High frequency repetitive transcranial magnetic stimulation (HF-rTMS) has gained interest as a neuromodulation treatment technique for alcohol dependence. Single sessions of HF-rTMS have consistently shown to decrease craving for substances. However, the results of randomized controlled clinical trials investigating the effect of multiple HF-rTMS sessions in alcohol dependence on abstinence rates and craving are inconsistent. Furthermore, they lack information on the effect of HF-rTMS on cognition and brain functioning.A single center, single blind, randomized controlled trial with 80 abstinent alcohol dependent subjects in treatment randomized (1:1) to either treatment as usual (TAU) plus ten sessions of active HF-rTMS or TAU plus 10 sessions of placebo/ sham HF-rTMS will be performed. The effects of ten HF-rTMS sessions on craving and neurocognitive functions are obtained. In addition a subset of participants will undergo an MR scanning session before the first and after the last HF-rTMS session in order to investigate the effect of ten HF-rTMS sessions on brain functioning. The primary outcome is the continued abstinence rate after the add-on HF-rTMS treatment.This study uses a randomized controlled trial to examine the clinical, neurocognitive and brain functioning effects of ten add-on HF-rTMS sessions in alcohol dependent individuals in treatment. If the add-on treatment is effective, this may add to the evidence needed for approval of this additional treatment method for alcohol dependence by regulatory authorities.NTR5291, 6-July-2015.The Netherlands National Trial Register (NTR),  Substance dependence is characterized by drug seeking and drug use which persists despite negative social and health consequences . In the High-frequency (HF) repetitive transcranial magnetic stimulation (rTMS)  is one oIndividuals suffering from alcohol dependence often experience an intense and abnormal desire for alcohol, also known as craving . Furthermultiple HF-rTMS sessions on craving are very scarce and inconsistent, with one study stimulating the right DLPFC that reported reduced craving [On a neurobiological level craving is associated with heightened striatal activity related to addiction-relevant stimuli, whereas diminished cognitive control is associated with decreased prefrontal activity . One of  craving  and one  craving . Furthermultiple sessions of HF-rTMS on abstinence, craving, cognition and brain functioning. We will conduct a single center, single blind, randomized controlled trial with 80 abstinent alcohol dependent subjects in treatment randomized (1:1) to either treatment as usual (TAU) plus 10 sessions of active HF-rTMS or TAU plus 10 sessions of placebo/ sham HF-rTMS. The effect of active versus sham HF-rTMS treatment will be investigated on measures of abstinence, craving, cognition and brain functioning. We expect higher abstinence rates and decreased perceived craving together with improved functioning on the neurocognitive tasks and brain functioning measures in the active HF-rTMS treated group compared with the sham HF-rTMS treated group.Altogether the effect of multiple HF-rTMS sessions on abstinence rates, perceived craving and the neurocognitive and neurobiological working mechanisms are poorly investigated . The cuWhat is the effect of ten sessions of active HF-rTMS on abstinence rates in alcohol dependent individuals in treatment, compared with ten sessions of sham HF-rTMS?Primary research question:What is the effect of 10 sessions of active add-on HF-rTMS treatment on total amount of alcohol consumed after treatment in alcohol dependent individuals, compared with 10 sessions of sham HF-rTMS?What is the effect of 10 sessions of active add-on HF-rTMS treatment on days until first relapse after treatment in alcohol dependent individuals, compared with 10 sessions of sham HF-rTMS?What is the effect of 10 sessions of active HF-rTMS on perceived craving levels in alcohol dependent individuals in treatment, compared with 10 sessions of sham HF-rTMS?Secondary research questions:What is the effect of 10 sessions of active HF-rTMS on performance on neurocognitive tasks measuring impulsivity, approach avoidance, spatial working memory and compulsivity in alcohol dependent individuals in treatment, compared with 10 sessions of sham HF-rTMS?What is the effect of 10 sessions of active HF-rTMS on brain functioning measures related to cognitive control and craving in alcohol dependent individuals in treatment, compared with 10 sessions of sham HF-rTMS?Additional research questions:This study aims to investigate the efficacy and working mechanisms of 10 add-on HF-rTMS sessions in a treatment-as-usual setup of alcohol dependence.The effectiveness of the HF-rTMS add-on treatment will be tested in a parallel, single center, single blind trial in abstinent alcohol dependent subjects, randomized (1:1) to either treatment as usual (TAU) plus 10 sessions of active HF-rTMS or TAU plus 10 sessions of sham HF-rTMS. A subset of the participants will undergo a magnetic resonance imaging (MRI) scan prior to the first and after the last stimulation session in order to investigate the effects of HF-rTMS on brain functioning (referred to as neuroimaging study part).This study is approved by the Medical Ethical Committee of the Academic Medical Centre Amsterdam (2015_064) and is registered in The Netherlands Trial Register (NTR) with trial number 5291. Written informed consent is obtained before screening for in and exclusion criteria takes place.All participants will be recruited when they are three to four weeks abstinent, and are recruited from the Jellinek Addiction Treatment Centre in Amsterdam, The Netherlands. Inclusion criteria are a recent (less than four months after detoxification) DSM-IV diagnosis of alcohol dependence and age between 20 and 65. Exclusion criteria are (1) insufficient knowledge of the Dutch language, (2) Montreal Cognitive Assessment (MOCA) score below 10, (3) current DSM-IV diagnosis of depression, schizophrenia or another psychotic disorder, (4) current recreational drug use, (5) rTMS contraindications , and ifThe intervention exists of 10 HF-rTMS sessions of the right DLPFC (rDLPFC) on 10 consecutive workdays. The HF-rTMS parameters of the active intervention are 60 10 Hz trains of five seconds at 110% of the motor threshold . The coi2 stimulator . The intervention will be applied by an rTMS trained researcher. The training exists of brain stimulation courses, practical rTMS tutorials, hands on training and first aid and emergency response training. During the stimulation session a predefined protocol will be executed.The motor threshold will be determined at rest before the first and sixth stimulation session, using single pulse TMS in combination with Motor Evoked Potentials. The muscular (left abductor pollicis brevis) response will be measured by visually observing a thumb muscular abduction. Stimulus intensity will be adjusted until there is an abduction in five out of ten trials. For the rTMS sessions we will use a 70 mm double air film coil  and a Magstim RapidThe HF-rTMS intervention will be added to the treatment as usual (TAU) provided by the Jellinek addiction treatment centre. This treatment consists of an intensive 3–5 days per week program with group sessions of cognitive behavioural therapy (CBT), emotion regulation training, and motivational enhancement therapy. Besides these sessions, every participant has individual sessions with a psychologist and a mentor every week. In the session with the psychologist, comorbidities and other problems of the patients that occur during treatment are discussed. During the mentor session supportive CBT focussing on remaining abstinent is given. Finally, some patients receive pharmacotherapy.The participants will start their TAU at a clinical or day detoxification unit where they stay for 7–10 days, and continue their treatment during an intensive 3–5 days treatment of at least three weeks. During the beginning of their treatment participants are informed of the study by the researcher. Individuals who are interested in participation are invited for an appointment to provide them with more information. If they are interested, informed consent is signed and the participant is screened for inclusion and exclusion criteria. If a patient meets all inclusion criteria and none of the exclusion criteria he/she will be included in the study by the researcher. After inclusion the patients can indicate whether they also want to participate in the neuroimaging study part. In that case a subsequent MR screening is performed. In order to assure concealed randomization, participants are assigned to the sham or active stimulation group after inclusion, based on the stratification factors anti-craving medication (yes / no) and age (20–40 / 41–65) using variable block sizes  of the randomisation module implemented in the data management system Castor EDC . After randomization participants start with the research procedure described below.For an overview of instruments, order of assessment and moment of assessment see Fig. The outcome measures concerning brain functioning will be obtained in the week before the first stimulation session (i.e. to measure baseline brain functioning) and in the week after the last stimulation session (i.e. to measure the effect of HF-rTMS). Before the participants enter the scanner they perform a urine alcohol and drug screening and practice the tasks that will be conducted in the MRI scanner.General patient characteristics such as age, handedness, educational level and use of medication will be assessed. Furthermore the digit span of the Wechsler Adult Intelligence Scale (WAIS) will be used to determine working memory capacity . In addiThe primary outcome of the study will be the abstinence rate after the add-on HF-rTMS treatment (in line with the guidelines of the European Medicines Agency ). This wTotal alcohol consumption after the add-on HF-rTMS treatment. This amount (g) of alcohol will be calculated using the TLFB at three months and six months follow-up.Days until the first relapse after the add-on HF-rTMS treatment, wherein relapse is defined as a heavy drinking day. This holds more than 60 g alcohol per day for men and more than 40 g alcohol per day for women , 33. ThiChange in craving levels after the add-on HF-rTMS treatment will be measured using the Alcohol Urge Questionnaire (AUQ)  assessedThe secondary outcome measures of the study will be described below.Full abstinence rate after the add- on HF-rTMS treatment, defined as the number of participants who did not consume any alcohol after the HF-rTMS treatment. This will be assessed using the TLFB at six months follow-up.Treatment success based on drinking status at 12 m follow up. The drinking status will be subdivided into the following categories: abstinence , non-excessive drinking  and excessive drinking (more than 21 drinking days per 30 days and more than 4 glasses per day) [The effect of HF-rTMS on alcohol consumption or abstinence will be assessed using the following explorative outcome measures.The effect of HF-rTMS on craving will also be assessed using the short (5-item) version of the Obsessive Compulsive Drinking Scale (OCDS) . This quTo assess the acute effect of HF-rTMS on craving two 100 mm visual analogue scale (VAS) ranging for 0  to 100 (very much) will be assessed prior and after every stimulation session. Participants will be asked to indicate (1) their current desire to consume alcohol: “How much do you feel like drinking alcohol right now?” and (2) their current urge to drink alcohol: “How strong is your urge to drink alcohol right now?” by drawing a line on a VAS scale.Alcohol consumption burden measured with the Alcohol Use Disorders Identification Test (AUDIT) .Confidence in abstaining from drinking as measured with the Alcohol Abstinence Self Efficacy (AASE) .Depressive symptoms as measured with The Beck Depression Inventory (BDI) .Impulsive behavior will be assessed using Barratt Impulsivity Scale-11 (BIS) , UPPS (uCurrent state anxiety as measured with the state trait anxiety inventory (STAI STATE) .Experience of positive and/ or negative affect measured with the Positive And Negative Affect Scale (PANAS) .Questionnaires that will be filled in during the study are:In order to get an overview of the drinking behavior during the testing period  participants are asked before every HF-rTMS session “whether (1) he/she consumed alcohol in the last 24 hours and if so (2) what kind of alcohol, (3) the alcohol percentage and (4) the amount consumed”. Furthermore participants are asked whether they were suffering from any side effects after the previous HF-rTMS session. The reported side effects will be listed.Go/ No-go Task (GNGT): This task (adapted from ) measureApproach Avoidance Task (AAT): This task (adapted from ) measureDelay Discounting Task (DDT): This task (based on ) measureSpatial Working Memory Task (SWMT): This task is part of the Cambridge Neuropsychological Test Automated Battery (CANTAB) test battery, and measures the ability of a subject to remember spatial information and manipulate this using working memory. During this task participants are presented with a number of colored squares (or boxes for the participant) shown on a screen. The participant is instructed to find a blue token hidden in each box, and use this token to fill up the black empty space on the right side of the screen Fig. . In ordeStop Signal Task (SST): This task (part of the CANTAB test battery) measures the ability of an individual to inhibit an ongoing action . ParticiIntra-dimensional/ Extra- dimensional Set Shift (IDED): This task (part from the CANTAB test battery) tests rule acquisition and reversal learning. Two stimuli are presented on a screen from which the participant has to choose one by pressing on the screen. These stimuli are made up of two artificial dimensions: color-filled shapes and/or white lines. The stimuli presented can be either simple (just one of the two dimensions) or compound (stimuli contain both the dimensions). The task contains 9 blocks increasing in difficulty Fig. . After pDespite the role of the prefrontal cortex in higher cognitive processes , the effCue reactivity task: This task (adapted from ) measureStroop task: This task (adapted from ) measureMonetary Incentive Delay Task (MIDT): This task adapted from , 56 measResting state task: During this scan (adopted from ) the neuBecause the effect of HF- rTMS over the rDLPFC of alcohol dependent individuals on brain functioning is poorly investigated , severalThe current study is the first to investigate the effect of multiple HF-rTMS sessions on abstinence rates measured six months after the last stimulation session. To the best of our knowledge no studies thus far have investigated the effect of rTMS on abstinence after a longer - clinically more relevant - period of time. Therefore no effect size is known or can be calculated, which makes it impossible to perform a scientifically correct power analysis. However, to estimate how many participants we would need, we based our population estimate on a previous study with a similar design , which uN = 27 [N = 20–30) [For the neuroimaging sub-study no information about the effect-size is available. However, studies of the statistical properties of one large fMRI cohort found that the sensitivity and reproducibility of group analyses reaches a plateau at N = 27 . With th= 20–30) . TherefoDescriptive analyses will be performed in order to see whether randomization resulted in two research groups with a similar distribution of demographic factors. Appropriate parametric and non-parametric statistical tests will be used to analyze descriptive statistics, and if required, multiple comparison corrections will be performed.Data of the primary outcome measure will be analyzed in accordance with the intention-to-treat principle. Missing data of subjects that are randomized and received at least one stimulation session will be imputed in order to achieve complete datasets. Subsequently an appropriate test for comparing two groups will be used depending on the distribution of the outcome measure. Additionally, data of the primary outcome measure will be analyzed with only treatment completers , again using an appropriate test depending on the distribution of the outcome measure.An appropriate test for comparison of two groups will be used depending on the distribution of the outcome measures ‘days until first relapse’ and ‘total alcohol consumption’.Multilevel models (capable of handling missing data points) with predictors time, treatment and time X treatment interaction will be used to test for the effect of the HF-rTMS add-on treatment on craving. The following time points will be included: post HF-rTMS, 3 months follow up and 6 months follow up.An appropriate test for comparison of two groups will be used depending on the distribution of the outcome measure ‘full abstinence rate’. An appropriate test for comparing a categorical variable between two groups will be used for the outcome measure ‘treatment success’.Multilevel models (capable of handling missing data points) with predictors time, treatment and time X treatment interaction will be used to test for the effect of the HF-rTMS add-on treatment on craving measured with the OCDS. The following time points will be included: post HF-rTMS, 3 months follow up and 6 months follow up. Data of the VAS scales will be analyzed by calculating difference scores between pre and post HF-rTMS. These scores will be used in a multilevel model (capable of handling missing data points) with predictors time, treatment and time X treatment interaction to test for the effect of the HF-rTMS add-on treatment.Multilevel models (capable of handling missing data points) with predictors time, treatment and time X treatment interaction will be used to test for the effect of the HF-rTMS add-on treatment on questionnaire scores. Total scores of baseline, intermediate and post-rTMS data will be compared.These measures will be compared between groups using an appropriate statistical test depending on the distribution of the outcome measure.Multilevel models (capable of handling missing data points) with predictors time, treatment and time X treatment interaction will be used to test for the effect of the HF-rTMS add-on treatment on neurocognitive measures. The following time points will be included: baseline, intermediate and post HF-rTMS.The software package Statistical Parametric Mapping (SPM)  will be used to analyze the task induced  activation patterns. First, images will be preprocessed, including motion correction, normalization to correct for individual differences in anatomy and smoothing. Thereafter, individual subject analyses will be performed within the context of the General Linear Model (GLM), using delta functions convolved with a synthetic hemodynamic response function to model events of interest. Contrast images containing parameter estimates for each comparison of interest will be entered into second level analyses to assess baseline versus HF-rTMS effects. For the resting state activation spatiotemporal independent component analysis will be employed.arterial = 1650 ms, labeling efficiency α = 0.8, labeling duration τ = 1650 ms [ASL scans will be analyzed using the Explore ASL toolbox in Matlab. First, the T1 images are normalized and segmented into grey and white matter. The probability maps and the gray matter tissue probability maps are then spatially normalized using the Diffeomorphic Anatomical Registration analysis using Exponentiated Lie algebra (DARTEL) algorithm . Then, f 1650 ms . FinallyThis paper presents a single blind randomized clinical trial protocol investigating whether 10 sessions of active HF-rTMS compared with 10 sessions of sham HF-rTMS improve the treatment outcomes of alcohol dependence. The aim of this study is to increase abstinence rates, decrease craving, and improve neurocognitive and brain functioning measures relevant for alcohol dependence treatment.The main challenge of this study will be completion of the entire follow-up procedure. Participants will be called three, six and twelve months after finishing the last stimulation session to assess the number of abstinent days. Within the population of alcohol dependent individuals, chances are high that participants will get lost at follow-up . There imultiple HF-rTMS session. The second strength of this study is that it takes into account several aspects that are important in the treatment of alcohol dependence. So far studies mainly looked at the effect of HF-rTMS on self-reported craving, although the most relevant clinical question is whether HF-rTMS has an effect on abstinence [The first strength of this study is that it investigates the effect of stinence . This ststinence .The limitation of this study is that the trial is not double blind controlled. However, a double blind controlled rTMS study with a sham condition similar to this study is impossible because the researcher needs to tilt the coil 90° relative to the skull . BecauseIf this study reveals higher abstinence rates and decreased craving in the active stimulation group compared with the sham stimulation group, and the active HF-rTMS induces negligible side effects, this may lead to larger clinical trials. If most of these trials find positive results of multiple HF-rTMS sessions on treatment outcomes, this eventually could result in approval by the regulatory authorities as additional treatment method for substance dependence, just as for instance the Food and Drug Administration has approved HF-rTMS for the treatment of depression.


