Currently, no approved antiviral drugs target dengue virus (DENV) infection, leaving treatment reliant on supportive care. DENV vaccine efficacy varies depending on the vaccine type, the circulating serotype, and vaccine coverage. We investigated defective interfering particles (DIPs) and lipid nanoparticles (LNPs) to deliver DI290, an anti-DENV DI RNA. Both DIPs and DI290-loaded LNPs (LNP-290) effectively suppressed DENV infection in human primary monocyte-derived macrophages (MDMs), THP-1 macrophages, and fibroblasts-natural DENV targets. Inhibiting interferon (IFN) signaling with a Janus kinase 1/2 inhibitor or an IFN-c/13 receptor 1 (IFNAR1)binding antibody blocked DIP and LNP-290 antiviral activity. LNP-290 demonstrated a greater than log10 inhibition of DENV viral loads in IFNAR-deficient (Ifnar-/-) and IFN regulatory factor (IRF) 3 and 7 double knockout (Irf3/7-/-) mice. Pathway analysis of RNA sequencing data from LNP-treated C57BL/6J mice, Ifnar-/- mice, and human MDMs treated with LNPs or DENV DIPs indicated DI290 treatment enhanced IFN responses, suggesting IFN-A and IFN-y provided antiviral activity when IFN-c/13 responses were diminished. While viral interference by DI290 is possible, results did not support RNA replication competition as an inhibition mechanism. These findings suggest that DI290 may be a promising DENV therapeutic by activating the innate immune system.

Presently, no approved antiviral drug targets dengue virus (DENV) infection. Treatment mainly relies on supportive measures, while DENV vaccines’ efficacy varies based on factors like vaccine type, circulating DENV serotypes and vaccinated population. This study explores using defective interfering particles (DIPs) and lipid nanoparticles (LNPs) to deliver an anti-DENV defective interfering RNA, known as DI290. Results showed that both DIPs and DI290 loaded LNPs (LNP-290) effectively suppressed DENV infection in human primary monocyte-derived macrophages (MDMs), THP-1 macrophages and human fibroblasts, representing cell types naturally targeted by DENV. Furthermore, LNP-290 demonstrated >log10 inhibition of DENV viral loads in IFNAR-deficient mice, which lack functional type I interferon (IFN) receptors. DI290-mediated inhibition was also effective in IFN regulatory factor 3 and 7 double knockout mice. RNA-Seq data from LNP-treated C57BL/6J mice, IFNAR-deficient mice and human MDMs treated with LNPs or DENV DIPs illustrated DI290 treatment heightened IFN responses, particularly IFNγ, as well as IFNα/β and IFNλ. DI290 thus induces a broad range of IFN responses, with IFNγ and IFNλ providing anti-viral activity when IFNα/β responses are absent. Mice administered LNP-290 also did not manifest acute overt clinical signs. In summary, these experiments suggest DI290’s potential as a therapeutic approach for combating DENV infection.### Competing Interest StatementThe authors have declared no competing interest.

Background and ObjectiveNeutrophil elastase (NE), is an important host defence against lung pathogens. Maintaining a homeostatic balance between proteases such as NE and anti-proteases such as secretory leukocyte protease inhibitor (SLPI), is important to prevent tissue damage. In the cystic fibrosis (CF) lung, elevated protease levels and impaired anti-protease defences contribute to tissue destruction. MethodsWe assessed lung function and sputum SLPI and NE levels from Pseudomonas aeruginosa infected and non-infected CF patients (median age 20 years at recruitment) during different phases of clinical disease. Healthy, never smokers served as healthy controls (HC). Sputum total cell counts (TCC) and colony forming units of P. aeruginosa were also determined in each sputum sample. ResultsCompared to HC, sputum SLPI was significantly reduced and NE increased in all CF subjects whether infected with P. aeruginosa or not, but the presence of P. aeruginosa worsened these parameters. Females with chronic P. aeruginosa infection had significantly lower sputum SLPI levels than males (p < 0.001). Higher sputum SLPI levels were associated with a significantly reduced rate of longitudinal decline in FEV1% predicted (p < 0.05). Antibiotic treatment in P. aeruginosa-infected patients significantly decreased sputum TCC and increased SLPI levels, which positively correlated with improved lung function. ConclusionAirway SLPI is deficient in CF, which appears more marked in P. aeruginosa-infected female patients. Importantly, a reduced anti-protease to protease ratio is associated with accelerated lung function decline. Treatment of an exacerbation is accompanied by partial recovery of anti-protease defences and significant improvement in lung function, an important clinical outcome.

