Cholangioscopic biopsy sample detection of bile duct invasion by hepatocellular carcinoma: an underappreciated entity

iGIE

2022 Dec 01

Nagaria, T;Raijman, I;Othman, M;Horn, G;Vierling, J;Mahadik, J;Dhingra, S;
| DOI: 10.1016/j.igie.2022.10.010

Background and Aims Bile duct invasion (BDI) by hepatocellular carcinoma (HCC) is rare and poorly characterized. Our aim was to elucidate clinical, cholangioscopic, and pathologic features of HCC with BDI and to compare them with features of cholangiocarcinoma (CC). Methods Seven cases of HCC with BDI (6 HCC and 1 combined HCC-CC) and 7 cases of CC diagnosed by cholangioscopic biopsy sampling between 2016 and 2020 were compared. Results The median age of HCC patients was 64 years (range, 49-77), and 6 patients were men. The median age of CC patients was 73 years (range, 58-75), and 4 patients were men. Obstructive jaundice was the presenting sign in 86% of HCC and 100% of CC cases. Cirrhosis was present in 77% of HCC cases but only 28% of CC cases. α-Fetoprotein was elevated in 57% of HCC cases and none of the CC cases. Both groups had biliary strictures; however, cholangioscopic features of HCC were more likely to show noncircumferential strictures with a mass and were less likely to include ulceration. Villiform formation and frond-like projections were more common in CC. Both showed increased vascularity and friability. Imaging showed a mass in 100% of HCC and in 57% of CC cases. The histopathology of HCC with BDI included trabecular or pseudoglandular architecture, granular eosinophilic cytoplasm, absence of mucin, and atypical nuclear features. Immunohistochemical staining in all HCC cases confirmed a hepatocyte phenotype. Immunohistochemical markers were required to distinguish cases of BDI caused by poorly differentiated HCC or CC because of overlapping clinicopathologic features between the 2 groups. Conclusions Cholangioscopic findings of a noncircumferential stricture with a luminal mass are indicative of HCC with BDI. Pathologists should routinely use a panel of hepatocyte and cholangiocyte biomarkers to differentiate BDI by HCC from CC and metastases in poorly differentiated carcinoma that lack mucin.

Spatial Proteomics for Further Exploration of Missing Proteins: A Case Study of the Ovary

Journal of proteome research

2022 Sep 15

Méar, L;Sutantiwanichkul, T;Östman, J;Damdimopoulou, P;Lindskog, C;
PMID: 36108145 | DOI: 10.1021/acs.jproteome.2c00392

In the quest for "missing proteins" (MPs), the proteins encoded by the human genome still lacking evidence of existence at the protein level, novel approaches are needed to detect this challenging group of proteins. The current count stands at 1,343 MPs, and it is likely that many of these proteins are expressed at low levels, in rare cell or tissue types, or the cells in which they are expressed may only represent a small minority of the tissue. Here, we used an integrated omics approach to identify and explore MPs in human ovaries. By taking advantage of publicly available transcriptomics and antibody-based proteomics data in the Human Protein Atlas (HPA), we selected 18 candidates for further immunohistochemical analysis using an exclusive collection of ovarian tissues from women and patients of reproductive age. The results were compared with data from single-cell mRNA sequencing, and seven proteins (CTXN1, MRO, RERGL, TTLL3, TRIM61, TRIM73, and ZNF793) could be validated at the single-cell type level with both methods. We present for the first time the cell type-specific spatial localization of 18 MPs in human ovarian follicles, thereby showcasing the utility of the HPA database as an important resource for identification of MPs suitable for exploration in specialized tissue samples. The results constitute a starting point for further quantitative and qualitative analysis of the human ovaries, and the novel data for the seven proteins that were validated with both methods should be considered as evidence of existence of these proteins in human ovary.

