Development of spirulina for the manufacture and oral delivery of protein therapeutics.

The use of the edible photosynthetic cyanobacterium Arthrospira platensis (spirulina) as a biomanufacturing platform has been limited by a lack of genetic tools. Here we report genetic engineering methods for stable, high-level expression of bioactive proteins in spirulina, including large-scale, indoor cultivation and downstream processing methods. Following targeted integration of exogenous genes into the spirulina chromosome (chr), encoded protein biopharmaceuticals can represent as much as 15% of total biomass, require no purification before oral delivery and are stable without refrigeration and protected during gastric transit when encapsulated within dry spirulina. Oral delivery of a spirulina-expressed antibody targeting campylobacter-a major cause of infant mortality in the developing world-prevents disease in mice, and a phase 1 clinical trial demonstrated safety for human administration. Spirulina provides an advantageous system for the manufacture of orally delivered therapeutic proteins by combining the safety of a food-based production host with the accessible genetic manipulation and high productivity of microbial platforms.

Rapid 3D Bioprinting of Glioblastoma Model Mimicking Native Biophysical Heterogeneity.

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor characterized by high cellular and molecular heterogeneity, hypervascularization, and innate drug resistance. Cellular components and extracellular matrix (ECM) are the two primary sources of heterogeneity in GBM. Here, biomimetic tri-regional GBM models with tumor regions, acellular ECM regions, and an endothelial region with regional stiffnesses patterned corresponding to the GBM stroma, pathological or normal brain parenchyma, and brain capillaries, are developed. Patient-derived GBM cells, human endothelial cells, and hyaluronic acid derivatives are used to generate a species-matched and biochemically relevant microenvironment. This in vitro study demonstrates that biophysical cues are involved in various tumor cell behaviors and angiogenic potentials and promote different molecular subtypes of GBM. The stiff models are enriched in the mesenchymal subtype, exhibit diffuse invasion of tumor cells, and induce protruding angiogenesis and higher drug resistance to temozolomide. Meanwhile, the soft models demonstrate enrichment in the classical subtype and support expansive cell growth. The three-dimensional bioprinting technology utilized in this study enables rapid, flexible, and reproducible patient-specific GBM modeling with biophysical heterogeneity that can be employed by future studies as a tunable system to interrogate GBM disease mechanisms and screen drug compounds.

Glial cell diversity and methamphetamine-induced neuroinflammation in human cerebral organoids.

Methamphetamine (METH) is a potent stimulant that induces a euphoric state but also causes cognitive impairment, neurotoxicity and neurodevelopmental deficits. Yet, the molecular mechanisms by which METH causes neurodevelopmental defects have remained elusive. Here we utilized human cerebral organoids and single-cell RNA sequencing (scRNA-seq) to study the effects of prenatal METH exposure on fetal brain development. We analyzed 20,758 cells from eight untreated and six METH-treated cerebral organoids and found that the organoids developed from embryonic stem cells contained a diverse array of glial and neuronal cell types. We further identified transcriptionally distinct populations of astrocytes and oligodendrocytes within cerebral organoids. Treatment of organoids with METH-induced marked changes in transcription in multiple cell types, including astrocytes and neural progenitor cells. METH also elicited novel astrocyte-specific gene expression networks regulating responses to cytokines, and inflammasome. Moreover, upregulation of immediate early genes, complement factors, apoptosis, and immune response genes suggests a neuroinflammatory program induced by METH regulating neural stem cell proliferation, differentiation, and cell death. Finally, we observed marked METH-induced changes in neuroinflammatory and cytokine gene expression at the RNA and protein levels. Our data suggest that human cerebral organoids represent a model system to study drug-induced neuroinflammation at single-cell resolution.

Zika virus depletes neural stem cells and evades selective autophagy by suppressing the Fanconi anemia protein FANCC.

