Relationships between genomic dissipation and de novo SNP evolution.

Patterns of single nucleotide polymorphisms (SNPs) in eukaryotic DNA are traditionally attributed to selective pressure, drift, identity descent, or related factors-without accounting for ways in which bias during de novo SNP formation, itself, might contribute. A functional and phenotypic analysis based on evolutionary resilience of DNA points to decreased numbers of non-synonymous SNPs in human and other genomes, with a predominant component of SNP depletion in the human gene pool caused by robust preferences during de novo SNP formation (rather than selective constraint). Ramifications of these findings are broad, belie a number of concepts regarding human evolution, and point to a novel interpretation of evolving DNA across diverse species.

Endoluminal Biopsy for Vein of Galen Malformation.

BACKGROUND AND OBJECTIVES: Vein of Galen malformation (VOGM), the result of arteriovenous shunting between choroidal and/or subependymal arteries and the embryologic prosencephalic vein, is among the most severe cerebrovascular disorders of childhood. We hypothesized that in situ analysis of the VOGM lesion using endoluminal tissue sampling (ETS) is feasible and may advance our understanding of VOGM genetics, pathogenesis, and maintenance. METHODS: We collected germline DNA (cheek swab) from patients and their families for genetic analysis. In situ VOGM "endothelial" cells (ECs), defined as CD31+ and CD45-, were obtained from coils through ETS during routine endovascular treatment. Autologous peripheral femoral ECs were also collected from the access sheath. Single-cell RNA sequencing of both VOGM and peripheral ECs was performed to demonstrate feasibility to define the transcriptional architecture. Comparison was also made with a published normative cerebrovascular transcriptome atlas. A subset of VOGM ECs was reserved for future DNA sequencing to assess for somatic and second-hit mutations. RESULTS: Our cohort contains 6 patients who underwent 10 ETS procedures from arterial and/or venous access during routine VOGM treatment (aged 12 days to ∼6 years). No periprocedural complications attributable to ETS occurred. Six unique coil types were used. ETS captured 98 ± 88 (mean ± SD; range 17-256) experimental ECs (CD31+ and CD45-). There was no discernible correlation between cell yield and coil type or route of access. Single-cell RNA sequencing demonstrated hierarchical clustering and unique cell populations within the VOGM EC compartment compared with peripheral EC controls when annotated using a publicly available cerebrovascular cell atlas. CONCLUSION: ETS may supplement investigations aimed at development of a molecular-genetic taxonomic classification scheme for VOGM. Moreover, results may eventually inform the selection of personalized pharmacologic or genetic therapies for VOGM and cerebrovascular disorders more broadly.

ST6GAL1 sialyltransferase promotes acinar to ductal metaplasia and pancreatic cancer progression.

The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that ST6 ß-galactoside α2,6 sialyltransferase 1 (ST6GAL1), which adds α2,6-linked sialic acids to N-glycosylated proteins, was upregulated in patients with early-stage PDAC and was further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft experiments with human PDAC cells. Additionally, we developed a genetically engineered mouse (GEM) model with transgenic expression of ST6GAL1 in the pancreas and found that mice with dual expression of ST6GAL1 and oncogenic KRASG12D had greatly accelerated PDAC progression compared with mice expressing KRASG12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1's role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We verified ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.

Cx3cr1 controls kidney resident macrophage heterogeneity.

Kidney macrophages are comprised of both monocyte-derived and tissue resident populations; however, the heterogeneity of kidney macrophages and factors that regulate their heterogeneity are poorly understood. Herein, we performed single cell RNA sequencing (scRNAseq), fate mapping, and parabiosis to define the cellular heterogeneity of kidney macrophages in healthy mice. Our data indicate that healthy mouse kidneys contain four major subsets of monocytes and two major subsets of kidney resident macrophages (KRM) including a population with enriched Ccr2 expression, suggesting monocyte origin. Surprisingly, fate mapping data using the newly developed Ms4a3Cre Rosa Stopf/f TdT model indicate that less than 50% of Ccr2+ KRM are derived from Ly6chi monocytes. Instead, we find that Ccr2 expression in KRM reflects their spatial distribution as this cell population is almost exclusively found in the kidney cortex. We also identified Cx3cr1 as a gene that governs cortex specific accumulation of Ccr2+ KRM and show that loss of Ccr2+ KRM reduces the severity of cystic kidney disease in a mouse model where cysts are mainly localized to the kidney cortex. Collectively, our data indicate that Cx3cr1 regulates KRM heterogeneity and niche-specific disease progression.

