Multidimensional ultrashort optical pulse manipulation using spatial light modulation.

Ultrashort optical pulse manipulation is one of the key techniques for applications such as high-speed imaging and high-precision laser processing. In this study, we demonstrate the multidimensional manipulation of ultrashort optical pulses by integrating spatial dispersion and spatial light modulation. Specifically, by modulating the phase of each wavelength, we achieve arbitrary adjustments in multiple dimensions, including number of sub pulses, time interval, intensity, and pulse width simultaneously and independently with a simple setup and few calculations. The performance of the optical pulse manipulation method is verified through both numerical simulations and experiments.

Optofluidic time-stretch imaging flow cytometry with a real-time storage rate beyond 5.9 GB/s.

Optofluidic time-stretch imaging flow cytometry (OTS-IFC) provides a suitable solution for high-precision cell analysis and high-sensitivity detection of rare cells due to its high-throughput and continuous image acquisition. However, transferring and storing continuous big data streams remains a challenge. In this study, we designed a high-speed streaming storage strategy to store OTS-IFC data in real-time, overcoming the imbalance between the fast generation speed in the data acquisition and processing subsystem and the comparatively slower storage speed in the transmission and storage subsystem. This strategy, utilizing an asynchronous buffer structure built on the producer-consumer model, optimizes memory usage for enhanced data throughput and stability. We evaluated the storage performance of the high-speed streaming storage strategy in ultra-large-scale blood cell imaging on a common commercial device. The experimental results show that it can provide a continuous data throughput of up to 5891 MB/s.

An optimized PDMS microfluidic device for ultra-fast and high-throughput imaging flow cytometry.

Imaging flow cytometry (IFC) is a powerful tool for cell detection and analysis due to its high throughput and compatibility in image acquisition. Optical time-stretch (OTS) imaging is considered as one of the most promising imaging techniques for IFC because it can realize cell imaging at a flow speed of around 60 m s-1. However, existing PDMS-based microchannels cannot function at flow velocities higher than 10 m s-1; thus the capability of OTS-based IFC is significantly limited. To overcome the velocity barrier for PDMS-based microchannels, we proposed an optimized design of PDMS-based microchannels with reduced hydraulic resistance and 3D hydrodynamic focusing capability, which can drive fluids at an ultra-high flow velocity (of up to 40 m s-1) by using common syringe pumps. To verify the feasibility of our design, we fabricated and installed the microchannel in an OTS IFC system. The experimental results first proved that the proposed microchannel can support a stable flow velocity of up to 40 m s-1 without any leakage or damage. Then, we demonstrated that the OTS IFC is capable of imaging cells at a velocity of up to 40 m s-1 with good quality. To the best of our knowledge, it is the first time that IFC has achieved such a high flow velocity just by using a PDMS-glass chip. Moreover, high velocity can enhance the focusing of cells on the optical focal plane, increasing the number of detected cells and the throughput. This work provides a promising solution for IFC to fully release its capability of advanced imaging techniques by operating at an extremely high screening throughput.

Differential Susceptibility of Aedes aegypti and Aedes albopictus Mosquitoes to Infection by Mayaro Virus Strains.

Mayaro virus (MAYV) is an arthropod-borne virus (arbovirus) belonging to the family Togaviridae, genus Alphavirus. In recent years, the geographic distribution of MAYV may have expanded north from South and Central America into the Caribbean Islands. Although Haemagogus janthinomys is considered the main vector for MAYV, the virus has also been isolated from other mosquitoes, including Aedes aegypti, a widespread species that serves as the main vector for highly epidemic viruses. Given the possible expansion and outbreaks of MAYV in Latin America, it is possible that MAYV might be adapting to be efficiently transmitted by urban vectors. Therefore, to investigate this possibility, we evaluated the vector competence of Ae. aegypti and Ae. albopictus mosquitoes to transmit MAYV isolated during a year of low or high MAYV transmission. Adult Ae. aegypti and Ae. albopictus were orally infected with the MAYV strains, and the infection, dissemination, and transmission rates were calculated to evaluate their vector competence. Overall, we found higher infection, dissemination, and transmission rates in both Ae. aegypti and Ae. albopictus mosquitoes infected with the strain isolated during a MAYV outbreak, whereas low/no transmission was detected with the strain isolated during a year of low MAYV activity. Our results confirmed that both Ae. aegypti and Ae. albopictus are competent vectors for the emergent MAYV. Our data suggest that strains isolated during MAYV outbreaks might be better fit to infect and be transmitted by urban vectors, raising serious concern about the epidemic potential of MAYV.

