Endoplasmic reticulum localized TMEM33 domain-containing protein is crucial for all life cycle stages of the malaria parasite.

Endoplasmic reticulum (ER) plays a pivotal role in the regulation of stress responses in multiple eukaryotic cells. However, little is known about the effector mechanisms that regulate stress responses in ER of the malaria parasite. Herein, we aimed to identify the importance of a transmembrane protein 33 (TMEM33)-domain-containing protein in life cycle of the rodent malaria parasite Plasmodium berghei. TMEM33 is an ER membrane-resident protein that is involved in regulating stress responses in various eukaryotic cells. A C-terminal tagged TMEM33 was localized in the ER throughout the blood and mosquito stages of development. Targeted deletion of TMEM33 confirmed its importance for asexual blood stages and ookinete development, in addition to its essential role for sporozoite infectivity in the mammalian host. Pilot scale analysis shows that the loss of TMEM33 results in the initiation of ER stress response and induction of autophagy. Our findings conclude an important role of TMEM33 in the development of all life cycle stages of the malaria parasite, which indicates its potential as an antimalarial target.

A recombinant Aspergillus oryzae fungus transmitted from larvae to adults of Anopheles stephensi mosquitoes inhibits malaria parasite oocyst development.

The control of malaria parasite transmission from mosquitoes to humans is hampered by decreasing efficacies of insecticides, development of drug resistance against the last-resort antimalarials, and the absence of effective vaccines. Herein, the anti-plasmodial transmission blocking activity of a recombinant Aspergillus oryzae (A. oryzae-R) fungus strain, which is used in human food industry, was investigated in laboratory-reared Anopheles stephensi mosquitoes. The recombinant fungus strain was genetically modified to secrete two anti-plasmodial effector peptides, MP2 (midgut peptide 2) and EPIP (enolase-plasminogen interaction peptide) peptides. The transstadial transmission of the fungus from larvae to adult mosquitoes was confirmed following inoculation of A. oryzae-R in the water trays used for larval rearing. Secretion of the anti-plasmodial effector peptides inside the mosquito midguts inhibited oocyst formation of P. berghei parasites. These results indicate that A. oryzae can be used as a paratransgenesis model carrying effector proteins to inhibit malaria parasite development in An. stephensi. Further studies are needed to determine if this recombinant fungus can be adapted under natural conditions, with a minimal or no impact on the environment, to target mosquito-borne infectious disease agents inside their vectors.

A Candidate Bacterial-Type Amino Acid Decarboxylase Is Essential for Male Gamete Exflagellation and Mosquito Transmission of the Malaria Parasite.

A frequent side effect of chemotherapy against malaria parasite blood infections is a dramatic induction of the sexual blood stages, thereby enhancing the risk of future malaria transmissions. The polyamine biosynthesis pathway has been suggested as a candidate target for transmission-blocking anti-malarial drug development. Herein, we describe the role of a bacterial-type amino acid decarboxylase (AAD) in the life cycle of the malaria model parasite Plasmodium yoelii. Hallmarks of AAD include a conserved catalytic lysine residue and high-level homology to arginine/lysine/ornithine decarboxylases of pathogenic bacteria. By targeted gene deletion, we show that AAD plays an essential role in the exflagellation of microgametes, resulting in complete absence of sporozoites in the mosquito vector. These data highlight the central role of the biosysthesis of polyamines in the final steps of male gamete sexual development of the malaria parasite and, hence, onward transmission to mosquitoes.

Iron affects localization of Ght5 in fission yeast.

Iron is an essential cofactor for eukaryotic cells, as well as a toxic metal under certain conditions. On the other hand, glucose is the preferred energy and carbon source by most organisms and is an important signaling molecule in the regulation of biological processes. In Schizosaccharomyces pombe, the Ght5 hexose transporter, known as a high affinity glucose transporter, is required for cell proliferation in low glucose concentrations. Herein, we aimed to investigate the effects of iron stress on the Ght5 hexose transporter under glucose repression and derepression conditions. The effect of iron stress on the expression profile of the ght5 gene was analyzed by RT-qPCR and western blot. The localization of the Ght5-mNeonGreen fusion protein examined with confocal microscopy. Our results revealed that iron stress had an inhibitory effect on ght5 expression, and it altered Ght5 localization on the cell surface, causing it to accumulate in the cytoplasm.

An efficient antenna system with improved radiation for multi-standard/multi-mode 5G cellular communications.

