Longitudinal changes in iron homeostasis in human experimental and clinical malaria.

BACKGROUND: The interaction between iron status and malaria is incompletely understood. We evaluated longitudinal changes in iron homeostasis in volunteers enrolled in malaria volunteer infection studies (VIS) and in Malaysian patients with falciparum and vivax malaria. METHODS: We retrieved data and samples from 55 participants (19 female) enrolled in malaria VIS, and 171 patients (45 female) with malaria and 30 healthy controls (13 female) enrolled in clinical studies in Malaysia. Ferritin, hepcidin, erythropoietin, and soluble transferrin receptor (sTfR) were measured by ELISA. FINDINGS: In the VIS, participants' parasitaemia was correlated with baseline mean corpuscular volume (MCV), but not iron status (ferritin, hepcidin or sTfR). Ferritin, hepcidin and sTfR all increased during the VIS. Ferritin and hepcidin normalised by day 28, while sTfR remained elevated. In VIS participants, baseline ferritin was associated with post-treatment increases in liver transaminase levels. In Malaysian patients with malaria, hepcidin and ferritin were elevated on admission compared to healthy controls, while sTfR increased following admission. By day 28, hepcidin had normalised; however, ferritin and sTfR both remained elevated. INTERPRETATION: Our findings demonstrate that parasitaemia is associated with an individual's MCV rather than iron status. The persistent elevation in sTfR 4 weeks post-infection in both malaria VIS and clinical malaria may reflect a causal link between malaria and iron deficiency. FUNDING: National Health and Medical Research Council (Program Grant 1037304, Project Grants 1045156 and 1156809; Investigator Grants 2016792 to BEB, 2016396 to JCM, 2017436 to MJG); US National Institute of Health (R01-AI116472-03); Malaysian Ministry of Health (BP00500420).

Diversity of protonated mixed pyrene-water clusters investigated by collision induced dissociation.

Protonated mixed pyrene-water clusters, (Py)m(H2O)nH+, where m = [1-3] and n = [1-10], are generated using a cryogenic molecular cluster source. Subsequently, the mass-selected mixed clusters undergo controlled collisions with rare gases, and the resulting fragmentation mass spectra are meticulously analyzed to discern distinct fragmentation channels. Notably, protonated water cluster fragments emerge for n ≥ 3, whereas they are absent for n = 1 and 2. The experimental results are complemented by theoretical calculations of structures and energetics for (Py)(H2O)nH+ with n = [1-4]. These calculations reveal a shift in proton localization, transitioning from the pyrene molecule for n = 1 and 2 to water molecules for n ≥ 3. The results support a formation scenario wherein water molecules attach to protonated pyrene PyH+ seeds, and, by extension, to (Py)2H+ and (Py)3H+ seeds. Various isomers are identified, corresponding to potential protonation sites on the pyrene molecule. Protonated polycyclic aromatic hydrocarbons are likely to be formed in cold, dense interstellar clouds and protoplanetary disks due to the high proton affinity of these species. Our findings show that the presence of protonated PAHs in these environments could lead to the formation of water clusters and mixed carbon-water nanograins, having a potential impact on the water cycle in regions of planet formation.

Chrysin inhibits adipogenesis by modulating PPARγ: in silico and in vitro studies.

Adipose tissue is the major storage site of lipids and plays a vital role in energy homeostasis. Adipogenesis is a well-regulated process wherein preadipocytes differentiate into adipocytes. It requires the sequential activation of numerous transcription factors, including peroxisome proliferator activated receptor-γ (PPAR-γ). Phytochemicals have been reported to regulate adipogenesis and flavonoids represent the most researched groups of phytochemicals with regard to their effect on adipogenesis. Chrysin is a naturally occurring flavone and is reported to have anti-inflammatory effects in obese conditions. The present study was aimed to examine the effect of chrysin on adipogenesis. In silico Molecular docking, dynamic simulation studies and in vitro cell-based assays showed that chrysin inhibited adipogenesis by modulating key adipogenic transcription factor PPARγ. Enhanced adipogenesis leads to obesity and targeting adipogenesis is potential in regulating adipose tissue development. So, these investigations may provide important information for designing therapeutic interventions to control adiposity.Communicated by Ramaswamy H. Sarma.

