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This protocol uses micropatterned co-cultures comprising 2D islands of primary human hepatocytes surrounded by supportive fibroblast cells to model liver infection by the hepatitis B and C viruses or Plasmodium pathogens in vitro . The development of therapies and vaccines for human hepatropic pathogens requires robust model systems that enable the study of host-pathogen interactions. However, in vitro liver models of infection typically use either hepatoma cell lines that exhibit aberrant physiology or primary human hepatocytes in culture conditions in which they rapidly lose their hepatic phenotype. To achieve stable and robust in vitro primary human hepatocyte models, we developed micropatterned cocultures (MPCCs), which consist of primary human hepatocytes organized into 2D islands that are surrounded by supportive fibroblast cells. By using this system, which can be established over a period of days, and maintained over multiple weeks, we demonstrate how to recapitulate in vitro hepatic life cycles for the hepatitis B and C viruses and the Plasmodium pathogens P. falciparum and P. vivax . The MPCC platform can be used to uncover aspects of host-pathogen interactions, and it has the potential to be used for drug and vaccine development.

Chronic disruption of the synchronous relationship between endogenous and exogenous circadian timing is associated with the development of obesity and metabolic disease. Social jetlag is a measure of circadian misalignment and has been identified as a risk factor for overweight and related diseases. However, the mechanisms involved in this relationship remain underexplored. The objective of this study was to investigate the association between social jetlag and food consumption at late meal timing in patients with obesity-related chronic diseases. This study included 792 individuals (73% female; age 55.9 ± 12.4 years) in which the prevalence of social jetlag (>1h) was 24.4% (n = 194). Participants with social jetlag reported late meal timing for breakfast, early afternoon snack and dinner. Individuals with social jetlag also reported a higher intake of total calories (kcal), protein, total fat, saturated fat, cholesterol, and servings of meat and eggs and sweets in relation to those without social jetlag. Regarding the consumption during each meal of the day, participants with social jetlag had consumed more calories, saturated fat and cholesterol during dinner; more protein, total fat, saturated fat, and cholesterol during lunch; and more total fat and saturated fat during morning snack. In addition, individuals with social jetlag had a higher risk of inadequate consumption of total fat, saturated fat and cholesterol intake when compared with those without social jetlag. We conclude that social jetlag is associated with a poor diet and later meal times, which should be avoided in individuals with obesity-related chronic diseases. More studies are needed to confirm these findings.

Key Points Malaria is a major health problem, mainly in sub-Saharan Africa and in some parts of Asia and south America. Each year there are about 600 million new clinical cases and at least one million individuals, mostly children, die from malaria; in other words there is a death caused by malaria every 30 seconds. Successful malaria transmission to the mammalian host is established through sporozoite infection of the liver. The importance of the liver stages, owing to its status as an obligatory step in infection, makes this stage an attractive target for both drug and vaccine development. In recent years, our understanding of Plasmodium sporozoite biology, the interactions of sporozoites with different host cells and their establishment in the liver, has increased through the implementation of new technologies that allow researchers to follow the progress of the parasites from the site of injection to the liver. Plasmodium sporozoites are deposited in the skin of their vertebrate host by the bite of an infected female Anopheles mosquito. A proportion of these parasites are drained to the lymph nodes, where they are trapped, whereas the majority find a blood vessel and travel in the peripheral blood circulation until they reach the liver sinusoids. Once arrested in the liver sinusoids, the sporozoites cross the sinusoidal wall and migrate through several hepatocytes by breaching their plasma membrane, before they infect a final hepatocyte with the formation of a parasitophorous vacuole. The intrahepatic form of the parasite grows and multiplies in this vacuole, producing thousands of merozoites per sporozoite. This Review summarizes our current knowledge on this stage of the Plasmodium life cycle. The liver stage of Plasmodium infection constitutes an appealing target for the development of vaccines or prophylatic drugs as this step in the life cycle is obligatory but occurs before the onset of pathology. This Review summarizes the current knowledge on this stage of the Plasmodium life cycle. Plasmodium sporozoites are deposited in the skin of their vertebrate hosts through the bite of an infected female Anopheles mosquito. Most of these parasites find a blood vessel and travel in the peripheral blood circulation until they reach the liver sinusoids. Once there, the sporozoites cross the sinusoidal wall and migrate through several hepatocytes before they infect a final hepatocyte, with the formation of a parasitophorous vacuole, in which the intrahepatic form of the parasite grows and multiplies. During this period, each sporozoite generates thousands of merozoites. As the development of Plasmodium sporozoites inside hepatocytes is an obligatory step before the onset of disease, understanding the parasite's requirements during this period is crucial for the development of any form of early intervention. This Review summarizes our current knowledge on this stage of the Plasmodium life cycle.