This paper introduces a multi-input multiple-output (MIMO) antenna array system that provides improved radiation diversity for multi-standard/multi-mode 5G communications. The introduced MIMO design contains four pairs of miniaturized self-complementary antennas (SCAs) fed by pairs of independently coupled structures which are symmetrically located at the edge corners of the smartphone mainboard with an overall size of 75 × 150 (mm2). Hence, in total, the design incorporates four pairs of horizontally and vertically polarized resonators. The elements have compact profiles and resonate at 3.6 GHz, the main candidate bands of the sub-6 GHz 5G spectrum. In addition, despite the absence of decoupling structures, adjacent elements demonstrate high isolation. To the best of the authors' knowledge, it is the first type of smartphone antenna design using dual-polarized self-complementary antennas that could possess anti-interference and diversity properties. In addition to exhibiting desirable radiation coverage, the presented smartphone antenna also supports dual polarizations on different sides of the printed circuit board (PCB). It also exhibits good isolation, high-gain patterns, improved radiation coverage, low ECC/TARC, and sufficient channel capacity. The introduced antenna design was manufactured on a standard smartphone board and its main characteristics were experimentally measured. Simulations and measurement results are generally in good agreement with each other. Moreover, the presented antenna system delivers low SAR with adequate efficiency when it comes to the appearance of the user. Hence, the design could be adapted to 5G hand-portable devices. As an additional feature, a new ultra-compact phased array millimeter-wave antenna with super-wide bandwidth and end-fire radiation is being introduced for integration into the MIMO antenna systems. As a result, the proposed antenna system design with improved radiation and multi-standard operation is a good candidate for future multi-mode 5G cellular applications.

A single dose of genetically-attenuated malaria blood-stage parasites protects against two Plasmodium species infections.

Genetically-growth-attenuated blood-stage parasites were generated inPlasmodium falciparumby targeted deletion of NT1 (Nucleoside Transporter-1) gene, and Pfnt1(-) parasites only grew after providing the culture with supra-physiological concentrations of purines. Genetically-attenuatedP. yoeliint1(-)parasites induced sterile-protection against homologous blood-stage infectious challenge after immunization with single subpatent doses, which remained subpatent even in immune-compromised mice. Here, we showed that immunizations with frozen-stocks of equally-mixedP. bergheiandP. yoelii nt1(-)parasites in single subcutaneous doses, which did not lead to patent blood-stage infection, conferred sterile protection against intravenous infectious blood-stage challenge with wild-type parasites ofP. bergheiANKA andP. yoelii17X-NL strains. This data highlights the possibility that a single subcutaneous sub-patent dose of two species of genetically-growth-attenuated parasites, which can protect humans against twoPlasmodiumspp. infections, could be developed in cultures provided with supra-physiological concentrations of purines, and shipped to endemic areas as frozen-stock doses.

Genetic disruption of nucleoside transporter 4 reveals its critical roles in malaria parasite sporozoite functions.

All protozoan parasites are lacking the pathway to synthesize purines de novo and therefore they depend on their host cells to provide purines. A number of highly conserved nucleoside transporter (NT) proteins are encoded in malaria parasite genomes, of which NT1 is characterized in Plasmodium falciparum and P. yoelii as a plasma membrane protein that is responsible for salvage of purines from the host, and NT2 is an endoplasmic membrane NT protein. Whereas NT3 is only present in primate malaria parasites, little is known about NT4, which is conserved in all malaria parasite species. Herein, we targeted NT4 gene for deletion in P. berghei. NT4 knockout parasites developed normally as blood stages, ookinetes and formed oocysts with sporozoites compared with wild-type (WT) P. berghei ANKA parasites. However, nt4(-) sporozoites showed significantly decreased egress from oocysts to hemolymph, significant reduction of colonization of the salivary glands, and complete abolishment of infection of the mammalian host by salivary gland and hemolymph sporozoites. Therefore, we identify NT4 as a NT that is important, not for replication and growth, but for sporozoite infectivity functions.

In vitro production of infectious Plasmodium falciparum sporozoites.

An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1-7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we report the production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity of cryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P. falciparum life cycle from infectious gametocyte to infectious gametocyte without mosquitoes or primates.

An Alternative Autophagy-Related Mechanism of Chloroquine Drug Resistance in the Malaria Parasite.

