Zika virus (ZIKV), which can cause devastating disease in fetuses of infected pregnant women, can be transmitted by mosquito inoculation and sexual routes. Little is known about immune protection against sexually transmitted ZIKV. In this study, we show that previous infection through intravaginal or subcutaneous routes with a contemporary Brazilian strain of ZIKV can protect against subsequent intravaginal challenge with a homologous strain. Both routes of inoculation induced high titers of ZIKV-specific and neutralizing antibody in serum and the vaginal lumen. Virus-specific T cells were recruited to and retained in the female reproductive tract after intravaginal and subcutaneous ZIKV infection. Studies in mice with genetic or acquired deficiencies in B and/or T cells demonstrated that both lymphocyte populations redundantly protect against intravaginal challenge in ZIKV-immune animals. Passive transfer of ZIKV immune IgG or T cells significantly limited intravaginal infection of naïve mice, although antibody more effectively prevented dissemination throughout the reproductive tract. Collectively, our experiments begin to establish the immune correlates of protection against intravaginal ZIKV infection, which should inform vaccination strategies in non-pregnant and pregnant women.IMPORTANCE The recent ZIKV epidemic resulted in devastating outcomes in fetuses and may affect reproductive health. Unlike other flaviviruses, ZIKV can be spread by sexual contact as well as a mosquito vector. While previous studies have identified correlates of protection for mosquito-mediated infection, few have focused on immunity against sexual transmission. As exposure to ZIKV via mosquito bite has likely occurred to many living in endemic areas, our study addresses whether this route of infection can protect against subsequent sexual exposure. We demonstrate that subcutaneous ZIKV infection can protect against subsequent vaginal infection by generating both local antiviral T cell and antibody responses. Our research begins to define the immune correlates of protection for ZIKV infection in the vagina and provides a foundation for testing ZIKV vaccines against sexual transmission

Coordinated changes in gene expression underlie the early patterning and cell-type specification of the central nervous system. However, much less is known about how such changes contribute to later stages of circuit assembly and refinement. In this study, we employ single-cell RNA sequencing to develop a detailed, whole-transcriptome resource of gene expression across four time points in the developing dorsal lateral geniculate nucleus (LGN), a visual structure in the brain that undergoes a well-characterized program of postnatal circuit development. This approach identifies markers defining the major LGN cell types, including excitatory relay neurons, oligodendrocytes, astrocytes, microglia, and endothelial cells. Most cell types exhibit significant transcriptional changes across development, dynamically expressing genes involved in distinct processes including retinotopic mapping, synaptogenesis, myelination, and synaptic refinement. Our data suggest that genes associated with synapse and circuit development are expressed in a larger proportion of nonneuronal cell types than previously appreciated. Furthermore, we used this single-cell expression atlas to identify the Prkcd-Cre mouse line as a tool for selective manipulation of relay neurons during a late stage of sensory-driven synaptic refinement. This transcriptomic resource provides a cellular map of gene expression across several cell types of the LGN, and offers insight into the molecular mechanisms of circuit development in the postnatal brain.

During human gastric carcinogenesis, intestinal metaplasia (IM) is frequently seen in the atrophic stomach. In mice, a distinct type of metaplasia known as spasmolytic polypeptide-expressing metaplasia (SPEM) is found in several inflammatory and genetically engineered models. Given the diversity of long- and short-term models of mouse SPEM, it remains unclear whether all models have a shared or distinct molecular mechanism. The origin of SPEM in mice is currently under debate. It is postulated that stem or progenitor cells acquire genetic alterations that then supply metaplastic cell clones, while the possibility of transdifferentiation or dedifferentiation from mature gastric chief cells has also been suggested. In this study, we report that loss of chief cells was sufficient to induce short-term regenerative SPEM-like lesions that originated from chief cell precursors in the gastric neck region. Furthermore, Lgr5+ mature chief cells failed to contribute to both short- and long-term metaplasia, whereas isthmus stem and progenitor cells efficiently contributed to long-term metaplasia. Interestingly, multiple administrations of high-dose pulsed tamoxifen induced expansion of Lgr5 expression and Lgr5-CreERT recombination within the isthmus progenitors apart from basal chief cells. Thus, we conclude that short-term SPEM represents a regenerative process arising from neck progenitors following chief cell loss, whereas true long-term SPEM originates from isthmus progenitors. Mature gastric chief cells may be dispensable for SPEM development.

