A novel kind of immobilized trypsin reactor based on organic-inorganic hybrid silica monoliths has been developed. With the presence of cetyltrimethyl ammonium bromide (CTAB) in the polymerization mixture, the hybrid silica monolithic support was prepared in a 100 microm i.d. capillary by the sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, the monolith was activated by glutaraldehyde, and trypsin was covalently immobilized. By monitoring the reaction of a decapeptide, C-myc (EQKLISEEDL), the enzymatic activity of the immobilized trypsin was calculated, and the results showed that the digestion speed was about 6600 times faster than that performed in free solution. The performance of such a microreactor was further demonstrated by digesting myoglobin, with the digested products analyzed by microflow reversed-phase liquid chromatography coupled with tandem mass spectrometry (microRPLC-MS/MS). With a stringent threshold for the unambiguous identification of the digests, the yielding sequence coverage for on-column digestion was 92%, the same as that obtained by in-solution digestion, whereas the residence time of myoglobin in the former case was only 30 s, about 1/1440 of that performed in the latter case (12 h). Moreover, such an immobilized trypsin reactor was also successfully applied to the digestion of a mixture of model proteins and proteins extracted from E. coli.

The innate immune response is highly conserved across all eukaryotes and has been studied in great detail in several model organisms. Hemocytes, the primary immune cell population in mosquitoes, are important components of the mosquito innate immune response, yet critical aspects of their biology have remained uncharacterized. Using a novel method of enrichment, we isolated phagocytic granulocytes and quantified their proteomes by mass spectrometry. The data demonstrate that phagocytosis, blood-feeding, and Plasmodium falciparum infection promote dramatic shifts in the proteomic profiles of An. gambiae granulocyte populations. Of interest, large numbers of immune proteins were induced in response to blood feeding alone, suggesting that granulocytes have an integral role in priming the mosquito immune system for pathogen challenge. In addition, we identify several granulocyte proteins with putative roles as membrane receptors, cell signaling, or immune components that when silenced, have either positive or negative effects on malaria parasite survival. Integrating existing hemocyte transcriptional profiles, we also compare differences in hemocyte transcript and protein expression to provide new insight into hemocyte gene regulation and discuss the potential that post-transcriptional regulation may be an important component of hemocyte gene expression. These data represent a significant advancement in mosquito hemocyte biology, providing the first comprehensive proteomic profiling of mosquito phagocytic granulocytes during homeostasis blood-feeding, and pathogen challenge. Together, these findings extend current knowledge to further illustrate the importance of hemocytes in shaping mosquito innate immunity and their principal role in defining malaria parasite survival in the mosquito host.

One of the critical gaps in malaria transmission biology and surveillance is our lack of knowledge about Plasmodium falciparum gametocyte biology, especially sexual dimorphic development and how sex ratios that may influence transmission from the human to the mosquito. Dissecting this process has been hampered by the lack of sex-specific protein markers for the circulating, mature stage V gametocytes. The current evidence suggests a high degree of conservation in gametocyte gene complement across Plasmodium, and therefore presumably for sex-specific genes as well. To better our understanding of gametocyte development and subsequent infectiousness to mosquitoes, we undertook a Systematic Subtractive Bioinformatic analysis (filtering) approach to identify sex-specific P. falciparum NF54 protein markers based on a comparison with the Dd2 strain, which is defective in producing males, and with syntenic male and female proteins from the reanalyzed and updated P. berghei (related rodent malaria parasite) gametocyte proteomes. This produced a short list of 174 male- and 258 female-enriched P. falciparum stage V proteins, some of which appear to be under strong diversifying selection, suggesting ongoing adaptation to mosquito vector species. We generated antibodies against three putative female-specific gametocyte stage V proteins in P. falciparum and confirmed either conserved sex-specificity or the lack of cross-species sex-partitioning. Finally, our study provides not only an additional resource for mass spectrometry-derived evidence for gametocyte proteins but also lays down the foundation for rational screening and development of novel sex-partitioned protein biomarkers and transmission-blocking vaccine candidates.

A novel kind of immobilized metal affinity chromatography (IMAC) column based on organic-inorganic hybrid silica monolith has been developed. The monolithic support was prepared in a 250 microm i.d. capillary by the sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, amine groups were functionalized by glutaraldehyde, and then activated with (aminomethyl) phosphonic acid, followed by Ti(4+) chelation. By such a hybrid silica monolithic Ti(4+)-IMAC column, 15 phosphopeptides were effectively isolated from the digest mixture of alpha-casein and BSA with the molar ratio as low as 1:200, illustrating its superior selectivity. With a synthetic phosphorylated peptide, YKVPQLEIVPNSpAEER, as the sample, the loading capacity and recovery of the Ti(4+)-IMAC monolithic column were measured to be 1.4 micromol/mL and 69%, respectively. Such an IMAC monolithic column was further applied to enrich phosphopeptides from rat liver mitochondria. In total, 224 unique phosphopeptides, corresponding to 148 phosphoprotein groups, were identified by duplicate nanoRPLC-LTQ MS/MS/MS runs with a false-positive rate of less than 1% at the peptide level. These results demonstrate that the hybrid silica monolith based Ti(4+)-IMAC column might provide a promising tool for large-scale phosphopeptide enrichment, facilitating the in-depth understanding of the biological functions of phosphoproteomes.