Dual RNA sequencing of group B Streptococcus-infected human monocytes reveals new insights into host-pathogen interactions and bacterial evasion of phagocytosis.

Streptococcus agalactiae, also known as Group B Streptococcus (GBS) is a frequent cause of infections, including bacteraemia and other acute diseases in adults and immunocompromised individuals. We developed a novel system to study GBS within human monocytes to define the co-transcriptome of intracellular GBS (iGBS) and host cells simultaneously using dual RNA-sequencing (RNA-seq) to better define how this pathogen responds to host cells. Using human U937 monocytes and genome-sequenced GBS reference strain 874,391 in antibiotic protection assays we validated a system for dual-RNA seq based on measures of GBS and monocyte viability to ensure that the bacterial and host cell co-transcriptome reflected mainly intracellular (iGBS) rather than extracellular GBS. Elucidation of the co-transcriptome revealed 1119 dysregulated transcripts in iGBS with most genes, including several that encode virulence factors (e.g., scpB, hvgA, ribD, pil2b) exhibiting activation by upregulated expression. Infection with iGBS resulted in significant remodelling of the monocyte transcriptome, with 7587 transcripts differentially expressed including 7040 up-regulated and 547 down-regulated. qPCR confirmed that the most strongly activated genes included sht, encoding Streptococcal Histidine Triad Protein. An isogenic GBS mutant strain deficient in sht revealed a significant effect of this gene on phagocytosis of GBS and survival of the bacteria during systemic infection in mice. Identification of a novel contribution of sht to GBS virulence shows the co-transcriptome responses elucidated in GBS-infected monocytes help to shape the host-pathogen interaction and establish a role for sht in the response of the bacteria to phagocytic uptake. This study provides comprehension of concurrent transcriptional responses that occur in GBS and human monocytes that shape the host-pathogen interaction.

Deletion in chromosome 6 spanning alpha-synuclein and multimerin1 loci in the Rab27a/b double knockout mouse.

We report an incidental 358.5 kb deletion spanning the region encoding for alpha-synuclein (αsyn) and multimerin1 (Mmrn1) in the Rab27a/Rab27b double knockout (DKO) mouse line previously developed by Tolmachova and colleagues in 2007. Western blot and RT-PCR studies revealed lack of αsyn expression at either the mRNA or protein level in Rab27a/b DKO mice. PCR of genomic DNA from Rab27a/b DKO mice demonstrated at least partial deletion of the Snca locus using primers targeted to exon 4 and exon 6. Most genes located in proximity to the Snca locus, including Atoh1, Atoh2, Gm5570, Gm4410, Gm43894, and Grid2, were shown not to be deleted by PCR except for Mmrn1. Using whole genomic sequencing, the complete deletion was mapped to chromosome 6 (60,678,870-61,037,354), a slightly smaller deletion region than that previously reported in the C57BL/6J substrain maintained by Envigo. Electron microscopy of cortex from these mice demonstrates abnormally enlarged synaptic terminals with reduced synaptic vesicle density, suggesting potential interplay between Rab27 isoforms and αsyn, which are all highly expressed at the synaptic terminal. Given this deletion involving several genes, the Rab27a/b DKO mouse line should be used with caution or with appropriate back-crossing to other C57BL/6J mouse substrain lines without this deletion.

Targeted massively parallel sequencing of candidate regions on chromosome 22q predisposing to multiple schwannomas: An analysis of 51 individuals in a single-center experience.