The tumor suppressor FBW7 targets oncoproteins such as c-MYC for ubiquitylation and is mutated in several human cancers. We noted that in a significant percentage of colon cancers, FBW7 protein is undetectable despite the presence of FBW7 mRNA. To understand the molecular mechanism of FBW7 regulation in these cancers, we employed proteomics and identified the deubiquitinase USP9X as an FBW7 interactor. USP9X antagonised FBW7 ubiquitylation, and Usp9x deletion caused Fbw7 destabilization. Mice lacking Usp9x in the gut showed reduced secretory cell differentiation and increased progenitor proliferation, phenocopying Fbw7 loss. In addition, Usp9x inactivation impaired intestinal regeneration and increased tumor burden in colitis-associated intestinal cancer. c-Myc heterozygosity abrogated increased progenitor proliferation and tumor burden in Usp9x-deficient mice, suggesting that Usp9x suppresses tumor formation by regulating Fbw7 protein stability and thereby reducing c-Myc. Thus, we identify a novel tumor suppressor mechanism in the mammalian intestine that arises from the posttranslational regulation of FBW7 by USP9X independent of somatic FBW7 mutations.

Stem cells reside in a niche, a complex cellular and molecular environment. In Drosophila ovaries, germline stem cells depend on cap cells for self-renewing signals and physical attachment. Germline stem cells also contact the anterior escort cells, and here we report that anterior escort cells are absolutely required for germline stem cell maintenance. When escort cells die from impaired Wnt signaling or hid expression, the loss of anterior escort cells causes consequent loss of germline stem cells. Anterior escort cells function as an integral niche component by promoting DE-cadherin anchorage and by transiently expressing the Dpp ligand to promote full-strength BMP signaling in germline stem cells. Anterior escort cells are maintained by Wnt6 ligands produced by cap cells; without Wnt6 signaling, anterior escort cells die leaving vacancies in the niche, leading to loss of germline stem cells. Our data identify anterior escort cells as constituents of the germline stem cellniche, maintained by a cap-cell produced Wnt6 survival signal.

The medial preoptic area (mPOA) differs between males and females in nearly all species examined to date, including humans. Here, using fiber photometry recordings of Ca2+ transients in freely behaving mice, we show ramping activities in the mPOA that precede and correlate with sexually dimorphic display of male-typical mounting and female-typical pup retrieval. Strikingly, optogenetic stimulation of the mPOA elicits similar display of mounting and pup retrieval in both males and females. Furthermore, by means of recording, ablation, optogenetic activation, and inhibition, we show mPOA neurons expressing estrogen receptor alpha (Esr1) are essential for the sexually biased display of these behaviors. Together, these results underscore the shared layout of the brain that can mediate sex-specific behaviors in both male and female mice and provide an important functional frame to decode neural mechanisms governing sexually dimorphic behaviors in the future.

ABASTRACT To determine PD1/PDL1 expression status in triple-negative breast cancer (TNBC) at both protein and mRNA levels, and to analyze the relationship between their expression and clinical parameters of the TNBC patients. Immunohistochemistry and RNAscope were used to semi quantitively evaluate PD1/PDL1 protein and mRNA expression in 195 TNBC cases on tissue microarrays. Tumor infiltrating lymphocyte (TILs) abundance was assessed using hematoxylin-eosin staining.Both tumor cells and TILs expressed PDL1. PDL1 protein and mRNA positivity was 6.7% and 74.4% respectively in tumor cells, and 31.3% and 50.9% respectively in TILs. PDL1 protein and mRNA expressions had no significant association with patient prognosis. PD1 protein was only detected in TILs (70.3% positivity). PD1 protein expression was significantly related to PDL1 expression, higher TIL abundance, Ki-67 index, basal-like subtypes, and distant metastasis. Furthermore, it was significantly associated with longer disease free survival (P<0.001) and overall survival (P = 0.004). There was no significant association between PD1 mRNA expression and clinicopathological characteristics. PD1/ PDL1 protein and mRNA expressions were inconsistent (kappa = 0.705 and 0.061, respectively). PD1 protein expression in TILs, but not PDL1 in tumor cells, was a favorable prognostic factor in TNBC. PD1/ PDL1 mRNA and protein expressions were inconsistent.

