Opposing Wnt signals regulate cervical squamocolumnar homeostasis and emergence of metaplasia

Nature cell biology

2021 Jan 18

Chumduri, C;Gurumurthy, RK;Berger, H;Dietrich, O;Kumar, N;Koster, S;Brinkmann, V;Hoffmann, K;Drabkina, M;Arampatzi, P;Son, D;Klemm, U;Mollenkopf, HJ;Herbst, H;Mangler, M;Vogel, J;Saliba, AE;Meyer, TF;
PMID: 33462395 | DOI: 10.1038/s41556-020-00619-0

The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages.

Hypoxia-induced suppression of alternative splicing of MBD2 promotes breast cancer metastasis via activation of FZD1

Cancer research

2021 Jan 05

Liu, Z;Sun, L;Cai, Y;Shen, S;Zhang, T;Wang, N;Wu, G;Ma, W;Li, ST;Suo, C;Hao, Y;Jia, WD;Semenza, GL;Gao, P;Zhang, H;
PMID: 33402389 | DOI: 10.1158/0008-5472.CAN-20-2876

Metastasis is responsible for the majority of breast cancer (BrCa) deaths; however, the mechanisms underlying metastasis in this disease remain largely elusive. Here we report that under hypoxic conditions, alternative splicing of MBD2 is suppressed, favoring the production of MBD2a which facilitates BrCa metastasis. Specifically, MBD2a promoted, whereas its lesser known short form MBD2c suppressed metastasis. Activation of HIF-1 under hypoxia facilitated MBD2a production via repression of SRSF2-mediated alternative splicing. As a result, elevated MBD2a outcompeted MBD2c for binding to promoter CpG islands to activate expression of FZD1, thereby promoting EMT and metastasis. Strikingly, clinical data reveals significantly correlated expression of MBD2a and MBD2c with the invasiveness of malignancy, indicating opposing roles for MBD2 splicing variants in regulating human BrCa metastasis. Collectively, our findings establish a novel link between MBD2 switching and tumor metastasis and provide a promising therapeutic strategy and predictive biomarkers for hypoxia-driven BrCa metastasis.

Neurod4 converts endogenous neural stem cells to neurons with synaptic formation after spinal cord injury

iScience

2021 Jan 01

Fukuoka, T;Kato, A;Hirano, M;Ohka, F;Aoki, K;Awaya, T;Adilijiang, A;Sachi, M;Tanahashi, K;Yamaguchi, J;Motomura, K;Shimizu, H;Nagashima, Y;Ando, R;Wakabayashi, T;Lee-Liu, D;Larrain, J;Nishimura, Y;Natsume, A;
| DOI: 10.1016/j.isci.2021.102074

The transcriptome analysis of injured Xenopus laevis tadpole and mice suggested that Neurod4L.S., a basic-helix-loop-helix transcription factor, was the most promising transcription factor to exert neuroregeneration after spinal cord injury (SCI) in mammals. We generated a pseudotyped retroviral vector with the neurotropic lymphocytic choriomeningitis virus (LCMV) envelope to deliver murine Neurod4 to mice undergoing SCI. SCI induced ependymal cells to neural stem cells (NSCs) in the central canal. The LCMV envelope-based pseudotypedvector preferentially introduced Neurod4 into activated NSCs, which converted to neurons with axonal regrowth and suppressed the scar-forming glial lineage. Neurod4-induced inhibitory neurons predominantly projected to the subsynaptic domains of motor neurons at the epicenter, and Neurod4-induced excitatory neurons predominantly projected to subsynaptic domains of motor neurons caudal to the injury site suggesting the formation of functional synapses. Thus, Neurod4 is a potential therapeutic factor that can improve anatomical and functional recovery after SCI.

Infectious titres of human papillomaviruses (HPVs) in patient lesions, methodological considerations in evaluating HPV infectivity and implications for the efficacy of high-level disinfectants

EBioMedicine

2021 Jan 01

Ozbun, MA;Bondu, V;Patterson, NA;Sterk, RT;Waxman, AG;Bennett, EC;McKee, R;Sharma, A;Yarwood, J;Rogers, M;Eichenbaum, G;
PMID: 33422988 | DOI: 10.1016/j.ebiom.2020.103165

