3: Multimodal Molecular Analysis Reveals Divergent Trajectories Of Wound Regeneration Versus Fibrosis

Plastic and Reconstructive Surgery - Global Open

2021 Jul 26

desJardins-Park, H;Mascharak, S;Januszyk, M;Chen, K;Davitt, M;Demeter, J;Henn, D;Griffin, M;Bonham, C;Mooney, N;Cheng, R;Jackson, P;Wan, D;Gurtner, G;Longaker, M;
| DOI: 10.1097/01.gox.0000769936.79898.fc

RESULTS: Pseudotime analysis (Monocle3) of pooled scRNA-seq data revealed that fibroblasts followed two distinct transcriptional trajectories, one characterized by mechanical activation (_En-1_ lineage-positive, “fibrotic” trajectory) and the other characterized by developmental and regenerative pathways (_En-1_ lineage-negative; Rspo1, Dkk2/3, Trps1). Cross-platform data integration confirmed that fibroblasts in the fibrotic trajectory correlated with myofibroblast proteomic signatures (Col1a1/2, Fn1, etc.) and fibrotic/scar ECM features. In contrast, fibroblasts in the regenerative trajectory negatively correlated with myofibroblast markers and were associated with a “basket-weave” ECM pattern quantitatively indistinguishable from that of unwounded skin. Our integrated dataset suggested an important role for Wnt pathway proteins in ENF-mediated skin regeneration, so we compared POD 14 scars and regenerated wounds by multiplexed _in situ_ hybridization (RNAScope) for _Rspo1_ (Wnt agonist), _Trps1_ (master hair follicle regulator), _Ank1_ (YAP target gene), and _Dpp4_ (EPF marker). Quantification of RNA granules across thousands of cells using a custom image analysis pipeline revealed that ENF-mediated healing (low _Dpp4_) in YAP-inhibited (low _Ank1_) wounds yielded regeneration of functional hair follicles through Wnt-mediated pathway activation (high _Rpos1_, _Trps1_). These data suggest that YAP inhibition unlocks wound regeneration via Wnt-active, _En-1_ lineage-negative fibroblasts.

Cyclic growth of dermal papilla and regeneration of follicular mesenchymal components during feather cycling

Development (Cambridge, England)

2021 Jul 16

Wu, P;Jiang, TX;Lei, M;Chen, CK;Li, SH;Widelitz, RB;Chuong, CM;
PMID: 34269796 | DOI: 10.1242/dev.198671

How dermis maintains tissue homeostasis in cyclic growth and wounding is a fundamental un-solved question. Here we study how dermal components of feather follicles undergo physiological (molting) and plucking injury-induced regeneration. Proliferation analyses reveal quiescent, transient-amplifying, and long-term label-retaining dermal cell (LRDC) states. In Growth phase, LRDCs are activated to make new dermal components with distinct cellular flows. Dermal transient amplifying (TA) cells, enriched in the proximal follicle, generate (i) peripheral pulp which extends distally to expand the epithelial-mesenchymal interactive interface for barb patterning, and (ii) central pulp which provides nutrition. Entering Resting phase, LRDCs, accompanying collar bulge epidermal LRC cells, descend to the apical dermal papilla. In the next cycle, these apical derma papilla LRDCs are re-activated to become new pulp progenitor TA cells. In growth phase, lower dermal sheath can generate dermal papilla and pulp. Transcriptome analyses identify marker genes and highlight molecular signaling associated with dermal specification. We compare cyclic topological changes with that of hair follicle, a convergently evolved follicle configuration. The work presents a model for analyzing homeostasis and tissue remodeling of mesenchymal progenitors.

Mother-to-Child Transmission of Arboviruses during Breastfeeding: From Epidemiology to Cellular Mechanisms

Viruses

2021 Jul 07

Desgraupes, S;Hubert, M;Gessain, A;Ceccaldi, P;Vidy, A;
| DOI: 10.3390/v13071312

Most viruses use several entry sites and modes of transmission to infect their host (parenteral, sexual, respiratory, oro-fecal, transplacental, transcutaneous, etc.). Some of them are known to be essentially transmitted via arthropod bites (mosquitoes, ticks, phlebotomes, sandflies, etc.), and are thus named arthropod-borne viruses, or arboviruses. During the last decades, several arboviruses have emerged or re-emerged in different countries in the form of notable outbreaks, resulting in a growing interest from scientific and medical communities as well as an increase in epidemiological studies. These studies have highlighted the existence of other modes of transmission. Among them, mother-to-child transmission (MTCT) during breastfeeding was highlighted for the vaccine strain of yellow fever virus (YFV) and Zika virus (ZIKV), and suggested for other arboviruses such as Chikungunya virus (CHIKV), dengue virus (DENV), and West Nile virus (WNV). In this review, we summarize all epidemiological and clinical clues that suggest the existence of breastfeeding as a neglected route for MTCT of arboviruses and we decipher some of the mechanisms that chronologically occur during MTCT via breastfeeding by focusing on ZIKV transmission process.