Neural mechanisms of comforting: Prosocial touch and stress buffering

Hormones and behavior

2023 Jun 08

Lim, KY;Hong, W;
PMID: 37301130 | DOI: 10.1016/j.yhbeh.2023.105391

Comforting is a crucial form of prosocial behavior that is important for maintaining social unity and improving the physical and emotional well-being of social species. It is often expressed through affiliative social touch toward someone in distress, providing relief for their distressed state. In the face of increasing global distress, these actions are paramount to the continued improvement of individual welfare and the collective good. Understanding the neural mechanisms responsible for promoting actions focused on benefitting others is particularly important and timely. Here, we review prosocial comforting behavior, emphasizing synthesizing recent studies carried out using rodent models. We discuss its underlying behavioral expression and motivations, and then explore both the neurobiology of prosocial comforting in a helper animal and the neurobiology of stress relief following social touch in a recipient as part of a feedback loop interaction.

Quanty-cFOS, a Novel ImageJ/Fiji Algorithm for Automated Counting of Immunoreactive Cells in Tissue Sections

Cells

2023 Feb 23

Beretta, C;Liu, S;Stegemann, A;Gan, Z;Wang, L;Tan, L;Kuner, R;
| DOI: 10.3390/cells12050704

Analysis of neural encoding and plasticity processes frequently relies on studying spatial patterns of activity-induced immediate early genes’ expression, such as c-fos. Quantitatively analyzing the numbers of cells expressing the Fos protein or c-fos mRNA is a major challenge owing to large human bias, subjectivity and variability in baseline and activity-induced expression. Here, we describe a novel open-source ImageJ/Fiji tool, called ‘Quanty-cFOS’, with an easy-to-use, streamlined pipeline for the automated or semi-automated counting of cells positive for the Fos protein and/or c-fos mRNA on images derived from tissue sections. The algorithms compute the intensity cutoff for positive cells on a user-specified number of images and apply this on all the images to process. This allows for the overcoming of variations in the data and the deriving of cell counts registered to specific brain areas in a highly time-efficient and reliable manner. We validated the tool using data from brain sections in response to somatosensory stimuli in a user-interactive manner. Here, we demonstrate the application of the tool in a step-by-step manner, with video tutorials, making it easy for novice users to implement. Quanty-cFOS facilitates a rapid, accurate and unbiased spatial mapping of neural activity and can also be easily extended to count other types of labelled cells.

Programmable peroxidase-assisted signal amplification enables flexible detection of nucleic acid targets in cellular and histopathological specimens

bioRxiv : the preprint server for biology

2023 Feb 01

Attar, S;Browning, VE;Liu, Y;Nichols, EK;Tsue, AF;Shechner, DM;Shendure, J;Lieberman, JA;Akilesh, S;Beliveau, BJ;
PMID: 36778496 | DOI: 10.1101/2023.01.30.526264

In situ hybridization (ISH) is a powerful tool for investigating the spatial arrangement of nucleic acid targets in fixed samples. ISH is typically visualized using fluorophores to allow high sensitivity and multiplexing or with colorimetric labels to facilitate co-visualization with histopathological stains. Both approaches benefit from signal amplification, which makes target detection effective, rapid, and compatible with a broad range of optical systems. Here, we introduce a unified technical platform, termed 'pSABER', for the amplification of ISH signals in cell and tissue systems. pSABER decorates the in situ target with concatemeric binding sites for a horseradish peroxidase-conjugated oligonucleotide which can then catalyze the massive localized deposition of fluorescent or colorimetric substrates. We demonstrate that pSABER effectively labels DNA and RNA targets, works robustly in cultured cells and challenging formalin fixed paraffin embedded (FFPE) specimens. Furthermore, pSABER can achieve 25-fold signal amplification over conventional signal amplification by exchange reaction (SABER) and can be serially multiplexed using solution exchange. Therefore, by linking nucleic acid detection to robust signal amplification capable of diverse readouts, pSABER will have broad utility in research and clinical settings.