Recent publications from a single research group have suggested that aldehyde-based high-level disinfectants (HLDs), such as ortho-phthalaldehyde (OPA), are not effective at inactivating HPVs and that therefore, patients may be at risk of HPV infection from medical devices. These results could have significant public health consequences and therefore necessitated evaluation of their reproducibility and clinical relevance. We developed methods and used standardised controls to: (1) quantify the infectious levels of clinically-sourced HPVs from patient lesions and compare them to laboratory-derived HPVs, (2) evaluate experimental factors that should be controlled to ensure consistent and reproducible infectivity measurements of different HPV genotypes, and (3) determine the efficacy of select HLDs. A novel focus forming unit (FFU) infectivity assay demonstrated that exfoliates from patient anogenital lesions and respiratory papillomas yielded infectious HPV burdens up to 2.7 × 103 FFU; therefore, using 2.2 × 102 to 1.0 × 104 FFU of laboratory-derived HPVs in disinfection assays provides a relevant range for clinical exposures. RNase and neutralising antibody sensitivities were used to ensure valid infectivity measures of tissue-derived and recombinant HPV preparations. HPV infectivity was demonstrated over a dynamic range of 4-5 log10; and disinfection with OPA and hypochlorite was achieved over 3 to >4 log10 with multiple genotypes of tissue-derived and recombinant HPV isolates. This work, along with a companion publication from an independent lab in this issue, address a major public health question by showing that HPVs are susceptible to HLDs. Advanced Sterilization Products; US NIH (R01CA207368, U19AI084081, P30CA118100).

Inadequate Analgesia in African Americans with Cancer Pain

The Journal of Pain

2022 May 01

Singh, N;Vallerand, A;
| DOI: 10.1016/j.jpain.2022.03.141

African Americans have been found to receive less medication and experience more pain than Caucasians. Unfortunately, many studies simply highlight the disparities that exist between African Americans and Caucasian in pain management, but there is a lack of understanding at the etiology of these disparities. The purpose of this study was to determine the adequacy of analgesia that was prescribed for African Americans with cancer pain and elucidate any potential characteristics that contributed to receiving appropriate analgesia. This was a secondary analysis of baseline data of an intervention study of African Americans with cancer pain. In order to determine the adequacy of analgesics received, the Pain Management Index (PMI) was calculated for 302 African Americans with cancer pain. Structure equation modeling was utilized to determine which patient factors (age, gender, presence of caregiver, employment status, educational level, perceived control over pain, pain-related distress, pain intensity, functional status) led to adequate analgesia. Forty percent of African Americans with cancer pain did not receive adequate analgesia based on the drug class alone. African Americans with cancer pain who reported higher levels of pain received higher levels of analgesics. Cancer metastasis (p = .03) was the only significant predictor of an increased likelihood of receiving adequate analgesia. Despite national attention to racial disparities, African Americans with cancer pain continue to receive inadequate analgesia. Forty percent of African Americans did not get the appropriate drug based on their reported pain score. The only significant predictor of receiving appropriate analgesia was cancer metastasis. Ensuing studies testing whether these results would be the same for other races will clarify if this disparity was due to race alone. Interventions should then be created and utilized to decrease these disparities.

Evidence of disrupted rhombic lip development in the pathogenesis of Dandy-Walker malformation

Acta neuropathologica

2021 Aug 04

Haldipur, P;Bernardo, S;Aldinger, KA;Sivakumar, T;Millman, J;Sjoboen, AH;Dang, D;Dubocanin, D;Deng, M;Timms, AE;Davis, BD;Plummer, JT;Mankad, K;Oztekin, O;Manganaro, L;Guimiot, F;Adle-Biassette, H;Russo, R;Siebert, JR;Kidron, D;Petrilli, G;Roux, N;Razavi, F;Glass, IA;Di Gioia, C;Silvestri, E;Millen, KJ;
PMID: 34347142 | DOI: 10.1007/s00401-021-02355-7