Assessing proviral competence: current approaches to evaluate HIV-1 persistence

Current opinion in HIV and AIDS

2021 Jul 01

Cicilionytė, A;Berkhout, B;Pasternak, AO;
PMID: 33993171 | DOI: 10.1097/COH.0000000000000687

Despite decades of suppressive antiretroviral therapy (ART), HIV-1 reservoirs persist and fuel viral rebound if therapy is interrupted. The persistence of viral reservoirs in infected individuals is the main obstacle to achieving HIV-1 eradication or a long-term remission. Accurate assessment of the viral reservoir size is necessary for monitoring the effectiveness of the curative interventions. Here, we review the recent progress in the development of assays to measure HIV-1 persistence, highlighting their key advantages and limitations.To estimate the viral reservoir size, a number of assays have been developed that assess different aspects of HIV-1 persistence in ART-treated individuals. These include viral outgrowth assays to measure proviral replication competence, sequencing-based assays to measure genetic intactness of HIV-1 proviruses, and diverse techniques that measure the ability of proviruses to produce viral RNA and/or proteins (transcription and translation competence), with or without ex vivo stimulation. Recent years have seen the development of next-generation reservoir assays that, in addition to measuring viral persistence markers, assess the proviral integration sites and characterize the HIV-1 reservoir cells on the single-cell level.Although no assay yet can measure the HIV-1 reservoir with 100% accuracy, recent technical advances allow reliable estimation of its size and composition.

Intestinal MYC modulates obesity-related metabolic dysfunction

Nature metabolism

2021 Jul 01

Luo, Y;Yang, S;Wu, X;Takahashi, S;Sun, L;Cai, J;Krausz, KW;Guo, X;Dias, HB;Gavrilova, O;Xie, C;Jiang, C;Liu, W;Gonzalez, FJ;
PMID: 34211180 | DOI: 10.1038/s42255-021-00421-8

MYC is a transcription factor with broad biological functions, notably in the control of cell proliferation. Here, we show that intestinal MYC regulates systemic metabolism. We find that MYC expression is increased in ileum biopsies from individuals with obesity and positively correlates with body mass index. Intestine-specific reduction of MYC in mice improves high-fat-diet-induced obesity, insulin resistance, hepatic steatosis and steatohepatitis. Mechanistically, reduced expression of MYC in the intestine promotes glucagon-like peptide-1 (GLP-1) production and secretion. Moreover, we identify Cers4, encoding ceramide synthase 4, catalysing de novo ceramide synthesis, as a MYC target gene. Finally, we show that administration of the MYC inhibitor 10058-F4 has beneficial effects on high-fat-diet-induced metabolic disorders, and is accompanied by increased GLP-1 and reduced ceramide levels in serum. This study positions intestinal MYC as a putative drug target against metabolic diseases, including non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

Pioneer axons utilize a dcc signaling-mediated invasion brake to precisely complete their pathfinding odyssey

The Journal of Neuroscience

2021 Jun 15

Kikel-Coury, N;Green, L;Nichols, E;Zellmer, A;Pai, S;Hedlund, S;Marsden, K;Smith, C;
| DOI: 10.1523/jneurosci.0212-21.2021

Axons navigate through the embryo to construct a functional nervous system. A missing part of the axon navigation puzzle is how a single axon traverses distinct anatomical choice-points through its navigation. The dorsal root ganglia neurons experience such choice-points; first they navigate to the dorsal root entry zone, then halt navigation in the peripheral nervous system to invade the spinal cord, and then reinitiate navigation inside the CNS. Here, we used time-lapse super-resolution imaging in zebrafish DRG pioneer neurons to investigate how embryonic axons control their cytoskeleton to navigate to and invade at the correct anatomical position. We found that invadopodia components form in the growth cone even during filopodia-based navigation, but only stabilize when the axon is at the spinal cord entry location. Further, we show that intermediate levels of DCC and cAMP, as well as Rac1 activation, subsequently engage an axon invasion brake. Our results indicate that actin-based invadopodia components form in the growth cone and disruption of the invasion brake causes axon entry defects and results in failed behavioral responses, thereby demonstrating the importance of regulating distinct actin populations during navigational challenges.

