Exploiting the intrinsic misfolding propensity of the KRAS oncoprotein.

Mutant KRAS is a major driver of oncogenesis in a multitude of cancers but remains a challenging target for classical small molecule drugs, motivating the exploration of alternative approaches. Here, we show that aggregation-prone regions (APRs) in the primary sequence of the oncoprotein constitute intrinsic vulnerabilities that can be exploited to misfold KRAS into protein aggregates. Conveniently, this propensity that is present in wild-type KRAS is increased in the common oncogenic mutations at positions 12 and 13. We show that synthetic peptides (Pept-ins™) derived from two distinct KRAS APRs could induce the misfolding and subsequent loss of function of oncogenic KRAS, both of recombinantly produced protein in solution, during cell-free translation and in cancer cells. The Pept-ins exerted antiproliferative activity against a range of mutant KRAS cell lines and abrogated tumor growth in a syngeneic lung adenocarcinoma mouse model driven by mutant KRAS G12V. These findings provide proof-of-concept that the intrinsic misfolding propensity of the KRAS oncoprotein can be exploited to cause its functional inactivation.

"Smart markets": harnessing the potential of new technologies for endemic and emerging infectious disease surveillance in traditional food markets.

Emerging and endemic zoonotic diseases continue to threaten human and animal health, our social fabric, and the global economy. Zoonoses frequently emerge from congregate interfaces where multiple animal species and humans coexist, including farms and markets. Traditional food markets are widespread across the globe and create an interface where domestic and wild animals interact among themselves and with humans, increasing the risk of pathogen spillover. Despite decades of evidence linking markets to disease outbreaks across the world, there remains a striking lack of pathogen surveillance programs that can relay timely, cost-effective, and actionable information to decision-makers to protect human and animal health. However, the strategic incorporation of environmental surveillance systems in markets coupled with novel pathogen detection strategies can create an early warning system capable of alerting us to the risk of outbreaks before they happen. Here, we explore the concept of "smart" markets that utilize continuous surveillance systems to monitor the emergence of zoonotic pathogens with spillover potential.IMPORTANCEFast detection and rapid intervention are crucial to mitigate risks of pathogen emergence, spillover and spread-every second counts. However, comprehensive, active, longitudinal surveillance systems at high-risk interfaces that provide real-time data for action remain lacking. This paper proposes "smart market" systems harnessing cutting-edge tools and a range of sampling techniques, including wastewater and air collection, multiplex assays, and metagenomic sequencing. Coupled with robust response pathways, these systems could better enable Early Warning and bolster prevention efforts.

Pestilence and famine: Continuing down the vicious cycle with COVID-19.

Despite the fact that we produce enough food to feed everyone on Earth, world hunger is on the rise. On the other side of the table, the obesity crisis also weighs heavily. Malnutrition is less about food than about socioeconomic factors such as conflict, poverty, and global disasters such as climate change and the novel Coronavirus Disease 2019 (COVID-19) pandemic. Nutrition and infectious disease exist in an intricate dance. Adequate and balanced nutrition is critical for appropriate response to infection and any changes in the balance can serve as a tipping point for the next pandemic. On the other hand, pandemics, such as COVID-19, lead to greater malnutrition. Both over- and undernutrition increase severity of disease, alter vaccine effectiveness, and potentially create conditions for viral mutation and adaptation-further driving the disease and famine vicious cycle. These long-term health and socioeconomic repercussions have direct effects at individual and global levels and lead to long-term consequences. Therefore, investing in and strengthening public health, pandemic prevention, and nutrition programs become vital at a much more complex systems level.

Detection of Clade 2.3.4.4b Avian Influenza A(H5N8) Virus in Cambodia, 2021.

In late 2021, highly pathogenic avian influenza A(H5N8) clade 2.3.4.4b viruses were detected in domestic ducks in poultry markets in Cambodia. Surveillance, biosafety, and biosecurity efforts should be bolstered along the poultry value chain to limit spread and infection risk at the animal-human interface.

Human Infection with Avian Influenza A(H9N2) Virus, Cambodia, February 2021.