1213P DKN-01 and tislelizumab + chemotherapy as first-line (1L) investigational therapy in advanced gastroesophageal adenocarcinoma (GEA): DisTinGuish trial

Annals of Oncology

2022 Sep 01

Klempner, S;Chao, J;Uronis, H;Sirard, C;Kagey, M;Baum, J;Song, J;Wang, J;Sonbol, M;Wainberg, Z;Ajani, J;
| DOI: 10.1016/j.annonc.2022.07.1331

Background Despite recent approval of anti-PD-1 antibodies as 1L therapy in advanced GEA, benefit is largely limited to PD-L1 combined positive scores (CPS) ≥5 patients (pts); novel therapeutic approaches are needed. DKN-01 is a targeted anti-DKK1 mAb which has demonstrated activity in GEA pts with elevated tumoral DKK1 expression, a subset of pts with more aggressive disease and shorter overall survival. Methods Phase IIa single arm trial investigating DKN-01 300 mg (D) + tislelizumab (TS) + CAPOX as 1L therapy in advanced HER2(-) GEA regardless of DKK1 status. Tumoral DKK1 mRNA expression was assessed by a chromogenic in situ hybridization RNAscope assay and assigned an H-score (0-300). Primary endpoint was ORR in modified intent to treat (mITT) population (>1 dose D); secondary endpoints included PFS and OS in intent to treat (ITT) population overall and by DKK1 expression: high (H-score ≥35) vs low. Results 25 pts enrolled (01 Sept 2020 - 08 Apr 2021). Median age 61 years (22, 80); 17 pts gastroesophageal junction adenocarcinoma; 8 pts gastric cancer. 21 GEA pts had RNAscope DKK1 expression; 57% were DKK1-high. 22 of 25 pts had vCPS: 73% were vCPS

Delayed effects of radiation in adipose tissue reflect progenitor damage and not cellular senescence

GeroScience

2022 Sep 22

Ruggiero, AD;Davis, MA;Davis, AT;DeStephanis, D;Williams, AG;Vemuri, R;Fanning, KM;Sherrill, C;Cline, JM;Caudell, DL;Kavanagh, K;
PMID: 36136223 | DOI: 10.1007/s11357-022-00660-x

The pathogenesis of many age-related diseases is linked to cellular senescence, a state of inflammation-inducing, irreversible cell cycle arrest. The consequences and mechanisms of age-associated cellular senescence are often studied using in vivo models of radiation exposure. However, it is unknown whether radiation induces persistent senescence, like that observed in ageing. We performed analogous studies in mice and monkeys, where young mice and rhesus macaques received sub-lethal doses of ionizing radiation and were observed for ~ 15% of their expected lifespan. Assessments of 8-hydroxy-2' -deoxyguanosine (8-OHdG), senescence-associated beta-galactosidase (SAβ-gal), and p16Ink4a and p21 were performed on mitotic and post-mitotic tissues - liver and adipose tissue - 6 months and 3 years post-exposure for the mice and monkeys, respectively. No elevations in 8-OHdG, SA-βgal staining, or p16 Ink4a or p21 gene or protein expression were found in mouse and monkey liver or adipose tissue compared to control animals. Despite no evidence of senescence, progenitor cell dysfunction persisted after radiation exposure, as indicated by lower in situ CD34+ adipose cells (p = 0.03), and deficient adipose stromal vascular cell proliferation (p < 0.05) and differentiation (p = 0.04) ex vivo. Our investigation cautions that employing radiation to study senescence-related processes should be limited to the acute post-exposure period and that stem cell damage likely underpins the dysfunction associated with delayed effects of radiation.