Evolution of Cell-Type-Specific RNA Aptamers via Live Cell-Based SELEX.

Live cell-based SELEX (Systematic Evolution of Ligand EXponential enrichment) is a promising approach for identifying aptamers that can selectively bind to a cell-surface receptor or recognize a particular target cell population. In particular, it offers a facile selection strategy for some special cell-surface proteins that are originally glycosylated or heavily posttranslationally modified and are unavailable in their native/active conformation after in vitro expression and purification. In this chapter, we describe a generalized procedure for evolution of cell type-specific RNA aptamers targeting a cell membrane bound target by combining the live cell-based SELEX strategy with high-throughput sequencing (HTS) and bioinformatics analysis.

Hybrid-plane spectrum slicing for sequentially timed all-optical mapping photography.

Sequentially timed all-optical mapping photography (STAMP) is an effective tool for observing ultrafast and non-repetitive events. In the classical design of STAMP, the spatial resolution of the acquired images is different in two directions, severely limiting the scalability of STAMP. Here, by introducing an asymmetric optical design, we make the slicing mirror locate in the hybrid plane of the system, i.e., the image plane in the direction of the short edge, while the Fourier plane is in the direction of the long edge. This avoids the loss of the high-frequency components of the images and hence offers the possibility to further extend the frame number of the system.

Alternative polyadenylation dysregulation contributes to the differentiation block of acute myeloid leukemia.

Posttranscriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among posttranscriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in myeloid leukemia. Furthermore, it is poorly understood how altered poly(A) site usage of individual genes contributes to malignancy or whether targeting global APA patterns might alter oncogenic potential. In this study, we examined global APA dysregulation in patients with acute myeloid leukemia (AML) by performing 3' region extraction and deep sequencing (3'READS) on a subset of AML patient samples along with healthy hematopoietic stem and progenitor cells (HSPCs) and by analyzing publicly available data from a broad AML patient cohort. We show that patient cells exhibit global 3' untranslated region (UTR) shortening and coding sequence lengthening due to differences in poly(A) site (PAS) usage. Among APA regulators, expression of FIP1L1, one of the core cleavage and polyadenylation factors, correlated with the degree of APA dysregulation in our 3'READS data set. Targeting global APA by FIP1L1 knockdown reversed the global trends seen in patients. Importantly, FIP1L1 knockdown induced differentiation of t(8;21) cells by promoting 3'UTR lengthening and downregulation of the fusion oncoprotein AML1-ETO. In non-t(8;21) cells, FIP1L1 knockdown also promoted differentiation by attenuating mechanistic target of rapamycin complex 1 (mTORC1) signaling and reducing MYC protein levels. Our study provides mechanistic insights into the role of APA in AML pathogenesis and indicates that targeting global APA patterns can overcome the differentiation block in patients with AML.

Clinical Characteristics, Treatment Effectiveness, and Predictors of Response to Pharmacotherapeutic Interventions Among Patients with Herpetic-Related Neuralgia: A Retrospective Analysis.