In February 2021, routine sentinel surveillance for influenza-like illness in Cambodia detected a human avian influenza A(H9N2) virus infection. Investigations identified no recent H9N2 virus infections in 43 close contacts. One chicken sample from the infected child's house was positive for H9N2 virus and genetically similar to the human virus.

Incorporating heterogeneous sampling probabilities in continuous phylogeographic inference - Application to H5N1 spread in the Mekong region.

MOTIVATION: The potentially low precision associated with the geographic origin of sampled sequences represents an important limitation for spatially explicit (i.e. continuous) phylogeographic inference of fast-evolving pathogens such as RNA viruses. A substantial proportion of publicly available sequences is geo-referenced at broad spatial scale such as the administrative unit of origin, rather than more precise locations (e.g. geographic coordinates). Most frequently, such sequences are either discarded prior to continuous phylogeographic inference or arbitrarily assigned to the geographic coordinates of the centroid of their administrative area of origin for lack of a better alternative. RESULTS: We here implement and describe a new approach that allows to incorporate heterogeneous prior sampling probabilities over a geographic area. External data, such as outbreak locations, are used to specify these prior sampling probabilities over a collection of sub-polygons. We apply this new method to the analysis of highly pathogenic avian influenza H5N1 clade data in the Mekong region. Our method allows to properly include, in continuous phylogeographic analyses, H5N1 sequences that are only associated with large administrative areas of origin and assign them with more accurate locations. Finally, we use continuous phylogeographic reconstructions to analyse the dispersal dynamics of different H5N1 clades and investigate the impact of environmental factors on lineage dispersal velocities. AVAILABILITY AND IMPLEMENTATION: Our new method allowing heterogeneous sampling priors for continuous phylogeographic inference is implemented in the open-source multi-platform software package BEAST 1.10. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Synthetic Pept-Ins as a Generic Amyloid-Like Aggregation-Based Platform for In Vivo PET Imaging of Intracellular Targets.

Amyloid-like aggregation of proteins is induced by short amyloidogenic sequence segments within a specific protein sequence resulting in self-assembly into ß-sheets. We recently validated a technology platform in which synthetic amyloid peptides ("Pept-ins") containing a specific aggregation-prone region (APR) are used to induce specific functional knockdown of the target protein from which the APR was derived, including bacterial, viral, and mammalian cell proteins. In this work, we investigated if Pept-ins can be used as vector probes for in vivo Positron Emission Tomography (PET) imaging of intracellular targets. The radiolabeled Pept-ins [68Ga]Ga-NODAGA-PEG4-vascin (targeting VEGFR2) and [68Ga]Ga-NODAGA-PEG2-P2 (targeting E. coli) were evaluated as PET probes. The Pept-in based radiotracers were cross-validated in a murine tumor and muscle infection model, respectively, and were found to combine target specificity with favorable in vivo pharmacokinetics. When the amyloidogenicity of the interacting region of the peptide is suppressed by mutation, cellular uptake and in vivo accumulation are abolished, highlighting the importance of the specific design of synthetic Pept-ins. The ubiquity of target-specific amyloidogenic sequence segments in natural proteins, the straightforward sequence-based design of the Pept-in probes, and their spontaneous internalization by cells suggest that Pept-ins may constitute a generic platform for in vivo PET imaging of intracellular targets.

Emergence of Influenza A(H7N4) Virus, Cambodia.

Active surveillance in high-risk sites in Cambodia has identified multiple low-pathogenicity influenza A(H7) viruses, mainly in ducks. None fall within the A/Anhui/1/2013(H7N9) lineage; however, some A(H7) viruses from 2018 show temporal and phylogenetic similarity to the H7N4 virus that caused a nonfatal infection in Jiangsu Province, China, in December 2017.

Changing Geographic Patterns and Risk Factors for Avian Influenza A(H7N9) Infections in Humans, China.

The fifth epidemic wave of avian influenza A(H7N9) virus in China during 2016-2017 demonstrated a geographic range expansion and caused more human cases than any previous wave. The factors that may explain the recent range expansion and surge in incidence remain unknown. We investigated the effect of anthropogenic, poultry, and wetland variables on all epidemic waves. Poultry predictor variables became much more important in the last 2 epidemic waves than they were previously, supporting the assumption of much wider H7N9 transmission in the chicken reservoir. We show that the future range expansion of H7N9 to northern China may increase the risk of H7N9 epidemic peaks coinciding in time and space with those of seasonal influenza, leading to a higher risk of reassortments than before, although the risk is still low so far.