Virus-Dependent Immune Conditioning of Tissue Microenvironments

SSRN Electronic Journal

2021 Jun 13

Jiang, S;Chan, C;Rovira-Clavé, X;Chen, H;Bai, Y;Zhu, B;McCaffrey, E;Greenwald, N;Liu, C;Barlow, G;Weirather, J;Oliveria, J;Phillips, D;Mukherjee, N;Busman-Sahay, K;Nekorchuk, M;Terry, M;Younger, S;Bosse, M;Demeter, J;Golstev, Y;McIlwain, D;Angelo, M;Estes, J;Nolan, G;
| DOI: 10.2139/ssrn.3860320

A thorough understanding of complex spatial host-disease interactions _in situ_ is necessary in order to develop effective preventative measures and therapeutic strategies. Here, we developed Protein And Nucleic acid IN situ Imaging (PANINI) and coupled it with Multiplexed Ion Beam Imaging (MIBI) to sensitively and simultaneously quantify DNA, RNA, and protein levels within the microenvironments of tissue compartments. The PANINI-MIBI approach was used to measure over 30 parameters simultaneously across large sections of archival lymphoid tissues from non-human primates that were healthy or infected with simian immunodeficiency virus (SIV), a model that accurately recapitulates human immunodeficiency virus infection (HIV). This enabled multiplexed dissection of cellular phenotypes, functional markers, viral DNA integration events, and viral RNA transcripts as resulting from viral infection. The results demonstrated immune coordination from an unexpected upregulation of IL10 in B cells in response to SIV infection that correlated with macrophage M2 polarization, thus conditioning a potential immunosuppressive environment that allows for viral production. This multiplexed imaging strategy also allowed characterization of the coordinated microenvironment around latently or actively infected cells to provide mechanistic insights into the process of viral latency. The spatial multi-modal framework presented here is applicable to deciphering tissue responses in other infectious diseases and tumor biology.

Leptin receptor-expressing pericytes mediate access of hypothalamic feeding centers to circulating leptin

Cell metabolism

2021 Jun 08

Butiaeva, LI;Slutzki, T;Swick, HE;Bourguignon, C;Robins, SC;Liu, X;Storch, KF;Kokoeva, MV;
PMID: 34129812 | DOI: 10.1016/j.cmet.2021.05.017

Knowledge of how leptin receptor (LepR) neurons of the mediobasal hypothalamus (MBH) access circulating leptin is still rudimentary. Employing intravital microscopy, we found that almost half of the blood-vessel-enwrapping pericytes in the MBH express LepR. Selective disruption of pericytic LepR led to increased food intake, increased fat mass, and loss of leptin-dependent signaling in nearby LepR neurons. When delivered intravenously, fluorescently tagged leptin accumulated at hypothalamic LepR pericytes, which was attenuated upon pericyte-specific LepR loss. Because a paracellular tracer was also preferentially retained at LepR pericytes, we pharmacologically targeted regulators of inter-endothelial junction tightness and found that they affect LepR neuronal signaling and food intake. Optical imaging in MBH slices revealed a long-lasting, tonic calcium increase in LepR pericytes in response to leptin, suggesting pericytic contraction and vessel constriction. Together, our data indicate that LepR pericytes facilitate localized, paracellular blood-brain barrier leaks, enabling MBH LepR neurons to access circulating leptin.

A locus coeruleus to dentate gyrus noradrenergic circuit modulates aversive contextual processing

Neuron

2021 May 26

Seo, DO;Zhang, ET;Piantadosi, SC;Marcus, DJ;Motard, LE;Kan, BK;Gomez, AM;Nguyen, TK;Xia, L;Bruchas, MR;
PMID: 34081911 | DOI: 10.1016/j.neuron.2021.05.006

Dysregulation in contextual processing is believed to affect several forms of psychopathology, such as post-traumatic stress disorder (PTSD). The dentate gyrus (DG), a subregion of the hippocampus, is thought to be an important brain region for disambiguating new experiences from prior experiences. Noradrenergic (NE) neurons in the locus coeruleus (LC) are more tonically active during stressful events and send dense projections to the DG, yet an understanding of their function in DG-dependent contextual discrimination has not been established. Here, we isolate a key function of the LC-NE-DG circuit in contextual aversive generalization using selective manipulations and in vivo single-cell calcium imaging. We report that activation of LC-NE neurons and terminal activity results in contextual generalization. We found that these effects required β-adrenergic-mediated modulation of hilar interneurons to ultimately promote aversive generalization, suggesting that disruption of noradrenergic tone may serve as an important avenue for treating stress-induced disorders.