Protein quality control in the early secretory pathway is a ubiquitous eukaryotic mechanism for adaptation to endoplasmic reticulum (ER) stress. An ER molecular chaperone, immunoglobulin heavy chain-binding protein (BiP), is one of the essential components in this process. BiP interacts with nascent proteins to facilitate their folding. BiP also plays an important role in preventing aggregation of misfolded proteins and regulating the ER stress response when cells suffer various injuries. BiP is a member of the 70-kDa heat shock protein (HSP70) family of molecular chaperones that resides in the ER. Interaction between BiP and unfolded proteins is mediated by a substrate-binding domain and a nucleotide-binding domain for ATPase activity, leading to protein folding and maturation. BiP also possesses a retrieval motif in its carboxyl terminal. When BiP is secreted from the ER, the Lys-Asp-Glu-Leu (KDEL) receptor in the post-ER compartments binds with the carboxyl terminal KDEL sequence of BiP and returns BiP to the ER via coat protein complex I (COPI) vesicular transport. Although yeast studies showed that BiP retrieval by the KDEL receptor is not essential in single cells, it is crucial for multicellular organisms, where some essential proteins require retrieval to facilitate folding and maturation. Experiments in knock-in mice expressing mutant BiP with the retrieval motif deleted revealed a unique role of BiP retrieval by the KDEL receptor in neuronal development and age-related neurodegeneration. The endoplasmic reticulum (ER) is the first organelle of the secretory pathway and plays an important role in synthesizing proteins and the lipid membrane. Newly synthesized polypeptides are inserted into the ER via translocon  proteins are formed due to extrinsic insults  or intrinsic insults . They accumulate in the ER, inducing the ER stress response or unfolded protein response (UPR). The UPR comprises three major events: (1) increased molecular chaperone production  gene are associated with familial amyotrophic lateral sclerosis , presenilin-1 (PSEN1), and presenilin-2  is the most common type of dementia and is neuropathologically characterized by intracellular neurofibrillary tangles of tau protein and extracellular senile plaques composed of amyloid β (Aβ). Aβ aggregated outside the cells is considered to cause toxicity with multiple mechanisms such as oxidative stress, mitochondrial dysfunction, synaptic dysfunction and effects on signaling pathways  II -coated vesicles and transported from the ER  gene is considered a major risk factor (Wisniewski et al., Late-onset AD seems to be sporadic, but the e4 allele of the apolipoprotein E (Kar2ΔHDEL gene (Beh and Rose, As single cells, embryonic fibroblasts from homozygous BiP mutants survive similar to mutant yeast cells with the The absence of the KDEL sequence from BiP may cause functional impairments in the KDEL receptor, in addition to defective protein folding in the early secretory pathway. Ligand recognition by KDEL receptors transduces the signal from the luminal side to the cytosol, which activates heterotrimeric G-protein signaling (Giannotta et al., Sporadic, age-associated neurodegenerative diseases may be due to a failure of quality control machinery in coping with chronic protein aggregation and accumulation. ER chaperone function and expression decrease with age (Brown and Naidoo, The KDEL receptors are not merely transport receptors. They regulate the UPR signaling. BiP is a major ER chaperone and contributes to the activation of the KDEL receptor. The first studies of quality control in the early secretory pathway were in yeast, and these results are still guiding clinical research and therapeutic applications in humans. Drugs and gene therapy could one day enhance protein quality control and treat a wide range of diseases.HJ, MK and TA edited the content and structure of the review. TA wrote the review and generated the figures.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 lung research field has pioneered the use of organoids for the study of cell-cell interactions.The use of organoids for airway basal cells is routine. However, the development of organoids for the other regions of the lung is still in its infancy. Such cultures usually rely on cell-cell interactions between the stem cells and a putative niche cell for their growth and differentiation.The use of co-culture organoid systems has facilitated the in vitro cultivation of previously inaccessible stem cell populations, providing a novel method for dissecting the molecular requirements of these cell-cell interactions. Future technology development will allow the growth of epithelial-only organoids in more defined media and also the introduction of specific non-epithelial cells for the study of cell interactions. These developments will require an improved understanding of the epithelial and non-epithelial cell types present in the lung and their lineage relationships. OrgaD structuem cells .Organoid growth requires the initiating stem cell population to self-renew, to increase organoid size, and to differentiate. Organoids have been successfully cultured from multiple endoderm-derived organs including the adult mouse stomach , mouse cLung organoids have been successfully grown from the embryonic lungs –24, mousLung diseases account for the third highest mortality of non-infectious disease deaths and lung cancer is the most common cancer worldwide . The morThe lung is a complex structure of branched epithelial-lined airways and endothelial-lined blood vessels which unite at the alveoli for gas exchange. The lung is surrounded by pleura; referred to as the mesothelium in mouse , 34. TheThe human tracheobronchial airway is mostly lined by pseudostratified epithelium in which each cell type makes contact with the basement membrane. Below the basement membrane, blood vessels, smooth muscle, cartilage, extracellular matrix-producing fibroblasts, and nerves are found. The height of luminal epithelial cells and the proportion and density of the different cell types vary along the proximal-distal axis and between human and mouse . The majThe alveolar epithelium consists of type I and type II alveolar cells AC1 and AC2) which are surrounded by capillaries and multiple different types of fibroblasts –40. AC1  and AC2 The lung is a complex organ with numerous distinct cell types. How these cells interact in development, homeostasis, and disease is a key research question and organoids provide a platform for investigating some of these cell-cell interactions Table .Table 1LIn the mouse, lung development starts after establishment of the primary germ layers with the two primary endoderm buds becoming visible at around E9.5 . MoleculWhole E12.5 mouse lungs can be cultured in vitro on an air-liquid interface in 2D where they will both grow and differentiate –54. ThesMixed cell populations obtained from dissociated whole E17.5 mouse lungs have been grown in 3D on various matrices –22. SuchA recent advance is the formation of mesenchymal organoids consisting of human foetal lung (18- to 20-week gestation) fibroblasts grown on collagen-coated alginate beads in 3D units . These w+ lung progenitors are subsequently differentiated into bronchiolar or alveolar cell fates using a stage-specific combination of growth factors. Recent efforts have been made to increase the maturity of iPS-derived differentiated cells by 3D differentiation as organoids [iPSCs are derived through reprogramming adult somatic cells by introducing pluripotency transcription factors . The difrganoids ••, 64. Trganoids  and bronrganoids .+ organoids contained small numbers of Aquaporin5+ AC1 cells and pro-SFTPC+ (SPC) AC2 cells, the latter of which contained some structures reminiscent of lamellar bodies when analysed by transmission electron microscopy. Interestingly, CPM+ iPSC-derived lung progenitors cultured without human foetal lung fibroblasts were unable to produce SPC+ cells, suggesting that the fibroblasts produce essential signal(s) for alveolar identity. Although the AC2-like cells were produced at low efficiency and are likely to be very immature, this co-culture is an encouraging strategy which could be improved upon by improved characterisation of the fibroblast cell type(s) and optimised culture conditions.The first published attempt at deriving alveolar organoids from iPSC-produced human lung progenitors used carboxypeptidase M (CPM) as a cell-surface marker to specifically isolate VAFE iPSC-derived lung progenitors. These were then seeded into Matrigel with human foetal lung fibroblasts obtained at 17.5 weeks of gestation and an alveolar-specific combination of growth factors . These N+ iPSC-derived lung progenitors followed by organoid culture to produce bronchiolar lung organoids. In this case, there was evidence of differentiation into ciliated cells with beating cilia, as well as neuroendocrine, secretory, and basal cells [The same group used a similar strategy of isolating CPMal cells . InteresAnother study differentiated human pluripotent cells into VAFE spheroids ••. TheseAll of these studies illustrate that bronchiolar and alveolar iPSC-derived organoids can be grown in vitro to a degree of maturity which equates to foetal lung tissue. The most successful studies were those which produced organoids consisting of both epithelial and mesenchymal lineages, suggesting that epithelial-mesenchymal cell interaction is crucial for differentiation. Similar results have been found in the differentiation of other organs from human PSCs, including the kidney and intestine –68. FurtOrganoid culture has also been used to investigate stem cell identity and dissect cellular and molecular interactions for the adult lung. Single basal cells isolated from the adult mouse trachea have been grown into organoids, known as tracheospheres, in which the basal cells expand and, following a change in medium composition, differentiate . These cThe smaller mouse airways consist of secretory club and ciliated cells. Attempts to grow club cells as spheroids rely exclusively on co-culture with underlying stromal populations. For example, isolated club cells could not be grown as spheres alone, only when co-cultured with an ill-defined fibroblast population . Subsequ+ fibroblast population, which possibly corresponds to the currently ill-defined alveolar lipofibroblasts [Multiple in vivo and in vitro experiments have shown that, at steady state, the AC2 cells are the primary stem cell for the alveolar epithelium , 51. Alvroblasts . This orroblasts . FurtherAn additional, less well-characterised, distal lung stem cell population contributes to mouse alveolar repair following severe injury, for example that caused by influenza infection. The steady-state characteristics of these cells are not thoroughly defined. However, they have been expanded in 2D, there is consensus that they upregulate the basal cell markers keratin5 (KRT5) and p63 following injury, they are likely to be Sox2-positive, and there is some evidence for multilineage differentiation in organoid culture in vitro –85. A reFor disease modelling, generating mature disease-specific human lung cells is particularly important because murine models often do not completely phenocopy human lung disease. Methods for culturing human airway basal stem cells at air-liquid interface to recapitulate airway epithelial organisation are well-established  and haveOrganoids have already proved extremely useful for lung research particularly for the study of adult mouse basal cells. In this case, they have provided an initial screening tool prior to pathway functional validation in vivo , 70, 72 Pseudomonas infections in lung organoids that model aspects of cystic fibrosis, or bronchiectasis, may be highly fruitful.One question which still needs to be answered is how complex do we need to make lung organoids for them to be useful? Throughout this review, we have highlighted the use of organoid technology for studying epithelial-epithelial and mesenchymal-epithelial cell interaction. But, should we be using these experiments to develop defined growth media which supports epithelial-only organoids? Or, should we be focusing on the inclusion of multiple cell types in a quest to more completely model the in vivo situation? The answers will most likely depend on the specific research question. However, we are particularly intrigued by the possibilities of using organoid technology to study the relationship between the epithelium and innate immune system or even to model the response to infection. Indeed, a recent report has used human intestinal organoids both as the first in vitro culture system for human norovirus and to study the epithelial response to infection . A similThe most significant limitations of current research using lung organoids are the lack of reliable validation by comparison to the in vivo cell types and the lack of defined starting populations. Particularly for human, differentiated organoids have been validated by marker expression from the mouse lung literature. An improved understanding of the numerous cell types in the developing and adult human lung is required to improve the validation and maturation attempts for lung organoids and will likely require more single-cell transcriptomics , 91. In


