Plasmodium translocon component EXP2 facilitates hepatocyte invasion

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

UHRF genes regulate programmed interdigital tissue regression and chondrogenesis in the embryonic limb

The primordium of the limb contains a number of progenitors far superior to those necessary to form the skeletal components of this appendage. During the course of development, precursors that do not follow the skeletogenic program are removed by cell senescence and apoptosis. The formation of the digits provides the most representative example of embryonic remodeling via cell degeneration. In the hand/foot regions of the embryonic vertebrate limb (autopod), the interdigital tissue and the zones of interphalangeal joint formation undergo massive degeneration that accounts for jointed and free digit morphology. Developmental senescence and caspase-dependent apoptosis are considered responsible for these remodeling processes. Our study uncovers a new upstream level of regulation of remodeling by the epigenetic regulators Uhrf1 and Uhrf2 genes. These genes are spatially and temporally expressed in the pre-apoptotic regions. UHRF1 and UHRF2 showed a nuclear localization associated with foci of methylated cytosine. Interestingly, nuclear labeling increased in cells progressing through the stages of degeneration prior to TUNEL positivity. Functional analysis in cultured limb skeletal progenitors via the overexpression of either UHRF1 or UHRF2 inhibited chondrogenesis and induced cell senescence and apoptosis accompanied with changes in global and regional DNA methylation. Uhrfs modulated canonical cell differentiation factors, such as Sox9 and Scleraxis, promoted apoptosis via up-regulation of Bak1, and induced cell senescence, by arresting progenitors at the S phase and upregulating the expression of p21. Expression of Uhrf genes in vivo was positively modulated by FGF signaling. In the micromass culture assay Uhrf1 was down-regulated as the progenitors lost stemness and differentiated into cartilage. Together, our findings emphasize the importance of tuning the balance between cell differentiation and cell stemness as a central step in the initiation of the so-called ?embryonic programmed cell death? and suggest that the structural organization of the chromatin, via epigenetic modifications, may be a precocious and critical factor in these regulatory events.Funding: We thank Montse Fernandez Calderon, Susana Dawalibi, and Sonia Perez Mantecon, for excellent technical assistance. This work was supported by a Grant (BFU2017-84046-P) from the Spanish Science and Innovation Ministry to J.A.M

The private life of malaria parasites:Strategies for sexual reproduction

Malaria parasites exhibit a complex lifecycle, requiring extensive asexual replication in the liver and blood of the vertebrate host, and in the haemocoel of the insect vector. Yet, they must also undergo a single round of sexual reproduction, which occurs in the vector’s midgut upon uptake of a blood meal. Sexual reproduction is obligate for infection of the vector and thus, is essential for onwards transmission to new hosts. Sex in malaria parasites involves several bottlenecks in parasite number, making the stages involved attractive targets for blocking disease transmission. Malaria parasites have evolved a suite of adaptations (“strategies”) to maximise the success of sexual reproduction and transmission, which could undermine transmission-blocking interventions. Yet, understanding parasite strategies may also reveal novel opportunities for such interventions. Here, we outline how evolutionary and ecological theories, developed to explain reproductive strategies in multicellular taxa, can be applied to explain two reproductive strategies (conversion rate and sex ratio) expressed by malaria parasites within the vertebrate host

Direct functional consequences of ZRS enhancer mutation combine with secondary long range SHH signalling effects to cause preaxial polydactyly

AbstractSonic hedgehog (SHH) plays a central role in patterning numerous embryonic tissues including, classically, the developing limb bud where it controls digit number and identity. This study utilises the polydactylous Silkie (Slk) chicken breed, which carries a mutation in the long range limb-specific regulatory element of SHH, the ZRS. Using allele specific SHH expression analysis combined with quantitative protein analysis, we measure allele specific changes in SHH mRNA and concentration of SHH protein over time. This confirms that the Slk ZRS enhancer mutation causes increased SHH expression in the posterior leg mesenchyme. Secondary consequences of this increased SHH signalling include increased FGF pathway signalling and growth as predicted by the SHH/GREM1/FGF feedback loop and the Growth/Morphogen models. Manipulation of Hedgehog, FGF signalling and growth demonstrate that anterior-ectopic expression of SHH and induction of preaxial polydactyly is induced secondary to increased SHH signalling and Hedgehog-dependent growth directed from the posterior limb. We predict that increased long range SHH signalling acts in combination with changes in activation of SHH transcription from the Slk ZRS allele. Through analysis of the temporal dynamics of anterior SHH induction we predict a gene regulatory network which may contribute to activation of anterior SHH expression from the Slk ZRS