Recapitulating folliculogenesis and oogenesis outside the body: encapsulated in vitro follicle growth

Biology of reproduction

2022 Sep 22

Converse, A;Zaniker, EJ;Amargant, F;Duncan, FE;
PMID: 36136744 | DOI: 10.1093/biolre/ioac176

Folliculogenesis is a tightly coordinated process essential for generating a fertilization-competent gamete while also producing gonadal hormones that sustain endocrine function. In vitro follicle growth systems have been critical to our understanding of key events in folliculogenesis, such as gonadotropin-independent and -dependent growth, steroid hormone production, and oocyte growth and maturation (cytoplasmic and meiotic). Although there are several successful follicle culture strategies, the following protocol details an encapsulated in vitro follicle growth (eIVFG) system for use with mouse ovarian follicles. eIVFG is performed with alginate hydrogels, which are biologically inert, maintain cell-to-cell interactions between granulosa cells and the oocyte, and preserve follicle architecture as found in the ovary. The system supports follicle growth, development, and differentiation from the early primary follicle to the antral follicle stage. Moreover, post-folliculogenesis events including meiotic maturation, ovulation, and luteinization are also supported. Importantly, the culture of secondary follicles has successfully resulted in viable pups after blastocyst transfer. This alginate-based eIVFG system is versatile and has broad applications as a tool for interrogating the fundamental biology of the ovarian follicle in a controlled manner, a screening platform for toxicity and bioactivity, and a potential fertility preservation method for endangered species as well as humans.

EWSR1-ATF1 dependent 3D connectivity regulates oncogenic and differentiation programs in Clear Cell Sarcoma

Nature communications

2022 Apr 27

Möller, E;Praz, V;Rajendran, S;Dong, R;Cauderay, A;Xing, YH;Lee, L;Fusco, C;Broye, LC;Cironi, L;Iyer, S;Rengarajan, S;Awad, ME;Naigles, B;Letovanec, I;Ormas, N;Finzi, G;La Rosa, S;Sessa, F;Chebib, I;Petur Nielsen, G;Digklia, A;Spentzos, D;Cote, GM;Choy, E;Aryee, M;Stamenkovic, I;Boulay, G;Rivera, MN;Riggi, N;
PMID: 35477713 | DOI: 10.1038/s41467-022-29910-4

Oncogenic fusion proteins generated by chromosomal translocations play major roles in cancer. Among them, fusions between EWSR1 and transcription factors generate oncogenes with powerful chromatin regulatory activities, capable of establishing complex gene expression programs in permissive precursor cells. Here we define the epigenetic and 3D connectivity landscape of Clear Cell Sarcoma, an aggressive cancer driven by the EWSR1-ATF1 fusion gene. We find that EWSR1-ATF1 displays a distinct DNA binding pattern that requires the EWSR1 domain and promotes ATF1 retargeting to new distal sites, leading to chromatin activation and the establishment of a 3D network that controls oncogenic and differentiation signatures observed in primary CCS tumors. Conversely, EWSR1-ATF1 depletion results in a marked reconfiguration of 3D connectivity, including the emergence of regulatory circuits that promote neural crest-related developmental programs. Taken together, our study elucidates the epigenetic mechanisms utilized by EWSR1-ATF1 to establish regulatory networks in CCS, and points to precursor cells in the neural crest lineage as candidate cells of origin for these tumors.

Pthlha and mechanical force control early patterning of growth zones in the zebrafish craniofacial skeleton

Development (Cambridge, England)

2022 Jan 15

Hoyle, DJ;Dranow, DB;Schilling, TF;
PMID: 34919126 | DOI: 10.1242/dev.199826

Secreted signals in patterning systems often induce repressive signals that shape their distributions in space and time. In developing growth plates (GPs) of endochondral long bones, Parathyroid hormone-like hormone (Pthlh) inhibits Indian hedgehog (Ihh) to form a negative-feedback loop that controls GP progression and bone size. Whether similar systems operate in other bones and how they arise during embryogenesis remain unclear. We show that Pthlha expression in the zebrafish craniofacial skeleton precedes chondrocyte differentiation and restricts where cells undergo hypertrophy, thereby initiating a future GP. Loss of Pthlha leads to an expansion of cells expressing a novel early marker of the hypertrophic zone (HZ), entpd5a, and later HZ markers, such as ihha, whereas local Pthlha misexpression induces ectopic entpd5a expression. Formation of this early pre-HZ correlates with onset of muscle contraction and requires mechanical force; paralysis leads to loss of entpd5a and ihha expression in the pre-HZ, mislocalized pthlha expression and no subsequent ossification. These results suggest that local Pthlh sources combined with force determine HZ locations, establishing the negative-feedback loop that later maintains GPs.

