Machine learning for predicting Plasmodium liver stage development in vitro using microscopy imaging.

Malaria, a significant global health challenge, is caused by Plasmodium parasites. The Plasmodium liver stage plays a pivotal role in the establishment of the infection. This study focuses on the liver stage development of the model organism Plasmodium berghei, employing fluorescent microscopy imaging and convolutional neural networks (CNNs) for analysis. Convolutional neural networks have been recently proposed as a viable option for tasks such as malaria detection, prediction of host-pathogen interactions, or drug discovery. Our research aimed to predict the transition of Plasmodium-infected liver cells to the merozoite stage, a key development phase, 15 hours in advance. We collected and analyzed hourly imaging data over a span of at least 38 hours from 400 sequences, encompassing 502 parasites. Our method was compared to human annotations to validate its efficacy. Performance metrics, including the area under the receiver operating characteristic curve (AUC), sensitivity, and specificity, were evaluated on an independent test dataset. The outcomes revealed an AUC of 0.873, a sensitivity of 84.6%, and a specificity of 83.3%, underscoring the potential of our CNN-based framework to predict liver stage development of P. berghei. These findings not only demonstrate the feasibility of our methodology but also could potentially contribute to the broader understanding of parasite biology

Variable density and viscosity, miscible displacements in horizontal Hele-Shaw cells. Part 2. Nonlinear simulations

Direct numerical simulations of the variable density and viscosity Navier-Stokes equations are employed, in order to explore three-dimensional effects within miscible displacements in horizontal Hele-Shaw cells. These simulations identify a number of mechanisms concerning the interaction of viscous fingering with a spanwise Rayleigh-Taylor instability. The dominant wavelength of the Rayleigh-Taylor instability along the upper, gravitationally unstable side of the interface generally is shorter than that of the fingering instability. This results in the formation of plumes of the more viscous resident fluid not only in between neighbouring viscous fingers, but also along the centre of fingers, thereby destroying their shoulders and splitting them longitudinally. The streamwise vorticity dipoles forming as a result of the spanwise Rayleigh-Taylor instability place viscous resident fluid in between regions of less viscous, injected fluid, thereby resulting in the formation of gapwise vorticity via the traditional, gap-averaged viscous fingering mechanism. This leads to a strong spatial correlation of both vorticity components. For stronger density contrasts, the streamwise vorticity component increases, while the gapwise component is reduced, thus indicating a transition from viscously dominated to gravitationally dominated displacements. Gap-averaged, time-dependent concentration profiles show that variable density displacement fronts propagate more slowly than their constant density counterparts. This indicates that the gravitational mixing results in a more complete expulsion of the resident fluid from the Hele-Shaw cell. This observation may be of interest in the context of enhanced oil recovery or carbon sequestration application

Transfer of carbon dioxide within cultures of microalgae: plain bubbling versus hollow-fiber modules

In attempts to improve the metabolic efficiency in closed photosynthetic reactors, availability of light and CO2 are often considered as limiting factors, as they are difficult to control in a culture. The carbon source is usually provided via bubbling of CO2- enriched air into the culture medium; however, this procedure is not particularly effective in terms of mass transfer. Besides, it leads to considerable waste of that gas to the open atmosphere, which adds to operation costs. Increase in the interfacial area of contact available for gas exchange via use of membranes might be a useful alternative; microporous membranes, in hollow-fiber form, were tested accordingly. Two hollow-fiber modules, different in both hydrophilicity and outer surface area, were tested and duly compared, in terms of mass transfer, versus traditional plain bubbling. Overall volumetric coefficients (KLa) for CO2 transfer were 1.48 10-2 min-1 for the hydrophobic membrane, 1.33 10-2 min-1 for the hydrophilic membrane, and 7.0 10-3 min-1 for plain bubbling. A model microalga, viz. Nannochloropsis sp., was cultivated using the two aforementioned membrane systems and plain bubbling. The produced data showed slight (but hardly significant) increases in biomass productivity when the hollow-fiber devices were used. However, hollow-fiber modules allow recirculation of unused CO2, thus reducing feedstock costs. Furthermore, such indirect way of supplying CO2 offers the additional possibility for use of lower gas pressures, as no need to counterbalance hydrostatic heads exists

TGF-b2 induction regulates invasiveness of theileria-transformed leukocytes and disease susceptibility

Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence

Expression Analysis of the Theileria parva Subtelomere-Encoded Variable Secreted Protein Gene Family