An increasing body of evidence points to significant spatio-temporal differences in early placental development between mouse and human, but a detailed comparison of placentae in these two species is missing. We set out to compare placentae from both species across gestation, with a focus on trophoblast progenitor markers. We found that CDX2 and ELF5, but not EOMES, are expressed in early post-implantation trophoblast subpopulations in both species. Genome-wide expression profiling of mouse and human placentae revealed clusters of genes with distinct co-expression patterns across gestation. Overall, there was a closer fit between patterns observed in the placentae when the inter-species comparison was restricted to human placentae through gestational week 16 (thus excluding term samples), suggesting that the developmental timeline in mouse runs parallel to the first half of human placental development. In addition, we identified VGLL1 as a human-specific marker of proliferative cytotrophoblast, where it is co-expressed with the transcription factor TEAD4. Since TEAD4 is involved in trophectoderm specification in the mouse, we posit a regulatory role for VGLL1 in early events during human placental development.

Mucoepidermoid carcinoma (MEC) arises in many glandular tissues and contributes to the most common malignant salivary gland cancers. MEC is specifically associated with a unique t(11;19) translocation and the resulting CRTC1-MAML2 fusion is a major oncogenic driver for MEC initiation and maintenance. However, the molecular basis underlying the CRTC1-MAML2 oncogenic functions remains elusive. Through gene expression profiling analysis, we observed that LINC00473, a long non-coding RNA (lncRNA), was the top down-regulated target in CRTC1-MAML2-depleted human MEC cells. LncRNAs belong to a new class of non-coding RNAs with emerging roles in tumorigenesis and progression, but remain poorly characterized. In this study, we investigated the role of LINC00473 in mediating CRTC1-MAML2 oncogenic activity in human MEC. We found that LINC00473 transcription was significantly induced in human CRTC1-MAML2-positive MEC cell lines and primary MEC tumors, and was tightly correlated with the CRTC1-MAML2 RNA level. LINC00473 induction was dependent on the ability of CRTC1-MAML2 to activate CREB-mediated transcription. Depletion of LINC00473 significantly reduced the proliferation and survival of human MEC cells in vitro and blocked the in vivo tumor growth in a human MEC xenograft model. RNA in situ hybridization analysis demonstrated a predominantly nuclear localization pattern for LINC00473 in human MEC cells. Furthermore, gene expression profiling revealed that LINC00473 depletion resulted in differential expression of genes important in cancer cell growth and survival. LINC00473 likely regulates gene expression in part through its ability to bind to a cAMP signaling pathway component NONO, enhancing the ability of CRTC1-MAML2 to activate CREB-mediated transcription. Our overall results demonstrate that LINC00473 is a downstream target and an important mediator of the CRTC1-MAML2 oncoprotein. Therefore, LINC00473 acts as a promising biomarker and therapeutic target for human CRTC1-MAML2-positive MECs.