Zika virus (ZIKV) is an emerging flavivirus, which when passed through vertical transmission from mother to developing fetus can lead to developmental abnormalities, including microcephaly. While there is mounting evidence that suggests a causal relationship between ZIKV infection and microcephaly, the mechanisms by which ZIKV induces these changes remain to be elucidated. Here, we demonstrate that ZIKV infection of neural stems cells, both in vitro and in vivo, induces macroautophagy to enhance viral replication. At the same time, ZIKV downregulates a number of essential selective autophagy genes, including the Fanconi anemia (FA) pathway genes. Bioinformatics analyses indicate that the transcription factor E2F4 promotes FANCC expression and is downregulated upon ZIKV infection. Gain and loss of function assays indicate that FANCC is essential for selective autophagy and acts as a negative regulator of ZIKV replication. Finally, we show that Fancc KO mice have increased ZIKV infection and autophagy protein levels in various brain regions. Taken together, ZIKV downregulates FANCC to modulate the host antiviral response and simultaneously attenuate neuronal growth.

Optimizing sequencing protocols for leaderboard metagenomics by combining long and short reads.

As metagenomic studies move to increasing numbers of samples, communities like the human gut may benefit more from the assembly of abundant microbes in many samples, rather than the exhaustive assembly of fewer samples. We term this approach leaderboard metagenome sequencing. To explore protocol optimization for leaderboard metagenomics in real samples, we introduce a benchmark of library prep and sequencing using internal references generated by synthetic long-read technology, allowing us to evaluate high-throughput library preparation methods against gold-standard reference genomes derived from the samples themselves. We introduce a low-cost protocol for high-throughput library preparation and sequencing.

Genome-wide Integrative Analysis of Zika-Virus-Infected Neuronal Stem Cells Reveals Roles for MicroRNAs in Cell Cycle and Stemness.

Zika virus (ZIKV) infection is implicated in severe fetal developmental disorders, including microcephaly. MicroRNAs (miRNAs) post-transcriptionally regulate numerous processes associated with viral infection and neurodegeneration, but their contribution to ZIKV pathogenesis is unclear. We analyzed the mRNA and miRNA transcriptomes of human neuronal stem cells (hNSCs) during infection with ZIKV MR766 and Paraiba strains. Integration of the miRNA and mRNA expression data into regulatory interaction networks showed that ZIKV infection resulted in miRNA-mediated repression of genes regulating the cell cycle, stem cell maintenance, and neurogenesis. Bioinformatics analysis of Argonaute-bound RNAs in ZIKV-infected hNSCs identified a number of miRNAs with predicted involvement in microcephaly, including miR-124-3p, which dysregulates NSC maintenance through repression of the transferrin receptor (TFRC). Consistent with this, ZIKV infection upregulated miR-124-3p and downregulated TFRC mRNA in ZIKV-infected hNSCs and mouse brain tissue. These data provide insights into the roles of miRNAs in ZIKV pathogenesis, particularly the microcephaly phenotype.

The long noncoding RNA ROCKI regulates inflammatory gene expression.

Long noncoding RNAs (lncRNAs) can regulate target gene expression by acting in cis (locally) or in trans (non-locally). Here, we performed genome-wide expression analysis of Toll-like receptor (TLR)-stimulated human macrophages to identify pairs of cis-acting lncRNAs and protein-coding genes involved in innate immunity. A total of 229 gene pairs were identified, many of which were commonly regulated by signaling through multiple TLRs and were involved in the cytokine responses to infection by group B Streptococcus We focused on elucidating the function of one lncRNA, named lnc-MARCKS or ROCKI (Regulator of Cytokines and Inflammation), which was induced by multiple TLR stimuli and acted as a master regulator of inflammatory responses. ROCKI interacted with APEX1 (apurinic/apyrimidinic endodeoxyribonuclease 1) to form a ribonucleoprotein complex at the MARCKS promoter. In turn, ROCKI-APEX1 recruited the histone deacetylase HDAC1, which removed the H3K27ac modification from the promoter, thus reducing MARCKS transcription and subsequent Ca2+ signaling and inflammatory gene expression. Finally, genetic variants affecting ROCKI expression were linked to a reduced risk of certain inflammatory and infectious disease in humans, including inflammatory bowel disease and tuberculosis. Collectively, these data highlight the importance of cis-acting lncRNAs in TLR signaling, innate immunity, and pathophysiological inflammation.

Zika virus infection reprograms global transcription of host cells to allow sustained infection.