Nuclear inclusion bodies of mutant and wild-type p53 in cancer: a hallmark of p53 inactivation and proteostasis remodelling by p53 aggregation.

Although p53 protein aggregates have been observed in cancer cell lines and tumour tissue, their impact in cancer remains largely unknown. Here, we extensively screened for p53 aggregation phenotypes in tumour biopsies, and identified nuclear inclusion bodies (nIBs) of transcriptionally inactive mutant or wild-type p53 as the most frequent aggregation-like phenotype across six different cancer types. p53-positive nIBs co-stained with nuclear aggregation markers, and shared molecular hallmarks of nIBs commonly found in neurodegenerative disorders. In cell culture, tumour-associated stress was a strong inducer of p53 aggregation and nIB formation. This was most prominent for mutant p53, but could also be observed in wild-type p53 cell lines, for which nIB formation correlated with the loss of p53's transcriptional activity. Importantly, protein aggregation also fuelled the dysregulation of the proteostasis network in the tumour cell by inducing a hyperactivated, oncogenic heat-shock response, to which tumours are commonly addicted, and by overloading the proteasomal degradation system, an observation that was most pronounced for structurally destabilized mutant p53. Patients showing tumours with p53-positive nIBs suffered from a poor clinical outcome, similar to those with loss of p53 expression, and tumour biopsies showed a differential proteostatic expression profile associated with p53-positive nIBs. p53-positive nIBs therefore highlight a malignant state of the tumour that results from the interplay between (1) the functional inactivation of p53 through mutation and/or aggregation, and (2) microenvironmental stress, a combination that catalyses proteostatic dysregulation. This study highlights several unexpected clinical, biological and therapeutically unexplored parallels between cancer and neurodegeneration. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

PP2A regulatory subunit Bα controls endothelial contractility and vessel lumen integrity via regulation of HDAC7.

To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens requires the Bα regulatory subunit of protein phosphatase 2A (PP2A). Deficiency of Bα in zebrafish precludes vascular lumen stabilization resulting in perfusion defects. Similarly, inactivation of PP2A-Bα in cultured ECs induces tubulogenesis failure due to alteration of cytoskeleton dynamics, actomyosin contractility and maturation of cell-extracellular matrix (ECM) contacts. Mechanistically, we show that PP2A-Bα controls the activity of HDAC7, an essential transcriptional regulator of vascular stability. In the absence of PP2A-Bα, transcriptional repression by HDAC7 is abrogated leading to enhanced expression of the cytoskeleton adaptor protein ArgBP2. ArgBP2 hyperactivates RhoA causing inadequate rearrangements of the EC actomyosin cytoskeleton. This study unravels the first specific role for a PP2A holoenzyme in development: the PP2A-Bα/HDAC7/ArgBP2 axis maintains vascular lumens by balancing endothelial cytoskeletal dynamics and cell-matrix adhesion.

FAF1, a gene that is disrupted in cleft palate and has conserved function in zebrafish.

Cranial neural crest (CNC) is a multipotent migratory cell population that gives rise to most of the craniofacial bones. An intricate network mediates CNC formation, epithelial-mesenchymal transition, migration along distinct paths, and differentiation. Errors in these processes lead to craniofacial abnormalities, including cleft lip and palate. Clefts are the most common congenital craniofacial defects. Patients have complications with feeding, speech, hearing, and dental and psychological development. Affected by both genetic predisposition and environmental factors, the complex etiology of clefts remains largely unknown. Here we show that Fas-associated factor-1 (FAF1) is disrupted and that its expression is decreased in a Pierre Robin family with an inherited translocation. Furthermore, the locus is strongly associated with cleft palate and shows an increased relative risk. Expression studies show that faf1 is highly expressed in zebrafish cartilages during embryogenesis. Knockdown of zebrafish faf1 leads to pharyngeal cartilage defects and jaw abnormality as a result of a failure of CNC to differentiate into and express cartilage-specific markers, such as sox9a and col2a1. Administration of faf1 mRNA rescues this phenotype. Our findings therefore identify FAF1 as a regulator of CNC differentiation and show that it predisposes humans to cleft palate and is necessary for lower jaw development in zebrafish.