Clinical translation of human pluripotent stem cells (hPSCs) requires advanced strategies that ensure safe and robust long-term growth and functional differentiation. Pluripotent cells are capable of extensive self-renewal, yet remain highly sensitive to environmental perturbations in vitro , posing challenges to their therapeutic use. Here, we deployed innovative high-throughput screening strategies to identify a small molecule cocktail that dramatically improves viability of hPSCs and their differentiated progeny. The combination of Chroman 1, Emricasan, Polyamines, and Trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT provided strong improvements for several key applications in stem cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and genome editing. Thus, CEPT represents a unique polypharmacology strategy for comprehensive cytoprotection, providing a new rationale for efficient and safe utilization of hPSCs. Conferring cell fitness by multi-target drug combinations may become a common approach in cryobiology, drug development, and regenerative medicine.

Mosquito-based malaria transmission-blocking vaccines (mTBVs) target midgut-surface antigens of the Plasmodium parasite's obligate vector, the Anopheles mosquito. The alanyl aminopeptidase N (AnAPN1) is the leading mTBV immunogen; however AnAPN1's role in Plasmodium infection of the mosquito and how anti-AnAPN1 antibodies functionally block parasite transmission remains elusive. Here we present the 2.65 Å crystal structure of AnAPN1 and the immunoreactivity and transmission-blocking profile of three AnAPN1 monoclonal antibodies (mAb), including mAb 4H5B7, which effectively block transmission of natural strains of Plasmodium falciparum. Utilizing the AnAPN1 structure we map the conformation-dependent 4H5B7 neo-epitope to a previously uncharacterized region on domain 1, and further demonstrate that non-human primate neo-epitope-specific IgG also block parasite transmission. We discuss the Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Correspondence should be addressed to: Natalie A. Borg, PhD., Department of Biochemistry and Molecular Biology, Monash University, Clayton, 3800, Victoria, Australia, natalie.borg@monash.edu, Tel: +613-9902-9369, Fax: +613-9902-9500; Rhoel R. HHS Public AccessAuthor manuscript Nat Struct Mol Biol. Author manuscript; available in PMC 2016 January 01. Published in final edited form as:Nat Struct Mol Biol. 2015 July ; 22(7): 532-539. doi:10.1038/nsmb.3048. Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript prospect of a novel biological function of AnAPN1 as a receptor for Plasmodium in the mosquito midgut and the implications for redesigning the AnAPN1 mTBV.Malaria exacts a deadly toll on human populations worldwide. A new era in the fight against the disease has seen multiple malaria vaccines entering or completing advanced clinical study 1 , including malaria transmission-blocking vaccines (mTBVs) 2 . Malaria transmission requires the establishment of Plasmodium in the Anopheles mosquito, which is contingent on the parasite's ookinete stage successfully traversing the midgut epithelium to initiate sporogonic development 3,4 . TBVs disrupt this obligatory step in the parasite life cycle 5 , limiting the number of infectious mosquito vectors and introducing local herd immunity 6 . The concept of mTBVs is simple: antibodies against specific, mosquito midgut antigens circulating in the peripheral blood are ingested by the mosquito while feeding on immunized hosts. These antibodies, as well as complement, can survive in the mosquito midgut for up to 24 hours post-blood feeding and prevent parasite access to host ligands that mediate midgut cell adhesion and invasion. Unable to establish infection in the vector, progression of parasite development and transmission to new human hosts is arrested or reduced.We have shown that AnAPN1, an alanyl aminopeptidase N present on...

The recent decline in global malaria burden has stimulated efforts toward Plasmodium falciparum elimination. Understanding the biology of malaria transmission stages may provide opportunities to reduce or prevent onward transmission to mosquitoes. Immature P. falciparum transmission stages, termed stages I to IV gametocytes, sequester in human bone marrow before release into the circulation as mature stage V gametocytes. This process likely involves interactions between host receptors and potentially immunogenic adhesins on the infected red blood cell (iRBC) surface. Here, we developed a flow cytometry assay to examine immune recognition of live gametocytes of different developmental stages by naturally exposed Malawians. We identified strong antibody recognition of the earliest immature gametocyte-iRBCs (giRBCs) but not mature stage V giRBCs. Candidate surface antigens (n = 30), most of them shared between asexual- and gametocyte-iRBCs, were identified by mass spectrometry and mouse immunizations, as well as correlations between responses by protein microarray and flow cytometry. Naturally acquired responses to a subset of candidate antigens were associated with reduced asexual and gametocyte density, and plasma samples from malaria-infected individuals were able to induce immune clearance of giRBCs in vitro. Infected RBC surface expression of select candidate antigens was validated using specific antibodies, and genetic analysis revealed a subset with minimal variation across strains. Our data demonstrate that humoral immune responses to immature giRBCs and shared iRBC antigens are naturally acquired after malaria exposure. These humoral immune responses may have consequences for malaria transmission potential by clearing developing gametocytes, which could be leveraged for malaria intervention.

A large proportion of ongoing malaria parasite transmission is attributed to low-density subclinical infections not readily detected by available rapid diagnostic tests (RDTs) or microscopy.Plasmodium falciparumgametocyte carriage is subclinical, but gametocytemic individuals comprise the parasite reservoir that leads to infection of mosquitoes and local transmission. Effective detection and quantification of these carriers can help advance malaria elimination strategies. However, no point-of-need (PON) RDTs for gametocyte detection exist, much less one that can perform noninvasive sampling of saliva outside a clinical setting. Here, we report on the discovery of 35 parasite markers from which we selected a single candidate for use in a PON RDT. We performed a cross-sectional, multi-omics study of saliva from 364 children with subclinical infection in Cameroon and Zambia and produced a prototype saliva-based PON lateral flow immunoassay test forP. falciparumgametocyte carriers. The test is capable of identifying submicroscopic carriage in both clinical and nonclinical settings and is compatible with archived saliva samples.