Pathophysiology of mood disorders and common pathways with heart failure : implications for an outpatient-setting program

Introduction: Cardiovascular disease (CVD) and mood disorders (MD) are two of the world’s leading health problems. Both conditions are chronic, debilitating, and extremely prevalent among the general population, often occurring together in the same individual. Epidemiological evidence has been growing suggesting that there is a two-way relationship between them. In fact, a significant proportion of people with no prior record of MD develop depression after a cardiovascular event. As well, physically healthy individuals who are diagnosed with depression are at increased risk of developing CVD compared to the general population. This link is particularly evident between depression and heart failure (HF). The HF population with depression is particularly known to have an increased risk of hospitalizations, emergency visits and re-admissions with higher mortality rates and worse health-related outcomes. The burden of these diseases is overwhelming when they occur individually and even greater when they occur in comorbidity, yet there is still no consensus or any particular guidelines for the best method to treat patients with comorbid MD and CVD. This may be partially due to the fact that MD remains poorly understood. To better clarify the pathophysiology behind this spectrum of disorders we should not be confined to certain conceptual boundaries. First, although there are clear clinical distinctions between depression and Bipolar Disorder (BD), experts still debate the possibility of some degree of continuum between these diseases. Second, the biological mechanisms behind MD are not yet fully understood; namely what separates them and what mechanisms they have in common. This is also true if we aim to identify common biological changes between CVD and MD, particularly between HF and depression. HF is one of the most common causes of hospitalization, mortality, and economic burden worldwide. It is crucial to develop new and improved models of care for this condition. A new program design at Health Sciences North (HSN) called the Heart Failure Disease Management Program (HFDMP) utilizes various outpatient strategies that aim to avoid emergency department (ED) visits, to decrease HF hospitalizations, to improve outcomes, and to decrease mortality and health care costs. The success of this outpatient program model needs to be evaluated. Furthermore, since the association of depression with HF seems to be extremely relevant the prevalence and impact of depression in an original program like the HFDMP needs to be clarified. The general aims of this work were: 1. To investigate pathophysiological mechanisms in mood disorders; 2. To investigate common pathophysiological mechanisms between mood disorders and cardiovascular disease, particularly depression and heart failure; 3. To further investigate the relationship between depression and heart failure from a clinical perspective. The following specific aims were established to address the general goals of this work: 1 To investigate pathophysiological changes in mood disorders, namely the role of mechanisms known to be relevant to the pathophysiology of cardiovascular disease. 1.1 To investigate oxidative stress changes across the life span of patients with MD, Specifically, to analyse the levels of oxidative damage in plasma from 185 subjects, consisting of 110 euthymic older BD patients (BD-I and BD-II with 35 years of evolution of disease) compared to 75 healthy controls; 1.2 To investigate oxidative changes in different mood episodes and mood polarities (depression, hypomania and euthymia) and their relationship with hippocampus changes in patients with MD; specifically, to analyse peripheral levels of oxidative stress markers and decreased hippocampal subvolume dentate gyrus–cornu ammonis (CA4) in correlation to depressive, hypomanic and euthymic episodes in 62 subjects consisting of 29 patients with BD-II and 33 healthy controls. 2 To investigate common pathophysiological mechanisms between mood disorders and cardiovascular disease, particularly between depression and heart failure: 2.1 To review the role of inflammation as a pathophysiological mechanism shared between depression and CVD; specifically, to discuss the evidence of the benefit ofanti-inflammatory drugs in both conditions in support of the hypothesis of a shared pathophysiology between these diseases through inflammation. 2.2 To investigate further common biological pathways between MD and CVD, in HF patients with depression; specifically, to investigate whether NT-proBNP mediated reduced ejection fraction (EF) and depressive symptoms in 124 subjects with HF and depression compared to HF patients without depression. 