Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors.

Studies of tissue regeneration and host-pathogen interactions using the model planarian Schmidtea mediterranea have been performed at an experimental temperature of 19 °C. S. mediterranea planarians exposed to 19 °C-32 °C were observed for survival, mobility, feeding and regeneration for three months and elimination of the Staphylococcus aureus pathogen over six days. S. mediterranea planarians died at 30 °C-32 °C after 18 days of observation but tolerated temperatures of 19 °C up to 28 °C with non-significant differences in mobility and feeding behavior. Genetic malleability tested by RNAi feeding was still efficient at 26 °C and 28 °C. Concerning the immune capacity of planarians, we reported an exacerbation of the immune response in worms infected by S. aureus at 26 °C and 28 °C. These observations suggest a temperature modulation of planarian stem cells and illustrate the importance of modulating experimental temperature when using planarians as model organisms to study regeneration and immune response.

Inherited axonopathies represent a spectrum of disorders unified by the common pathological mechanism of length-dependent axonal degeneration. Progressive axonal degeneration can lead to both Charcot-Marie-Tooth type 2 (CMT2) and Hereditary Spastic Paraplegia (HSP) depending on the affected neurons: peripheral motor and sensory nerves or central nervous system axons of the corticospinal tract and dorsal columns, respectively. Inherited axonopathies display an extreme degree of genetic heterogeneity of Mendelian high-penetrance genes. High locus heterogeneity is potentially advantageous to deciphering disease etiology by providing avenues to explore biological pathways in an unbiased fashion. Here, we investigate 'gene modules' in inherited axonopathies through a network-based analysis of the Human Integrated Protein-Protein Interaction rEference (HIPPIE) database. We demonstrate that CMT2 and HSP disease proteins are significantly more connected than randomly expected. We define these connected disease proteins as 'proto-modules' and show the topological relationship of these proto-modules by evaluating their overlap through a shortest-path based measurement. In particular, we observe that the CMT2 and HSP proto-modules significantly overlapped, demonstrating a shared genetic etiology. Comparison of both modules with other diseases revealed an overlapping relationship between HSP and hereditary ataxia and between CMT2 + HSP and hereditary ataxia. We then use the DIseAse Module Detection (DIAMOnD) algorithm to expand the proto-modules into comprehensive disease modules. Analysis of disease modules thus obtained reveals an enrichment of ribosomal proteins and pathways likely central to inherited axonopathy pathogenesis, including protein processing in the endoplasmic reticulum, spliceosome, and mRNA processing. Furthermore, we determine pathways specific to each axonopathy by analyzing the difference of the axonopathy modules. CMT2-specific pathways include glycolysis and gluconeogenesis-related processes, while HSP-specific pathways include processes involved in viral infection response. Unbiased characterization of inherited axonopathy disease modules will provide novel candidate disease genes, improve interpretation of candidate genes identified through patient data, and guide therapy development.

The triterpene oil squalene is an essential component of nanoemulsion vaccine adjuvants. It is most notably in the MF59 adjuvant, a component in some seasonal influenza vaccines, in stockpiled, emulsion-based adjuvanted pandemic influenza vaccines, and with demonstrated efficacy for vaccines to other pandemic viruses, such as SARS-CoV-2. Squalene has historically been harvested from shark liver oil, which is undesirable for a variety of reasons. In this study, we have demonstrated the use of a Synthetic Biology (yeast) production platform to generate squalene and novel triterpene oils, all of which are equally as efficacious as vaccine adjuvants based on physiochemical properties and immunomodulating activities in a mouse model. These Synthetic Biology adjuvants also elicited similar IgG1, IgG2a, and total IgG levels compared to marine and commercial controls when formulated with common quadrivalent influenza antigens. Injection site morphology and serum cytokine levels did not suggest any reactogenic effects of the yeast-derived squalene or novel triterpenes, suggesting their safety in adjuvant formulations. These results support the advantages of yeast produced triterpene oils to include completely controlled growth conditions, just-in-time and scalable production, and the capacity to produce novel triterpenes beyond squalene.

Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase constitutively expressed by acute myeloid leukaemia (AML) blasts. In addition, 25% of AML patients harbour a FLT3-ITD mutation, associated with inferior outcome due to increased relapse rate. Relapse might be propagated by interactions between AML blasts and the bone marrow microenvironment. Besides cellular elements of the microenvironment (e.g. mesenchymal stromal cells), bone marrow hypoxia has emerged as an additional crucial component. Hence, effects of hypoxia on FLT3 expression and biology could provide novel insight into AML biology. Here we show that 25% of AML patients down-regulate FLT3 expression on blasts in response to in vitro hypoxia (1% O2), which was independent of its mutational state. While virtually no AML cell lines regulate FLT3 in response to hypoxia, the down-regulation could be observed in Ba/F3 cells stably transfected with different FLT3 mutants. Hypoxia-mediated down-regulation was specific for FLT3, reversible and proteasome-dependent; with FLT3 half-life being significantly shorter at hypoxia. Also, PI-3K inhibition could partially abrogate down-regulation of FLT3. Hypoxia-mediated down-regulation of FLT3 conferred resistance against cytarabine in vitro. In conclusion, FLT3 expression in AML is dependent on the oxygen partial pressure, but response to hypoxia differs.

Campylobacter jejuni is a major pathogen in bacterial gastroenteritis worldwide and can cause bacteremia in severe cases. C. jejuni is highly structured into clonal lineages of which the ST677CC lineage has been overrepresented among C. jejuni isolates derived from blood. In this study, we characterized the genomes of 31 C. jejuni blood isolates and 24 faecal isolates belonging to ST677CC in order to study the genome biology related to C. jejuni invasiveness. We combined the genome analyses with phenotypical evidence on serum resistance which was associated with phase variation of wcbK; a GDP-mannose 4,6-dehydratase involved in capsular biosynthesis. We also describe the finding of a Type III restriction-modification system unique to the ST-794 sublineage. However, features previously considered to be related to pathogenesis of C. jejuni were either absent or disrupted among our strains. Our results refine the role of capsule features associated with invasive disease and accentuate the possibility of methylation and restriction enzymes in the potential of C. jejuni to establish invasive infections. Our findings underline the importance of studying clinically relevant well-characterized bacterial strains in order to understand pathogenesis mechanisms important in human infections.

Droplet microfluidics enables massively-parallel analysis of single cells, biomolecules, and chemicals, making it valuable for high-throughput screens. However, many hydrophobic analytes are soluble in carrier oils, preventing their quantitative analysis with the method. We apply Printed Droplet Microfluidics to construct defined reactions with chemicals and cells incubated under air on an open array. The method interfaces with most bioanalytical tools and retains hydrophobic compounds in compartmentalized reactors, allowing their quantitation.

Microphysiological systems (MPSs) are in vitro models that capture facets of in vivo organ function through use of specialized culture microenvironments, including 3D matrices and microperfusion. Here, we report an approach to co-culture multiple different MPSs linked together physiologically on re-useable, open-system microfluidic platforms that are compatible with the quantitative study of a range of compounds, including lipophilic drugs. We describe three different platform designs - "4-way", "7-way", and "10-way" - each accommodating a mixing chamber and up to 4, 7, or 10 MPSs. Platforms accommodate multiple different MPS flow configurations, each with internal re-circulation to enhance molecular exchange, and feature on-board pneumatically-driven pumps with independently programmable flow rates to provide precise control over both intra- and inter-MPS flow partitioning and drug distribution. We first developed a 4-MPS system, showing accurate prediction of secreted liver protein distribution and 2-week maintenance of phenotypic markers. We then developed 7-MPS and 10-MPS platforms, demonstrating reliable, robust operation and maintenance of MPS phenotypic function for 3 weeks (7-way) and 4 weeks (10-way) of continuous interaction, as well as PK analysis of diclofenac metabolism. This study illustrates several generalizable design and operational principles for implementing multi-MPS "physiome-on-a-chip" approaches in drug discovery.

Telomere Biology Disorders (TBDs) are characterized by mutations in telomere-related genes leading to short telomeres and premature aging but with no strict correlation between telomere length and disease severity. Epigenetic alterations are also markers of aging and we aimed to evaluate whether DNA methylation (DNAm) could be part of the pathogenesis of TBDs. In blood from 35 TBD cases, genome-wide DNAm were analyzed and the cases were grouped based on relative telomere length (RTL): short (S), with RTL close to normal controls, and extremely short (ES). TBD cases had increased epigenetic age and DNAm alterations were most prominent in the ES-RTL group. Thus, the differentially methylated (DM) CpG sites could be markers of short telomeres but could also be one of the mechanisms contributing to disease phenotype since DNAm alterations were observed in symptomatic, but not asymptomatic, cases with S-RTL. Furthermore, two or more DM-CpGs were identified in four genes previously linked to TBD or telomere length (PRDM8, SMC4, VARS, and WNT6) and in three genes that were novel in telomere biology (MAS1L, NAV2, and TM4FS1). The DM-CpGs in these genes could be markers of aging in hematological cells, but they could also be of relevance for the progression of TBD.

