Targeting the Cell Stress Response of Plasmodium falciparum to Overcome Artemisinin Resistance

Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART resistance.     

Hypoxia-inducible factor-1α mediates oral squamous cell carcinoma invasion via upregulation of α5 integrin and fibronectin

With progressive and rapid growth of malignant tumors, cancer cells in an ischemic condition are expected to develop an increased potential for local invasive growth. To address this hypothesis, we first examined the effect of hypoxia on the invasiveness of oral squamous cell carcinoma (OSCC) cells using the Matrigel invasion assay. We then investigated the effect of hypoxia on the protein and mRNA expression of α5 integrin and fibronectin, which are major factors involved in tumor cell invasion. We showed that (i) hypoxia increased the invasiveness of OSCC cells, (ii) α5 integrin and fibronectin protein and mRNA expression levels were increased in OSCC cells under hypoxic conditions, (iii) hypoxia stimulated autocrine secretion of fibronectin in OSCC cells, (iv) administration of siRNA_HIF-1α caused a significant decrease in α5 integrin and fibronectin protein, confirming that HIF-1α plays a role in their induction, and (v) siRNA_HIF-1α abrogated hypoxia-induced cell invasion. Collectively, these data suggest that hypoxia promotes OSCC cell invasion that is elicited by HIF-1α-dependent α5 integrin and fibronectin induction.     

The <Emphasis Type="Italic">COL1A1</Emphasis> gene and high myopia susceptibility in Japanese

The collagen type Ι alpha Ι (COL1A1) gene encodes the extracellular matrix component, collagen, and resides in candidate MYP5 for high myopia on the chromosome 17q22–q23.3. This locus has recently been implicated in playing an important role in the pathogenesis of experimental myopia. We investigated the association of disruptions of COL1A1 gene with high myopia by analyzing the frequency of ten SNPs in a Japanese population of 330 subjects with high myopia of −9.25 D or less and 330 randomized controls without high myopia. Two SNPs (rs2075555 and rs2269336) were significantly associated with high myopia (P < 0.05, Pc < 0.1). Two different haplotype blocks in COL1A1 were observed by the pair-wise linkage disequilibrium between the SNPs. The frequency of GGC/GGC diplotype constructed by the three SNPs (rs2075555, rs2269336, rs1107946) was significantly high (OR = 1.6) and associated with high myopia (P = 0.028, Pc< 0.084). Together our results provide the first evidence for COL1A1 as a gene associated with high myopia.     

Folate immobilized and PEGylated adenovirus for retargeting to tumor cells

The surface of adenovirus (ADV) was modified with folate-poly(ethylene glycol) (FOL-PEG) conjugate to overcome the inherent problems associated with viral gene therapy. The ADV coding the green fluorescent protein (GFP) gene was used to immobilize PEG or FOL-PEG conjugate on the viral surface to comparatively evaluate their extents of retargeting and immune response, as compared to those of naked ADV. The FOL-PEG immobilized ADV exhibited a greatly enhanced level of GFP expression than naked or PEG immobilized ADV for a folate receptor overexpressing cell line (KB cells), but not for a folate receptor deficient cell line (A549 cells), suggesting that the retargeting of ADV could be achieved by immobilizing FOL-PEG conjugate. The ADV immobilized with PEG or FOL-PEG also significantly lowered innate immune response, as judged from the amount of interleukin 6 released from macrophage cells.     

Characterization of BARD1 targeting and dynamics at the centrosome: the role of CRM1, BRCA1 and the Q564H mutation

BARD1 heterodimerizes with BRCA1, forming an E3 ubiquitin ligase that functions at nuclear foci to repair DNA damage and the centrosome to regulate mitosis. We compared BARD1 recruitment at these structures using fluorescence recovery after photobleaching assays to measure YFP-BARD1 dynamics in live cells. In nuclei at ionizing radiation-induced foci, 20% of the BARD1 pool was immobile and 80% of slow mobility exhibiting a recovery time > 500 s. In contrast, at centrosomes 83% of BARD1 was rapidly mobile with extremely fast turnover (recovery time ~ 20 s). The ~ 25-fold faster exchange of BARD1 at centrosomes correlated with BRCA1-independent recruitment. We mapped key targeting sequences to a combination of the N and C-termini, and showed that mutation of the nuclear export signal reduced centrosome localization by 50%, revealing a role for CRM1. Deletion of the sequence 128-550 increased BARD1 turnover at the centrosome, consistent with a role in transient associations. Conversely, the cancer mutation Q564H reduced turnover by 25%. BARD1 is one of the most highly mobile proteins yet detected at the centrosome, and in contrast to its localization at DNA repair foci, which requires dimerization with BRCA1, targeting of BARD1 to the centrosome occurs prior to heterodimerization and its rapid turnover may provide a mechanism to regulate dimer formation.     

AMP-activated protein kinase β-subunit requires internal motion for optimal carbohydrate binding

AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the β-subunit, for which there are two isoforms (β(1) and β(2)). Muscle-specific β(2)-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous β(1)-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of β(2)-CBM is concurrent with an increase in flexibility in β(2)-CBM, which may account for the affinity differences between the two isoforms. In contrast to β(1)-CBM, unbound β(2)-CBM showed microsecond-to-millisecond motion at the base of a β-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this β-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from β(2)-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the β(2)-CBM mutant. Insertion of a threonine into the background of β(1)-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms.     

Modelling the potential impact of climate variability and change on species regeneration potential in the temperate forests of South‐Eastern Australia

The sensitivity of early plant regeneration to environmental change makes regeneration a critical stage for understanding species response to climate change. We investigated the spatial and temporal response of eucalypt trees in the Central Highland region of south eastern Australia to high and low climate change scenarios. We developed a novel mechanistic model incorporating germination processes, TACA‐GEM, to evaluate establishment probabilities of five key eucalypt species, Eucalyptus pauciflora, Eucalyptus delegatensis, Eucalyptus regnans, Eucalyptus nitens and Eucalyptus obliqua. Changes to regeneration potential at landscape and site levels were calculated to determine climate thresholds. Model results demonstrated that climate change is likely to impact plant regeneration. We observed increases and decreases in regeneration potential depending on the ecosystem, indicating that some species will increase in abundance in some forest types, whilst other forest types will become inhabitable. In general, the dry forest ecosystems were most impacted, whilst the wet forests were least impacted. We also observed that species with seed dormancy mechanisms, like E. pauciflora and E. delegatensis, are likely to be at higher risk than those without. Landscape‐ and site‐level analysis revealed heterogeneity in species response at different scales. On a landscape scale, a 4.3 °C mean temperature increase and 22% decline in precipitation (predicted for 2080) is predicted to be a threshold for large spatial shifts in species regeneration niches across the study region, while a 2.6 °C increase and 15% decline in precipitation (predicted for 2050) will likely result in local site‐level shifts. Site‐level analysis showed that considerable declines in regeneration potential for E. delegatensis, E. pauciflora and E. nitens were modelled to occur in some ecosystems by 2050. While overall model performance and accuracy was good, better understanding of effects from extreme events and other underlying processes on regeneration will improve modelling and development of species conservation strategies.