Constitutional LZTR1 or SMARCB1 pathogenic variants (PVs) have been found in ∼86% of familial and ∼40% of sporadic schwannomatosis cases. Hence, we performed massively parallel sequencing of the entire LZTR1, SMARCB1, and NF2 genomic loci in 35 individuals with schwannomas negative for constitutional first-hit PVs in the LZTR1/SMARCB1/NF2 coding sequences; however, with 22q deletion and/or a different NF2 PV in each tumor, including six cases with only one tumor available. Furthermore, we verified whether any other LZTR1/SMARCB1/NF2 (likely) PVs could be found in 16 cases carrying a SMARCB1 constitutional variant in the 3'-untranslated region (3'-UTR) c.*17C>T, c.*70C>T, or c.*82C>T. As no additional variants were found, functional studies were performed to clarify the effect of these 3'-UTR variants on the transcript. The 3'-UTR variants c.*17C>T and c.*82C>T showed pathogenicity by negatively affecting the SMARCB1 transcript level. Two novel deep intronic SMARCB1 variants, c.500+883T>G and c.500+887G>A, resulting in out-of-frame missplicing of intron 4, were identified in two unrelated individuals. Further resequencing of the entire repeat-masked genomics sequences of chromosome 22q in individuals negative for PVs in the SMARCB1/LZTR1/NF2 coding- and noncoding regions revealed five potential schwannomatosis-predisposing candidate genes, that is, MYO18B, NEFH, SGSM1, SGSM3, and SBF1, pending further verification.

Genome-Wide Analysis on Transcriptome and Methylome in Prevention of Mammary Tumor Induced by Early Life Combined Botanicals.

Breast cancer (BC) is the most common malignancy and the second leading cause of cancer death among women in the United States. The consumption of natural dietary components such as broccoli sprouts (BSp) and green tea polyphenols (GTPs) has demonstrated exciting potential in reducing the risk of BC through the regulation of epigenetic mechanisms. However, little is known about their impacts on reversing epigenomic aberrations that are centrally involved in the initiation and progression of BC. Previously, we have determined the efficacy of combined BSp and GTPs treatment on the inhibition of the growth of a mammary tumor in a transgenic Her2/neu mouse model. We sought to extend our previous study to identify universal biomarkers that represent common mechanistic changes among different mouse models in response to this dietary regime by including a new transgenic mouse model, C3(1)-SV40 TAg (SV40). As a result, we identified novel target genes that were differentially expressed and methylated in response to dietary botanicals when administered singly (BSp and GTPs) and in combination (BSp + GTPs) in both mouse models. We discovered more differentially expressed and methylated genes in the combination treatment group compared to the singly administered groups. Subsequently, several biological pathways related to epigenetic regulations were identified in response to the combination treatment. Furthermore, when compared to the BSp and GTPs treatment alone, the combinatorial treatment showed a more significant impact on the regulation of the epigenetic modifier activities involved in DNA methylation and histone modifications. Our study provides key insights about the impact of the combined administration of BSp and GTPs on BC using a multi-omics analysis, suggesting a combinatorial approach is more efficacious in preventing and inhibiting BC by impacting key tumor-related genes at transcriptomic and methylomic levels. Our findings could be further extrapolated as a comprehensive source for understanding the epigenetic modifications that are associated with the effects of these dietary botanicals on BC prevention.

Therapeutic Effects of Dietary Soybean Genistein on Triple-Negative Breast Cancer via Regulation of Epigenetic Mechanisms.

Consumption of dietary natural components such as genistein (GE) found in soy-rich sources is strongly associated with a lower risk of breast cancer. However, bioactive dietary component-based therapeutic strategies are largely understudied in breast cancer treatment. Our investigation sought to elucidate the potential mechanisms linking bioactive dietary GE to its breast cancer chemotherapeutic potential in a special subtype of aggressive breast cancer-triple-negative breast cancer (TNBC)-by utilizing two preclinical patient-derived xenograft (PDX) orthotopic mouse models: BCM-3204 and TM00091. Our study revealed that administration of GE resulted in a delay of tumor growth in both PDX models. With transcriptomics analyses in TNBC tumors isolated from BCM-3204 PDXs, we found that dietary soybean GE significantly influenced multiple tumor-regulated gene expressions. Further validation assessment of six candidate differentially expressed genes (DEGs)-Cd74, Lpl, Ifi44, Fzd9, Sat1 and Wwc1-demonstrated a similar trend at gene transcriptional and protein levels as observed in RNA-sequencing results. Mechanistically, GE treatment-induced Cd74 downregulation regulated the NF-κB/Bcl-xL/TAp63 signal pathway, which may contribute to soybean GE-mediated therapeutic effects on TNBC tumors. Additionally, our findings revealed that GE can modify expression levels of key epigenetic-associated genes such as DNA methyltransferases (Dnmt3b), ten-eleven translocation (Tet3) methylcytosine dioxygenases and histone deacetyltransferase (Hdac2), and their enzymatic activities as well as genomic DNA methylation and histone methylation (H3K9) levels. Collectively, our investigation shows high significance for potential development of a novel therapeutic approach by using bioactive soybean GE for TNBC patients who have few treatment options.