Multiple sclerosis (MS) is a chronic autoimmune disorder characterized by inflammatory-demyelinating events in the central nervous system. Despite more than 40 years of MS research its aetiology remains unknown. This study aims to identify the most frequently reported and consistently regulated molecules in MS in order to generate molecular interaction networks and thereby leading to the identification of deregulated processes and pathways which could give an insight of the underlying molecular mechanisms of MS. Driven by an integrative systems biology approach, gene-expression profiling datasets were combined and stratified into "Non-treated" and "Treated" groups and additionally compared to other disease patterns. Molecular identifiers from dataset comparisons were matched to our Multiple Sclerosis database (MuScle; www.padb.org/muscle ). From 5079 statistically significant molecules, correlation analysis within groups identified a panel of 16 high-confidence genes unique to the naïve MS phenotype, whereas the "Treated" group reflected a common pattern associated with autoimmune disease. Pathway and gene-ontology clustering identified the Interferon gamma signalling pathway as the most relevant amongst all significant molecules, and viral infections as the most likely cause of all down-stream events observed. This hypothesis-free approach revealed the most significant molecular events amongst different MS phenotypes which can be used for further detailed studies.

Here we demonstrate localization of the isoform3 of DNA Methyltransferase1 (DNMT1) enzyme to mitochondria, instead of isoform1 as reported earlier. The fused DNMT1-isoform1, reported earlier to localize in mitochondria, surprisingly showed its exclusive presence inside the nucleus after its ectopic expression; and failed to localize in mitochondria. On the other hand, ectopically expressed DNMT1-isoform3 targeted itself to mitochondria and subsequently methylated CpG regions in the mitochondrial genome. In addition, overexpression of DNMT1-isoform3 affected mitochondrial biology and regulated its function. Under different conditions of oxidative and nutritional stress, this isoform was down-regulated, resulting in hypomethylation of mitochondrial genome. Our study reveals how DNMT1-isoform3, instead of isoform1, is responsible for mtDNA methylation, influencing its biology.

Parkinson's disease (PD) arises as neurodegenerative disorder and characterized by progressive deterioration of motor functions due to forfeiture of dopamine-releasing neurons. During PD, neurons at stake loss their functionality that results into cognition impairment and forgetfulness, commonly called as dementia. Recently, nanoparticles (NPs) have been reported for easy drug delivery through blood-brain barrier (BBB) into the central nervous system (CNS) against the conventional drug delivery systems. However, present study attempted to elucidate the α-synuclein activity, a major factor casing PD, in presence of its inhibitor cerium oxide (CeO2) nanoparticle via computational biology approach. A computational analysis was also conducted for the α-synuclein activity with biocompatible metal NPs such as GOLD NPs and SPIONs to scrutinize the efficacy and degree of inhibition induced by the CeO2 NP. The obtained results concluded that CeO2 NP fit best in the active site of α-synuclein with good contacts and interaction, and potentially inhibited the PD against L-DOPA drug selected as positive control in the designed PD biochemical pathway. Hence, CeO2 NP has been purposed as potential inhibitor of α-synuclein and can be employed as nano-drug against the PD.