Reassortant avian influenza A(H5N1) viruses with H9N2-PB1 gene in poultry, Bangladesh.

Bangladesh has reported a high number of outbreaks of highly pathogenic avian influenza (HPAI) (H5N1) in poultry. We identified a natural reassortant HPAI (H5N1) virus containing a H9N2-PB1 gene in poultry in Bangladesh. Our findings highlight the risks for prolonged co-circulation of avian influenza viruses and the need to monitor their evolution.

Influx of Backyard Farming with Limited Biosecurity Due to the COVID-19 Pandemic Carries an Increased Risk of Zoonotic Spillover in Cambodia.

Backyard farming with limited biosecurity creates a massive potential for zoonotic spillover. Cambodia, a developing nation in Southeast Asia, is a hub for emerging and endemic infectious diseases. Due to pandemic-induced job losses in the tourism sector, rumors suggest that many former Cambodian tour guides have turned to backyard farming as a source of income and food security. A cross-sectional study including 331 tour guides and 69 poultry farmers in Cambodia before and during the novel coronavirus disease 2019 (COVID-19) pandemic was conducted. Participants were administered a survey to assess food security, income, and general farming practices. Survey data were collected to evaluate the risk perceptions for avian influenza virus (AIV), antimicrobial resistance (AMR), and general biosecurity management implemented on these poultry farms. Overall, food security decreased for 80.1% of the tour guides during the COVID-19 pandemic. Approximately 21% of the tour guides interviewed used backyard poultry farming to supplement losses of income and food insecurity during the COVID-19 pandemic, with a significantly higher risk than for traditional poultry farmers. Agricultural intensification in Cambodia due to the COVID-19 pandemic has caused an influx of makeshift farms with limited biosecurity. Inadequate biosecurity measures in animal farms can facilitate spillover and contribute to future pandemics. Improved biosecurity and robust viral surveillance systems are critical for reducing the risk of spillover from backyard farms. IMPORTANCE While this study highlights COVID-19-associated changes in poultry production at a small scale in Cambodia, poultry production is expected to expand due to an increase in the global demand for poultry protein during the pandemic, changes in urbanization, and the reduction of the global pork supply caused by African swine fever (ASF). The global demand and surge in poultry products, combined with inadequate biosecurity methods, can lead to an increased risk of domestic animal and human spillovers of zoonotic pathogens such as avian influenza. Countries in regions of endemicity are often plagued by complex emergency situations (i.e., food insecurity and economic fallouts) that hinder efforts to effectively address the emergence (or reemergence) of zoonotic diseases. Thus, novel surveillance strategies for endemic and emerging infectious diseases require robust surveillance systems and biosecurity training programs to prevent future global pandemics.

Reduced Levels of Misfolded and Aggregated Mutant p53 by Proteostatic Activation.

In malignant cancer, excessive amounts of mutant p53 often lead to its aggregation, a feature that was recently identified as druggable. Here, we describe that induction of a heat shock-related stress response mediated by Foldlin, a small-molecule tool compound, reduces the protein levels of misfolded/aggregated mutant p53, while contact mutants or wild-type p53 remain largely unaffected. Foldlin also prevented the formation of stress-induced p53 nuclear inclusion bodies. Despite our inability to identify a specific molecular target, Foldlin also reduced protein levels of aggregating SOD1 variants. Finally, by screening a library of 778 FDA-approved compounds for their ability to reduce misfolded mutant p53, we identified the proteasome inhibitor Bortezomib with similar cellular effects as Foldlin. Overall, the induction of a cellular heat shock response seems to be an effective strategy to deal with pathological protein aggregation. It remains to be seen however, how this strategy can be translated to a clinical setting.

Role of synectin in lymphatic development in zebrafish and frogs.