BACKGROUND: The treatment for herpetic-related neuralgia focuses on symptom control by use of antiviral drugs, anticonvulsants, and tricyclic antidepressants. We aimed to explore the clinical characteristics associated with medication responsiveness, and to build a classifier for identification of patients who have risk of inadequate pain management. METHODS: We recruited herpetic-related neuralgia patients during a 3-year period. Patients were stratified into a medication-resistant pain (MRP) group when the pain decrease in the visual analogue scale (VAS) is < 3 points, and otherwise a medication-sensitive pain (MSP) group. Multivariate logistic regression was performed to determine the factors associated with MRP. We fitted four machine learning (ML) models, namely logistic regression, random forest, supporting vector machines (SVM), and naïve Bayes with clinical characteristics gathered at admission to identify patients with MRP. RESULTS: A total of 213 patients were recruited, and 132 (61.97%) patients were diagnosed with MRP. Subacute herpes zoster (HZ) (vs. acute, OR 8.95, 95% CI 3.15-29.48, p = 0.0001), severe lesion (vs. mild lesion, OR 3.84, 95% CI 1.44-10.81, p = 0.0084), depressed mood (unit increase OR 1.10, 95% CI 1.00-1.20, p = 0.0447), and hypertension (hypertension, vs. no hypertension, OR 0.36, 95% CI 0.14-0.87, p = 0.0266) were significantly associated with MRP. Among four ML models, SVM had the highest accuracy (0.917) and receiver operating characteristic-area under the curve (0.918) to discriminate MRP from MSP. Phase of disease is the most important feature when fitting ML models. CONCLUSIONS: Clinical characteristics collected before treatment could be adopted to identify patients with MRP.

MicroRNA let-7b downregulates AML1-ETO oncogene expression in t(8;21) AML by targeting its 3'UTR.

BACKGROUND: Acute myeloid leukemia (AML) with the t(8;21)(q22;q22) chromosomal translocation is among the most common subtypes of AML and produces the AML1-ETO (RUNX1-ETO, RUNX1-RUNX1T1) oncogenic fusion gene. AML1-ETO functions as an aberrant transcription factor which plays a key role in blocking normal hematopoiesis. Thus, the expression of AML1-ETO is critical to t(8;21) AML leukemogenesis and maintenance. Post-transcriptional regulation of gene expression is often mediated through interactions between trans-factors and cis-elements within transcript 3'-untranslated regions (UTR). AML1-ETO uses the 3'UTR of the ETO gene, which is not normally expressed in hematopoietic cells. Therefore, the mechanisms regulating AML1-ETO expression via the 3'UTR are attractive therapeutic targets. METHODS: We used RNA-sequencing of t(8;21) patients and cell lines to examine the 3'UTR isoforms used by AML1-ETO transcripts. Using luciferase assay approaches, we test the relative contribution of 3'UTR cis elements to AML1-ETO expression. We further use let-7b microRNA mimics and anti-let-7b sponges for functional studies of t(8;21) AML cell lines. RESULTS: In this study, we examine the regulation of AML1-ETO via the 3'UTR. We demonstrate that AML1-ETO transcripts primarily use a 3.7 kb isoform of the ETO 3'UTR in both t(8;21) patients and cell lines. We identify a negative regulatory element within the AML1-ETO 3'UTR. We further demonstrate that the let-7b microRNA directly represses AML1-ETO through this site. Finally, we find that let-7b inhibits the proliferation of t(8;21) AML cell lines, rescues expression of AML1-ETO target genes, and promotes differentiation. CONCLUSIONS: AML1-ETO is post-transcriptionally regulated by let-7b, which contributes to the leukemic phenotype of t(8;21) AML and may be important for t(8;21) leukemogenesis and maintenance.

Synthesis and use of an amphiphilic dendrimer for siRNA delivery into primary immune cells.