Microbial Composition and Genes for Key Metabolic Attributes in the Gut Digesta of Sea Urchins Lytechinus variegatus and Strongylocentrotus purpuratus Using Shotgun Metagenomics.

This paper describes the microbial community composition and genes for key metabolic genes, particularly the nitrogen fixation of the mucous-enveloped gut digesta of green (Lytechinus variegatus) and purple (Strongylocentrotus purpuratus) sea urchins by using the shotgun metagenomics approach. Both green and purple urchins showed high relative abundances of Gammaproteobacteria at 30% and 60%, respectively. However, Alphaproteobacteria in the green urchins had higher relative abundances (20%) than the purple urchins (2%). At the genus level, Vibrio was dominant in both green (~9%) and purple (~10%) urchins, whereas Psychromonas was prevalent only in purple urchins (~24%). An enrichment of Roseobacter and Ruegeria was found in the green urchins, whereas purple urchins revealed a higher abundance of Shewanella, Photobacterium, and Bacteroides (q-value < 0.01). Analysis of key metabolic genes at the KEGG-Level-2 categories revealed genes for amino acids (~20%), nucleotides (~5%), cofactors and vitamins (~6%), energy (~5%), carbohydrates (~13%) metabolisms, and an abundance of genes for assimilatory nitrogen reduction pathway in both urchins. Overall, the results from this study revealed the differences in the microbial community and genes designated for the metabolic processes in the nutrient-rich sea urchin gut digesta, suggesting their likely importance to the host and their environment.

Natural and Recombinant SARS-CoV-2 Isolates Rapidly Evolve In Vitro to Higher Infectivity through More Efficient Binding to Heparan Sulfate and Reduced S1/S2 Cleavage.

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include (i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increases the positive charge of the surface of this domain, (ii) insertions into the NTD of heterologous peptides containing positively charged amino acids, and (iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide, and makes viruses less capable of syncytium formation. These viral adaptations result in higher affinity of viral particles to heparin, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers, and 2 orders of magnitude higher infectivity. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA-positive (RNA+) viruses, evolution to HS binding may result in virus attenuation in vivo. IMPORTANCE The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to the ACE2 receptor and, later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the NTD and in the P1' position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells, and decrease the genome equivalent to PFU (GE/PFU) ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high-affinity interaction of the spikes with the ACE2 receptor.

Natural isolate and recombinant SARS-CoV-2 rapidly evolve in vitro to higher infectivity through more efficient binding to heparan sulfate and reduced S1/S2 cleavage.

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant, cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increase the positive charge of the surface of this domain, ii) insertions into NTD of heterologous peptides, containing positively charged amino acids, and iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide and makes viruses less capable of syncytia formation. These viral adaptations result in higher affinity of viral particles to heparin sepharose, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers and two orders of magnitude lower GE:PFU ratios. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA+ viruses, evolution to HS binding may result in virus attenuation in vivo . IMPORTANCE: The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to ACE2 receptor and later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the N-terminal domain (NTD) and in P1â€Ëœ position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells and decrease GE:PFU ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high affinity interaction of the spikes with the ACE2 receptor.

Systematic integrated analyses of methylomic and transcriptomic impacts of early combined botanicals on estrogen receptor-negative mammary cancer.