Zika virus (ZIKV) is an emerging virus causally linked to neurological disorders, including congenital microcephaly and Guillain-Barré syndrome. There are currently no targeted therapies for ZIKV infection. To identify novel antiviral targets and to elucidate the mechanisms by which ZIKV exploits the host cell machinery to support sustained replication, we analyzed the transcriptomic landscape of human microglia, fibroblast, embryonic kidney and monocyte-derived macrophage cell lines before and after ZIKV infection. The four cell types differed in their susceptibility to ZIKV infection, consistent with differences in their expression of viral response genes before infection. Clustering and network analyses of genes differentially expressed after ZIKV infection revealed changes related to the adaptive immune system, angiogenesis and host metabolic processes that are conducive to sustained viral production. Genes related to the adaptive immune response were downregulated in microglia cells, suggesting that ZIKV effectively evades the immune response after reaching the central nervous system. Like other viruses, ZIKV diverts host cell resources and reprograms the metabolic machinery to support RNA metabolism, ATP production and glycolysis. Consistent with these transcriptomic analyses, nucleoside metabolic inhibitors abrogated ZIKV replication in microglia cells.

miR-1298 Inhibits Mutant KRAS-Driven Tumor Growth by Repressing FAK and LAMB3.

Global miRNA functional screens can offer a strategy to identify synthetic lethal interactions in cancer cells that might be exploited therapeutically. In this study, we applied this strategy to identify novel gene interactions in KRAS-mutant cancer cells. In this manner, we discovered miR-1298, a novel miRNA that inhibited the growth of KRAS-driven cells both in vitro and in vivo Using miR-TRAP affinity purification technology, we identified the tyrosine kinase FAK and the laminin subunit LAMB3 as functional targets of miR-1298. Silencing of FAK or LAMB3 recapitulated the synthetic lethal effects of miR-1298 expression in KRAS-driven cancer cells, whereas coexpression of both proteins was critical to rescue miR-1298-induced cell death. Expression of LAMB3 but not FAK was upregulated by mutant KRAS. In clinical specimens, elevated LAMB3 expression correlated with poorer survival in lung cancer patients with an oncogenic KRAS gene signature, suggesting a novel candidate biomarker in this disease setting. Our results define a novel regulatory pathway in KRAS-driven cancers, which offers a potential therapeutic target for their eradication. Cancer Res; 76(19); 5777-87. ©2016 AACR.

1,2,3-Triazoles as Amide Bioisosteres: Discovery of a New Class of Potent HIV-1 Vif Antagonists.

RN-18 based viral infectivity factor (Vif), Vif antagonists reduce viral infectivity by rescuing APOBEC3G (A3G) expression and enhancing A3G-dependent Vif degradation. Replacement of amide functionality in RN-18 (IC50 = 6 µM) by isosteric heterocycles resulted in the discovery of a 1,2,3-trizole, 1d (IC50 = 1.2 µM). We identified several potent HIV-1 inhibitors from a 1d based library including 5ax (IC50 = 0.01 µM), 5bx (0.2 µM), 2ey (0.4 µM), 5ey (0.6 µM), and 6bx (0.2 µM).

Polycomb Group Protein Pcgf6 Acts as a Master Regulator to Maintain Embryonic Stem Cell Identity.

The polycomb repressive complex 1 (PRC1) is a multi-subunit complex that plays critical roles in the epigenetic modulation of gene expression. Here, we show that the PRC1 component polycomb group ring finger 6 (Pcgf6) is required to maintain embryonic stem cell (ESC) identity. In contrast to canonical PRC1, Pcgf6 acts as a positive regulator of transcription and binds predominantly to promoters bearing active chromatin marks. Pcgf6 is expressed at high levels in ESCs, and knockdown reduces the expression of the core ESC regulators Oct4, Sox2, and Nanog. Conversely, Pcgf6 overexpression prevents downregulation of these factors and impairs differentiation. In addition, Pcgf6 enhanced reprogramming in both mouse and human somatic cells. The genomic binding profile of Pcgf6 is highly similar to that of trithorax group proteins, but not of PRC1 or PRC2 complexes, suggesting that Pcgf6 functions atypically in ESCs. Our data reveal novel roles for Pcgf6 in directly regulating Oct4, Nanog, Sox2, and Lin28 expression to maintain ESC identity.

Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate Immune Receptor TLR3.