Background The intracellular protozoan parasite Theileria parva transforms bovine lymphocytes inducing uncontrolled proliferation. Proteins released from the parasite are assumed to contribute to phenotypic changes of the host cell and parasite persistence. With 85 members, genes encoding subtelomeric variable secreted proteins (SVSPs) form the largest gene family in T. parva. The majority of SVSPs contain predicted signal peptides, suggesting secretion into the host cell cytoplasm. Methodology/Principal Findings We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Microarray and quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was largely defined by the parasite genotype and not by host background or cell type, and found to be relatively stable in vitro over a period of two months. Interestingly, immunofluorescence analysis carried out on cell lines established from a cloned parasite showed that expression of a single SVSP encoded by TP03_0882 is limited to only a small percentage of parasites. Epitope-tagged TP03_0882 expressed in mammalian cells was found to translocate into the nucleus, a process that could be attributed to two different nuclear localisation signals. Conclusions Our analysis reveals a complex pattern of Theileria SVSP mRNA expression, which depends on the parasite genotype. Whereas in cell lines established from a cloned parasite transcripts can be found corresponding to a wide range of SVSP genes, only a minority of parasites appear to express a particular SVSP protein. The fact that a number of SVSPs contain functional nuclear localisation signals suggests that proteins released from the parasite could contribute to phenotypic changes of the host cell. This initial characterisation will facilitate future studies on the regulation of SVSP gene expression and the potential biological role of these enigmatic proteins

A bovine lymphosarcoma cell line infected with theileria annulata exhibits an irreversible reconfiguration of host cell gene expression

Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. Fifty percent of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of chromatin modification and gene expression. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and largely irreversible manner

Photosensitized INA-Labelled protein 1 (PhIL1) is novel component of the inner membrane complex and is required for Plasmodium parasite development.

Plasmodium parasites, the causative agents of malaria, possess a distinctive membranous structure of flattened alveolar vesicles supported by a proteinaceous network, and referred to as the inner membrane complex (IMC). The IMC has a role in actomyosin-mediated motility and host cell invasion. Here, we examine the location, protein interactome and function of PhIL1, an IMC-associated protein on the motile and invasive stages of both human and rodent parasites. We show that PhIL1 is located in the IMC in all three invasive (merozoite, ookinete-, and sporozoite) stages of development, as well as in the male gametocyte and locates both at the apical and basal ends of ookinete and sporozoite stages. Proteins interacting with PhIL1 were identified, showing that PhIL1 was bound to only some proteins present in the glideosome motor complex (GAP50, GAPM1-3) of both P. falciparum and P. berghei. Analysis of PhIL1 function using gene targeting approaches indicated that the protein is required for both asexual and sexual stages of development. In conclusion, we show that PhIL1 is required for development of all zoite stages of Plasmodium and it is part of a novel protein complex with an overall composition overlapping with but different to that of the glideosome

Analysis of common genetic variation and rare CNVs in the Australian Autism Biobank.

BackgroundAutism spectrum disorder (ASD) is a complex neurodevelopmental condition whose biological basis is yet to be elucidated. The Australian Autism Biobank (AAB) is an initiative of the Cooperative Research Centre for Living with Autism (Autism CRC) to establish an Australian resource of biospecimens, phenotypes and genomic data for research on autism.MethodsGenome-wide single-nucleotide polymorphism genotypes were available for 2,477 individuals (after quality control) from 546 families (436 complete), including 886 participants aged 2 to 17 years with diagnosed (n = 871) or suspected (n = 15) ASD, 218 siblings without ASD, 1,256 parents, and 117 unrelated children without an ASD diagnosis. The genetic data were used to confirm familial relationships and assign ancestry, which was majority European (n = 1,964 European individuals). We generated polygenic scores (PGS) for ASD, IQ, chronotype and height in the subset of Europeans, and in 3,490 unrelated ancestry-matched participants from the UK Biobank. We tested for group differences for each PGS, and performed prediction analyses for related phenotypes in the AAB. We called copy-number variants (CNVs) in all participants, and intersected these with high-confidence ASD- and intellectual disability (ID)-associated CNVs and genes from the public domain.ResultsThe ASD (p = 6.1e-13), sibling (p = 4.9e-3) and unrelated (p = 3.0e-3) groups had significantly higher ASD PGS than UK Biobank controls, whereas this was not the case for height-a control trait. The IQ PGS was a significant predictor of measured IQ in undiagnosed children (r = 0.24, p = 2.1e-3) and parents (r = 0.17, p = 8.0e-7; 4.0% of variance), but not the ASD group. Chronotype PGS predicted sleep disturbances within the ASD group (r = 0.13, p = 1.9e-3; 1.3% of variance). In the CNV analysis, we identified 13 individuals with CNVs overlapping ASD/ID-associated CNVs, and 12 with CNVs overlapping ASD/ID/developmental delay-associated genes identified on the basis of de novo variants.LimitationsThis dataset is modest in size, and the publicly-available genome-wide-association-study (GWAS) summary statistics used to calculate PGS for ASD and other traits are relatively underpowered.ConclusionsWe report on common genetic variation and rare CNVs within the AAB. Prediction analyses using currently available GWAS summary statistics are largely consistent with expected relationships based on published studies. As the size of publicly-available GWAS summary statistics grows, the phenotypic depth of the AAB dataset will provide many opportunities for analyses of autism profiles and co-occurring conditions, including when integrated with other omics datasets generated from AAB biospecimens (blood, urine, stool, hair)