We generated highly chloroquine (CQ)-resistant (ResCQ) Plasmodium yoelii parasites by stepwise exposure to increasing concentrations of CQ and CQ-sensitive parasites (SenCQ) by parallel mock treatments. No mutations in genes that are associated with drug resistance were detected in ResCQ clones. Autophagy-related genes were highly upregulated in SenCQ compared to ResCQ parasites during CQ treatment. This indicates that CQ resistance can be developed in the malaria parasite by the inhibition of autophagy as an alternative drug resistance mechanism.

Subcutaneous Immunization with Unaltered Axenic Malaria Parasite Liver Stages Induces Sterile Protection against Infectious Sporozoite Challenge.

Host cell-free, axenic development of liver stages (LS) of the malaria parasite has been demonstrated. Here we explored axenic liver stages as a novel live whole parasite malaria vaccine platform, which is unaltered and not prone to human-error, compared to the immunization with live-attenuated sporozoites that must be done intravenously. We show that in contrast to live sporozoites, axenic LS are not infectious to the immunized host. Subcutaneous immunizations of mice with Plasmodium yoelii axenic LS, developed from wild-type (WT) sporozoites or WT sporozoites expressing enhanced-GFP, conferred sterile protection against P. yoelii infectious sporozoite challenge. Thus, axenic liver stages of P. falciparum and P. vivax might constitute an attractive alternative to live sporozoite immunization.

Mitochondrial Spermidine Synthase is Essential for Blood-stage growth of the Malaria Parasite.

Positively-charged polyamines are essential molecules for the replication of eukaryotic cells and are particularly important for the rapid proliferation of parasitic protozoa and cancer cells. Unlike in Trypanosoma brucei, the inhibition of the synthesis of intermediate polyamine Putrescine caused only partial defect in malaria parasite blood-stage growth. In contrast, reducing the intracellular concentrations of Spermidine and Spermine by polyamine analogs caused significant defects in blood-stage growth in Plasmodium yoelii and P. falciparum. However, little is known about the synthesizing enzyme of Spermidine and Spermine in the malaria parasite. Herein, malaria parasite conserved Spermidine Synthase (SpdS) gene was targeted for deletion/complementation analyses by knockout/knock-in constructs in P. yoelii. SpdS was found to be essential for blood-stage growth. Live fluorescence imaging in blood-stages and sporozoites confirmed a specific mitochondrial localization, which is not known for any polyamine-synthesizing enzyme so far. This study identifies SpdS as an excellent drug targeting candidate against the malaria parasite, which is localized to the parasite mitochondrion.

Automated wide-field malaria parasite infection detection using Fourier ptychography on stain-free thin-smears.

Diagnosis of malaria in endemic areas is hampered by the lack of a rapid, stain-free and sensitive method to directly identify parasites in peripheral blood. Herein, we report the use of Fourier ptychography to generate wide-field high-resolution quantitative phase images of erythrocytes infected with malaria parasites, from a whole blood sample. We are able to image thousands of erythrocytes (red blood cells) in a single field of view and make a determination of infection status of the quantitative phase image of each segmented cell based on machine learning (random forest) and deep learning (VGG16) models. Our random forest model makes use of morphology and texture based features of the quantitative phase images. In order to label the quantitative images of the cells as either infected or uninfected before training the models, we make use of a Plasmodium berghei strain expressing GFP (green fluorescent protein) in all life cycle stages. By overlaying the fluorescence image with the quantitative phase image we could identify the infected subpopulation of erythrocytes for labelling purposes. Our machine learning model (random forest) achieved 91% specificity and 72% sensitivity while our deep learning model (VGG16) achieved 98% specificity and 57% sensitivity. These results highlight the potential for quantitative phase imaging coupled with artificial intelligence to develop an easy to use platform for the rapid and sensitive diagnosis of malaria.

An Attenuated HSV-1-Derived Malaria Vaccine Expressing Liver-Stage Exported Proteins Induces Sterilizing Protection against Infectious Sporozoite Challenge.

Here, we present the construction of an attenuated herpes simplex virus type-1 (HSV-1)-vectored vaccine, expressing three liver-stage (LS) malaria parasite exported proteins (EXP1, UIS3 and TMP21) as fusion proteins with the VP26 viral capsid protein. Intramuscular and subcutaneous immunizations of mice with a pooled vaccine, composed of the three attenuated virus strains expressing each LS antigen, induced sterile protection against the intravenous challenge of Plasmodium yoelii 17X-NL salivary gland sporozoites. Our data suggest that this malaria vaccine may be effective in preventing malaria parasite infection using practical routes of immunization in humans.