Emerging evidence from the current outbreak of Zika virus (ZIKV) indicates a strong causal link between Zika and microcephaly. To investigate how ZIKV infection leads to microcephaly, we used human embryonic stem cell-derived cerebral organoids to recapitulate early stage, first trimester fetal brain development. Here we show that a prototype strain of ZIKV, MR766, efficiently infects organoids and causes a decrease in overall organoid size that correlates with the kinetics of viral copy number. The innate immune receptor Toll-like-Receptor 3 (TLR3) was upregulated after ZIKV infection of human organoids and mouse neurospheres and TLR3 inhibition reduced the phenotypic effects of ZIKV infection. Pathway analysis of gene expression changes during TLR3 activation highlighted 41 genes also related to neuronal development, suggesting a mechanistic connection to disrupted neurogenesis. Together, therefore, our findings identify a link between ZIKV-mediated TLR3 activation, perturbed cell fate, and a reduction in organoid volume reminiscent of microcephaly.

Enhancing Induced Pluripotent Stem Cell Generation by MicroRNA.

Somatic reprogramming to generate induced pluripotent stem cells, or iPSC, is a powerful tool in developmental biology, disease modeling, and regenerative medicine. microRNAs have been shown to regulate many key pathways in iPSC induction. Here we describe a microRNA mimic enhanced somatic reprogramming process starting from mouse embryonic fibroblast isolation to iPSC induction to colony derivation and characterization.

Haunting the HOXA Locus: Two Faces of lncRNA Regulation.

Long non-coding RNAs (lncRNAs) regulate diverse biological functions through mechanisms ascribed to the lncRNA transcript itself. Now in Cell Stem Cell, Yin et al. (2015) use CRISPR/Cas9-mediated genome editing to demonstrate discrete and opposing roles for the lncRNA Haunt transcript and DNA at the HOXA locus during ESC differentiation.

Therapeutic targeting of polo-like kinase 1 using RNA-interfering nanoparticles (iNOPs) for the treatment of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) remains the most common cause of cancer death worldwide due its resistance to chemotherapy and aggressive tumor growth. Polo-like kinase 1 (PLK1) is a serine-threonine protein kinase which is overexpressed in cancer cells, and plays a major role in regulating tumor growth. A number of PLK1 inhibitors are in clinical trial; however, poor tumor bioavailability and off-target effects limit their efficacy. Short-interfering-RNA (siRNA) holds promise as a class of therapeutics, which can selectively silence disease-causing genes. However, siRNA cannot enter cells without a delivery vehicle. Herein, we investigated whether RNAi-interfering nanoparticles could deliver siRNA to NSCLC cells and silence PLK1 expression in vitro and in vivo. iNOP-7 was non-toxic, and delivered siRNA with high efficiency to NSCLC cells. iNOP-7-PLK1 siRNA silenced PLK1 expression and reduced NSCLC growth in vitro. Notably, iNOP-7 delivered siRNA to orthotopic lung tumors in mice, and administration of iNOP-7-PLK1 siRNA reduced lung tumor burden. These novel data show that iNOP-7 can deliver siRNA against PLK1 to NSCLC cells, and decrease cell proliferation both in vitro and in vivo. iNOP-7-PLK1 siRNA may provide a novel therapeutic strategy for the treatment of NSCLC as well as other cancers which aberrantly express this gene.

MicroRNA-mediated regulation of extracellular matrix formation modulates somatic cell reprogramming.

Somatic cells can be reprogrammed to reach an embryonic stem cell-like state by overexpression of defined factors. Recent studies have greatly improved the efficiency of the reprogramming process but the underlying mechanisms regulating the transition from a somatic to a pluripotent state are still relatively unknown. MicroRNAs (miRs) are small noncoding RNAs that primarily regulate target gene expression post-transcriptionally. Here we present a systematic and comprehensive study of microRNAs in mouse embryonic fibroblasts (MEFs) during the early stage of cell fate decisions and reprogramming to a pluripotent state, in which significant transcriptional and epigenetic changes occur. One microRNA found to be highly induced during this stage of reprogramming, miR-135b, targeted the expression of extracellular matrix (ECM) genes including Wisp1 and Igfbp5. Wisp1 was shown to be a key regulator of additional ECM genes that serve as barriers to reprogramming. Regulation of Wisp 1 is likely mediated through biglycan, a glycoprotein highly expressed in MEFs that is silenced in reprogrammed cells. Collectively, this report reveals a novel link between microRNA-mediated regulation of ECM formation and somatic cell reprogramming, and demonstrates that microRNAs are powerful tools to dissect the intracellular and extracellular molecular mechanisms of reprogramming.

