Recent Advances in Aptamer Discovery and Applications.

Aptamers are short, single-stranded DNA, RNA, or synthetic XNA molecules that can be developed with high affinity and specificity to interact with any desired targets. They have been widely used in facilitating discoveries in basic research, ensuring food safety and monitoring the environment. Furthermore, aptamers play promising roles as clinical diagnostics and therapeutic agents. This review provides update on the recent advances in this rapidly progressing field of research with particular emphasis on generation of aptamers and their applications in biosensing, biotechnology and medicine. The limitations and future directions of aptamers in target specific delivery and real-time detection are also discussed.

Molecular target validation, antimicrobial delivery, and potential treatment of Toxoplasma gondii infections.

Toxoplasma gondii persistently infects over two billion people worldwide. It can cause substantial morbidity and mortality. Existing treatments have associated toxicities and hypersensitivity and do not eliminate encysted bradyzoites that recrudesce. New, improved medicines are needed. Transductive peptides carry small molecule cargos across multiple membranes to enter intracellular tachyzoites and encysted bradyzoites. They also carry cargos into retina when applied topically to eyes, and cross blood brain barrier when administered intravenously. Phosphorodiamidate morpholino oligomers (PMO) inhibit gene expression in a sequence-specific manner. Herein, effect of transductive peptide conjugated PMO (PPMO) on tachyzoite protein expression and replication in vitro and in vivo was studied. Initially, sequence-specific PPMO successfully reduced transfected T. gondii's fluorescence and luminescence. PPMO directed against T. gondii's dihydrofolate reductase (DHFR), an enzyme necessary for folate synthesis, limited tachyzoite replication. Rescue with exogenous folate demonstrated DHFR PPMO's specificity. PPMO directed against enoyl-ACP reductase (ENR), an enzyme of type II fatty acid synthesis that is structurally distinct in T. gondii from ENR in mammalian cells was investigated. PPMO directed against plant-like Apetela 2 (AP2) domain transcription factor XI-3 (AP2XI-3), not present in human cells, was characterized. ENR and AP2XI-3 PPMO each restricted intracellular parasite replication validating these molecular targets in tachyzoites. DHFR-specific PPMO administered to infected mice diminished parasite burden. Thus, these antisense oligomers are a versatile approach to validate T. gondii molecular targets, reduce essential T. gondii proteins in vitro and in vivo, and have potential for development as curative medicines.

Spiroindolone that inhibits PfATPase4 is a potent, cidal inhibitor of Toxoplasma gondii tachyzoites in vitro and in vivo.

Here, we show that spiroindolone, an effective treatment for plasmodia, is also active against Toxoplasma gondii tachyzoites. In vitro, spiroindolone NITD609 is cidal for tachyzoites (50% inhibitory concentration [IC50], 1µM) and not toxic to human cells at ≥10µM. Two daily oral doses of 100 mg/kg of body weight reduced the parasite burden in mice by 90% (P=0.002), measured 3 days after the last dose. This inhibition of T. gondii tachyzoites in vitro and in vivo indicates that spiroindolone is a promising lead candidate for further medicine development.

Design, synthesis, and biological activity of diaryl ether inhibitors of Toxoplasma gondii enoyl reductase.

Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan's poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC50 value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the B-ring modifications have additional interactions with the strongly conserved Asn130.

Deep learning for diagnosis of acute promyelocytic leukemia via recognition of genomically imprinted morphologic features.

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML), classified by a translocation between chromosomes 15 and 17 [t(15;17)], that is considered a true oncologic emergency though appropriate therapy is considered curative. Therapy is often initiated on clinical suspicion, informed by both clinical presentation as well as direct visualization of the peripheral smear. We hypothesized that genomic imprinting of morphologic features learned by deep learning pattern recognition would have greater discriminatory power and consistency compared to humans, thereby facilitating identification of t(15;17) positive APL. By applying both cell-level and patient-level classification linked to t(15;17) PML/RARA ground-truth, we demonstrate that deep learning is capable of distinguishing APL in both discovery and prospective independent cohort of patients. Furthermore, we extract learned information from the trained network to identify previously undescribed morphological features of APL. The deep learning method we describe herein potentially allows a rapid, explainable, and accurate physician-aid for diagnosing APL at the time of presentation in any resource-poor or -rich medical setting given the universally available peripheral smear.

Deactivation and mislocalization of Toxoplasma gondii rhoptry protein 18 induced by a single amino acid mutation on the proton transport catalytic aspartic acid.

Rhoptry protein 18 (ROP18) is a major determinant of strain-specific virulence in Toxoplasma gondii. The kinase activity of ROP18 is required for acute virulence, while the aspartate in the catalytic loop of ROP18 is considered essential for phosphoryl transfer. We showed that a single amino acid mutation at the catalytic aspartate residue (D409A mutation) significantly altered ROP18 kinase activity in vitro, and abolished ROP18-mediated ATF6ß degradation. Furthermore, the investigated single amino acid mutation in ROP18 led to alternation of subcellular localization of ROP18 protein. Our findings demonstrate that a single amino acid mutation on the proton transport catalytic aspartic acid induced alternations associated with ROP18 protein.