Potent antiplasmodial alkaloids from the rhizobacterium Pantoea agglomerans as hemozoin modulators.

Global health concern regarding malaria has increased since the first report of artemisinin-resistant Plasmodium falciparum (Pf) two decades ago. The current therapies suffer various drawbacks such as low efficacy and significant side effects, alarming for an urgent need of more effective and less toxic drugs with higher patient compliance. Chemical entities with natural origins become progressively attractive as new drug leads due to their structural diversity and bio-compatibility. This study initially aimed at the targeted isolation of hydroxyquinoline derivatives following our published genomics and metabolomics study of Pantoea agglomerans (Pa). Fermentation of Pa on a pre-selected medium followed by chromatographic isolation, NMR and HRMS analyses led to the characterisation of one new hydroxyquinoline alkaloid together with another six known congeners and two known hydroxyquinolone derivatives. When screened for their antimalarial activity by high throughput screening against asexual blood-stage parasites, almost all compounds showed potent and selective sub-micromolar activities. Computational investigation was performed to identify the antiplasmodial potential targets. Ligand-based similarity search predicted the tested compounds to act as hemozoin inhibitors. Computational target identification results were further validated by competitive hemozoin inhibitory properties of hydroxyquinoline and hydroxyquinolone derivatives in vitro. The overall results suggest this natural scaffold is of potential to be developed as antimalarial drug lead.

Marine Sulfated Polysaccharides as Promising Antiviral Agents: A Comprehensive Report and Modeling Study Focusing on SARS CoV-2.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) is a novel coronavirus strain that emerged at the end of 2019, causing millions of deaths so far. Despite enormous efforts being made through various drug discovery campaigns, there is still a desperate need for treatments with high efficacy and selectivity. Recently, marine sulfated polysaccharides (MSPs) have earned significant attention and are widely examined against many viral infections. This article attempted to produce a comprehensive report about MSPs from different marine sources alongside their antiviral effects against various viral species covering the last 25 years of research articles. Additionally, these reported MSPs were subjected to molecular docking and dynamic simulation experiments to ascertain potential interactions with both the receptor-binding domain (RBD) of SARS CoV-2's spike protein (S-protein) and human angiotensin-converting enzyme-2 (ACE2). The possible binding sites on both S-protein's RBD and ACE2 were determined based on how they bind to heparin, which has been reported to exhibit significant antiviral activity against SARS CoV-2 through binding to RBD, preventing the virus from affecting ACE2. Moreover, our modeling results illustrate that heparin can also bind to and block ACE2, acting as a competitor and protective agent against SARS CoV-2 infection. Nine of the investigated MSPs candidates exhibited promising results, taking into consideration the newly emerged SARS CoV-2 variants, of which five were not previously reported to exert antiviral activity against SARS CoV-2, including sulfated galactofucan (1), sulfated polymannuroguluronate (SPMG) (2), sulfated mannan (3), sulfated heterorhamnan (8), and chondroitin sulfate E (CS-E) (9). These results shed light on the importance of sulfated polysaccharides as potential SARS-CoV-2 inhibitors.

Flavonoid-Coated Gold Nanoparticles as Efficient Antibiotics against Gram-Negative Bacteria-Evidence from In Silico-Supported In Vitro Studies.

Flavonoids are a class of bioactive plant-derived natural products that exhibit a broad range of biological activities, including antibacterial ones. Their inhibitory activity toward Gram-positive bacterial was found to be superior to that against Gram-negative ones. In the present study, a number of flavonoid-coated gold nanoparticles (GNPs) were designed to enhance the antibacterial effects of chrysin, kaempferol, and quercetin against a number of Gram-negative bacteria. The prepared GNPs were able to conjugate to these three flavonoids with conjugation efficiency ranging from 41% to 80%. Additionally, they were able to exert an enhanced antibacterial activity in comparison with the free flavonoids and the unconjugated GNPs. Quercetin-coated GNPs were the most active nano-conjugates and were able to penetrate the cell wall of E. coli. A number of in silico experiments were carried out to explain the conjugation efficiency and the antibacterial mechanisms of these flavonoids as follows: (i) these flavonoids can efficiently bind to the glutathione linker on the surface of GNPs via H-bonding; (ii) these flavonoids, particularly quercetin, were able to increase the bacterial membrane rigidity, and hence decrease its functionality; (iii) these flavonoids can inhibit E. coli's DNA gyrase (Gyr-B) with IC50 values ranging from 0.9 to 3.9 µM. In conclusion, these bioactive flavonoid-based GNPs are considered to be very promising antibiotic candidates for further development and evaluation.