Kinome-wide functional analysis highlights the role of cytoskeletal remodeling in somatic cell reprogramming.

The creation of induced pluripotent stem cells (iPSCs) from somatic cells by ectopic expression of transcription factors has galvanized the fields of regenerative medicine and developmental biology. Here, we report a kinome-wide RNAi-based analysis to identify kinases that regulate somatic cell reprogramming to iPSCs. We prepared 3,686 small hairpin RNA (shRNA) lentiviruses targeting 734 kinase genes covering the entire mouse kinome and individually examined their effects on iPSC generation. We identified 59 kinases as barriers to iPSC generation and characterized seven of them further. We found that shRNA-mediated knockdown of the serine/threonine kinases TESK1 or LIMK2 promoted mesenchymal-to-epithelial transition, decreased COFILIN phosphorylation, and disrupted Actin filament structures during reprogramming of mouse embryonic fibroblasts. Similarly, knockdown of TESK1 in human fibroblasts also promoted reprogramming to iPSCs. Our study reveals the breadth of kinase networks regulating pluripotency and identifies a role for cytoskeletal remodeling in modulating the somatic cell reprogramming process.

An evolutionarily conserved long noncoding RNA TUNA controls pluripotency and neural lineage commitment.

Here, we generated a genome-scale shRNA library targeting long intergenic noncoding RNAs (lincRNAs) in the mouse. We performed an unbiased loss-of-function study in mouse embryonic stem cells (mESCs) and identified 20 lincRNAs involved in the maintenance of pluripotency. Among these, TUNA (Tcl1 Upstream Neuron-Associated lincRNA, or megamind) was required for pluripotency and formed a complex with three RNA-binding proteins (RBPs). The TUNA-RBP complex was detected at the promoters of Nanog, Sox2, and Fgf4, and knockdown of TUNA or the individual RBPs inhibited neural differentiation of mESCs. TUNA showed striking evolutionary conservation of both sequence- and CNS-restricted expression in vertebrates. Accordingly, knockdown of tuna in zebrafish caused impaired locomotor function, and TUNA expression in the brains of Huntington's disease patients was significantly associated with disease grade. Our results suggest that the lincRNA TUNA plays a vital role in pluripotency and neural differentiation of ESCs and is associated with neurological function of adult vertebrates.

Staged miRNA re-regulation patterns during reprogramming.

BACKGROUND: MiRNAs often operate in feedback loops with transcription factors and represent a key mechanism for fine-tuning gene expression. In transcription factor-induced reprogramming, miRNAs play a critical role; however, detailed analyses of miRNA expression changes during reprogramming at the level of deep sequencing have not been previously reported. RESULTS: We use four factor reprogramming to induce pluripotent stem cells from mouse fibroblasts and isolate FACS-sorted Thy1- and SSEA1+ intermediates and Oct4-GFP+ induced pluripotent stem cells (iPSCs). Small RNAs from these cells, and two partial-iPSC lines, another iPSC line, and mouse embryonic stem cells (mES cells) were deep sequenced. A comprehensive resetting of the miRNA profile occurs during reprogramming; however, analysis of miRNA co-expression patterns yields only a few patterns of change. Dlk1-Dio3 region miRNAs dominate the large pool of miRNAs experiencing small but significant fold changes early in reprogramming. Overexpression of Dlk1-Dio3 miRNAs early in reprogramming reduces reprogramming efficiency, suggesting the observed downregulation of these miRNAs may contribute to reprogramming. As reprogramming progresses, fewer miRNAs show changes in expression, but those changes are generally of greater magnitude. CONCLUSIONS: The broad resetting of the miRNA profile during reprogramming that we observe is due to small changes in gene expression in many miRNAs early in the process, and large changes in only a few miRNAs late in reprogramming. This corresponds with a previously observed transition from a stochastic to a more deterministic signal.