Using siRNAs to genetically manipulate immune cells is important to both basic immunological studies and therapeutic applications. However, siRNA delivery is challenging because primary immune cells are often sensitive to the delivery materials and generate immune responses. We have recently developed an amphiphilic dendrimer that is able to deliver siRNA to a variety of cells, including primary immune cells. We provide here a protocol for the synthesis of this dendrimer, as well as siRNA delivery to immune cells such as primary T and B cells, natural killer cells, macrophages, and primary microglia. The dendrimer synthesis entails straightforward click coupling followed by an amidation reaction, and the siRNA delivery protocol requires simple mixing of the siRNA and dendrimer in buffer, with subsequent application to the primary immune cells to achieve effective and functional siRNA delivery. This dendrimer-mediated siRNA delivery largely outperforms the standard electroporation technique, opening a new avenue for functional and therapeutic studies of the immune system. The whole protocol encompasses the dendrimer synthesis, which takes 10 days; the primary immune cell preparation, which takes 3-10 d, depending on the tissue source and cell type; the dendrimer-mediated siRNA delivery; and subsequent functional assays, which take an additional 3-6 d.

Early Treatment with Temporary Spinal Cord Stimulation Effectively Prevents Development of Postherpetic Neuralgia.

BACKGROUND: Some 7.7% of the Chinese population suffer from herpes zoster each year, with 29.8% proceeding on to develop postherpetic neuralgia (PHN). This amounts to over 32 million people per year. PHN is preceded by 2 phases of pain: acute herpetic neuralgia (AHN), and subacute herpetic neuralgia (SHN). Considering the large individual and economic burden, preventing the transition of AHN/SHN to PHN is crucial. However, to date this has been difficult. OBJECTIVES: To evaluate the efficacy of temporary spinal cord stimulation (tSCS) treatment and prevention of PHN. STUDY DESIGN: A retrospective, observational study. SETTING: Department of Pain Medicine. METHODS: From 2013 to 2017, 99 patients with AHN (n = 42), SHN (n = 34), and PHN (n = 23) underwent tSCS treatment (7-14 days) after failed pharmacologic and interventional therapies. Visual analog scale (VAS), Pittsburgh Sleep Quality Index (PSQI), and analgesic consumption were recorded at baseline, post-tSCS, and 1, 3, 6, and 12 months after tSCS treatment. RESULTS: Pooled results demonstrated statistically significant decreases in VAS scores and PSQI post-tSCS and at 1, 3, 6, and 12 months follow-up (P < 0.001). When compared with the PHN group, both AHN and SHN groups were clinically and statistically improved in VAS scores and PSQI (P < 0.001). Analgesic consumption decreased in all 3 groups after tSCS treatment, and downward linear gradient of medication in the AHN group was more significant than that in the SHN and PHN groups. At 12 months follow-up, 2.5% (1/40) patients in the AHN group, 16.0% (4/25) in the SHN group, and 62.5% (10/16) in the PHN group had ongoing pain >= 3/10 VAS score requiring analgesia. Expressed differently, at 12 months, 97.5% of the AHN group and 84% of the SHN group had pain of 2/10 VAS score or less versus only 37.5% of the PHN group. LIMITATIONS: This was a single-center, retrospective study, which made it difficult to collect complete data for all variables. The therapeutic effect of tSCS could not be studied independently. CONCLUSIONS: This retrospective analyses of 99 patients treated with tSCS (7-14 days) suggests that tSCS may be effective for treating and preventing PHN. Early treatment within 4 to 8 weeks was more likely to result in pain <= 2/10 VAS score, improvement in sleep, and no requirement for analgesia at 12 months. Early tSCS may be a promising prevention strategy against the development of chronic neuropathic pain following herpes zoster infection. Further research is justified. KEY WORDS: Herpes zoster, zoster-related pain, postherpetic neuralgia, temporary spinal cord stimulation.

Tissue-Specific Delivery of Oligonucleotides.