Slotted E-Shaped Meta-Material Decoupling Slab for Densely Packed MIMO Antenna Arrays.

In contemporary wireless communication systems, the multiple-input and multiple-output systems are extensively utilized due to their enhanced spectral efficiency and diversity. Densely packed antenna arrays play an important role in such systems to enhance their spatial diversity, array gain, and beam scanning capabilities. In this article, a slotted meta-material decoupling slab (S-MTM-DS) with dual reflexes slotted E-shapes and an inductive stub is proposed. Its function was validated when located between two microstrip patch antenna elements to reduce the inter-element spacing, the mutual coupling, the return losses, and manufacturing costs due to size reduction. A prototype is simply fabricated in a volume of 67.41 × 33.49 × 1.6 mm3 and frequency-span measured from 8.4:11 GHz. At 9.4 GHz frequency, the spaces between the transmitting elements are decreased to 0.57 of the free space wavelength. When the proposed isolation S-MTM-DS is applied, the average isolation among them is measured to be -36 dB, the operational bandwidth is enhanced to be 1.512 GHz, the fractional bandwidth improved to be 16.04%, and the return losses are decreased to be -26.5 dB at 9.4 GHz center frequency. Consequently, the proposed design has the potential to be implemented simply in wireless contemporary communication schemes.

Antimicrobial and Antibiofilm Activities of the Fungal Metabolites Isolated from the Marine Endophytes Epicoccum nigrum M13 and Alternaria alternata 13A.

Epicotripeptin (1), a new cyclic tripeptide along with four known cyclic dipeptides (2-5) and one acetamide derivative (6) were isolated from seagrass-associated endophytic fungus Epicoccum nigrum M13 recovered from the Red Sea. Additionally, two new compounds, cyclodidepsipeptide phragamide A (7) and trioxobutanamide derivative phragamide B (8), together with eight known compounds (9-16), were isolated from plant-derived endophyte Alternaria alternata 13A collected from a saline lake of Wadi El Natrun depression in the Sahara Desert. The structures of the isolated compounds were determined based on the 1D and 2D NMR spectroscopic data, HRESIMS data, and a comparison with the reported literature. The absolute configurations of 1 and 7 were established by advanced Marfey's and Mosher's ester analyses. The antimicrobial screening indicated that seven of the tested compounds exhibited considerable (MIC range of 2.5-5 µg/mL) to moderate (10-20 µg/mL) antibacterial effect against the tested Gram-positive strains and moderate to weak (10-30 µg/mL) antibacterial effect against Gram-negative strains. Most of the compounds exhibited weak or no activity against the tested Gram-negative strains. On the other hand, four of the tested compounds showed considerable antibiofilm effects against biofilm forming Gram-positive and Gram-negative strains.

Synthetic DNA Vaccines Adjuvanted with pIL-33 Drive Liver-Localized T Cells and Provide Protection from Plasmodium Challenge in a Mouse Model.

The need for a malaria vaccine is indisputable. A single vaccine for Plasmodium pre-erythrocytic stages targeting the major sporozoite antigen circumsporozoite protein (CSP) has had partial success. Additionally, CD8+ T cells targeting liver-stage (LS) antigens induced by live attenuated sporozoite vaccines were associated with protection in human challenge experiments. To further evaluate protection mediated by LS antigens, we focused on exported pre-erythrocytic proteins (exported protein 1 (EXP1), profilin (PFN), exported protein 2 (EXP2), inhibitor of cysteine proteases (ICP), transmembrane protein 21 (TMP21), and upregulated in infective sporozoites-3 (UIS3)) expressed in all Plasmodium species and designed optimized, synthetic DNA (synDNA) immunogens. SynDNA antigen cocktails were tested with and without the molecular adjuvant plasmid IL-33. Immunized animals developed robust T cell responses including induction of antigen-specific liver-localized CD8+ T cells, which were enhanced by the co-delivery of plasmid IL-33. In total, 100% of mice in adjuvanted groups and 71%-88% in non-adjuvanted groups were protected from blood-stage disease following Plasmodium yoelii sporozoite challenge. This study supports the potential of synDNA LS antigens as vaccine components for malaria parasite infection.