Abstract Background Defective chloride transport in airway epithelial cells (AECs) and the associated lung disease are the main causes of morbidity and early mortality in cystic fibrosis (CF). Abnormal airway iron homeostasis and the presence of lipid peroxidation products, indicative of oxidative stress, are features of CF lung disease. Results Here, we report that CF AECs (IB3-1) are susceptible to ferroptosis, a type of cell death associated with iron accumulation and lipid peroxidation. Compared to isogenic CFTR corrected cells (C38), the IB3-1 cells showed increased susceptibility to cell death upon exposure to iron in the form of ferric ammonium citrate (FAC) and the ferroptosis inducer, erastin. This phenotype was accompanied by accumulation of intracellular ferrous iron and lipid peroxides and the extracellular release of malondialdehyde, all indicative of redox stress, and increased levels of lactate dehydrogenase in the culture supernatant, indicating enhanced cell injury. The ferric iron chelator deferoxamine (DFO) and the lipophilic antioxidant ferrostatin-1 inhibited FAC and erastin induced ferroptosis in IB3-1 cells. Glutathione peroxidase 4 (GPX4) expression was decreased in IB3-1 cells treated with FAC and erastin, but was unchanged in C38 AECs. Necroptosis appeared to be involved in the enhanced susceptibility of IB3-1 AECs to ferroptosis, as evidenced by partial cell death rescue with necroptosis inhibitors and enhanced mixed lineage kinase domain-like (MLKL) localisation to the plasma membrane. Conclusion These studies suggest that the increased susceptibility of CF AECs to ferroptosis is linked to abnormal intracellular ferrous iron accumulation and reduced antioxidant defences. In addition, the process of ferroptotic cell death in CF AECs does not appear to be a single entity and for the first time we describe necroptosis as a potential contributory factor. Iron chelation and antioxidant treatments may be promising therapeutic interventions in cystic fibrosis. Graphical Abstract

The Neural Tube Defects Research Group of University of Malaya was approached to analyze a tablet named TELSE, which may have resulted in a baby born with central nervous system malformation at the University of Malaya Medical Centre. In this animal experimental study, we investigated the content of TELSE and exposure of its contents that resulted in failure of primary neurulation.

Burkholderia pseudomallei, the etiologic agent of melioidosis is a common cause of sepsis mainly in diabetic individuals in South East Asia. Glycogen synthase kinase-3β (GSK3β) plays a pivotal role in modulating inflammatory balance in Gram-negative bacterial infections. In this study, we demonstrate that inhibition of GSK3β significantly improved survival of hyperglycaemic mice acutely infected with B. pseudomallei. With GSK3β inhibition, we found significant modulation between pro- (IL-12, TNF-alpha) and anti-inflammatory (IL10) serum cytokines which may have contributed to bacterial clearance in multiple organs of B. pseudomallei-infected hyperglycaemic mice. Concurrently, an increase in phosphorylation of GSK3β at Ser-9 was observed in the liver of B. pseudomallei-infected hyperglycaemic mice. Likewise, B. pseudomallei-infected non-hyperglycaemic mice upon GSK3β inhibition showed similar trends of bacterial clearance and modulation of serum cytokines; however, the effect of enhanced survival was less substantial than in infected hyperglycaemic mice. Taken together, we demonstrate that inhibition of GSK3β confers survival advantage of hyperglycaemic mice infected with B. pseudomallei and offers a potential therapeutic strategy for the treatment of diabetic patients with melioidosis.