Dietary botanicals such as the cruciferous vegetable broccoli sprouts (BSp) as well as green tea polyphenols (GTPs) have shown exciting potential in preventing or delaying breast cancer (BC). However, little is known about their impact on epigenomic aberrations that are centrally involved in the initiation and progression of estrogen receptor-negative [ER(-)] BC. We have investigated the efficacy of combined BSp and GTPs diets on mammary tumor inhibition in transgenic Her2/neu mice that were administered the diets from prepubescence until adulthood. Herein, we present an integrated DNA methylome and transcriptome analyses for defining the early-life epigenetic impacts of combined BSp and GTPs on mammary tumors and our results indicate that a combinatorial administration of BSp and GTPs have a stronger impact at both transcriptome and methylome levels in comparison to BSp or GTPs administered alone. We also demonstrated a streamlined approach by performing an extensive preprocessing, quality assessment and downstream analyses on the genomic dataset. Our identification of differentially methylated regions in response to dietary botanicals administered during early-life will allow us to identify key genes and facilitate implementation of the subsequent downstream functional analyses on a genomic scale and various epigenetic modifications that are crucial in preventing ER(-) mammary cancer. Furthermore, our realtime PCR results were also found to be consistent with our genome-wide analysis results. These results could be exploited as a comprehensive resource for understanding understudied genes and their associated epigenetic modifications in response to these dietary botanicals.

Human gut microbial communities dictate efficacy of anti-PD-1 therapy in a humanized microbiome mouse model of glioma.

BACKGROUND: Although immunotherapy works well in glioblastoma (GBM) preclinical mouse models, the therapy has not demonstrated efficacy in humans. To address this anomaly, we developed a novel humanized microbiome (HuM) model to study the response to immunotherapy in a preclinical mouse model of GBM. METHODS: We used 5 healthy human donors for fecal transplantation of gnotobiotic mice. After the transplanted microbiomes stabilized, the mice were bred to generate 5 independent humanized mouse lines (HuM1-HuM5). RESULTS: Analysis of shotgun metagenomic sequencing data from fecal samples revealed a unique microbiome with significant differences in diversity and microbial composition among HuM1-HuM5 lines. All HuM mouse lines were susceptible to GBM transplantation, and exhibited similar median survival ranging from 19 to 26 days. Interestingly, we found that HuM lines responded differently to the immune checkpoint inhibitor anti-PD-1. Specifically, we demonstrate that HuM1, HuM4, and HuM5 mice are nonresponders to anti-PD-1, while HuM2 and HuM3 mice are responsive to anti-PD-1 and displayed significantly increased survival compared to isotype controls. Bray-Curtis cluster analysis of the 5 HuM gut microbial communities revealed that responders HuM2 and HuM3 were closely related, and detailed taxonomic comparison analysis revealed that Bacteroides cellulosilyticus was commonly found in HuM2 and HuM3 with high abundances. CONCLUSIONS: The results of our study establish the utility of humanized microbiome mice as avatars to delineate features of the host interaction with gut microbial communities needed for effective immunotherapy against GBM.

Human papillomavirus sequencing reveals its usefulness for the management of HIV-infected women at risk for developing cervical cancer.

AIM: Next-generation sequencing (NGS) is able to describe the composition of human papillomaviruses (HPVs) as percent (%) reads rather than positive/negative results. Therefore, we used this unique approach to assess the prevalence of cervical HPVs of HIV infected (HIV+) in order to understand the determinants of being infected with higher % reads of high risk (HR)-HPVs and cervical abnormalities of atypical squamous cells of unknown significance or higher (ASCUS+). METHODS: Study included 66 women characterized for relevant risk factors/cytology. Receiver-operating curve curve was used to derive the optimal % read cut point to identify ASCUS+ in relation to any HR-HPV genotype or other specific HPV genotypes. The determinants of ASCUS+ and HR-HPVs were tested using logistic regression. RESULTS: Women with >20% reads of any HR-HPV or >12% any HR-HPV other than HPV 16/18 were 5.7 and 12.6 times more likely to be diagnosed with ASCUS+, respectively. Lower CD4 count was a significant determinant of >20% reads of HR-HPV (odds ratio [OR] = 4.1) or >12% any HR-HPV other than HPV 16/18 (OR = 4.5). CONCLUSION: We envision that the NGS-based HPV detection will be more accurate for screening and management of HIV+ at risk for developing cervical cancer (CC). We raise concerns regarding the limitations of 16/18-based HPV testing for triage and the efficacy of current HPV vaccines for preventing CC in HIV+.

Targeting the HuR Oncogenic Role with a New Class of Cytoplasmic Dimerization Inhibitors.