Relationship of human papillomavirus with seborrheic keratosis of the female genital tract- a case-series and literature review

Histology and histopathology

2021 May 25

Dasgupta, S;van Eersel, R;Morrel, B;van den Munckhof, HAM;de Geus, VA;van der Hoeven, NMA;van de Sandt, MM;Piso-Jozwiak, M;Quint, WGV;van der Avoort, IAM;Koljenović, S;Ewing-Graham, PC;van Kemenade, FJ;
PMID: 34170001 | DOI: 10.14670/HH-18-357

Seborrheic keratoses (SKs) are benign lesions of uncertain etiology, which can develop in both genital and extra-genital locations. For genital SKs, there has been conjecture about the pathogenic role of human papillomavirus (HPV), in view of the frequent association of this virus with genital lesions. In light of the potential consequences on patient management, we investigated the relationship between HPV and SKs of the female genital tract (FGT). For this, we evaluated the current evidence on this relationship by performing an in-depth review of the literature. Furthermore, to add to the evidence on this association, we investigated the presence of HPV in a series of vulvar SKs (n=15), using a novel multimodal approach. This involved whole tissue section-polymerase chain reaction (WTS-PCR) using SPF10-DEIA-LipA25 for HPV detection and genotyping. In addition, immunohistochemistry (IHC) was performed with cellular biomarkers p16 and MIB-1, and viral biomarker E4, to augment HPV-testing. Finally, laser-capture microdissection-PCR (LCM-PCR) was performed to locate HPV to specific lesional cells, and to rule out incidental detection of resident HPV with WTS-PCR. Our findings from the literature review, as well as, the case-series are presented.

Localization and characterization of human papillomavirus-16 in oral squamous cell carcinoma

Oral diseases

2021 May 22

Saleh, W;Cha, S;Banasser, A;Fitzpatrick, SG;Bhattacharyya, I;Youssef, JM;Anees, MM;Elzahaby, IA;Katz, J;
PMID: 34022097 | DOI: 10.1111/odi.13920

The role of Human papillomavirus (HPV) in the oral squamous cell carcinoma (OSCC) has not been completely elucidated. The purpose of the present study was to investigate the prevalence and localization of HPV-16 virus in OSCC and to correlate HPV-16 positivity and p16INK4A expression with the clinical and pathological features of OSCC. The archives of Oral Pathology at University of Florida, College of Dentistry were accessed for demographic, clinical, histopathological data and slides of 114 OSCC patients. HPV-16 positivity of OSCC was evaluated by p16INK4A immunohistochemistry (IHC) and HPV-16 E6/E7mRNA by in situ hybridization (ISH). Out of 114 consecutive pathological slides of OSCC, 16 samples (14%) showed positivity for p16INK4A by IHC and 14 samples (12%) were positive for HPV-16 E6/E7mRNA ISH and the Positivity showed a significant correlation with the patients' age, alcohol consumption, and the degree of OSSC differentiation. The hard palate showed the highest positivity of p16INK4A IHC and HPV-16 mRNA ISH (38%,36% respectively). HPV-16 is a significant factor in oral carcinogenesis. We recommend using p16INK4A as a surrogate marker for HPV detection in OSCC, which can be complemented by RNA ISH for the identification of HPV subtype. This article is protected by

VEGF receptor 2 (KDR) protects airways from mucus metaplasia through a Sox9-dependent pathway

Developmental cell

2021 May 11

Jiang, M;Fang, Y;Li, Y;Huang, H;Wei, Z;Gao, X;Sung, HK;Hu, J;Qiang, L;Ruan, J;Chen, Q;Jiang, D;Whitsett, JA;Ai, X;Que, J;
PMID: 34010630 | DOI: 10.1016/j.devcel.2021.04.027

Mucus-secreting goblet cells are the dominant cell type in pulmonary diseases, e.g., asthma and cystic fibrosis (CF), leading to pathologic mucus metaplasia and airway obstruction. Cytokines including IL-13 are the major players in the transdifferentiation of club cells into goblet cells. Unexpectedly, we have uncovered a previously undescribed pathway promoting mucous metaplasia that involves VEGFa and its receptor KDR. Single-cell RNA sequencing analysis coupled with genetic mouse modeling demonstrates that loss of epithelial VEGFa, KDR, or MEK/ERK kinase promotes excessive club-to-goblet transdifferentiation during development and regeneration. Sox9 is required for goblet cell differentiation following Kdr inhibition in both mouse and human club cells. Significantly, airway mucous metaplasia in asthmatic and CF patients is also associated with reduced KDR signaling and increased SOX9 expression. Together, these findings reveal an unexpected role for VEGFa/KDR signaling in the defense against mucous metaplasia, offering a potential therapeutic target for this common airway pathology.

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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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