Abnormal tumor hemodynamics are a critical determinant of a tumor's microenvironment (TME), and profoundly affect drug delivery, therapeutic efficacy and the emergence of drug and radio-resistance. Since multiple hemodynamic variables can simultaneously exhibit transient and spatiotemporally heterogeneous behavior, there is an exigent need for analysis tools that employ multiple variables to characterize the anomalous hemodynamics within the TME. To address this, we developed a new toolkit called HemoSYS for quantifying the hemodynamic landscape within angiogenic microenvironments. It employs multivariable time-series data such as in vivo tumor blood flow (BF), blood volume (BV) and intravascular oxygen saturation (Hbsat) acquired concurrently using a wide-field multicontrast optical imaging system. The HemoSYS toolkit consists of propagation, clustering, coupling, perturbation and Fourier analysis modules. We demonstrate the utility of each module for characterizing the in vivo hemodynamic landscape of an orthotropic breast cancer model. With HemoSYS, we successfully described: (i) the propagation dynamics of acute hypoxia; (ii) the initiation and dissolution of distinct hemodynamic niches; (iii) tumor blood flow regulation via local vasomotion; (iv) the hemodynamic response to a systemic perturbation with carbogen gas; and (v) frequency domain analysis of hemodynamic heterogeneity in the TME. HemoSYS (freely downloadable via the internet) enables vascular phenotyping from multicontrast in vivo optical imaging data. Its modular design also enables characterization of non-tumor hemodynamics (e.g. brain), other preclinical disease models (e.g. stroke), vascular-targeted therapeutics, and hemodynamic data from other imaging modalities (e.g. MRI).

PRK1 and PRK2 are two closely related AGC-family serine/threonine protein kinases. Here we demonstrate novel roles for them at cilia and in cancer biology. In both instances serum withdrawal leads to increased activating PRK1 and PRK2 phosphorylation (pPRK1/pPRK2) and their depletion results in reduced spheroid growth. pPRK1/pPRK2 localise to the transition zone of cilia and their co-depletion results in reduced cilia size, impaired planer polarity and impaired cilia associated signalling. High PRK2 (but not PRK1) expression correlates with poor outcome in patients with basal-like/Triple Negative (TN) Breast Cancer (BC) where there is also higher expression relative to other BC tumour subtypes. In agreement, depletion of PRK1 and PRK2 in mouse TNBC cells, or CRISPR/Cas9 mediated deletion of PRK2 alone, significantly reduces cell proliferation and spheroid growth. Finally proteomic analysis to identify PRK2 binding partners in mouse TNBC cells revealed proteins that are important for both cilia and BC biology. Taken together these data demonstrate novel roles for PRK1 and PRK2 at cilia and in BC biology and in the case of PRK2 in particular, identifies it as a novel TNBC therapeutic target.

The striped venus (Chamelea gallina) is an important economic resource in the Mediterranean Basin; this species has exhibited a strong quantitative decline in the Adriatic Sea. The aim of this work was to provide a comprehensive view of the biological status of C. gallina to elucidate the bioecological characteristics and genetic diversity of wild populations. To the best of our knowledge, this investigation is the first to perform a multidisciplinary study on C. gallina based on two omics approaches integrated with histological, ecotoxicological, and chemical analyses and with the assessment of environmental parameters. The results obtained through RNA sequencing indicated that the striped venus has a notable ability to adapt to different environmental conditions. Moreover, the stock reduction exhibited by this species in the last 2 decades seems not to have negatively affected its genetic diversity. Indeed, the high level of genetic diversity that emerged from our ddRAD dataset analyses is ascribable to the high larval dispersal rate, which might have played a "compensatory role" on local fluctuations, conferring to this species a good adaptive potential to face the environmental perturbations. These findings may facilitate the efforts of conservation biologists to adopt ad hoc management plans for this fishery resource.

Quantum biology is the study of quantum effects on biochemical mechanisms and biological function. We show that the biological production of reactive oxygen species (ROS) in live cells can be influenced by coherent electron spin dynamics, providing a new example of quantum biology in cellular regulation. ROS partitioning appears to be mediated during the activation of molecular oxygen (O2) by reduced flavoenzymes, forming spin-correlated radical pairs (RPs). We find that oscillating magnetic fields at Zeeman resonance alter relative yields of cellular superoxide (O2•-) and hydrogen peroxide (H2O2) ROS products, indicating coherent singlet-triplet mixing at the point of ROS formation. Furthermore, the orientation-dependence of magnetic stimulation, which leads to specific changes in ROS levels, increases either mitochondrial respiration and glycolysis rates. Our results reveal quantum effects in live cell cultures that bridge atomic and cellular levels by connecting ROS partitioning to cellular bioenergetics.