3 To evaluate an original outpatient program for heart failure management and the relationship of depression with patients as well as the program’s outcomes: 3.1 To investigate the safety and effectiveness of the program HFDMP in managing HF in an outpatient setting; specifically, to investigate the ability of the program to reduce emergency department visits, to decrease HF admissions and readmissions, to improve HF outcome, and to decrease mortality in 138 patients enrolled and followed in the program over a period of 12 months; 3.2 To investigate the relationship between depression, quality of life (QoL) and HF outcomes in the HFDMP; specifically, to investigate the prevalence of depression in the HFDMP and the impact of depression in the QoL and clinical symptoms and outcomes of 124 patients enrolled and followed in the program over a period of 12-months. Results and discussion: The pathophysiological mechanisms responsible for MD are complex and multifaceted. The multiple studies conducted in this work and its respective findings support the idea of that complexity. The hypothesis of a central role for oxidative stress in MD is supported and highlighted by the findings (*1.1) of increased levels of an early component of the peroxidation chain, the lipid hydroperoxide (LPH), in euthymic patients with BD-I and BD-II into late life. This suggests a persistent effect of reactive species of oxygen throughout a BD patient’s life. Further proof for this hypothesis can be gleaned from the findings (*1.2) of increased peripheral levels of two lipid peroxidation markers, the 4-hydroxy-2-nonenal (4-HNE) and LPH, which is significantly correlated with depressive episodes and with decreased dentate gyrus–CA4 volume. It was shown that a larger number of depressive episodes predict a greater volume loss in patient’s hippocampus. Furthermore, it was shown that 4-HNE is negatively associated with left and right dentate gyrus–CA4 volumes. Altogether, these results are consistent in proving the by oxidative stress in the pathophysiology of MD. Furthermore, they suggest that depressive episodes and elevated oxidative stress might contribute to hippocampal volume loss in these patients. In addition, these findings provide further support for the hypothesis that peripheral lipid peroxidation markers may reflect brain alterations and may one day represent a biomarker for these disorders. Oxidative stress is a mechanism well established in CVD and in our findings it appears to also play a central role in MD pathophysiology. Therefore, oxidative stress is an extremely likely biological interface between mood and heart problems. Oxidative stress and inflammation are closely related pathophysiological mechanisms. One can be easily induced by the other, therefore both processes are simultaneously found in many pathological conditions. Similar to oxidative stress, inflammation also seems to be implicated in the pathophysiology of both depression and CVD. Inflammation also seems to have a central role in MD and to be a central candidate mediating the link between depression and CVD, as suggested by the results of our review (*2.1). Taking this hypothesis further, we have found original and seemingly important biological pathways shared between depression and HF, highlighted by the findings in our study (*2.2) that suggest that NT-proBNP is a potential mediator between reduced EF and depression in patients with HF. This indicates that NT-proBNP may be a potential biomarker for HF patients with reduced EF and depression who are at higher risk in terms of disease prognosis and mortality. In this work we have also investigated the original approach established at HSN for HF management named HFDMP. This program was shown (*3.1) to be a safe and effective way to manage HF and avoid ED visits and decrease HF hospitalizations and mortality, all while improving clinical symptoms and decreasing health care expenditures. Finally, we have shown that depression is extremely prevalent in this program’s HF population (*3.2) and is associated with worse QoL levels in patients and worse outcomes in the program. On the other hand, this program was shown to be highly effective in improving patients’ QoL over time, both physically and emotionally. Altogether, these studies provide further evidence of the strong association between HF and depression, from molecular to clinical perspectives. Conclusion: Altogether this work leads to several conclusions. Firstly, oxidative stress and inflammation appear to have a central role in the pathophysiology of MD. Oxidative stress and a decreased volume in hippocampus seem to be directly implicated in the pathophysiology of depressive episodes in MD. Furthermore, oxidative stress and inflammation are central candidates for proving a shared pathophysiology between MD and CVD. Clinical and biological pathways were found between HF and depression in patients enrolled in the HFDMP. This original program for HF management – demonstrated also in this work to be an efficient and safe way to manage HF in an outpatient setting – was shown to have a population where depression is extremely prevalent and where depression influences HF patients’ QoL and outcomes. Finally, in this work we have found an important indicator for the existence of a shared pathophysiology between depression and HF: depressive symptoms and reduced ejection fraction in HF seem to be mediated by NT-proBNP, a gold-standard indicator of the clinical status, diagnosis and severity of HF. We have found that this biological marker, extremely well established in HF, reacts differently with the existence of depressive symptoms in HF patients. This finding provides a strong hint at the common pathways between these diseases. Altogether, this work contributed to clarifications on the pathophysiology of MD as well as the biological connections between MD and CVD. Furthermore, this work has shown important indicators of common pathways between depression and HF, two highly impactful comorbid diseases. It is expected that these findings may in the future contribute to the development of better therapeutic approaches and biomarkers that will help in the diagnosis of disease progression and the evaluation of treatment response in patients with this comorbidity