Urine metabolic profiles were assessed using non-targeted nuclear magnetic resonance spectroscopy and targeted liquid chromatography-tandem mass spectrometry analysis. Multivariate statistical models with data from either analytical approach showed clear separation between the metabolic profiles of septic neonates (both possible and confirmed) and the controls. Metabolic changes appeared to be related to disease progression. Overall, neonates with confirmed or possible LOS exhibited comparable metabolic profiles indicating similar metabolic alternations upon the onset of clinical manifestations. This methodology therefore enabled the discrimination of neonates with LOS from non-septic individuals, providing potential for further research toward the discovery of LOS-related biomarkers.Although late-onset sepsis (LOS) is a major cause of neonatal morbidity and mortality, biomarkers evaluated in LOS lack high diagnostic accuracy. In this prospective, case-control, pilot study, we aimed to determine the metabolic profile of neonates with LOS. Urine samples were collected at the day of initial LOS evaluation, the 3 Late-onset sepsis (LOS), variably defined for epidemiological purposes as occurring after 72 hours to 6 days of life124546Early diagnosis of sepsis and timely, prompt initiation of treatment have been shown to improve survival and functional outcomeMetabolomics is a bio-analytical approach aiming toward the comprehensive profiling of small molecule content of the examined sample. Urine metabolomics has been increasingly used for the study of biochemistry and biomarker discovery in disease as it can provide important information on the overall metabolic status of an individual. Moreover, urine seems to be more suitable for metabolic analysis in the neonatal population as it offers significant methodological advantages compared to e.g., blood-based sampling; the risk of iatrogenic anemia in critically-ill infants is minimized and issues associated with the need for invasive procedures and relatively large sample volumes in these tiny patients are eliminatedIn this context, metabolomics has also been applied for the assessment of critical illness such as sepsis. To date, metabolomics studies in sepsis have been conducted in animals12131H-NMR) spectroscopy and liquid chromatography-tandem mass spectrometry (LC-MS/MS).We hypothesized that metabolomics could provide potential biomarkers of neonatal sepsis. Thus, the aim of the present study was to investigate metabolic changes related to LOS, employing two analytical platforms: proton nuclear magnetic resonance (Klebsiella pneumonia (n = 4), Klebsiella oxytoca (n = 1), Enterobacter cloace (n = 2)]; Group B Streptococcus and Candida famata were isolated in two cases. Results of the initial laboratory investigation for LOS are also shown in Sixteen septic neonates (9 with confirmed and 7 with possible LOS) and 16 controls were studied. The two groups were comparable regarding demographic-perinatal characteristics . One neo1H-NMR spectroscopic profiling-data using Chenomx software, a local spectral library and 2D spectra for identification, 110 peaks were selected and their signals were integrated. This approach enabled the elimination of interfering signals deriving from pharmaceuticals and/or their metabolites.After an extensive search of the 1H NMR signals, and the p-values of T-tests for significant metabolites/peaks. In total 10 peaks were found to pass all evaluation filters for D0 and these are given in 1H NMR spectroscopy for D3 and D10 showed no significant statistical alteration to these metabolites. Analysis of the spectral data by multivariate analysis showed that, even by unsupervised Principal Component Analysis (PCA), septic infants (confirmed and possible) could be discriminated from non-septic ones (controls) at the time point of sepsis suspicion (D0). 1NMR spectral data, cases of possible sepsis (D0) were found to cluster together with of those with confirmed sepsis (D0) in the PCA plots. It should be stressed that in general, cases of confirmed and possible sepsis could not be discriminated by multivariate analysis. Even when models were built only for these two classes of samples, PCA did not show any separation, while no valid Partial Least Square-Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Square-Discriminant Analysis (OPLS-DA) models could be built.Differentiation of septic (both confirmed and possible) from control patients was revealed by PCA analysis. PCA scores plots  show patSubsequently, OPLS-DA models were built to allow the identification of metabolites contributing to group differentiation (D0). An OPLS-DA scores plot showing the two separate classes of samples is illustrated in OPLS-DA models of D3 urine  again showed clear differentiation between septic neonates and controls. 1H NMR spectral data and are noted with asterisk in With regard to the contribution of specific metabolites to group classification, in D0 samples were found to significantly differ in the signals of 17 metabolites measured by LC-MS between septic neonates and controls. Among the significant metabolites, three were also found significant in the ROC curves/values and box plots for significant metabolites are given in In this study, the potential of metabolomics to identify biomarkers related to LOS in the urine of neonates was investigated. The results show that the metabolic profile of neonates with proven and possible LOS was markedly different compared to those without sepsis. The metabolic alternations seen were mainly relevant to energy producing biosynthetic pathways and basic structural components of the organism, contributing significantly to the clear discrimination of septic neonates using metabolomic analysis. Neonates with confirmed and possible sepsis exhibited similar metabolic profiles under the applied methods, thus, indicating similar underlying biological derangements. This fact lends further support to the notion that septic neonates, and those fulfilling the accepted criteria for LOS, but not confirmed by culture should be treated in the same manner, irrespective of the blood culture results.1H-NMR spectroscopy for the analysis of the urine samples in an attempt to obtain an overall view of the metabolic profile, we were able to document a clear separation between neonates fulfilling the diagnostic criteria for LOS and non-septic controls. Non-targeted metabolomics investigation revealed significant changes in 10 urine metabolites. With the targeted metabolomics approach (LC-MS/MS) that was applied next for the quantification of additional important metabolites, the selection of which was based on the existing literature, we found statistically significant alternation in 17 metabolites in the urine, three of which were also found to be statistically significant by 1H-NMR spectroscopy.The triage of septic neonates from those with other severe conditions is a major challenge in everyday clinical practice. In this investigation, initially using 1H-NMR spectroscopy and LC-MS/MS  out of the 16 neonates evaluated had possible (blood culture negative) LOS, whereas the disease was strongly indicated by the clinical signs as well as laboratory indicators of sepsis. This subgroup of neonates could be clearly distinguished from controls using metabolomics. Interestingly, however, no discrimination could be made between neonates with possible and confirmed LOS on the clinical onset of the disease and the third day thereafter. Nevertheless, the finding of common metabolites in the neonates with confirmed and possible LOS on day 0 is suggestive of similar underlying pathophysiological mechanisms, regardless of the blood culture positivity. Positive blood culture is the gold standard for the diagnosis of sepsis, but novel diagnostic methods demonstrate a suboptimal diagnostic accuracy of blood cultures for the detection of LOS195LC-MS/MS . In a prAn interesting hypothesis in biomarker research including sepsis is the potential of predicting disease development considerably sooner, even days before the appearance of clinical signs. Circulating sepsis indicators have been reported to increase in septic neonates up to 2 days before clinical suspicionet al. reported significantly higher taurine concentrations in adult patients with SIRS and sepsisOur data show  that sevGlutamine and glutamate metabolism also seems to play a crucial role during sepsis in neonates. Critically-ill adult patients characteristically have depleted glutamine, presumably due to increased metabolic demandsAltered glucose metabolism is a major metabolic disturbance in septic neonates manifesting hypo- or hyperglycemia. In this study, the urinary glucose concentration was higher in septic neonates compared to controls, although this was significantly increased only with metabolomics. We also documented significantly increased amounts of pyruvate and lactate in the urine of sepsis cases possibly representing sepsis-associated hypoperfusion and/or hypoxia favoring lactate production or reduced lactate clearanceInosine, a product of the purine degradation pathway was found to be present in increased amounts in samples from septic neonates in the present study, while hypoxanthine was also increased but marginally failed the evaluation criteria. Accumulation of purines is believed to result from the cellular destruction following sepsis and the degradation of high-energy phosphate compounds. Xanthine oxidase activation is a leading factor for the oxidant stress and a marker of poor prognosis in patients with sepsis37We found several amino acids to be significantly increased in samples from septic vs. non-septic neonates. Previous studies in adults with sepsis have also documented altered plasma amino acids concnetrations2628et al., also reported on TMAO in the serum of adult patients with septic shockper seAdditionally, we noted decreased quantities of trimethylamine N-oxide (TMAO) in urine from septic neonates. Mickiewicz Finally, in our study, septic neonates had significantly lower amounts of certain vitamins of the B complex such as riboflavin (vitamin B2) and nicotinamide (vitamin B3) as compared to non-septic ones. These vitamins are coenzymes or precursors of cofactors involved in several important biochemical pathways , and sepsis predisposition to deficiency in several micronutrientsst urine sampling was specifically taken into consideration at study design because of their possible influence on urine metabolic profiles and, therefore, cases were matched to controls for these factors to the best possible level. Nevertheless, the association between risk factors and outcome  is difficult to evaluate in case-control studies particularly when the patient number is relatively small. Moreover, over-matching can make it difficult to find enough controls in the clinical setting. For