The molecular basis of lymphangiogenesis remains incompletely characterized. Here, we document a novel role for the PDZ domain-containing scaffold protein synectin in lymphangiogenesis using genetic studies in zebrafish and tadpoles. In zebrafish, the thoracic duct arises from parachordal lymphangioblast cells, which in turn derive from secondary lymphangiogenic sprouts from the posterior cardinal vein. Morpholino knockdown of synectin in zebrafish impaired formation of the thoracic duct, due to selective defects in lymphangiogenic but not angiogenic sprouting. Synectin genetically interacted with Vegfr3 and neuropilin-2a in regulating lymphangiogenesis. Silencing of synectin in tadpoles caused lymphatic defects due to an underdevelopment and impaired migration of Prox-1(+) lymphatic endothelial cells. Molecular analysis further revealed that synectin regulated Sox18-induced expression of Prox-1 and vascular endothelial growth factor C-induced migration of lymphatic endothelial cells in vitro. These findings reveal a novel role for synectin in lymphatic development.

Sero-epidemiological evaluation of changes in Plasmodium falciparum and Plasmodium vivax transmission patterns over the rainy season in Cambodia.

BACKGROUND: In Cambodia, malaria transmission is low and most cases occur in forested areas. Sero-epidemiological techniques can be used to identify both areas of ongoing transmission and high-risk groups to be targeted by control interventions. This study utilizes repeated cross-sectional data to assess the risk of being malaria sero-positive at two consecutive time points during the rainy season and investigates who is most likely to sero-convert over the transmission season. METHODS: In 2005, two cross-sectional surveys, one in the middle and the other at the end of the malaria transmission season, were carried out in two ecologically distinct regions in Cambodia. Parasitological and serological data were collected in four districts. Antibodies to Plasmodium falciparum Glutamate Rich Protein (GLURP) and Plasmodium vivax Merozoite Surface Protein-1(19) (MSP-1(19)) were detected using Enzyme Linked Immunosorbent Assay (ELISA). The force of infection was estimated using a simple catalytic model fitted using maximum likelihood methods. Risks for sero-converting during the rainy season were analysed using the Classification and Regression Tree (CART) method. RESULTS: A total of 804 individuals participating in both surveys were analysed. The overall parasite prevalence was low (4.6% and 2.0% for P. falciparum and 7.9% and 6.0% for P. vivax in August and November respectively). P. falciparum force of infection was higher in the eastern region and increased between August and November, whilst P. vivax force of infection was higher in the western region and remained similar in both surveys. In the western region, malaria transmission changed very little across the season (for both species). CART analysis for P. falciparum in the east highlighted age, ethnicity, village of residence and forest work as important predictors for malaria exposure during the rainy season. Adults were more likely to increase their antibody responses to P. falciparum during the transmission season than children, whilst members of the Charay ethnic group demonstrated the largest increases. DISCUSSION: In areas of low transmission intensity, such as in Cambodia, the analysis of longitudinal serological data enables a sensitive evaluation of transmission dynamics. Consecutive serological surveys allow an insight into spatio-temporal patterns of malaria transmission. The use of CART enabled multiple interactions to be accounted for simultaneously and permitted risk factors for exposure to be clearly identified.

Unexpected Rift Valley fever outbreak, northern Mauritania.

During September-October 2010, an unprecedented outbreak of Rift Valley fever was reported in the northern Sahelian region of Mauritania after exceptionally heavy rainfall. Camels probably played a central role in the local amplification of the virus. We describe the main clinical signs (hemorrhagic fever, icterus, and nervous symptoms) observed during the outbreak.

Selective regulation of arterial branching morphogenesis by synectin.

Branching morphogenesis is a key process in the formation of vascular networks. To date, little is known regarding the molecular events regulating this process. We investigated the involvement of synectin in this process. In zebrafish embryos, synectin knockdown resulted in a hypoplastic dorsal aorta and hypobranched, stunted, and thin intersomitic vessels due to impaired migration and proliferation of angioblasts and arterial endothelial cells while not affecting venous development. Synectin(-/-) mice demonstrated decreased body and organ size, reduced numbers of arteries, and an altered pattern of arterial branching in multiple vascular beds while the venous system remained normal. Murine synectin(-/-) primary arterial, but not venous, endothelial cells showed decreased in vitro tube formation, migration, and proliferation and impaired polarization due to abnormal localization of activated Rac1. We conclude that synectin is involved in selective regulation of arterial, but not venous, growth and branching morphogenesis and that Rac1 plays an important role in this process.