From the initial discovery of short-interfering RNA (siRNA) and antisense oligonucleotides for specific gene knockdown at the posttranscriptional level to the current CRISPR-Cas9 system offering gene editing at the genomic level, oligonucleotides, in addition to their biological functions in storing and conveying genetic information, provide the most prominent solutions to targeted gene therapies. Nonetheless, looking into the future of curing cancer and acute diseases, researchers are only cautiously optimistic as the cellular delivery of these polyanionic biomacromolecules is still the biggest hurdle for their therapeutic realization. To overcome the delivery obstacle, oligonucleotides have been encapsulated within or conjugated with delivery vehicles for enhanced membrane penetration, improved payload, and tissue-specific delivery. Such delivery systems include but not limited to virus-based vehicles, gold-nanoparticle vehicles, formulated liposomes, and synthetic polymers. In this chapter, delivery challenges imposed by biological barriers are briefly discussed; followed by recent advances in tissue-specific oligonucleotide delivery utilizing both viral and nonviral delivery vectors, discussing their advantages, and how judicious design and formulation could improve and expand their potential as delivery vehicles.

Dual Mechanisms of Action of Self-Delivering, Anti-HIV-1 FANA Oligonucleotides as a Potential New Approach to HIV Therapy.

Currently, the most effective and durable therapeutic option for HIV-1 infection is combination antiretroviral therapy (cART). Although cART is powerful and can delay viral evolution of drug resistance for decades, it is associated with limitations, including an inability to eradicate the virus and a potential for adverse effects. Therefore, it is imperative to discover new HIV therapeutic modalities. In this study, we designed, characterized, and evaluated the in vitro potency of 2'-deoxy-2'-fluoroarabinonucleotide (FANA) modified antisense oligonucleotides (ASOs) targeting highly conserved regions in the HIV-1 genome. Carrier-free cellular internalization of FANA ASOs resulted in strong suppression of HIV-1 replication in HIV-1-infected human primary cells. In vitro mechanistic studies suggested that the inhibitory effect of FANA ASOs can be attributed to RNase H1 activation and steric hindrance of dimerization. Using 5'-RACE PCR and sequencing analysis, we confirmed the presence of human RNase H1-mediated target RNA cleavage products in cells treated with FANA ASOs. We observed no overt cytotoxicity or immune responses upon FANA ASO treatment. Together, our results strongly suggest that FANA ASOs hold great promise for antiretroviral therapy. The dual ability of FANA ASOs to target RNA by recruiting RNase H1 and/or sterically blocking RNA dimerization further enhances their therapeutic potential.

Anti-inflammatory Activity of MTL-CEBPA, a Small Activating RNA Drug, in LPS-Stimulated Monocytes and Humanized Mice.

Excessive or inappropriate inflammatory responses can cause serious and even fatal diseases. The CCAAT/enhancer-binding protein alpha (CEBPA) gene encodes C/EBPα, a transcription factor that plays a fundamental role in controlling maturation of the myeloid lineage and is also expressed during the late phase of inflammatory responses when signs of inflammation are decreasing. MTL-CEBPA, a small activating RNA targeting for upregulation of C/EBPα, is currently being evaluated in a phase 1b trial for treatment of hepatocellular carcinoma. After dosing, subjects had reduced levels of pro-inflammatory cytokines, and we therefore hypothesized that MTL-CEBPA has anti-inflammatory potential. The current study was conducted to determine the effects of C/EBPα saRNA - CEBPA-51 - on inflammation in vitro and in vivo after endotoxin challenge. CEBPA-51 led to increased expression of the C/EBPα gene and inhibition of pro-inflammatory cytokines in THP-1 monocytes previously stimulated by E. coli-derived lipopolysaccharide (LPS). Treatment with MTL-CEBPA in an LPS-challenged humanized mouse model upregulated C/EBPα mRNA, increased neutrophils, and attenuated production of several key pro-inflammatory cytokines, including TNF-α, IL-6, IL-1ß, and IFN-γ. In addition, a Luminex analysis of mouse serum revealed that MTL-CEBPA reduced pro-inflammatory cytokines and increased the anti-inflammatory cytokine IL-10. Collectively, the data support further investigation of MTL-CEBPA in acute and chronic inflammatory diseases where this mechanism has pathogenic importance.

Humanized NOD/SCID/IL2rγnull (hu-NSG) Mouse Model for HIV Replication and Latency Studies.