Human BBB-on-a-chip reveals barrier disruption, endothelial inflammation, and T cell migration under neuroinflammatory conditions.

The blood-brain barrier (BBB) is a highly selective barrier that ensures a homeostatic environment for the central nervous system (CNS). BBB dysfunction, inflammation, and immune cell infiltration are hallmarks of many CNS disorders, including multiple sclerosis and stroke. Physiologically relevant human in vitro models of the BBB are essential to improve our understanding of its function in health and disease, identify novel drug targets, and assess potential new therapies. We present a BBB-on-a-chip model comprising human brain microvascular endothelial cells (HBMECs) cultured in a microfluidic platform that allows parallel culture of 40 chips. In each chip, a perfused HBMEC vessel was grown against an extracellular matrix gel in a membrane-free manner. BBBs-on-chips were exposed to varying concentrations of pro-inflammatory cytokines tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1ß) to mimic inflammation. The effect of the inflammatory conditions was studied by assessing the BBBs-on-chips' barrier function, cell morphology, and expression of cell adhesion molecules. Primary human T cells were perfused through the lumen of the BBBs-on-chips to study T cell adhesion, extravasation, and migration. Under inflammatory conditions, the BBBs-on-chips showed decreased trans-endothelial electrical resistance (TEER), increased permeability to sodium fluorescein, and aberrant cell morphology in a concentration-dependent manner. Moreover, we observed increased expression of cell adhesion molecules and concomitant monocyte adhesion. T cells extravasated from the inflamed blood vessels and migrated towards a C-X-C Motif Chemokine Ligand 12 (CXCL12) gradient. T cell adhesion was significantly reduced and a trend towards decreased migration was observed in presence of Natalizumab, an antibody drug that blocks very late antigen-4 (VLA-4) and is used in the treatment of multiple sclerosis. In conclusion, we demonstrate a high-throughput microfluidic model of the human BBB that can be used to model neuroinflammation and assess anti-inflammatory and barrier-restoring interventions to fight neurological disorders.

Longitudinal changes in iron homeostasis in human experimental and clinical malaria.

Background: The interaction between iron deficiency and malaria is incompletely understood. We evaluated longitudinal changes in iron homeostasis in volunteers enrolled in malaria volunteer infection studies (VIS) and in Malaysian patients with falciparum and vivax malaria. Methods: We retrieved samples and associated data from 55 participants enrolled in malaria VIS, and 171 malaria patients and 30 healthy controls enrolled in clinical studies in Malaysia. Ferritin, hepcidin, erythropoietin, and soluble transferrin receptor (sTfR) were measured by ELISA. Results: In the VIS, participants' parasitaemia was correlated with baseline mean corpuscular volume (MCV), but not iron status (ferritin, hepcidin or sTfR). Ferritin, hepcidin and sTfR all increased during the VIS. Ferritin and hepcidin normalised by day 28, while sTfR remained elevated. In VIS participants, baseline iron status (ferritin) was associated with post-treatment increases in liver transaminase levels. In Malaysian malaria patients, hepcidin and ferritin were elevated on admission compared to healthy controls, while sTfR increased following admission. Hepcidin normalised by day 28; however, ferritin and sTfR both remained elevated 4 weeks following admission. Conclusion: Our findings demonstrate that parasitaemia is associated with an individual's MCV rather than iron status. The persistent elevation in sTfR 4 weeks post-infection in both malaria VIS and clinical malaria may reflect a causal link between malaria and iron deficiency.

Antimicrobial Nanomaterials for Food Packaging.