The development of novel therapeutics that exploit alterations in the activation state of key cellular signaling pathways due to mutations in upstream regulators has generated the field of personalized medicine. These first-generation efforts have focused on actionable mutations identified by deep sequencing of large numbers of tumor samples. We propose that a second-generation opportunity exists by exploiting key downstream "nodes of control" that contribute to oncogenesis and are inappropriately activated due to loss of upstream regulation and microenvironmental influences. The RNA-binding protein HuR represents such a node. Because HuR functionality in cancer cells is dependent on HuR dimerization and its nuclear/cytoplasmic shuttling, we developed a new class of molecules targeting HuR protein dimerization. A structure-activity relationship algorithm enabled development of inhibitors of HuR multimer formation that were soluble, had micromolar activity, and penetrated the blood-brain barrier. These inhibitors were evaluated for activity validation and specificity in a robust cell-based assay of HuR dimerization. SRI-42127, a molecule that met these criteria, inhibited HuR multimer formation across primary patient-derived glioblastoma xenolines (PDGx), leading to arrest of proliferation, induction of apoptosis, and inhibition of colony formation. SRI-42127 had favorable attributes with central nervous system penetration and inhibited tumor growth in mouse models. RNA and protein analysis of SRI-42127-treated PDGx xenolines across glioblastoma molecular subtypes confirmed attenuation of targets upregulated by HuR. These results highlight how focusing on key attributes of HuR that contribute to cancer progression, namely cytoplasmic localization and multimerization, has led to the development of a novel, highly effective inhibitor. SIGNIFICANCE: These findings utilize a cell-based mechanism of action assay with a structure-activity relationship compound development pathway to discover inhibitors that target HuR dimerization, a mechanism required for cancer promotion.

Glycosyltransferase ST6Gal-I promotes the epithelial to mesenchymal transition in pancreatic cancer cells.

ST6Gal-I, an enzyme upregulated in numerous malignancies, adds α2-6-linked sialic acids to select membrane receptors, thereby modulating receptor signaling and cell phenotype. In this study, we investigated ST6Gal-I's role in epithelial to mesenchymal transition (EMT) using the Suit2 pancreatic cancer cell line, which has low endogenous ST6Gal-I and limited metastatic potential, along with two metastatic Suit2-derived subclones, S2-013 and S2-LM7AA, which have upregulated ST6Gal-I. RNA-Seq results suggested that the metastatic subclones had greater activation of EMT-related gene networks than parental Suit2 cells, and forced overexpression of ST6Gal-I in the Suit2 line was sufficient to activate EMT pathways. Accordingly, we evaluated expression of EMT markers and cell invasiveness (a key phenotypic feature of EMT) in Suit2 cells with or without ST6Gal-I overexpression, as well as S2-013 and S2-LM7AA cells with or without ST6Gal-I knockdown. Cells with high ST6Gal-I expression displayed enrichment in mesenchymal markers (N-cadherin, slug, snail, fibronectin) and cell invasiveness, relative to ST6Gal-I-low cells. Contrarily, epithelial markers (E-cadherin, occludin) were suppressed in ST6Gal-I-high cells. To gain mechanistic insight into ST6Gal-I's role in EMT, we examined the activity of epidermal growth factor receptor (EGFR), a known EMT driver. ST6Gal-I-high cells had greater α2-6 sialylation and activation of EGFR than ST6Gal-I-low cells. The EGFR inhibitor, erlotinib, neutralized ST6Gal-I-dependent differences in EGFR activation, mesenchymal marker expression, and invasiveness in Suit2 and S2-LM7AA, but not S2-013, lines. Collectively, these results advance our understanding of ST6Gal-I's tumor-promoting function by highlighting a role for ST6Gal-I in EMT, which may be mediated, at least in part, by α2-6-sialylated EGFR.

An individualized mosaic of maternal microbial strains is transmitted to the infant gut microbial community.