all the above reasons, the validation of our results in the context of larger multi-center studies would be justified, especially in relation to important clinical outcomes such as those aforementioned.Beyond the combined application of two analytical platforms, the present study also has other strengths. As far as we know, this is the largest metabolomic study to have been performed in septic neonates, where subjects were serially evaluated at three time-points during the acute phase and upon recovery from the disease. Moreover, we evaluated urine samples, with the aforementioned methodological advantages of the specific biological fluid in neonates. However, an obvious limitation is that it represents a pilot study conducted in a single center, thus limiting the number of the septic neonates that could have been enrolled. Although the sample size was adequate to reliably document differences in the metabolic profile between septic neonates, irrespective proven or suspected LOS status, and controls, the number of the enrolled subjects did not allow the evaluation of other important variables. For example, Gram (+) and Gram (−) bacteria may have elicited different metabolic responses, but we failed to prove this as the majority of cases suffered from Gram (−) sepsis. Similarly, we could not construct models based on mortality due to the very small number of cases (only two infants) with this unfavorable outcome. Interestingly, one could expect an altered metabolic profile related to neonatal maturation. Previous investigations have clearly documented developmental differences in inflammatory response to sepsis1H-NMR and LC-MS/MS-based urine analysis. Results of this pilot study support the usefulness of metabolomics in exploring underlying biochemical mechanisms of the disease, that could -after validation- provide novel candidate mechanisms to confirm or follow-up the progression of neonatal LOS.In conclusion, neonates with confirmed and possible sepsis at the onset of clinical manifestations showed a different metabolic profile compared to those without sepsis allowing their clear discrimination with the use of The study protocol was approved by the School of Medicine ethics committee of the Aristotle University of Thessaloniki. An informed written consent was obtained from all parents/guardians before enrolling their child in the study. All experiments were performed in accordance with relevant guidelines and regulations.This prospective, case-control, pilot study was performed in a single, tertiary-level neonatal intensive care unit during a two-year period (September 2013-August 2015). Eligible for the study were preterm and term neonates who needed evaluation for LOS (sepsis presenting after the first 72 hours of life). Cases included neonates with confirmed (positive blood cultures for microbes or fungi) or possible LOS . For LOS diagnosis, we adopted the criteria defined in 2010 by an Expert Meeting of the European Medicines Agency on Neonatal and Paediatric Sepsisst urine sampling. Controls were identified from hospitalized neonates who had no medical problems or were recovering from minor ones  but required sepsis screening, thereafter, for an event such as apnea or mottled skin, proved to have no laboratory evidence of sepsis and, thus, judged as not fulfilling sepsis criteria. The latter infants received either no antibiotics or were given a short course (3–4 days) during the time period in which blood culture results were pending according to our center protocol.In this study, we compared septic neonates with a group of non-septic ones (controls) receiving intensive care who were stable, less-severely ill and, also, we considered the probable influence of gestational and post-natal age on urine metabolic profile. To this end, each septic neonate was matched to one control of the same gestational age (±one week) and day of life  at the 1Neonates born to mothers with clinical chorioamnionitis or those who had early-onset sepsis (≤day 3 of life), known congenital infections and anomalies, medium/severe perinatal asphyxia or inborn errors of metabolism were excluded a priori from enrollment. Prior LOS episode, refusal of parental consent, inadequate selection of urine samples, and physical absence of the researcher were also exclusion criteria. Demographic, perinatal and clinical-laboratory data related to LOS were recorded in all neonates.rd (D3) and 10th day (D10), thereafter, using plastic bags or through a bladder catheter placed for clinical reasons. Samples were centrifuged and supernatants were stored at −80 °C until analysis. Further details on sample processing are provided in Urine samples were collected from cases and controls at the day of the initial evaluation for LOS and enrollment in the study, respectively, representing day 0 (D0) and on the 31H-NMR spectroscopy was performed on an Avance III 600 MHz spectrometer (Bruker BioSpin GmbH) using standard 1D NOESY pulse sequence (noesypr1d). For each sample, 128 scans were collected in 64-k data points over a spectral width of 14 ppm. MNova NMR was used for peak integration purposes and Chenomx software for signal identifications. Subsequently samples were analysed by LC-MS/MS based on an in-house method described previouslyThe MetSizeR R-package was used for the calculation of the sample size needed to find significant changes in 10–30% out of a total of 90–110 metabolites studied. The target false discovery rate (FDR) was set at 0.05. A sample size of 15 subjects per group was found adequate for the goals of our study.MedCalc software  was used for statistical analysis of patient characteristics. Continuous variables were expressed as mean ± SD. The t-test was used for comparisons between two study groups and the Fisher’s exact test was used for the categorical variables.Multivariate statistical analysis  was performed using SIMCA 13  with UV scaling. Coefficient plots, VIP plots, loading plots, Receiver Operating Characteristic (ROC) curves and univariate statistics (t-test) were used to find the significant metabolites differentiating the groups. Differences were considered statistically significant when the p value was <0.05.Associated biochemical pathways were investigated using MetaboAnalyst 3.0How to cite this article: Sarafidis, K. et al. Urine metabolomics in neonates with late-onset sepsis in a case-control study. Sci. Rep.7, 45506; doi: 10.1038/srep45506 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Hydrodynamic injection transfecting the hepatocytes in the gene-disrupted mouse model with the HBV replicative genome DNA has revealed that IFNAR and IRF3/7 are indispensable in HBV eradication in the mice liver but not the RNA sensing pathways. Interestingly, accumulating evidence of the recent studies has demonstrated that HBV markedly interfered with IFN-β induction and antiviral immunity mediated by the Stimulator of interferon genes (STING), which has been identified as a central factor in foreign DNA recognition and antiviral innate immunity. This review will present the current understanding of innate immunity in HBV infection and of the challenges for clearing of the HBV infection.Cellular innate immune system recognizing pathogen infection is critical for the host defense against viruses. Hepatitis B virus (HBV) is a DNA virus with a unique life cycle whereby the DNA and RNA intermediates present at different phases. However, it is still unclear whether the viral DNA or RNA templates are recognized by the pattern-recognition receptors (PRRs) to trigger host antiviral immune response. Here in this article, we review the recent advances in the progress of the HBV studies, focusing on the nucleic acid sensors and the pathways involved in the recognition of HBV in the liver–specific  Hepatitis B virus (HBV) is one of the significant health burdens worldwide as more than 240 million people are chronically infected . HBV infThe role of the adaptive immune responses in the clearance of HBV infection is widely accepted. However, the role of innate immune response and capacity of HBV to induce innate responses remain controversial. Type I interferon (IFN), a representative output of antiviral innate signaling, seems undetectable or missed in most cases of HBV patients . Thus faNevertheless, some recent studies suggested that in the early phases of infection HBV could activate some of pattern-recognition receptors (PRRs), which in turn limited the infection and drove adaptive immune responses crucial to the virus clearance ,7. For iin vivo is available because analysis on the gene expression and effectors required for elimination of the replicative template has been especially difficult. Therefore, it remains skeptical for the assumption that the HBV RNA is the PAMP and caution is necessary in the evaluation of the physiological role of this HBV-mediated IFN-inducing mechanism since the agonists PAMPs in these studies are originated from various viruses or synthetic double-stranded (ds) RNA (such as Poly I:C). In addition to this TLR3, RIG-I and MDA5, there are a number of cytoplasmic RNA sensors, including PKR, LRRFIP1, DEAD box helicase proteins , some RLRs and dicer-associated RNA-binding proteins. It remains untested whether these RNA sensors are associated with detection of HBV RNA in human hepatocytes. Since viral clearance is a multifaceted process, the full spectrum of immunological requirements for HBV clearance is not completely defined with the current in vitro studies using hepatocytes derived cell lines. A mouse model was developed by Yang et al. to provide a better insight into the host and viral interaction, in which hydrodynamic injection of a naked plasmid DNA encoding a supergenomic HBV1.3-length transgene into inbred mice initiates high-titer HBV replication in the liver that is rapidly terminated if the mice are immunocompetent [−/−Mavs, −/−Ticam1 or −/−Mavs/−/−Ticam1 double KO mice [−/−Irf-3/7 or the IFN-α/β receptor pathway, −/−Ifnar, led to a greatly elevated virus titers in the KO mice even though the intrahepatic DNA template was eventually eliminated [Viral RNA is sensed by the innate immune system via either Toll-like receptor 3 (TLR3) or cytoplasmic sensors such as retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). RIG-I and MDA5 mainly participate in type I interferon (IFN) induction in conjunction with the adaptor molecule, mitochondrial antiviral signaling protein . The Toll/IL-1R homology domain-containing adaptor molecule 1  is the adaptor of TLR3, which senses viral RNA on the endosomal membrane. A few reports have suggested that the antiviral response against HBV is mediated by the RIG-I/MAVS pathway in the cytosol and its activation is blocked by HBV polymerase in infected cells ,16. Howeompetent ,18. Usin KO mice . On the iminated . These rInterestingly, our attention has also been called to the fact that lacking the MyD88 adaptor molecule which participates in the TLR9 pathway leads to the failure of the mice to eliminate the HBV viral template . MyD88 iIn recent years, the cytosolic DNA sensors which signal through STING have been demonstrated to be essential in eliciting innate immune responses against foreign DNA . Most rei.e., cGAS and STING) may have a rather peripheral role in sensing HBV replication in the hepatocyte lines, and that host cells other than hepatocytes contribute to regulation of an HBV infectious state. However, such responses need to be validated in other infection systems and in primary human hepatocytes.It may however, be noted that the endogenous expression level of the cGAS-STING axis is rather low in the hepatocyte-derived cell lines, particularly the commonly used Huh7 and HepG2 in comparison to the macrophages or dendritic cells . This maThere have been reported many other cytoplasmic and membrane-associated DNA sensors , some ofThe liver is an immunological organ with non-parenchymal cells  and parenchymal cells  with unique immunosuppressive characteristics . Besideset al., shows that RIG-I can counteract the interaction of HBV polymerase (P protein) with the 5'-ε region of pgRNA in an RNA-binding dependent manner, resulting in the suppression of HBV replication using a HepG2 culture and a chimeric mice with humanized livers [The life cycle of HBV consisted of a replication strategy that is thought to facilitate the HBV evasion of innate immunity. During HBV infection, the HBV genome is delivered into the nucleus. Infection is defined by the formation of covalently closed circular DNA. Following formation of covalently closed circular DNA, viral mRNA and pregenomic RNA is transcribed, which are to be capped and polyadenylated by host machinery ,30. The d livers . IFN-λ id livers . The resAlthough these observations from mouse models reflect some direct and indirect ways of the host and viral interactions, the real situation in the virus-infected patients needs to be assessed and further validation in relevant infection models in the near future is required. Identification of entry receptors for HBV (including NTCP) will facilitate the analysis of HBV infection in animal models. Comprehensive screening of host proteins that interact with the HBV proteins will give us valuable hints for HBV persistent infection.Meanwhile, increasing studies suggest that the growing family of PRRs able to discriminate intracellular pathogen DNA might be the answer to the nucleic acid sensor to HBV. DNA sensors may take a two-pronged attack on the life-cycle of HBV, while RNA sensors target viral RNA. It remains undetermined which is more implicated in the elimination of HBV. Our current knowledge on the nucleic acid-sensing pathways of HBV based on the mouse hydrodynamic studies is schematically summarized in