The development of next-generation antimalarials that are efficacious against the human liver and asexual blood stages is recognized as one of the world’s most pressing public health challenges. In recent years, aminoacyl-tRNA synthetases, including prolyl-tRNA synthetase, have emerged as attractive targets for malaria chemotherapy. We describe the development of a single-step biochemical assay for Plasmodium and human prolyl-tRNA synthetases that overcomes critical limitations of existing technologies and enables quantitative inhibitor profiling with high sensitivity and flexibility. Supported by this assay platform and co-crystal structures of representative inhibitor-target complexes, we develop a set of high-affinity prolyl-tRNA synthetase inhibitors, including previously elusive aminoacyl-tRNA synthetase triple-site ligands that simultaneously engage all three substrate-binding pockets. Several compounds exhibit potent dual-stage activity against Plasmodium parasites and display good cellular host selectivity. Our data inform the inhibitor requirements to overcome existing resistance mechanisms and establish a path for rational development of prolyl-tRNA synthetase-targeted anti-malarial therapies. The development of antimalarials against the human liver and asexual blood stages is one of the top public health challenges. Here, the authors report a single-step biochemical assay for the characterization of prolyl-tRNA synthetase inhibitors, and develop high-affinity inhibitors for the enzyme, including elusive triple-site ligands.

Plasmodium parasites possess a translocon that exports parasite proteins into the infected erythrocyte. Although the translocon components are also expressed during the mosquito and liver stage of infection, their function remains unexplored. Here, using a combination of genetic and chemical assays, we show that the translocon component Exported Protein 2 (EXP2) is critical for invasion of hepatocytes. EXP2 is a pore-forming protein that is secreted from the sporozoite upon contact with the host cell milieu. EXP2-deficient sporozoites are impaired in invasion, which can be rescued by the exogenous administration of recombinant EXP2 and alpha-hemolysin (an S. aureus pore-forming protein), as well as by acid sphingomyelinase. The latter, together with the negative impact of chemical and genetic inhibition of acid sphingomyelinase on invasion, reveals that EXP2 pore-forming activity induces hepatocyte membrane repair, which plays a key role in parasite invasion. Overall, our findings establish a novel and critical function for EXP2 that leads to an active participation of the host cell in Plasmodium sporozoite invasion, challenging the current view of the establishment of liver stage infection. While the role of Plasmodium EXP2 protein as translocon component of blood stage parasites is established, its functional role in liver stage parasites remains unclear. Here, Mello-Vieira et al. reveal that EXP2 pore-forming activity induces hepatocyte membrane repair and hence is critical for hepatocyte invasion.

Human malaria infection begins with a one-time asymptomatic liver stage followed by a cyclic symptomatic blood stage. All high-throughput malaria drug discovery efforts have focused on the cyclic blood stage, which has limited potential for the prophylaxis, transmission blocking, and eradication efforts that will be needed in the future. To address these unmet needs, a high-throughput phenotypic liver-stage Plasmodium parasite screen was developed to systematically identify molecules with liver-stage efficacy. The screen recapitulates liver-stage infection by isolating luciferase-expressing Plasmodium berghei parasites directly from the salivary glands of infected mosquitoes, adding them to confluent human liver cells in 384-well plates, and measuring luciferase activity after a suitable incubation period. Screening 5,375 known bioactive compounds identified 37 liver-stage malaria inhibitors with diverse modes of action, as shown by inhibition time course experiments. Further analysis of the hits in the Food and Drug Administration-approved drug subset revealed compounds that seem to act specifically on the liver stage of infection, suggesting that this phase of the parasite’s life cycle presents a promising area for new drug discovery. Notably, many active compounds in this screen have molecular structures and putative targets distinctly different from those of known antimalarial agents.