Toxoplasma gondii secretory proteins and their role in invasion and pathogenesis.

T. gondii is a major opportunistic pathogen chronically infecting nearly one third of the world's population. Due to the high infection and mortality rates in immunocompromised patients and newborns, the extent or magnitude of T. gondii pathogenesis is determined mainly by host-pathogen interactions. T. gondii utilizes specialized secretory proteins to modify host cellular factors and facilitate invasion and replication. This review provides update on the recent progress in this field of research with particular emphasis on the T. gondii secretory proteins and their role in invasion and pathogenesis.

Multilayer network analysis of miRNA and protein expression profiles in breast cancer patients.

MiRNAs and proteins play important roles in different stages of breast tumor development and serve as biomarkers for the early diagnosis of breast cancer. A new algorithm that combines machine learning algorithms and multilayer complex network analysis is hereby proposed to explore the potential diagnostic values of miRNAs and proteins. XGBoost and random forest algorithms were employed to screen the most important miRNAs and proteins. Maximal information coefficient was applied to assess intralayer and interlayer connection. A multilayer complex network was constructed to identify miRNAs and proteins that could serve as biomarkers for breast cancer. Proteins and miRNAs that are nodes in the network were subsequently categorized into two network layers considering their distinct functions. The betweenness centrality was used as the first measurement of the importance of the nodes within each single layer. The degree of the nodes was chosen as the second measurement to map their signalling pathways. By combining these two measurements into one score and comparing the difference of the same candidate between normal tissue and cancer tissue, this novel multilayer network analysis could be applied to successfully identify molecules associated with breast cancer.

Point-of-care testing for Toxoplasma gondii IgG/IgM using Toxoplasma ICT IgG-IgM test with sera from the United States and implications for developing countries.

BACKGROUND: Congenital toxoplasmosis is a serious but preventable and treatable disease. Gestational screening facilitates early detection and treatment of primary acquisition. Thus, fetal infection can be promptly diagnosed and treated and outcomes can be improved. METHODS: We tested 180 sera with the Toxoplasma ICT IgG-IgM point-of-care (POC) test. Sera were from 116 chronically infected persons (48 serotype II; 14 serotype I-III; 25 serotype I-IIIa; 28 serotype Atypical, haplogroup 12; 1 not typed). These represent strains of parasites infecting mothers of congenitally infected children in the U.S. 51 seronegative samples and 13 samples from recently infected persons known to be IgG/IgM positive within the prior 2.7 months also were tested. Interpretation was confirmed by two blinded observers. A comparison of costs for POC vs. commercial laboratory testing methods was performed. RESULTS: We found that this new Toxoplasma ICT IgG-IgM POC test was highly sensitive (100%) and specific (100%) for distinguishing IgG/IgM-positive from negative sera. Use of such reliable POC tests can be cost-saving and benefit patients. CONCLUSIONS: Our work demonstrates that the Toxoplasma ICT IgG-IgM test can function reliably as a point-of-care test to diagnose Toxoplasma gondii infection in the U.S. This provides an opportunity to improve maternal-fetal care by using approaches, diagnostic tools, and medicines already available. This infection has serious, lifelong consequences for infected persons and their families. From the present study, it appears a simple, low-cost POC test is now available to help prevent morbidity/disability, decrease cost, and make gestational screening feasible. It also offers new options for improved prenatal care in low- and middle-income countries.

Modification of triclosan scaffold in search of improved inhibitors for enoyl-acyl carrier protein (ACP) reductase in Toxoplasma gondii.

Through our focused effort to discover new and effective agents against toxoplasmosis, a structure-based drug design approach was used to develop a series of potent inhibitors of the enoyl-acyl carrier protein (ACP) reductase (ENR) enzyme in Toxoplasma gondii (TgENR). Modifications to positions 5 and 4' of the well-known ENR inhibitor triclosan afforded a series of 29 new analogues. Among the resulting compounds, many showed high potency and improved physicochemical properties in comparison with the lead. The most potent compounds 16 a and 16 c have IC50 values of 250 nM against Toxoplasma gondii tachyzoites without apparent toxicity to the host cells. Their IC50 values against recombinant TgENR were found to be 43 and 26 nM, respectively. Additionally, 11 other analogues in this series had IC50 values ranging from 17 to 130 nM in the enzyme-based assay. With respect to their excellent in vitro activity as well as improved drug-like properties, the lead compounds 16 a and 16 c are deemed to be excellent starting points for the development of new medicines to effectively treat Toxoplasma gondii infections.