Tripple negative breast cancer (TNBC) accounts for approximately 15% of breast cancers. It is defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and HER-2 Over expression. Expression of ER, PR and HER-2 plays an important role in therapeutic assessment of patients with breast cancer. TNBC is not one disease, but a family of diseases, some of which are highly aggressive with limited treatment options. Triple-negative breast cancers Patients are not benefiting from currently available receptor-targeted systemic therapy. At present, there is no single agent that targets triple-negative breast cancer. However, researchers are presently investigating large number of potential therapies that may eventually improve outcomes in these patients. In this review article, we discussed about tripple negative breast cancer, also the role of BRCA gene mutations and targeted therapeutic options available to triple negative breast cancer patients. Triple-negative breast cancers are characterized by the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor -3. An esMultiple studies of gene expression profiling have advanced the understanding of the molecular diagnosis of breast cancer. Perou et al were the first to describe the various molecular subtypes or molecular profiles of breast cancers . More reMetastasis, the major cause of mortality in patients with breast cancer, is caused by tumor cells that escape from the primary tumor into the bloodstream and travel through the circulation to distant sites where they develop into secondary tumors . Triple-Hereditary breast cancers account for only 5-10% of all breast cancer cases. The functions of BRCA1 are the repair of double stranded DNA breaks by the potentially error-free mechanism of homologous recombination. Lack of BRCA1 could result in DNA repair by more error-prone mechanisms such as nonhomologous end-joining and single-strand annealing, resulting in genomic instability and therefore cancer predisposition  However,Triple-negative breast cancer Patients do not benefit from hormonal or trastuzumab-based therapies because of the loss of target receptors such as ER, PR, and Her-2. Hence, surgery and chemotherapy, individually or in combination, appear to be the only available modalities. Chemotherapy improves the outcome to a greater extent when used in patients with triple-negative breast cancer than when used in patients with the much more common ER-positive subtype. Currently, there is no preferred standard form of chemotherapy for triple-negative breast cancer. Chemotherapy Drugs for example carboplatin, cisplatin, parp1 inhibitors, and docetaxel could be very useful in the administration of patients with advanced triple-negative cancers. The addition of docetaxel or paclitaxel to anthracycline-containing adjuvant regimens may be of greater benefit for the treatment of ER-negative and HER2-negative cancers than for the treatment of ER-positive, HER2-negative cancers, which are much more common . To imprThe most interesting clinical target in triple-negative breast cancer is the enzyme poly (adenosine diphosphate- Ribose) polymerase (PARP), which is involved in base-excision repair after DNA damage. Several PARP1 inhibitors are currently in clinical development and hold promise in TNBC. PARP inhibitors have recently shown very encouraging clinical activity in early trials of tumors arising in BRCA mutation carriers. One of these inhibitors, iniparib (BSI-201), which was recently used in a randomized phase 2 trial involving patients with triple-negative cancer. When this inhibitor was added to a chemotherapy combination of gemcitabine and carboplatin, there were significant improvements in the rate of tumor regression. A phase II study with a combination of Carboplatin and Gemcitabine in metastatic triple-negative patients determined the activity in response rates from 26% to 34%. This study was conducted to observe if Iniparib could effectively increase the antiproliferative and cytotoxic nature of the two chemotherapeutic drugs which gave a positive output -26. The EGFR inhibition in breast cancer has been another interesting story. The EGFR signaling pathway is another area of investigation in TNBC, since EGFR expression may be more apparent in basal-cell type breast cancers. EGFR is expressed approximately in 60% of the triple negative breast cancer and is a marker of poor prognosis irrespective of the lymph node involvement and tumor size and may also contain mutations in the p53 gene . Due to The mTOR  is associated with cell cycle regulation and an effector of the common pathway of phosphatidylinositol 3-phosphate phosphatase and PTEN/AKT pathway. Breast cancers are associated with the impairment of this pathway. Loss of PTEN tumor suppressor gene is a common event in triple negative breast cancers which also leads to the activation of mTOR . As a reHigh expression of src has been observed in breast cancer tissues compared to that of the normal breast samples indicating an activation of src kinase in breast cancer tissues. Activation of src is associated with the activation of EGFR pathway which is frequently expressed in triple negative breast cancers . Due to Tripple negative breast cancer is harder to treat as the tumors don’t have the receptors that the common drugs such as oestrogen, progesterone or HER2 that are targeted by common treatments such as hormone therapy or Herceptin can target blocking the growth of the tumor. Breast tumor cells with BRCA1 mutation carriers may have particular sensitivity to platinum agents and relatively less sensitivity to taxanes. Therefore, the identification of novel molecular biomarkers to predict response to specific chemotherapy is required to further improve treatment strategies.


Cryptococcal meningitis is the most common central nervous system infection in the world today. It occurs primarily, but not exclusively, in immunocompromised individuals and despite substantial improvement in management of clinical events like AIDS, the numbers of cases of cryptococcosis remain very high. Unfortunately, despite several antifungal agents available for treatment, morbidity and mortality rates remain high with this fungal infection. In this Review, we will describe the treatments and strategies for success, identify the failures, and provide insights into the future developments / improvements for management. This sugar-coated yeast can play havoc within the human brain. Our goals must be to either prevent or diagnose disease early and treat aggressively with all our clinical tools when disease is detected. Cryptococcus spp. frequently causes fungal meningitis. In fact, this encapsulated yeast remains the most common cause of meningitis in HIV-infected individuals living in sub-Saharan Africa. It is estimated that in 2014 there were over 220,000 new cases of cryptococcal meningitis globally resulting in more than 180,000 deaths and is responsible for 15% of all AIDS-related deaths. Although the annual rate of cryptococcal disease has decreased after the widespread use of highly active anti-retroviral therapy (HAART) in developed countries, the prevalence of cryptococcal infection remains at a high level in low and middle-income countries despite the availability of HAART , of HAART . The oneof HAART .Furthermore, non-HIV patient populations are also at risk of cryptococcal infection, notably transplant recipients and patients on immunosuppressive therapies. For example, approximately 2-3% of solid organ recipients have been found to develop cryptococcal infection with most patients presenting with disseminated infection . With ovAntifungal drug therapy remains the mainstay of treatment of these cryptococcal infections. This review aims at highlighting the drugs and strategies utilized for the management of this life- threatening infection, as well as the new developments in treatment.Therapeutic principles for cryptococcal meningitis - Before examining the details of our arsenal, several therapeutic principles for management of cryptococcal meningitis should be listed: (1) early diagnosis is helpful to a successful outcome of treatment with a lower burden of yeasts and less destruction from a persistent, dysregulated immune system; (2) identification of new, old, and changing risk factors is necessary to properly utilize our outstanding biomarkers for disease; (3) a fungicidal anticryptococcal regimen that rapidly clears viable yeasts from the subarachnoid space is optimal management; (4) major complications of cryptococcal meningitis should be carefully identified and managed. These include (a) increased intracranial pressure and (b) development of the immune reconstitution inflammatory syndrome (IRIS); (5) further understanding of in vitro anticryptococcal yeast susceptibility testing for resistance  and strain evaluation genetically for identification of possible “bad actor” strains will be helpful. This area requires further research to become more clinically relevant and precise; (6) we must control the concomitant underlying diseases at all costs and this will likely demand attention to the “Goldilocks’s Paradigm of Immunology, not too much and not too little but must get it just right”; (7) our goal is to reduce mortality but it must also be accompanied by a reduction in morbidity, which is less chronicled in present reviews.Amphotericin B & flucytosine (5-FC) - The polyene, amphotericin B, has been the mainstay of treatment of cryptococcal meningitis in HIV-infected individuals and transplant recipients as well as non-HIV and non-transplant patients for several decades. Using a polyene-based regimen has been associated with significant reduction of the yeast burden within the CNS and is correlated with improved survival  . FluconaFor other cryptococcal infections, such as pulmonary cryptococcosis, fluconazole monotherapy is recommended as treatment for mild to moderate disease .Itraconazole, voriconazole and posaconazole - The extended-spectrum azoles have a limited experience in treatment of cryptococcosis. Itraconazole has successfully treated cryptococcal meningitis but its poor absorption and limited CNS penetration makes it unreliable  success  can be detected in serum more than three weeks prior to onset of symptoms of cryptococcal meningitis. Global cryptococcal antigenemia in 2014 was estimated at 6% of HIV-infected patients with CD4 counts less than 100 cells/µL, translating to approximately 280,000 