Increased susceptibility of diabetics to melioidosis, a disease caused by the Burkholderia pseudomallei bacterium is believed to be attributed to dysfunction of the innate immune system. However, the underlying mechanism of the innate susceptibility is not well-understood. Glycogen synthase kinase-3β (GSK3β) plays an important role in the innate inflammatory response caused by bacterial pathogens. The present study was conducted to investigate the effects of GSK3β inhibition by LiCl on levels of pro- and anti-inflammatory cytokines; and the activity of transcription factor NF-κB in B. pseudomallei-infected peripheral blood mononuclear cells (PBMC) derived from diabetic-induced and normal Sprague Dawley rats. In addition, the effects of LiCl on intracellular bacterial counts were also investigated. Infection of PBMC from diabetic and normal rats with B. pseudomallei resulted in elevated levels of cytokines (TNF-α, IL-12 and IL-10) and phosphorylation of NF-κB in both cell types. Intracellular bacterial counts decreased with time in both cell types during infection. However bacterial clearance was less prominent in diabetic PBMC. Burkholderia pseudomallei infection also caused inactivation (Ser9 phosphorylation) of GSK3β in normal PBMC, an effect absent in infected diabetic PBMC. Inhibition of GSK3β by LiCl lowered the levels of pro-inflammatory cytokines (TNF-α and IL-12) in both normal and diabetic PBMC. Similarly, phosphorylated NF- κB (pNF-κB) levels in both cell types were decreased with LiCl treatment. Also, LiCl was able to significantly decrease the intracellular bacterial count in normal as well as diabetic PBMC. Interestingly, the levels of anti-inflammatory cytokine IL-10 in both normal and diabetic PBMC were further elevated with GSK3β inhibition. More importantly, GSK3β in infected diabetic PBMC was inactivated as in their non-diabetic counterparts upon LiCl treatment. Taken together, our results suggest that inhibition of dysregulated GSK3β in diabetic PBMC resulted in the inactivation of NF-κB and modulation of inflammatory cytokine levels. This is evidence that dysregulation of GSK3β is a contributing factor in the molecular basis of innate dysfunction and susceptibility of diabetic host to melioidosis infection.

Burkholderia pseudomallei, the causative agent of melioidosis, is an intracellular pathogen capable of invading and multiplying in both phagocytic and non-phagocytic cells. Infection results in an inflammatory response involving production of both pro-and anti-inflammatory cytokines. The cellular mechanism regulating this response, believed to play an important role in the pathogenesis of meliodoisis, is not fully understood. In recent years, glycogen synthase kinase-3 (GSK3) has been shown to assume a pivotal role in regulating production of these cytokines. Bacterial infection of host cells activates Toll-like receptors (TLRs) and results in the phosphorylation of GSK3 beta through activation of the phosphoinositide 3-kinase (PI3K) pathway. In this study, we investigated the effects of GSK3 inhibition in regulating B. pseudomallei-induced inflammatory response in macrophages and A549 epithelial lung cells. Our results showed that infection of cells with B. pseudomallei resulted in the increase of anti-inflammatory cytokine, IL-10 and pro-inflammatory cytokine, TNF-alpha. Pre-treatment of infected cells with GSK3 inhibitors caused further increase in the level of IL-10 but a significant decrease in TNF-alpha. These changes corresponded with the detection of phosphorylated GSK3 beta in infected cells treated with LiCl; suggesting that modulation of inflammatory response in B. pseudomallei infection involves phosphorylation of GSK3 beta (Ser 9). This could explain our observations from the invasion assays that pre-treatment of B. pseudomallei-infected cells with GSK3 inhibitors resulted in decreased intracellular replication of bacteria within macrophages and A549 epithelial lung cells. In summary, our results demonstrate a regulatory function of GSK3 in the modulation of cytokine levels during B. pseudomallei infection.

Hepatic phosphoprotein levels are altered in mouse liver as a manifestation of bacteria, virus or parasite infection. Identification of signaling pathways mediated by these hepatic proteins contribute to the current understanding of the mechanism of pathogenesis in malarial infection. The present study was undertaken to evaluate the changes in hepatic phosphoprotein levels during Plasmodium berghei infection. Our study revealed changes in levels of three hepatic phosphoproteins following P. berghei infection compared to non-infected controls. Peptide fragment sequence analysis using tandem mass spectrometry (mS/MS) showed these hepatic proteins to be homologs to haemoglobin beta (HBB), class Pi glutathione S-tranferase (GSTPi) and carbonic anhydrase III (CAIII) proteins of Mus musculus species respectively from the NCBInr sequence database. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis predicted the involvement of these proteins in specific pathways in Mus musculus species; GSTPi in glutathione and drug metabolism and CAIII in nitrogen metabolism. This shows that P. berghei infection affects similar signaling pathways as those reported in other pathogenic injections such as that related to GSTPi and Dull in response to oxidative stress.