration of harmine  as a predictor of EEG power spectral changes, the alpha cluster was virtually identical to that of DMT Figs  whereas Harmine has been shown previously to be psychoactive in i.v. doses of 200 mg or more, even though qualitatively different from LSD or mescaline . In anotTHH plasma levels were associated with changes in alpha, slow- and fast-gamma bands Figs . THH wasThe only available report about the psychoactive properties of pure THH we are aware of was done with only one subject. After oral ingestion of 300 mg of racemic THH the subjective effects were rated as similar to 100 mg harmaline , inhibitLevels of harmaline in the blood were significantly associated with strong effects in the alpha, slow- and fast-gamma bands . These wPeganum harmala, which contains high concentrations of harmaline [Furthermore, harmaline's Tmax significantly correlated with the time for emesis  while DMarmaline . Also, iarmaline , Bigwoodarmaline ), while armaline . Thus haGiven that nausea and vomiting can be undesired and challenging in palliative care , their okamarampi, literally "vomiting medicine" [In contrast with biomedical concepts, nausea and vomiting assume central status among indigenous cultures who developed and use ayahuasca and also do so in ayahuasca religions. A striking example are the Matsigenka, whose name for ayahuasca is edicine" . Particiedicine" . Phenomeedicine" .It is therefore crucial to consider that nausea and vomiting can be psychogenic in origin and thus can be involved in the emotional experience induced by ayahuasca ingestion. Awareness of visceral stimuli has been proposed as part of the formation of emotional and affective states ,90 and tOne possible limitation of the present study is the absence of blinding to control for placebo effects. Although the lyophilizate was developed and employed in order to account for placebo effects in pharmacological research with ayahuasca , the strThe present design thus seems to make feasible a more ecologically valid approach to the neuroscientific study of ayahuasca. As was recently argued, it is important for biomedical and pharmacological studies to thoroughly consider the social, cultural and complex chemical composition of ayahuasca to avoid altering the object of their inquiry as a function of methodological imperatives . In thisBanisteriopsis rusbyana and Psychotria viridis. The MAO inhibitors harmine and harmaline may enhance the effects of tryptamines. It should, however, be borne in mind that the narcotic is still hallucinogenic even when, as often is the case, it is prepared exclusively from species of Banisteriopsis without the tryptamine additives." [The present results reveal acute biphasic effects of ayahuasca in the brain. Changes were mostly constituted of alpha decreases in the left centro-parieto-occipital cortex after 50 minutes and gamma band increases in frontal, parietal and temporal areas 75 to a 125 minutes after ingestion. These effects are associated with concentrations of tryptamines and beta-carbolines in the blood, suggesting a modified state of consciousness induced by interactions among many ayahuasca compounds. This is in agreement with the understanding of Schultes and Hofmann that "The hallucinogenic effects of ayahuasca preparations may be the result of the combined activity of harmine and its derivatives with dimethyltryptamine and other tryptamine derivatives which were found in certain admixtures to ayahuasca such as itives." . This co