To understand the origins of the infant gut microbial community, we have used a published metagenomic dataset of the faecal microbiome of mothers and their related infants at early (4, 7 and 21 days) and late times (6-15 months) following birth. Using strain-tracking analysis, individual-specific patterns of microbial strain sharing were found between mothers and infants following vaginal birth. Overall, three mother-infant pairs showed only related strains, while 12 infants of mother-infant pairs contained a mosaic of maternal-related and unrelated microbes. Analysis of a second dataset from nine women taken at different times of pregnancy revealed individual-specific faecal microbial strain variation that occurred in seven women. To model transmission in the absence of environmental microbes, we analysed the microbial strain transmission to F1 progenies of human faecal transplanted gnotobiotic mice bred with gnotobiotic males. Strain-tracking analysis of five different dams and their F1 progeny revealed both related and unrelated microbial strains in the mother's faeces. The results of our analysis demonstrate that multiple strains of maternal microbes, some that are not abundant in the maternal faecal community, can be transmitted during birth to establish a diverse infant gut microbial community.

Therapeutically actionable PAK4 is amplified, overexpressed, and involved in bladder cancer progression.

Muscle-invasive bladder carcinomas (MIBCs) are aggressive genitourinary malignancies. Metastatic urothelial carcinoma of the bladder is generally incurable by current chemotherapy and leads to early mortality. Recent studies have identified molecular subtypes of MIBCs with different sensitivities to frontline therapy, suggesting tumor heterogeneity. We have performed multi-omic profiling of the kinome in bladder cancer patients with the goal of identify therapeutic targets. Our analyses revealed amplification, overexpression, and elevated kinase activity of P21 (RAC1) activated kinase 4 (PAK4) in a subset of Bladder cancer (BLCA). Using bladder cancer cells, we confirmed the role of PAK4 in BLCA cell proliferation and invasion. Furthermore, we observed that a PAK4 inhibitor was effective in curtailing growth of BLCA cells. Transcriptomic analyses identified elevated expression of another kinase, protein tyrosine kinase 6 (PTK6), upon treatment with a PAK4 inhibitor and RNA interference of PAK4. Treatment with a combination of kinase inhibitors (vandetanib and dasatinib) showed enhanced sensitivity compared with either drug alone. Thus, PAK4 may be therapeutically actionable for a subset of MIBC patients with amplified and/or overexpressed PAK4 in their tumors. Our results also indicate that combined inhibition of PAK4 and PTK6 may overcome resistance to PAK4. These observations warrant clinical investigations with selected BLCA patients.

The impact of Lactococcus lactis (probiotic nasal rinse) co-culture on growth of patient-derived strains of Pseudomonas aeruginosa.

BACKGROUND: The Lactococcus strain of bacteria has been introduced as a probiotic nasal rinse for alleged salubrious effects on the sinonasal bacterial microbiome. However, data regarding interactions with pathogenic bacteria within the sinuses are lacking. The purpose of this study is to assess the interaction between L. lactis and patient-derived Pseudomonas aeruginosa, an opportunistic pathogen in recalcitrant chronic rhinosinusitis (CRS). METHODS: Commercially available probiotic suspension containing L. lactis W136 was grown in an anaerobic chamber and colonies were isolated. Colonies were co-cultured with patient-derived P. aeruginosa strains in the presence of porcine gastric mucin (mimicking human mucus) for 72 hours. P. aeruginosa cultures without L. lactis served as controls. Colony forming units (CFUs) were compared. RESULTS: Six P. aeruginosa isolates collected from 5 CRS patients (3 isolates from cystic fibrosis [CF], 1 mucoid strain) and laboratory strain PAO1 were co-cultured with L. lactis. There was no statistical difference in CFUs of 5 P. aeruginosa isolates grown with L. lactis compared to CFUs without presence of L. lactis. CFU counts were much higher when the mucoid strain was co-cultured with L. lactis (CFU+L.lactis = 1.9 × 108 ± 1.44 × 107, CFU-L.lactis = 1.3 × 108 ± 8.9 × 106, p = 0.01, n = 7). L. lactis suppressed the growth of 1 P. aeruginosa strain (CFU+L.lactis = 2.15 × 108 ± 2.9 × 107, CFU-L.lactis = 3.95 × 108 ± 4.8 × 106, p = 0.03, n = 7). CONCLUSION: L. lactis suppressed the growth of 1 patient P. aeruginosa isolate and induced growth of another (a mucoid strain) in in vitro co-culture setting in the presence of mucin. Further experiments are required to assess the underlying interactions between L. lactis and P. aeruginosa.