Food packaging plays a key role in offering safe and quality food products to consumers by providing protection and extending shelf life. Food packaging is a multifaceted field based on food science and engineering, microbiology, and chemistry, all of which have contributed significantly to maintaining physicochemical attributes such as color, flavor, moisture content, and texture of foods and their raw materials, in addition to ensuring freedom from oxidation and microbial deterioration. Antimicrobial food packaging systems, in addition to their function as conventional food packaging, are designed to arrest microbial growth on food surfaces, thereby enhancing food stability and quality. Nanomaterials with unique physiochemical and antibacterial properties are widely explored in food packaging as preservatives and antimicrobials, to extend the shelf life of packed food products. Various nanomaterials that are used in food packaging include nanocomposites composing nanoparticles such as silver, copper, gold, titanium dioxide, magnesium oxide, zinc oxide, mesoporous silica and graphene-based inorganic nanoparticles; gelatin; alginate; cellulose; chitosan-based polymeric nanoparticles; lipid nanoparticles; nanoemulsion; nanoliposomes; nanosponges; and nanofibers. Antimicrobial nanomaterial-based packaging systems are fabricated to exhibit greater efficiency against microbial contaminants. Recently, smart food packaging systems indicating the presence of spoilage and pathogenic microorganisms have been investigated by various research groups. The present review summarizes recent updates on various nanomaterials used in the field of food packaging technology, with potential applications as antimicrobial, antioxidant equipped with technology conferring smart functions and mechanisms in food packaging.

Nanoparticles-Attractive Carriers of Antimicrobial Essential Oils.

Microbial pathogens are the most prevalent cause of chronic infections and fatalities around the world. Antimicrobial agents including antibiotics have been frequently utilized in the treatment of infections due to their exceptional outcomes. However, their widespread use has resulted in the emergence of multidrug-resistant strains of bacteria, fungi, viruses, and parasites. Furthermore, due to inherent resistance to antimicrobial drugs and the host defence system, the advent of new infectious diseases, chronic infections, and the occurrence of biofilms pose a tougher challenge to the current treatment line. Essential oils (EOs) and their biologically and structurally diverse constituents provide a distinctive, inexhaustible, and novel source of antibacterial, antiviral, antifungal, and antiparasitic agents. However, due to their volatile nature, chemical susceptibility, and poor solubility, their development as antimicrobials is limited. Nanoparticles composed of biodegradable polymeric and inorganic materials have been studied extensively to overcome these limitations. Nanoparticles are being investigated as nanocarriers for antimicrobial delivery, antimicrobial coatings for food products, implantable devices, and medicinal materials in dressings and packaging materials due to their intrinsic capacity to overcome microbial resistance. Essential oil-loaded nanoparticles may offer the potential benefits of synergism in antimicrobial activity, high loading capacity, increased solubility, decreased volatility, chemical stability, and enhancement of the bioavailability and shelf life of EOs and their constituents. This review focuses on the potentiation of the antimicrobial activity of essential oils and their constituents in nanoparticulate delivery systems for a wide range of applications, such as food preservation, packaging, and alternative treatments for infectious diseases.

Evaluation of physicochemical and biological properties of nonreconstituted MYL-1401O vials, reconstituted MYL-1401O suspension in vial, and diluted MYL-1401O suspension in infusion bags (0.9% saline) for extended duration.

BACKGROUND: MYL-1401O; trastuzumab-dkst (Ogivri™; Mylan Inc.) is a biosimilar to the trastuzumab reference product (Herceptin®; Genentech, USA). Assessment of physicochemical stability and biological activity for the non-reconstituted, reconstituted, and infused solution over an extended, clinically relevant duration is critical for ensuring optimal patient outcomes and health resource utilization. METHODS: The physicochemical and biological stability of MYL-1401O was assessed in non-reconstituted vials stored at 25 °C ± 2 °C/60% ± 5% relative humidity (RH) for 6 months, reconstituted 21 mg/mL solution in vials stored at 2 °C to 8 °C for 10 days, and diluted in 0.9% saline-containing infusion bags at 0.3 mg/mL and 4.0 mg/mL stored for 77 days at 2 °C to 8 °C, plus an additional 2 days at 25 °C ± 2 °C/60% ± 5% RH. RESULTS: At all storage conditions tested, MYL-1401O was physicochemically and biologically stable for extended duration and under various temperature and humidity conditions. CONCLUSIONS: MYL-1401O retained its physicochemical and biological stability under different storage conditions, which supports advanced preparation of MYL-1401O, better efficiency, less wastage, and cost-savings for better patient management.