Watch me grow integrated (WMG-I): protocol for a cluster randomised controlled trial of a web-based surveillance approach for developmental screening in primary care settings

Introduction The increasing prevalence of developmental disorders in early childhood poses a significant global health burden. Early detection of developmental problems is vital to ensure timely access to early intervention, and universal developmental surveillance is recommended best practice for identifying issues. Despite this, there is currently considerable variation in developmental surveillance and screening between Australian states and territories and low rates of developmental screening uptake by parents. This study aims to evaluate an innovative web-based developmental surveillance programme and a sustainable approach to referral and care pathways, linking primary care general practice (GP) services that fall under federal policy responsibility and state government-funded child health services. Methods and analysis The proposed study describes a longitudinal cluster randomised controlled trial (c-RCT) comparing a â € Watch Me Grow Integrated' (WMG-I) approach for developmental screening, to Surveillance as Usual (SaU) in GPs. Forty practices will be recruited across New South Wales and Queensland, and randomly allocated into either the (1) WMG-I or (2) SaU group. A cohort of 2000 children will be recruited during their 18-month vaccination visit or opportunistic visit to GP. At the end of the c-RCT, a qualitative study using focus groups/interviews will evaluate parent and practitioner views of the WMG-I programme and inform national and state policy recommendations. Ethics and dissemination The South Western Sydney Local Health District (2020/ETH01625), UNSW Sydney (2020/ETH01625) and University of Queensland (2021/HE000667) Human Research Ethics Committees independently reviewed and approved this study. Findings will be reported to the funding bodies, study institutes and partners; families and peer-reviewed conferences/publications

Biophysics of Malarial Parasite Exit from Infected Erythrocytes

Upon infection and development within human erythrocytes, P. falciparum induces alterations to the infected RBC morphology and bio-mechanical properties to eventually rupture the host cells through parasitic and host derived proteases of cysteine and serine families. We used previously reported broad-spectrum inhibitors (E64d, EGTA-AM and chymostatin) to inhibit these proteases and impede rupture to analyze mechanical signatures associated with parasite escape. Treatment of late-stage iRBCs with E64d and EGTA-AM prevented rupture, resulted in no major RBC cytoskeletal reconfiguration but altered schizont morphology followed by dramatic re-distribution of three-dimensional refractive index (3D-RI) within the iRBC. These phenotypes demonstrated several-fold increased iRBC membrane flickering. In contrast, chymostatin treatment showed no 3D-RI changes and caused elevated fluctuations solely within the parasitophorous vacuole. We show that E64d and EGTA-AM supported PV breakdown and the resulting elevated fluctuations followed non-Gaussian pattern that resulted from direct merozoite impingement against the iRBC membrane. Optical trapping experiments highlighted reduced deformability of the iRBC membranes upon rupture-arrest, more specifically in the treatments that facilitated PV breakdown. Taken together, our experiments provide novel mechanistic interpretations on the role of parasitophorous vacuole in maintaining the spherical schizont morphology, the impact of PV breakdown on iRBC membrane fluctuations leading to eventual parasite escape and the evolution of membrane stiffness properties of host cells in which merozoites were irreversibly trapped, recourse to protease inhibitors. These findings provide a comprehensive, previously unavailable, body of information on the combined effects of biochemical and biophysical factors on parasite egress from iRBCs.Singapore. Agency for Science, Technology and ResearchSingapore-MIT AllianceGlobal Enterprise for Micro-Mechanics and Molecular MedicineNational University of SingaporeNational Institutes of Health (U.S.) (Grant R01 HL094270-01A1)National Institutes of Health (U.S.) (Grant 1-R01-GM076689-01)National Institutes of Health (U.S.) (P41-RR02594-18-24