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2rγnull (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

ZIKV Demonstrates Minimal Pathologic Effects and Mosquito Infectivity in Viremic Cynomolgus Macaques.

To evaluate the effects of ZIKV infection on non-human primates (NHPs), as well as to investigate whether these NHPs develop sufficient viremia to infect the major urban vector mosquito, Aedes aegypti, four cynomolgus macaques (Macaca fascicularis) were subcutaneously infected with 5.0 log10 focus-forming units (FFU) of DNA clone-derived ZIKV strain FSS13025 (Asian lineage, Cambodia, 2010). Following infection, the animals were sampled (blood, urine, tears, and saliva), underwent daily health monitoring, and were exposed to Ae. aegypti at specified time points. All four animals developed viremia, which peaked 3⁻4 days post-infection at a maximum value of 6.9 log10 genome copies/mL. No virus was detected in urine, tears, or saliva. Infection by ZIKV caused minimal overt disease: serum biochemistry and CBC values largely fell within the normal ranges, and cytokine elevations were minimal. Strikingly, the minimally colonized population of Ae. aegypti exposed to viremic animals demonstrated a maximum infection rate of 26% during peak viremia, with two of the four macaques failing to infect a single mosquito at any time point. These data indicate that cynomolgus macaques may be an effective model for ZIKV infection of humans and highlights the relative refractoriness of Ae. aegypti for ZIKV infection at the levels of viremia observed.

A tRNA-derived RNA Fragment Plays an Important Role in the Mechanism of Arsenite -induced Cellular Responses.

Chronic exposure to environmental heavy metals is a worldwide health concern. It is acknowledged to be an important cause of lower respiratory tract damage in children. However, the molecular mechanisms underlying the heavy metal-induced cellular stress/toxicity are not completely understood. Small non-coding RNAs (sncRNAs), such as microRNAs (miRNA) and more recently identified tRNA-derived RNA fragments (tRFs), are critical to the posttranscriptional control of genes. We used deep sequencing to investigate whether cellular sncRNA profiles are changed by environmental heavy metals. We found that the treatment of arsenite, an important groundwater heavy metal, leads to abundant production of tRFs, that are ~30 nucleotides (nts) long and most of which correspond to the 5'-end of mature tRNAs. It is unlikely for these tRFs to be random degradation by-products, as the type of induced tRFs is heavy metal-dependent. Three most inducible tRFs and their roles in arsenite-induced cellular responses were then investigated. We identified that p65, an important transcription factor belonging to NF-κB family and also a key factor controlling inflammatory gene expression, is a regulated target of a tRF derived from 5'-end of mature tRNA encoding AlaCGC (tRF5-AlaCGC). tRF5-AlaCGC activates p65, subsequently leading to enhanced secretion of IL-8 in arsenite response. In this study, we also identified that endonuclease Dicer and angiogenin temporally control the induction of tRF5-AlaCGC, providing an insight into the control of tRF biogenesis and subsequently the prevention of cellular damage.

Receptor-targeted aptamer-siRNA conjugate-directed transcriptional regulation of HIV-1.