Modeling ischemic stroke in a triculture neurovascular unit on-a-chip.

BACKGROUND: In ischemic stroke, the function of the cerebral vasculature is impaired. This vascular structure is formed by the so-called neurovascular unit (NVU). A better understanding of the mechanisms involved in NVU dysfunction and recovery may lead to new insights for the development of highly sought therapeutic approaches. To date, there remains an unmet need for complex human in vitro models of the NVU to study ischemic events seen in the human brain. METHODS: We here describe the development of a human NVU on-a-chip model using a platform that allows culture of 40 chips in parallel. The model comprises a perfused vessel of primary human brain endothelial cells in co-culture with induced pluripotent stem cell derived astrocytes and neurons. Ischemic stroke was mimicked using a threefold approach that combines chemical hypoxia, hypoglycemia, and halted perfusion. RESULTS: Immunofluorescent staining confirmed expression of endothelial adherens and tight junction proteins, as well as astrocytic and neuronal markers. In addition, the model expresses relevant brain endothelial transporters and shows spontaneous neuronal firing. The NVU on-a-chip model demonstrates tight barrier function, evidenced by retention of small molecule sodium fluorescein in its lumen. Exposure to the toxic compound staurosporine disrupted the endothelial barrier, causing reduced transepithelial electrical resistance and increased permeability to sodium fluorescein. Under stroke mimicking conditions, brain endothelial cells showed strongly reduced barrier function (35-fold higher apparent permeability) and 7.3-fold decreased mitochondrial potential. Furthermore, levels of adenosine triphosphate were significantly reduced on both the blood- and the brain side of the model (4.8-fold and 11.7-fold reduction, respectively). CONCLUSIONS: The NVU on-a-chip model presented here can be used for fundamental studies of NVU function in stroke and other neurological diseases and for investigation of potential restorative therapies to fight neurological disorders. Due to the platform's relatively high throughput and compatibility with automation, the model holds potential for drug compound screening.

Parallelizable Microfluidic Platform to Model and Assess In Vitro Cellular Barriers: Technology and Application to Study the Interaction of 3D Tumor Spheroids with Cellular Barriers.

Endothelial and epithelial cellular barriers play a vital role in the selective transport of solutes and other molecules. The properties and function of these barriers are often affected in case of inflammation and disease. Modelling cellular barriers in vitro can greatly facilitate studies of inflammation, disease mechanisms and progression, and in addition, can be exploited for drug screening and discovery. Here, we report on a parallelizable microfluidic platform in a multiwell plate format with ten independent cell culture chambers to support the modelling of cellular barriers co-cultured with 3D tumor spheroids. The microfluidic platform was fabricated by microinjection molding. Electrodes integrated into the chip in combination with a FT-impedance measurement system enabled transepithelial/transendothelial electrical resistance (TEER) measurements to rapidly assess real-time barrier tightness. The fluidic layout supports the tubeless and parallelized operation of up to ten distinct cultures under continuous unidirectional flow/perfusion. The capabilities of the system were demonstrated with a co-culture of 3D tumor spheroids and cellular barriers showing the growth and interaction of HT29 spheroids with a cellular barrier of MDCK cells.

Quantification of host-mediated parasite clearance during blood-stage Plasmodium infection and anti-malarial drug treatment in mice.