Malignant transformation is often accompanied by significant metabolic changes. To identify drivers underlying these changes, we calculated metabolic flux states for the NCI60 cell line collection and correlated the variance between metabolic states of these lines with their other properties. The analysis revealed a remarkably consistent structure underlying high flux metabolism. The three primary uptake pathways, glucose, glutamine and serine, are each characterized by three features: (1) metabolite uptake sufficient for the stoichiometric requirement to sustain observed growth, (2) overflow metabolism, which scales with excess nutrient uptake over the basal growth requirement, and (3) redox production, which also scales with nutrient uptake but greatly exceeds the requirement for growth. We discovered that resistance to chemotherapeutic drugs in these lines broadly correlates with the amount of glucose uptake. These results support an interpretation of the Warburg effect and glutamine addiction as features of a growth state that provides resistance to metabolic stress through excess redox and energy production. Furthermore, overflow metabolism observed may indicate that mitochondrial catabolic capacity is a key constraint setting an upper limit on the rate of cofactor production possible. These results provide a greater context within which the metabolic alterations in cancer can be understood.

Tumor proliferative capacity is a major biological correlate of breast tumor metastatic potential. In this paper, we developed a systems approach to investigate associations among gene expression patterns, representative protein-protein interactions, and the potential for clinical metastases, to uncover novel survival-related subnetwork signatures as a function of tumor proliferative potential. Based on the statistical associations between gene expression patterns and patient outcomes, we identified three groups of survival prognostic subnetwork signatures (SPNs) corresponding to three proliferation levels. We discovered 8 SPNs in the high proliferation group, 8 SPNs in the intermediate proliferation group, and 6 SPNs in the low proliferation group. We observed little overlap of SPNs between the three proliferation groups. The enrichment analysis revealed that most SPNs were enriched in distinct signaling pathways and biological processes. The SPNs were validated on other cohorts of patients, and delivered high accuracy in the classification of metastatic vs non-metastatic breast tumors. Our findings indicate that certain biological networks underlying breast cancer metastasis differ in a proliferation-dependent manner. These networks, in combination, may form the basis of highly accurate prognostic classification models and may have clinical utility in guiding therapeutic options for patients.

The widespread failure of anthelmintic drugs against nematodes of veterinary interest requires novel control strategies. Selective treatment of the most susceptible individuals could reduce drug selection pressure but requires appropriate biomarkers of the intrinsic susceptibility potential. To date, this has been missing in livestock species. Here, we selected Welsh ponies with divergent intrinsic susceptibility (measured by their egg excretion levels) to cyathostomin infection and found that their divergence was sustained across a 10-year time window. Using this unique set of individuals, we monitored variations in their blood cell populations, plasma metabolites and faecal microbiota over a grazing season to isolate core differences between their respective responses under worm-free or natural infection conditions. Our analyses identified the concomitant rise in plasma phenylalanine level and faecal Prevotella abundance and the reduction in circulating monocyte counts as biomarkers of the need for drug treatment (egg excretion above 200 eggs/g). This biological signal was replicated in other independent populations. We also unravelled an immunometabolic network encompassing plasma beta-hydroxybutyrate level, short-chain fatty acid producing bacteria and circulating neutrophils that forms the discriminant baseline between susceptible and resistant individuals. Altogether our observations open new perspectives on the susceptibility of equids to strongylid infection and leave scope for both new biomarkers of infection and nutritional intervention.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with a 5-year survival rate of <8%. Its dismal prognosis stems from inefficient therapeutic modalities owing to the lack of understanding about pancreatic cancer pathogenesis. Considering the molecular complexity and heterogeneity of PDAC, identification of novel molecular contributors involved in PDAC onset and progression using global "omics" analysis will pave the way to improved strategies for disease prevention and therapeutic targeting. Meta-analysis of multiple miRNA microarray datasets containing healthy controls (HC), chronic pancreatitis (CP) and PDAC cases, identified 13 miRNAs involved in the progression of PDAC. These miRNAs showed dysregulation in both tissue as well as blood samples, along with progressive decrease in expression from HC to CP to PDAC. Gene-miRNA interaction analysis further elucidated 5 miRNAs (29a/b, 27a, 130b and 148a) that are significantly downregulated in conjunction with concomitant upregulation of their target genes throughout PDAC progression. Among these, miRNA-29a/b targeted genes were found to be most significantly altered in comparative profiling of HC, CP and PDAC, indicating its involvement in malignant evolution. Further, pathway analysis suggested direct involvement of miRNA-29a/b in downregulating the key pathways associated with PDAC development and metastasis including focal adhesion signaling and extracellular matrix organization. Our systems biology data analysis, in combination with real-time PCR validation indicates direct functional involvement of miRNA-29a in PDAC progression and is a potential prognostic marker and therapeutic candidate for patients with progressive disease.