IL-1α promotes liver inflammation and necrosis during blood-stage Plasmodium chabaudi malaria.

Malaria causes hepatic inflammation and damage, which contribute to disease severity. The pro-inflammatory cytokine interleukin (IL)-1α is released by non-hematopoietic or hematopoietic cells during liver injury. This study established the role of IL-1α in the liver pathology caused by blood-stage P. chabaudi malaria. During acute infection, hepatic inflammation and necrosis were accompanied by NLRP3 inflammasome-independent IL-1α production. Systemically, IL-1α deficiency attenuated weight loss and hypothermia but had minor effects on parasitemia control. In the liver, the absence of IL-1α reduced the number of TUNEL+ cells and necrotic lesions. This finding was associated with a lower inflammatory response, including TNF-α production. The main source of IL-1α in the liver of infected mice was inflammatory cells, particularly neutrophils. The implication of IL-1α in liver inflammation and necrosis caused by P. chabaudi infection, as well as in weight loss and hypothermia, opens up new perspectives for improving malaria outcomes by inhibiting IL-1 signaling

A non-canonical sensing pathway mediates Plasmodium adaptation to amino acid deficiency

Eukaryotes have canonical pathways for responding to amino acid (AA) availability. Under AA-limiting conditions, the TOR complex is repressed, whereas the sensor kinase GCN2 is activated. While these pathways have been highly conserved throughout evolution, malaria parasites are a rare exception. Despite auxotrophic for most AA, Plasmodium does not have either a TOR complex nor the GCN2-downstream transcription factors. While Ile starvation has been shown to trigger eIF2α phosphorylation and a hibernation-like response, the overall mechanisms mediating detection and response to AA fluctuation in the absence of such pathways has remained elusive. Here we show that Plasmodium parasites rely on an efficient sensing pathway to respond to AA fluctuations. A phenotypic screen of kinase knockout mutant parasites identified nek4, eIK1 and eIK2—the last two clustering with the eukaryotic eIF2α kinases—as critical for Plasmodium to sense and respond to distinct AA-limiting conditions. Such AA-sensing pathway is temporally regulated at distinct life cycle stages, allowing parasites to actively fine-tune replication and development in response to AA availability. Collectively, our data disclose a set of heterogeneous responses to AA depletion in malaria parasites, mediated by a complex mechanism that is critical for modulating parasite growth and survival

Association of Plasmodium berghei With the Apical Domain of Hepatocytes Is Necessary for the Parasite's Liver Stage Development.

Plasmodium parasites undergo a dramatic transformation during the liver stage of their life cycle, amplifying over 10,000-fold inside infected hepatocytes within a few days. Such a rapid growth requires large-scale interactions with, and manipulations of, host cell functions. Whereas hepatocyte polarity is well-known to be critical for liver function, little is presently known about its involvement during the liver stage of Plasmodium development. Apical domains of hepatocytes are critical components of their polarity machinery and constitute the bile canalicular network, which is central to liver function. Here, we employed high resolution 3-D imaging and advanced image analysis of Plasmodium-infected liver tissues to show that the parasite associates preferentially with the apical domain of hepatocytes and induces alterations in the organization of these regions, resulting in localized changes in the bile canalicular architecture in the liver tissue. Pharmacological perturbation of the bile canalicular network by modulation of AMPK activity reduces the parasite's association with bile canaliculi and arrests the parasite development. Our findings using Plasmodium-infected liver tissues reveal a host-Plasmodium interaction at the level of liver tissue organization. We demonstrate for the first time a role for bile canaliculi, a central component of the hepatocyte polarity machinery, during the liver stage of Plasmodium development