Dandy-Walker malformation (DWM) and Cerebellar vermis hypoplasia (CVH) are commonly recognized human cerebellar malformations diagnosed following ultrasound and antenatal or postnatal MRI. Specific radiological criteria are used to distinguish them, yet little is known about their differential developmental disease mechanisms. We acquired prenatal cases diagnosed as DWM and CVH and studied cerebellar morphobiometry followed by histological and immunohistochemical analyses. This was supplemented by laser capture microdissection and RNA-sequencing of the cerebellar rhombic lip, a transient progenitor zone, to assess the altered transcriptome of DWM vs control samples. Our radiological findings confirm that the cases studied fall within the accepted biometric range of DWM. Our histopathological analysis points to reduced foliation and inferior vermian hypoplasia as common features in all examined DWM cases. We also find that the rhombic lip, a dorsal stem cell zone that drives the growth and maintenance of the posterior vermis is specifically disrupted in DWM, with reduced proliferation and self-renewal of the progenitor pool, and altered vasculature, all confirmed by transcriptomics analysis. We propose a unified model for the developmental pathogenesis of DWM. We hypothesize that rhombic lip development is disrupted through either aberrant vascularization and/or direct insult which causes reduced proliferation and failed expansion of the rhombic lip progenitor pool leading to disproportionate hypoplasia and dysplasia of the inferior vermis. Timing of insult to the developing rhombic lip (before or after 14 PCW) dictates the extent of hypoplasia and distinguishes DWM from CVH.

Identification of potential key genes and miRNAs involved in Hepatoblastoma pathogenesis and prognosis

Journal of cell communication and signaling

2021 Mar 01

Aghajanzadeh, T;Tebbi, K;Talkhabi, M;
PMID: 33051830 | DOI: 10.1007/s12079-020-00584-1

Hepatoblastoma (HB) is one of the most common liver malignancies in children, while the molecular basis of the disease is largely unknown. Therefore, this study aims to explore the key genes and molecular mechanisms of the pathogenesis of HB using a bioinformatics approach. The gene expression dataset GSE131329 was used to find differentially expressed genes (DEGs). Functional and enrichment analyses of the DEGs were performed by the EnrichR. Then, the protein-protein interaction (PPI) network of the up-regulated genes was constructed and visualized using STRING database and Cytoscape software, respectively. MCODE was used to detect the significant modules of the PPI network, and cytoHubba was utilized to rank the important nodes (genes) of the PPI modules. Overall, six ranking methods were employed and the results were validated by the Oncopression database. Moreover, the upstream regulatory network and the miRNA-target interactions of the up-regulated DEGs were analyzed by the X2K web and the miRTarBase respectively. A total of 594 DEGs, including 221 up- and 373 down-regulated genes, were obtained, which were enriched in different cellular and metabolic processes, human diseases, and cancer. Furthermore, 15 hub genes were screened, out of which, 11 were validated. Top 10 transcription factors, kinases, and miRNAs were also determined. To the best of our knowledge, the association of RACGAP1, MKI67, FOXM1, SIN3A, miR-193b, and miR-760 with HB was reported for the first time. Our findings may be used to shed light on the underlying mechanisms of HB and provide new insights for better prognosis and therapeutic strategies.

The end of the beginning: application of single-cell sequencing to chronic lymphocytic leukemia

Blood

2023 Jan 26

Nagler, A;Wu, CJ;
PMID: 36095842 | DOI: 10.1182/blood.2021014669

Single-cell analysis has emerged over the past decade as a transformative technology informative for the systematic analysis of complex cell populations such as in cancers and the tumor immune microenvironment. The methodologic and analytical advancements in this realm have evolved rapidly, scaling from but a few cells at its outset to the current capabilities of processing and analyzing hundreds of thousands of individual cells at a time. The types of profiling attainable at individual cell resolution now range from genetic and transcriptomic characterization and extend to epigenomic and spatial analysis. Additionally, the increasing ability to achieve multiomic integration of these data layers now yields ever richer insights into diverse molecular disease subtypes and the patterns of cellular circuitry on a per-cancer basis. Over the years, chronic lymphocytic leukemia (CLL) consistently has been at the forefront of genomic investigation, given the ready accessibility of pure leukemia cells and immune cells from circulating blood of patients with this disease. Herein, we review the recent forays into the application of single-cell analysis to CLL, which are already revealing a new understanding of the natural progression of CLL, the impact of novel therapies, and the interactions with coevolving nonmalignant immune cell populations. As we emerge from the end of the beginning of this technologic revolution, CLL stands poised to reap the benefits of single-cell analysis from the standpoints of uncovering fresh fundamental biological knowledge and of providing a path to devising regimens of personalized diagnosis, treatment, and monitoring.