A major mechanism of host-mediated control of blood-stage Plasmodium infection is thought to be removal of parasitized red blood cells (pRBCs) from circulation by the spleen or phagocytic system. The rate of parasite removal is thought to be further increased by anti-malarial drug treatment, contributing to the effectiveness of drug therapy. It is difficult to directly compare pRBC removal rates in the presence and absence of treatment, since in the absence of treatment the removal rate of parasites is obscured by the extent of ongoing parasite proliferation. Here, we transfused a single generation of fluorescently-labelled Plasmodium berghei pRBCs into mice, and monitored both their disappearance from circulation, and their replication to produce the next generation of pRBCs. In conjunction with a new mathematical model, we directly estimated host removal of pRBCs during ongoing infection, and after drug treatment. In untreated mice, pRBCs were removed from circulation with a half-life of 15.1 h. Treatment with various doses of mefloquine/artesunate did not alter the pRBC removal rate, despite blocking parasite replication effectively. An exception was high dose artesunate, which doubled the rate of pRBC removal (half-life of 9.1 h). Phagocyte depletion using clodronate liposomes approximately halved the pRBC removal rate during untreated infection, indicating a role for phagocytes in clearance. We next assessed the importance of pRBC clearance for the decrease in the parasite multiplication rate after high dose artesunate treatment. High dose artesunate decreased parasite replication ∼46-fold compared with saline controls, with inhibition of replication contributing 23-fold of this, and increased pRBC clearance contributing only a further 2.0-fold. Thus, in our in vivo systems, drugs acted primarily by inhibiting parasite replication, with drug-induced increases in pRBC clearance making only minor contributions to overall drug effect.

IFN Regulatory Factor 3 Balances Th1 and T Follicular Helper Immunity during Nonlethal Blood-Stage Plasmodium Infection.

Differentiation of CD4+ Th cells is critical for immunity to malaria. Several innate immune signaling pathways have been implicated in the detection of blood-stage Plasmodium parasites, yet their influence over Th cell immunity remains unclear. In this study, we used Plasmodium-reactive TCR transgenic CD4+ T cells, termed PbTII cells, during nonlethal P. chabaudi chabaudi AS and P. yoelii 17XNL infection in mice, to examine Th cell development in vivo. We found no role for caspase1/11, stimulator of IFN genes, or mitochondrial antiviral-signaling protein, and only modest roles for MyD88 and TRIF-dependent signaling in controlling PbTII cell expansion. In contrast, IFN regulatory factor 3 (IRF3) was important for supporting PbTII expansion, promoting Th1 over T follicular helper (Tfh) differentiation, and controlling parasites during the first week of infection. IRF3 was not required for early priming by conventional dendritic cells, but was essential for promoting CXCL9 and MHC class II expression by inflammatory monocytes that supported PbTII responses in the spleen. Thereafter, IRF3-deficiency boosted Tfh responses, germinal center B cell and memory B cell development, parasite-specific Ab production, and resolution of infection. We also noted a B cell-intrinsic role for IRF3 in regulating humoral immune responses. Thus, we revealed roles for IRF3 in balancing Th1- and Tfh-dependent immunity during nonlethal infection with blood-stage Plasmodium parasites.

Single-cell RNA-seq and computational analysis using temporal mixture modelling resolves Th1/Tfh fate bifurcation in malaria.

Differentiation of naïve CD4+ T cells into functionally distinct T helper subsets is crucial for the orchestration of immune responses. Due to extensive heterogeneity and multiple overlapping transcriptional programs in differentiating T cell populations, this process has remained a challenge for systematic dissection in vivo. By using single-cell transcriptomics and computational analysis using a temporal mixtures of Gaussian processes model, termed GPfates, we reconstructed the developmental trajectories of Th1 and Tfh cells during blood-stage Plasmodium infection in mice. By tracking clonality using endogenous TCR sequences, we first demonstrated that Th1/Tfh bifurcation had occurred at both population and single-clone levels. Next, we identified genes whose expression was associated with Th1 or Tfh fates, and demonstrated a T-cell intrinsic role for Galectin-1 in supporting a Th1 differentiation. We also revealed the close molecular relationship between Th1 and IL-10-producing Tr1 cells in this infection. Th1 and Tfh fates emerged from a highly proliferative precursor that upregulated aerobic glycolysis and accelerated cell cycling as cytokine expression began. Dynamic gene expression of chemokine receptors around bifurcation predicted roles for cell-cell in driving Th1/Tfh fates. In particular, we found that precursor Th cells were coached towards a Th1 but not a Tfh fate by inflammatory monocytes. Thus, by integrating genomic and computational approaches, our study has provided two unique resources, a database www.PlasmoTH.org, which facilitates discovery of novel factors controlling Th1/Tfh fate commitment, and more generally, GPfates, a modelling framework for characterizing cell differentiation towards multiple fates.