A Selective Peptidomimetic Modulator Of Cav2.2 (N-Type) Voltage-Gated Calcium Channels For Chronic Pain

The Journal of Pain

2023 Apr 01

Gomez, K;Santiago, U;Calderon-Rivera, A;Duran, P;Loya-Lopez, S;Ran, D;Perez-Miller, S;Handoko, H;Arora, P;Patek, M;King, T;Hu, H;Camacho, C;Khanna, R;
| DOI: 10.1016/j.jpain.2023.02.106

Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated pain targets. Clinical block of Cav2.2 (e.g., with Prialt) or indirect modulation (e.g., with gabapentinoids) mitigates chronic pain but is constrained by side effects. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release and pain. Homology-guided mutagenesis of CBD3 revealed an antinociceptive core in the N-terminal A1RSR4. Here, we developed and applied a novel molecular dynamics approach to identify the Cav2.2 recognition motif of the core CBD3 peptide as the A1R2 dipeptide and used its presenting motif to design pharmacophore models to screen 27 million compounds in the open access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization‐evoked calcium influx in rat dorsal root ganglion (DRG) neurons. Nine compounds were tested using electrophysiology and one compound (CBD3063) was evaluated biochemically, electrophysiologically, and behaviorally in models of experimental pain. CBD3063 reduced membrane Cav2.2 expression and currents, uncoupled the Cav2.2-CRMP2 interaction, inhibited neuronal excitability, decreased spinal cord transmission, induced analgesia in naïve rats and reversed mechanical allodynia in rats with spared nerve injury. These results identify CBD3063, as a selective, first-in-class, CRMP2-based peptidomimetic, which allosterically regulates Cav2.2 to achieve analgesia.

Ribonuclease inhibitor 1 (RNH1) deficiency cause congenital cataracts and global developmental delay with infection-induced psychomotor regression and anemia

European journal of human genetics : EJHG

2023 Mar 20

Hedberg-Oldfors, C;Mitra, S;Molinaro, A;Visuttijai, K;Fogelstrand, L;Oldfors, A;Sterky, FH;Darin, N;
PMID: 36935417 | DOI: 10.1038/s41431-023-01327-7

Ribonuclease inhibitor 1, also known as angiogenin inhibitor 1, encoded by RNH1, is a ubiquitously expressed leucine-rich repeat protein, which is highly conserved in mammalian species. Inactivation of rnh1 in mice causes an embryonically lethal anemia, but the exact biological function of RNH1 in humans remains unknown and no human genetic disease has so far been associated with RNH1. Here, we describe a family with two out of seven siblings affected by a disease characterized by congenital cataract, global developmental delay, myopathy and psychomotor deterioration, seizures and periodic anemia associated with upper respiratory tract infections. A homozygous splice-site variant (c.615-2A > C) in RNH1 segregated with the disease. Sequencing of RNA derived from patient fibroblasts and cDNA analysis of skeletal muscle mRNA showed aberrant splicing with skipping of exon 7. Western blot analysis revealed a total lack of the RNH1 protein. Functional analysis revealed that patient fibroblasts were more sensitive to RNase A exposure, and this phenotype was reversed by transduction with a lentivirus expressing RNH1 to complement patient cells. Our results demonstrate that loss-of-function of RNH1 in humans is associated with a multiorgan developmental disease with recessive inheritance. It may be speculated that the infection-induced deterioration resulted from an increased susceptibility toward extracellular RNases and/or other inflammatory responses normally kept in place by the RNase inhibitor RNH1.

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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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