individuals -γlb in HIV patients with cryptococcal meningitis showed accelerated clearance of CSF cultures but results did not achieve statistical significance . Second,Cryptococcus spp. such as capsule, as well as radiolabelled monoclonal antibodies to deliver radiation to cryptococcal cells and induce an apoptosis-like cell death  - Studies have shown that raised intracranial pressure (ICP) in cryptococcal meningitis is associated with increased mortality  - ACTA is a phase III trial aimed at finding more feasibly implemented regimens for resource-limited healthcare systems and to determine if 5-FC or fluconazole is better adjuvant treatment. Three treatment strategies were compared: (1) an all oral strategy, of high-dose fluconazole and 5-FC for two weeks; (2) amphotericin B plus either high-dose oral fluconazole/5-FC for one week only; (3) amphotericin B plus high dose fluconazole / 5-FC for two weeks (control). An early presentation from the trial revealed that the short course arms of amphotericin B and the all-oral fluconazole/5-FC were non-inferior to the control group. Furthermore, using 5-FC as adjuvant therapy with amphotericin B led to lower mortality compared with fluconazole. The one-week amphotericin B plus 5-FC arm had the lowest mortality among the treatment arms  - AMBITION-cm is a phase II randomized, controlled, non-inferiority trial that has recently shed some light on the use of a few large doses of liposomal amphotericin B with high dose fluconazole for the treatment of cryptococcal meningitis in HIV patients. The short-course high-dose polyene arms received one, two or three doses of liposomal amphotericin B while being given a high-dose fluconazole backbone regimen. Data has shown the rate of clearance of CSF cultures in all the short-course, high-dose arms to be non-inferior to the standard two-week course of daily polyene, with none of the participants necessitating any treatment interruptions. The single dose (10 mg/kg/day) liposomal amphotericin B regimen is being taken to a phase III trial  using a membrane with pores ≤ 5 μm in diameter to trap the fungus. A peristaltic pump circulates the CSF of rabbits with cryptococcal meningitis through this filter and reintroduces it back into the SAS. After six hours of cycling through the filter, the fungal colony forming units in the CSF were reduced by up to 99%. Further studies may aim at combining neurapheresis with direct antifungal drug delivery such as amphotericin B intrathecally to create high efficacy regimens along with systemic antifungal agents (l agents .In conclusion - Mortality and morbidity still remain high for cryptococcal meningitis despite several strategies for management. Infections in the CNS can be unforgiving and therefore, all tools available must be utilized. Complications such as increased intracranial pressure and IRIS must be recognized and treated despite unclear guidelines. Early diagnosis, expert care (ert care  and rapi


Since the development of capsule endoscopy technology, medical device companies and research groups have made significant progress to turn passive capsule endoscopes into robotic active capsule endoscopes. However, the use of robotic capsules in endoscopy still has some challenges. One such challenge is the precise localization of the actively controlled robot in real-time. In this paper, we propose a non-rigid map fusion based direct simultaneous localization and mapping method for endoscopic capsule robots. The proposed method achieves high accuracy for extensive evaluations of pose estimation and map reconstruction performed on a non-rigid, realistic surgical EsophagoGastroDuodenoscopy Simulator and outperforms state-of-the art methods. In the past decade, advances in microsensors and microelectronics have enabled small, low cost devices in a variety of high impact applications. Following these advances, untethered pill-size, swallowable capsule endoscopes with an on-board camera and wireless image transmission device have been developed and used in hospitals for screening the gastrointestinal (GI) tract and diagnosing diseases such as the inflammatory bowel disease, the ulcerative colitis, and the colorectal cancer. Unlike standard endoscopy, endoscopic capsule robots are non-invasive, painless, and more appropriate to be employed for long-duration screening purposes. Moreover, they can access difficult body parts that were not possible to reach before with standard endoscopy . Such advantages make pill-size capsule endoscopes a significant alternative screening method over standard endoscopy  have attracted the attention for small-scale medical devices. With their low cost and small size, cameras are frequently used in localization applications where weight and power consumption are limiting factors, such as in the case of small-scale robots. However, many challenges posed by the GI tract and low quality cameras of the endoscopic capsule robots cause further difficulties in front of a vSLAM technique to be applied in a medical operation. Self-repetitiveness of the GI tract texture, non-rigid organ deformations, heavy reflections caused by the organ fluids, and lack of distinctive feature points on the GI tract tissue are further challenges in front of a reliable robotic operation. Moreover, the low frame rate and limited resolution of the current capsule camera systems also restrict the applicability of computer vision methods inside the GI tract. Especially feature tracking based visual localization methods have poor performance in the abdomen region compared to outdoor or indoor large scale environments where unique features can be found easier.Figure In that section, we first summarize the contributions of our paper and give details of the proposed method.Create depth image from RGB image based on shading;Divide visited organ parts into active and inactive areas. Only active areas are used for pose tracking and map fusion. Areas that do not appear in the scene for a certain period of time are assigned as inactive and not used in the estimation.For every new frame, search for its intersection with the active model and fuse them;In case there exists an intersection of the active model with inactive model within the current frame, fuse the intersecting parts using loop closure and reactivate corresponding inactive parts.The contributions of the approach described in this paper include:A vSLAM approach able to deal with specularities typically occurring in images of inner organs tissues;A direct vSLAM method able to handle non-rigid structures, including performing their non-sparse 3D reconstruction;A direct vSLAM approach jointly minimizing photometric-geometric constraints, including depth;Inspired from large-scale RGB Depth SLAM approaches , where K is the camera intrinsics matrix and u is the homogeneous form of u. Geometric pose estimation is performed by minimizing the energy cost function k-th vertex in The input for pose estimation is the RGB image k such that Due to strict real-time concerns of the approach, we use surfel-based scene reconstruction. Each surfel has a position, normal, color, weight, radius, initialization timestamp and last updated timestamp. We also define a deformation graph consisting of a set of nodes and edges to detect non-rigid deformations throughout the frame sequence. Each node To ensure a globally consistent surface reconstruction, the framework closes loops with the existing map as those areas are revisited. This loop closure is performed by fusing reactivated parts of the inactive model into the active model and simultaneously deactivating surfels which have not appeared for a period of time.We evaluate the performance of our system both quantitatively and qualitatively in terms of trajectory estimation, surface reconstruction and computational performance.Figure Trajectory 1 is an uncomplicated path with slow incremental translations and rotations.Trajectory 2 follows a comprehensive scan of the stomach with many local loop closures.Trajectory 3 contains an extensive scan of the stomach with more complicated local loop closures.Trajectory 4 consists of more challenging motions including faster rotational and translational movements.Trajectory 5 consists of very loopy and complex motions.Trajectory 6 is the same as trajectory 5 but included added synthetic noise to allow checking the robustness of the system against noise.Before capturing trajectory 7, we added more paraffin oil into the simulator tissue to have stronger reflections. Similarly to trajectory 6, trajectory 7 consists of very loopy and complex motions including very fast rotations, translations and drifting.Qualitative tracking results of the proposed direct medical SLAM compared to ORB SLAM and to ground truth are shown in Fig. Table We scanned the non-rigid EGD (Esophagogastroduodenoscopy) simulator to obtain the ground truth 3D data. Reconstructed 3D surface and ground truth 3D data were aligned using iterative closest point algorithm (ICP). RMSE for the reconstructed surface was calculated using the absolute trajectory (ATE) RMSE measuring the root-mean-square of the Euclidean distances between estimated depth values and the corresponding ground truth values. RMSE results in Table To analyze the computational performance of the system, we observed the average frame processing time across trajectories 1–4. The test platform was a desktop PC with an Intel Xeon E5-1660v3- CPU at 3.00, 8 cores, 32 GB of RAM and an NVIDIA Quadro K1200 GPU with 4 GB of memory. The execution time of the system depended on the number of surfels in the map, with an overall average of 48 ms per frame scaling to a peak average of 53 ms implying a worst case processing frequency of 18 Hz.ORB SLAM is based on feature matching while direct medical SLAM uses joint photometric- geometric pose estimation. In our evaluation, we observed that for endoscopic images, direct pose estimation is advantageous as compared to feature-based methods because specularity, noise and presence of fewer robustly identifiable features reduce the matching accuracy across frames.Direct medical SLAM needs a good initialization for pose estimation to avoid local minima while ORB SLAM does not require initialization.Direct medical SLAM employs a frame-to-model alignment strategy, which is robust to unexpected severe drifts inside GI tract. ORB SLAM on the other hand, performs frame-to-frame alignment and may have difficulties recovering from such drifts.Direct medical SLAM is computationally heavy while ORB SLAM can run on standard CPU in real-time. However, modern GPUs can be used to accelerate direct medical SLAM to near real-time as well.Direct medical SLAM tolerates larger motions between successive frames, while ORB SLAM expects smaller motions. However, we observed that both methods fail for very large inter-frame motion that leads to small overlap between successive frames.ORB SLAM’s reconstruction is in the form of a sparse point cloud of the scanned inner organ, whereas direct medical SLAM creates a dense and high quality 3D map of the organ.Qualitative and quantitative comparisons depicted in Figs. We compared the proposed method with ORB SLAM using our endoscopic capsule dataset. We chose ORB SLAM due to its state-of-the-art performance in various tasks, publicly available code and its recent use in endoscopic applications. We make the following observations after a detailed theoretical and practical evaluation of the differences between the proposed medical SLAM and ORB SLAM:In this paper, we presented a direct and dense visual SLAM method for endoscopic capsule robots. Our system makes use of surfel-based dense data fusion in combination with frame-to-model tracking and non-rigid deformation. Experimental results suggest the effectiveness of the proposed system, both quantitatively and qualitatively, in occasionally looping endoscopic capsule robot trajectories and comprehensive inner organ scanning tasks. In future, we aim to extend our work into stereo capsule endoscopy applications to achieve even more accurate localization and mapping.