MEK/ERK signaling is a critical regulator of high-risk human papillomavirus oncogene expression revealing therapeutic targets for HPV-induced tumors

PLoS pathogens

2021 Jan 22

Luna, AJ;Sterk, RT;Griego-Fisher, AM;Chung, JY;Berggren, KL;Bondu, V;Barraza-Flores, P;Cowan, AT;Gan, GN;Yilmaz, E;Cho, H;Kim, JH;Hewitt, SM;Bauman, JE;Ozbun, MA;
PMID: 33481911 | DOI: 10.1371/journal.ppat.1009216

Intracellular pathogens have evolved to utilize normal cellular processes to complete their replicative cycles. Pathogens that interface with proliferative cell signaling pathways risk infections that can lead to cancers, but the factors that influence malignant outcomes are incompletely understood. Human papillomaviruses (HPVs) predominantly cause benign hyperplasia in stratifying epithelial tissues. However, a subset of carcinogenic or "high-risk" HPV (hr-HPV) genotypes are etiologically linked to nearly 5% of all human cancers. Progression of hr-HPV-induced lesions to malignancies is characterized by increased expression of the E6 and E7 oncogenes and the oncogenic functions of these viral proteins have been widely studied. Yet, the mechanisms that regulate hr-HPV oncogene transcription and suppress their expression in benign lesions remain poorly understood. Here, we demonstrate that EGFR/MEK/ERK signaling, influenced by epithelial contact inhibition and tissue differentiation cues, regulates hr-HPV oncogene expression. Using monolayer cells, epithelial organotypic tissue models, and neoplastic tissue biopsy materials, we show that cell-extrinsic activation of ERK overrides cellular control to promote HPV oncogene expression and the neoplastic phenotype. Our data suggest that HPVs are adapted to use the EGFR/MEK/ERK signaling pathway to regulate their productive replicative cycles. Mechanistic studies show that EGFR/MEK/ERK signaling influences AP-1 transcription factor activity and AP-1 factor knockdown reduces oncogene transcription. Furthermore, pharmacological inhibitors of EGFR, MEK, and ERK signaling quash HPV oncogene expression and the neoplastic phenotype, revealing a potential clinical strategy to suppress uncontrolled cell proliferation, reduce oncogene expression and treat HPV neoplasia.

Expression profiles of potentially angio-modulative microRNAs in the mouse model of oxygen-induced retinopathy (OIR)

Investigative Ophthalmology & Visual Science

2023 Jan 01

Busch, M;Wenzel, A;Pfeil, JM;Stahl, A;

METHODS : To induce OIR, C57BL/6J mice were exposed to 75% oxygen from postnatal day (p) 7 to p12 and then maintained under normal room air conditions. Control mice were kept under room air conditions throughout. At p12, p17, and p25, one eye of each mouse was harvested to prepare retinal flatmounts to analyze retinal vascular changes. From the contralateral eye, total RNA was isolated and reverse transcribed into cDNA for relative quantification of miRNA expression using qRT-PCR. An in situ hybridization technique (miRNAscopeTM) was used to visualize miR-21-5p expression on formalin-fixed, paraffin-embedded mouse eye tissue sections.

Fluorescence Microscopy in Adeno-Associated Virus Research

Viruses

2023 May 16

Golm, SK;Hübner, W;Müller, KM;
PMID: 37243260 | DOI: 10.3390/v15051174

Research on adeno-associated virus (AAV) and its recombinant vectors as well as on fluorescence microscopy imaging is rapidly progressing driven by clinical applications and new technologies, respectively. The topics converge, since high and super-resolution microscopes facilitate the study of spatial and temporal aspects of cellular virus biology. Labeling methods also evolve and diversify. We review these interdisciplinary developments and provide information on the technologies used and the biological knowledge gained. The emphasis lies on the visualization of AAV proteins by chemical fluorophores, protein fusions and antibodies as well as on methods for the detection of adeno-associated viral DNA. We add a short overview of fluorescent microscope techniques and their advantages and challenges in detecting AAV.

Analysis of target mRNAs in the fixed-frozen human brain using a modified BaseScope-ISH Assay protocol

STAR Protocols

2022 Dec 01

Carisì, M;Howell, O;Morgan, A;Davies, J;
| DOI: 10.1016/j.xpro.2022.101896

We describe a modified BaseScope Assay protocol (ACDBio) for RNA in situ hybridization on fixed-frozen human brain tissue. The original protocol caused tissue detachment due to harsh tissue pre-treatment. We therefore optimized it to improve tissue stability while providing high stain quality in fragile post-mortem tissue from aged donors with advanced neurodegeneration. The main changes include two additional fixation steps and modifications to the pre-treatment protocol. We also describe tissue imaging and stain quantification using the open-source QuPath software. For complete details on the use and execution of this protocol, please refer to Hornsby et al. (2020).

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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