Chemokine (C-C motif) ligand 5 (CCL5) belongs to a group of chemokines that play a role in the peripheral immune system, mostly as chemoattractant molecules, and mediate tactile allodynia. In the central nervous system (CNS), CCL5 and its receptors have multiple functions, including promoting neuroinflammation, insulin signaling, neuromodulator of synaptic activity and neuroprotection against a variety of neurotoxins. Evidence has also suggested that this chemokine may regulate opioid response. The multifunctional profile of CCL5 might correlate with its ability to bind different chemokine receptors, as well as with its unique cellular expression. In this work, we have used fluorescence in situ hybridization combined with immunohistochemistry to examine the expression profile of CCL5 mRNA in the adult rat brain and provide evidence of its cellular localization. We have observed that the highest expression of CCL5 mRNA occurs in all major fiber tracts, including the corpus callosum, anterior commissure, and cerebral peduncle. In these tracts, CCL5 mRNA was localized in oligodendrocytes, astrocytes and microglia. Astrocytic and microglial expression was also evident in several brain areas including the cerebral cortex, caudate/putamen, hippocampus, and thalamus. Furthermore, using a specific neuronal marker, we observed CCL5 mRNA expression in discrete layers of the cortex and hippocampus. Interestingly, in the midbrain, CCL5 mRNA co-localized with tyrosine hydroxylase (TH) positive cells of the ventral tegmental area, suggesting that CCL5 might be expressed by a subset of dopaminergic neurons of the mesolimbic system. The expression of CCL5 mRNA and protein, together with its receptors, in selected brain cell populations proposes that this chemokine could be involved in neuronal/glial communication.

Background and Aims

Human intestinal organoids derived from induced pluripotent stem cells have tremendous potential to elucidate the intestinal epithelium’s role in health and disease, but it is difficult to directly assay these complex structures. This study sought to make this technology more amenable for study by obtaining epithelial cells from induced pluripotent stem cell–derived human intestinal organoids and incorporating them into small microengineered Chips. We then investigated if these cells within the Chip were polarized, had the 4 major intestinal epithelial subtypes, and were biologically responsive to exogenous stimuli.

Methods

Epithelial cells were positively selected from human intestinal organoids and were incorporated into the Chip. The effect of continuous media flow was examined. Immunocytochemistry and in situ hybridization were used to demonstrate that the epithelial cells were polarized and possessed the major intestinal epithelial subtypes. To assess if the incorporated cells were biologically responsive, Western blot analysis and quantitative polymerase chain reaction were used to assess the effects of interferon (IFN)-γ, and fluorescein isothiocyanate–dextran 4 kDa permeation was used to assess the effects of IFN-γ and tumor necrosis factor-α on barrier function.

Results

The optimal cell seeding density and flow rate were established. The continuous administration of flow resulted in the formation of polarized intestinal folds that contained Paneth cells, goblet cells, enterocytes, and enteroendocrine cells along with transit-amplifying and LGR5+ stem cells. Administration of IFN-γ for 1 hour resulted in the phosphorylation of STAT1, whereas exposure for 3 days resulted in a significant upregulation of IFN-γ related genes. Administration of IFN-γ and tumor necrosis factor-α for 3 days resulted in an increase in intestinal permeability.

Conclusions

We demonstrate that the Intestine-Chip is polarized, contains all the intestinal epithelial subtypes, and is biologically responsive to exogenous stimuli. This represents a more amenable platform to use organoid technology and will be highly applicable to personalized medicine and a wide range of gastrointestinal conditions.

Despite recent efforts to dissect the inter-tumor heterogeneity of pancreatic ductal adenocarcinoma (PDAC) by determining prognosis-predictive gene expression signatures for specific subtypes, their functional differences remain elusive. Here, we established a pancreatic tumor organoid library encompassing 39 patient-derived PDACs and identified 3 functional subtypes based on their stem cell niche factor dependencies on Wnt and R-spondin. A Wnt-non-producing subtype required Wnt from cancer-associated fibroblasts, whereas a Wnt-producing subtype autonomously secreted Wnt ligands and an R-spondin-independent subtype grew in the absence of Wnt and R-spondin. Transcriptome analysis of PDAC organoids revealed gene-expression signatures that associated Wnt niche subtypes with GATA6-dependent gene expression subtypes, which were functionally supported by genetic perturbation of GATA6. Furthermore, CRISPR-Cas9-based genome editing of PDAC driver genes (KRAS, CDKN2A, SMAD4, and TP53) demonstrated non-genetic acquisition of Wnt niche independence during pancreas tumorigenesis. Collectively, our results reveal functional heterogeneity of Wnt niche independency in PDAC that is non-genetically formed through tumor progression.