Iron is an essential micronutrient but is also highly toxic. In yeast and plant cells, a key detoxifying mechanism involves iron sequestration into intracellular storage compartments, mediated by members of the vacuolar iron-transporter (VIT) family of proteins. Here we study the VIT homologue from the malaria parasites Plasmodium falciparum (PfVIT) and Plasmodium berghei (PbVIT). PfVIT-mediated iron transport in a yeast heterologous expression system is saturable ( K m ∼14.7 μM), and selective for Fe 2+ over other divalent cations. PbVIT-deficient P. berghei lines ( Pbvit − ) show a reduction in parasite load in both liver and blood stages of infection in mice. Moreover, Pbvit − parasites have higher levels of labile iron in blood stages and are more sensitive to increased iron levels in liver stages, when compared with wild-type parasites. Our data are consistent with Plasmodium VITs playing a major role in iron detoxification and, thus, normal development of malaria parasites in their mammalian host. Iron is an essential nutrient but, in high concentrations, it is also toxic to cells. Here, Slavic et al . identify an iron transporter in malaria parasites that plays a major role in iron detoxification and is required for the parasite’s normal development.

Malaria infection involves an obligatory, yet clinically silent liver stage 1 , 2 . Hepatocytes operate in repeating units termed lobules, exhibiting heterogeneous gene expression patterns along the lobule axis 3 , but the effects of hepatocyte zonation on parasite development at the molecular level remain unknown. Here we combine single-cell RNA sequencing 4 and single-molecule transcript imaging 5 to characterize the host and parasite temporal expression programmes in a zonally controlled manner for the rodent malaria parasite Plasmodium berghei ANKA. We identify differences in parasite gene expression in distinct zones, including potentially co-adaptive programmes related to iron and fatty acid metabolism. We find that parasites develop more rapidly in the pericentral lobule zones and identify a subpopulation of periportally biased hepatocytes that harbour abortive infections, reduced levels of Plasmodium transcripts and parasitophorous vacuole breakdown. These ‘abortive hepatocytes’, which appear predominantly with high parasite inoculum, upregulate immune recruitment and key signalling programmes. Our study provides a resource for understanding the liver stage of Plasmodium infection at high spatial resolution and highlights the heterogeneous behaviour of both the parasite and the host hepatocyte. Single-cell RNA sequencing and single-molecule RNA transcript imaging have been used to characterize spatially and temporally resolved mouse liver and parasite expression programmes during infection with the rodent malaria parasite Plasmodium berghei ANKA.

In this work, polyaniline (PANI) is synthesized via oxidative polymerization of aniline and purified using organic solvents where the emeraldine phase is isolated by employing a phase separation system. The above contributes to the increase in the percentage yield compared to previous works and the possibility of being used as a single phase. In addition, the PANI/AgNPs composite is prepared in situ at the polymerization of aniline, adding silver nitrate and glycine to create the AgNPs inside the PANI matrix by controlling the pH, temperature, time of reaction and incorporating a new purification technique.

This study reports the optical, structural, electrical and dielectric properties of Poly (vinyl alcohol) thin films membranes with embedded ZnO nanoparticles (PVA/ZnO) obtained by the solution casting method at low temperature of deposition. Fourier Transform Infrared spectra showed the characteristics peaks, which correspond to O–H and Zn–O bonds present in the hybrid material. The X-ray diffraction patterns indicated the presence of ZnO structure into the films. The composite material showed low absorbance and a wide band of gap energy from 5.5 to 5.83 eV. The surface morphology for the thin films of PVA/ZnO was studied by Atomic Force Microscopy and Scanning Electron Microscopy. The electrical properties of the membranes were also characterized by current-voltage characteristics and the DC conductivity showed Arrhenius behavior with values of activation energy from 0.62 to 0.78 eV and maximum conductivity at 2.4 × 10−12 S/cm. The dielectric properties of the nanocomposites were measured from low to high frequencies, and the results showed a high dielectric constant (ε) in the order of 104 at low frequency and values from ε ≈ 2000 to 100 in the range of 1 KHz–1 MHz respectively. The properties of PVA/ZnO such as the high permittivity and the low temperature of processing make it a suitable material for potential applications in the development of flexible electronic devices.