Background: Suffering from tinnitus causes mental distress in most patients. Recent findings point toward a diminished activity of the brain's default-mode network (DMN) in subjects with mental disorders including depression or anxiety and also recently in subjects with tinnitus-related distress. We recently developed a therapeutic intervention, namely the Heidelberg Neuro-Music Therapy (HNMT), which shows an effective reduction of tinnitus-related distress following a 1-week short-term treatment. This approach offers the possibility to evaluate the neural changes associated with the improvements in tinnitus distress. We previously reported gray matter (GM) reorganization in DMN regions and in primary auditory areas following HNMT in cases of recent-onset tinnitus. Here we evaluate on the same patient group, using functional MRI (fMRI), the activity of the DMN following the improvements tinnitus-related distress related to the HNMT intervention.Methods: The DMN activity was estimated by the task-negative activation (TNA) during long inter-trial intervals in a word recognition task. The level of TNA was evaluated twice, before and after the 1-week study period, in 18 treated tinnitus patients , 21 passive tinnitus controls (PTC), and 22 active healthy controls (AC). During the study, the participants in TG and AC groups were treated with HNMT, whereas PTC patients did not receive any tinnitus-specific treatment. Therapy-related effects on DMN activity were assessed by comparing the pairs of fMRI records from the TG and PTC groups.Results: Treatment of the TG group with HNMT resulted in an augmented DMN activity in the PCC by 2.5% whereas no change was found in AC and PTC groups. This enhancement of PCC activity correlated with a reduction in tinnitus distress .Conclusion: Our findings show that an increased DMN activity, especially in the PCC, underlies the improvements in tinnitus-related distress triggered by HNMT and identify the DMN as an important network involved in therapeutic improvements. Suffering from tinnitus causes mental distress in most patients. Especially in situations with reduced external sensory input , the tinnitus percept comes to the fore and can cause emotional distress Møller, . FocusedOver the past 15 years, great effort has been made to clarify the origin and tinnitus sensitivity of the so-called “task-negative activation” (TNA) during the brains “resting state” when less sensory input needs to be processed : growth of gray matter (GM) density has been found in the PCC and the precuneus, suggesting a potential influence of HNMT onto DMN activity  were invited to participate in the HNMT study after a treatment following the standard clinical protocol for acute tinnitus in the University Hospital for Ear, Nose, and Throat at the University of Heidelberg. Audiograms and psychological examination were conducted before the decision for study inclusion. All patients had age-appropriate hearing levels (audiograms are attached as SD 1.85) weeks in the TG group and 8.10 (SD 1.45) weeks in the PTC group .The general task-negative activity was measured by contrasting the brain activity during the ITI period against the brain activity during the word recognition condition using scans from T1. The task-negative activities from all participants  tinnitus-related effect on the DMN, (2) therapy-related effects on the DMN and (3) a possible intersection between therapy-related and tinnitus-related effects.To assess therapy related effects, the activity change among the tinnitus patients was compared using “group” and “time” as factors of a 2 × 2 ANOVA. The TG group revealed an increase in task-negative activity from T1 to T2, especially in PCC/precuneus, LP, and MPF  as well as between the TG and AC groups .Tinnitus-related effects were evaluated by comparing the two groups of “treated” participants: the TG and AC groups. Results indicated that the treated tinnitus patients in TG exhibited an increase in the DMN activity compared to the treated healthy participants in AC Table . In contThe conjunction of therapy-related and tinnitus-related effects was assessed by contrasting task-negative activity between the TG and AC groups within the spatial mask for therapy-related effects (TG > PTC). It focused on a cluster in the PCC/precuneus area Figure  and isoln = 39; Spearman-Rho: −0.5; p < 0.005; Figure Using SPSS, the therapy-specific change in the DMN activity was further consolidated by a significant correlation with improvements in tinnitus distress: lower levels of tinnitus distress led to rising DMN activity  over the study period (Figure For this synoptic presentation, structural alterations due to HNMT (Krick et al., We used TNAs to observe the DMN in the context of the HNMT tinnitus-therapy control task. The general DMN pattern was similar to findings from previous studies (Fox et al., The PCC is one of the most interlinked anatomical areas of the brain (Hagmann et al., Lanting et al.  observedThe modulation of the tinnitus percept by a short-term intervention lasting 5 days only afforded an opportunity to have an insight into the relationship between therapy-induced tinnitus modulation and related DMN activity. The data revealed a rising task-negative activity in tinnitus patients following HNMT, especially in the ventral PCC. The increase of PCC activity was accompanied by rising GM density in this region (Krick et al., Since it is known that precuneus and PCC are also involved in the perception of musically induced emotions (Blood et al., On the level of auditory processing and attention control, there is evidence that music based stimulation has positive effects on the tinnitus percept. Sound therapies that focus on “notched music listening” distributed lateral inhibition into the tinnitus region and have demonstrated an increase in responsiveness of the PCC (Pape et al., Functional measurements of brain activity can only depict the relative levels of hemodynamic blood flow between defined conditions, in this case in task-positive and task-negative states. Although, we observed some changes with the interaction of participant group and time, it is safe to say that this cannot originate from TNA. One can only argue that identical tasks performed at two time points may primarily evoke similar responses in all participants. This led to the logical consequence that the task-negative responses might be changed.Although, the DMN-specific RSN seems to interplay with the so-called “tinnitus core” (Husain and Schmidt, Unlike other auditory procedures, HNMT commits the patients to take an active role in overcoming their tinnitus distress. HNMT aims to target some the neuronal hubs that possibly trigger tinnitus distress. These assumed mechanisms were reflected by a distinct correlation between reduced tinnitus distress and increased DMN activity. As a diminished connectivity to the precuneus seems to be a characteristic pattern for non-pulsatile tinnitus patients (Han et al., This study was carried out in accordance with the recommendations of the ethic board of Saarland/Germany with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the ethic board of Saarland.CK: MRI measurements, analysis of MRI data, data interpretation. HA: Development of Heidelberg Neuro-Music Therapy, therapy management, statistics. MG: therapist, analysis of clinical data, statistics. PP: tinnitus diagnostics, clinical therapy control. WR: neuroradiological screening, coordinator between the facilities, study coordinator.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 high cost, complexity and reliance on electricity, specialized equipment and supplies associated with conventional diagnostic methods limit the scope and sustainability of newborn screening for sickle cell disease (SCD) in sub-Saharan Africa and other resource-limited areas worldwide. Here we describe the development of a simple, low-cost, rapid, equipment- and electricity-free paper-based test capable of detecting sickle hemoglobin (HbS) in newborn blood samples with a limit of detection of 2% HbS. We validated this newborn paper-based test in a cohort of 159 newborns at an obstetric hospital in Cabinda, Angola. Newborn screening results using the paper-based test were compared to conventional isoelectric focusing (IEF). The test detected the presence of HbS with 81.8% sensitivity and 83.3% specificity, and identified SCD newborns with 100.0% sensitivity and 70.7% specificity. The use of the paper-based test in a two-stage newborn screening process could have excluded about 70% of all newborns from expensive confirmatory testing by IEF, without missing any of the SCD newborns in the studied cohort. This study demonstrates the potential utility of the newborn paper-based test for reducing the overall cost of screening newborns for SCD and thus increasing the practicality of universal newborn SCD screening programs in resource-limited settings. Universal newborn screening, in combination with early intervention for affected infants, has nearly eliminated early childhood mortality due to sickle cell disease  in high-income developed countries124The major barriers limiting the expansion of these highly successful pilot screening programs are the high cost and technical complexity of conventional diagnostics methods  in the bottom chamber of a disposable syringe-less filter . After 5 minutes, the filter top is compressed until the solution is filtered into the top chamber. An approximately equal volume (75 μL) of the deoxygenation buffer (200 g/L sodium hydrosulfite in phosphate buffer) is then added to the top chamber and mixed with the filtered solution. After 10 minutes, a 20 μL drop of the mixture is deposited onto chromatography paper  and allowed to dry for up to 25 minutes  and should therefore capture all newborns at risk for either disease or trait for confirmatory IEF testing. We trained local health workers to collect capillary blood samples from newborns via heel-stick, process the samples using the paper-based test, and score the result of the test visually as either HbS-positive or HbS-negative . The resThe paper-based test deployed in Angola employed a reusable pipette with disposable pipette tips (4 per test) for liquid metering. The cost per test for all test specific consumables  of the paper-based test deployed in Angola was $2.16 . Additionally, the per test cost of the finalized test kit could potentially be lower than the current version as it will employ integrated sample collection and processing components, produced by a contract manufacturing partner, which are projected to be less expensive than the sum of the current retail prices for the multiple commercially available components comprising the current test. Secondly, the paper-based test is lightweight, completely electricity-free, requires no specialized equipment or instrumentation and is simple enough for a user with no previous experience performing the test to learn to operate and interpret the test with expert proficiency in under one hour. Therefore the paper-based test would not be limited to centralized laboratories with specialized equipment and trained technicians, but rather could be deployed to remote facilities and operated by health workers with any level of experience. In this study the results of the test were typically available within one hour or less of initiating the test. This rapid turnaround time, compared to conventional laboratory methods, could therefore potentially enable counseling of families with high-risk newborns before postpartum discharge . Finally, our data show that a two-stage approach consisting of preliminary screening of all infants and confirmatory testing of only the high-risk subset could reduce the number of newborns requiring IEF testing by at least 70%, thus significantly reducing the overall cost of the screening program. More importantly, accurate identification of newborns at the highest risk for SCD could help focus limited available resources on establishing proper longitudinal care for this much smaller cohort.TM) are not sensitive enough to detect the low levels of HbS typically found in newborn blood samples, are notoriously difficult to standardize and interpret, and are confounded by numerous comorbidities which effect the turbidity of blood samples222526The paper-based test offers important advantages over existing technologies designed to enable low-cost SCD diagnostics in resource-limited settings. Conventional hemoglobin solubility tests  or later in life using the previously developed adult version of the paper-based test13There are three major limitations to the paper-based newborn SCD screening test. The first is that the paper-based test cannot yet distinguish between SCT and SCD in newborns, and therefore must be used as a In summary, we demonstrate that the paper-based newborn SCD screening test, optimized to detect the low levels of sickle hemoglobin present in newborn blood, enables the direct screening of newborns for sickle cell trait and sickle cell disease. We also demonstrate that this test is feasible and has a high diagnostic accuracy when performed by local health workers in a resource-limited clinical setting. These results demonstrate the potential utility of the newborn paper-based test for identifying high-risk newborns in the immediate postnatal period and reducing the overall cost of screening newborns for sickle cell disease, thus increasing the practicality and effectiveness of universal newborn SCD screening programs in resource-limited settings.All experimental protocols involving human blood samples were approved by the institutional review boards at the Universidade Onze de Novembro Medical School , Baylor College of Medicine  and University of Houston . Informed consent was obtained from all subjects. All experiments were performed in accordance with guidelines and regulations established by the University of Houston, Baylor College of Medicine and the U.S. Department of Health and Human Services for the protection of human study subjects. After obtaining informed consent from the newborns’ mothers, local health workers collected blood samples from newborns at the obstetric center of the Primero de Maio hospital in Cabinda. Eligibility criteria included gestation of greater than 30 weeks and uncomplicated delivery . Samples were collected from n = 160 newborns, a convenience sample. We successfully screened 160 newborn samples with the paper-based test, and performed successful IEF testing on 159 of these samples .2EDTA, BD Diagnostics, USA) using standard venipuncture technique. Samples were stored at 4 °C until use. Artificially reconstituted samples with a range of HbS levels were created by mixing ABO/Rh-matched, equal-hematocrit HbAA and HbSS blood samples at various ratios.For initial development of the paper-based newborn SCD screening test, blood samples were collected from SCD (HbSS) patients at the Texas Children’s Hematology Center and from healthy volunteers (HbAA) into Vacutainer vials . Eluted dried blood spot samples were tested with isoelectric focusing (IEF) following existing standard operating proceduresFor test validation in Cabinda, blood samples were collected from newborns by heel-stick onto blood collection cards  and into capillary blood collection tubes . 95% confidence intervals (CI) were calculated using the Wilson methodHow to cite this article: Piety, N. Z. et al. A Paper-Based Test for Screening Newborns for Sickle Cell Disease. Sci. Rep.7, 45488; doi: 10.1038/srep45488 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.