Gene-based therapies represent a promising therapeutic paradigm for the treatment of HIV-1, as they have the potential to maintain sustained viral inhibition with reduced treatment interventions. Such an option may represent a long-term treatment alternative to highly active antiretroviral therapy. Methods: We previously described a therapeutic approach, referred to as transcriptional gene silencing (TGS), whereby small noncoding RNAs directly inhibit the transcriptional activity of HIV-1 by targeting sites within the viral promoter, specifically the 5' long terminal repeat (LTR). TGS differs from traditional RNA interference (RNAi) in that it is characterized by concomitant silent-state epigenetic marks on histones and DNA. To deliver TGS-inducing RNAs, we developed functional RNA conjugates based on the previously reported dual function of the gp120 (A-1) aptamer conjugated to 27-mer Dicer-substrate anti-HIV-1 siRNA (dsiRNA), LTR-362. Results: We demonstrate here that high levels of processed guide RNAs localize to the nucleus in infected T lymphoblastoid CEM cell line and primary human CD4+ T-cells. Treatment of the aptamer-siRNA conjugates induced TGS with an ~10-fold suppression of viral p24 levels as measured at day 12 post infection. To explore the silencing efficacy of aptamer-siRNA conjugates in vivo, HIV-1-infected humanized NOD/SCID/IL2 rγnull mice (hu-NSG) were treated with the aptamer-siRNA conjugates. Systemic delivery of the A-1-stick-LTR-362 27-mer siRNA conjugates suppressed HIV-1 infection and protected CD4+ T cell levels in viremia hu-NSG mice. Principle conclusions: Collectively these data suggest that the gp120 aptamer-dsiRNA conjugate design is suitable for systemic delivery of small RNAs that can be used to suppress HIV-1.

Current Advances in Aptamers for Cancer Diagnosis and Therapy.

Nucleic acid aptamers are single-stranded oligonucleotides that interact with target molecules with high affinity and specificity in unique three-dimensional structures. Aptamers are generally isolated by a simple selection process called systematic evolution of ligands by exponential enrichment (SELEX) and then can be chemically synthesized and modified. Because of their high affinity and specificity, aptamers are promising agents for biomarker discovery, as well as cancer diagnosis and therapy. In this review, we present recent progress and challenges in aptamer and SELEX technology and highlight some representative applications of aptamers in cancer therapy.

HIV Replication and Latency in a Humanized NSG Mouse Model during Suppressive Oral Combinational Antiretroviral Therapy.

Although current combinatorial antiretroviral therapy (cART) is therapeutically effective in the majority of HIV patients, interruption of therapy can cause a rapid rebound in viremia, demonstrating the existence of a stable reservoir of latently infected cells. HIV latency is therefore considered a primary barrier to HIV eradication. Identifying, quantifying, and purging the HIV reservoir is crucial to effectively curing patients and relieving them from the lifelong requirement for therapy. Latently infected transformed cell models have been used to investigate HIV latency; however, these models cannot accurately represent the quiescent cellular environment of primary latently infected cells in vivo For this reason, in vivo humanized murine models have been developed for screening antiviral agents, identifying latently infected T cells, and establishing treatment approaches for HIV research. Such models include humanized bone marrow/liver/thymus mice and SCID-hu-thy/liv mice, which are repopulated with human immune cells and implanted human tissues through laborious surgical manipulation. However, no one has utilized the human hematopoietic stem cell-engrafted NOD/SCID/IL2rγnull (NSG) model (hu-NSG) for this purpose. Therefore, in the present study, we used the HIV-infected hu-NSG mouse to recapitulate the key aspects of HIV infection and pathogenesis in vivo Moreover, we evaluated the ability of HIV-infected human cells isolated from HIV-infected hu-NSG mice on suppressive cART to act as a latent HIV reservoir. Our results demonstrate that the hu-NSG model is an effective surgery-free in vivo system in which to efficiently evaluate HIV replication, antiretroviral therapy, latency and persistence, and eradication interventions.IMPORTANCE HIV can establish a stably integrated, nonproductive state of infection at the level of individual cells, known as HIV latency, which is considered a primary barrier to curing HIV. A complete understanding of the establishment and role of HIV latency in vivo would greatly enhance attempts to develop novel HIV purging strategies. An ideal animal model for this purpose should be easy to work with, should have a shortened disease course so that efficacy testing can be completed in a reasonable time, and should have immune correlates that are easily translatable to humans. We therefore describe a novel application of the hematopoietic stem cell-transplanted humanized NSG model for dynamically testing antiretroviral treatment, supporting HIV infection, establishing HIV latency in vivo The hu-NSG model could be a facile alternative to humanized bone marrow/liver/thymus or SCID-hu-thy/liv mice in which laborious surgical manipulation and time-consuming human cell reconstitution is required.