ROC Analysis of p16 Expression in Cell Blocks of Metastatic Head and Neck Squamous Cell Carcinoma

Journal of the American Society of Cytopathology

2021 Apr 01

Wilson, B;Israel, A;Ettel, M;Lott Limbach, A;
| DOI: 10.1016/j.jasc.2021.03.004

Background Oropharyngeal squamous cell carcinoma is associated with human papillomavirus (HPV) and often presents with early metastasis to cervical neck lymph nodes which are amenable to fine needle aspiration (FNA). The most common method of HPV status determination is p16 immunohistochemistry (IHC). The literature suggests that a lower threshold is needed for p16 positivity on cell block. We examined and quantified p16 IHC staining on cell block and used ROC curve analysis to determine an optimal cut off value with high sensitivity and specificity. Methods Thirty-six FNAs of metastatic squamous cell carcinoma from cervical lymph nodes with p16 IHC were evaluated. The p16 stain was quantified in 5% increments and HR-HPV mRNA ISH was performed as a gold standard test. Statistical analysis was performed. Results Interobserver variability was evaluated and was shown to be low with an intraclass correlation coefficient of 0.857. Receiver operating characteristics (ROC) analysis was performed and showed that a cell block p16 IHC cut-off of 15% yielded the highest sensitivity (80%) and specificity (81.8%). Conclusion Our data shows that a threshold of 15% p16 staining in cell block maximizes sensitivity and specificity.

Double-stranded RNA immunohistochemistry as a screening tool for viral encephalitis

American journal of clinical pathology

2023 May 04

Piantadosi, A;Shariatzadeh, N;Bombin, A;Arkun, K;Alexandrescu, S;Kleinschmidt-DeMasters, BK;Solomon, IH;
PMID: 37141170 | DOI: 10.1093/ajcp/aqad039

Viral infections of the central nervous system can be challenging to diagnose because of the wide range of causative agents and nonspecific histologic features. We sought to determine whether detection of double-stranded RNA (dsRNA), produced during active RNA and DNA viral infections, could be used to select cases for metagenomic next-generation sequencing (mNGS) from formalin-fixed, paraffin-embedded brain tissue.Eight commercially available anti-dsRNA antibodies were optimized for immunohistochemistry (IHC) and the top antibody tested in a series of cases with confirmed viral infections (n = 34) and cases with inflammatory brain lesions of unclear etiology (n = 62).Among known positives, anti-dsRNA IHC produced a strong cytoplasmic or nuclear staining pattern for Powassan virus, West Nile virus, rabies virus, JC polyoma virus, and adenovirus while failing to detect Eastern equine encephalitis virus, Jamestown Canyon virus, or any herpesvirus. All the unknown cases were negative by anti-dsRNA IHC, while mNGS detected rare viral reads (0.3-1.3 reads per million total reads) in 2 cases (3%), with only 1 having potential clinical significance.Anti-dsRNA IHC can effectively identify a subset of clinically relevant viral infections but not all. The absence of staining should not exclude cases from mNGS if sufficient clinical and histologic suspicion exists.

Single-cell transcriptomic landscape of the developing human spinal cord

Nature neuroscience

2023 Apr 24

Andersen, J;Thom, N;Shadrach, JL;Chen, X;Onesto, MM;Amin, ND;Yoon, SJ;Li, L;Greenleaf, WJ;Müller, F;Pașca, AM;Kaltschmidt, JA;Pașca, SP;
PMID: 37095394 | DOI: 10.1038/s41593-023-01311-w

Understanding spinal cord assembly is essential to elucidate how motor behavior is controlled and how disorders arise. The human spinal cord is exquisitely organized, and this complex organization contributes to the diversity and intricacy of motor behavior and sensory processing. But how this complexity arises at the cellular level in the human spinal cord remains unknown. Here we transcriptomically profiled the midgestation human spinal cord with single-cell resolution and discovered remarkable heterogeneity across and within cell types. Glia displayed diversity related to positional identity along the dorso-ventral and rostro-caudal axes, while astrocytes with specialized transcriptional programs mapped into white and gray matter subtypes. Motor neurons clustered at this stage into groups suggestive of alpha and gamma neurons. We also integrated our data with multiple existing datasets of the developing human spinal cord spanning 22 weeks of gestation to investigate the cell diversity over time. Together with mapping of disease-related genes, this transcriptomic mapping of the developing human spinal cord opens new avenues for interrogating the cellular basis of motor control in humans and guides human stem cell-based models of disease.

Pain-causing stinging nettle toxins target TMEM233 to modulate NaV1.7 function

Nature communications

2023 Apr 28

Jami, S;Deuis, JR;Klasfauseweh, T;Cheng, X;Kurdyukov, S;Chung, F;Okorokov, AL;Li, S;Zhang, J;Cristofori-Armstrong, B;Israel, MR;Ju, RJ;Robinson, SD;Zhao, P;Ragnarsson, L;Andersson, Å;Tran, P;Schendel, V;McMahon, KL;Tran, HNT;Chin, YK;Zhu, Y;Liu, J;Crawford, T;Purushothamvasan, S;Habib, AM;Andersson, DA;Rash, LD;Wood, JN;Zhao, J;Stehbens, SJ;Mobli, M;Leffler, A;Jiang, D;Cox, JJ;Waxman, SG;Dib-Hajj, SD;Gregory Neely, G;Durek, T;Vetter, I;
PMID: 37117223 | DOI: 10.1038/s41467-023-37963-2

Voltage-gated sodium (NaV) channels are critical regulators of neuronal excitability and are targeted by many toxins that directly interact with the pore-forming α subunit, typically via extracellular loops of the voltage-sensing domains, or residues forming part of the pore domain. Excelsatoxin A (ExTxA), a pain-causing knottin peptide from the Australian stinging tree Dendrocnide excelsa, is the first reported plant-derived NaV channel modulating peptide toxin. Here we show that TMEM233, a member of the dispanin family of transmembrane proteins expressed in sensory neurons, is essential for pharmacological activity of ExTxA at NaV channels, and that co-expression of TMEM233 modulates the gating properties of NaV1.7. These findings identify TMEM233 as a previously unknown NaV1.7-interacting protein, position TMEM233 and the dispanins as accessory proteins that are indispensable for toxin-mediated effects on NaV channel gating, and provide important insights into the function of NaV channels in sensory neurons.

Anti-HERV-K Drugs and Vaccines, Possible Therapies against Tumors

Vaccines

2023 Mar 28

Hosseiniporgham, S;Sechi, LA;
PMID: 37112663 | DOI: 10.3390/vaccines11040751

The footprint of human endogenous retroviruses (HERV), specifically HERV-K, has been found in malignancies, such as melanoma, teratocarcinoma, osteosarcoma, breast cancer, lymphoma, and ovary and prostate cancers. HERV-K is characterized as the most biologically active HERV due to possession of open reading frames (ORF) for all Gag, Pol, and Env genes, which enables it to be more infective and obstructive towards specific cell lines and other exogenous viruses, respectively. Some factors might contribute to carcinogenicity and at least one of them has been recognized in various tumors, including overexpression/methylation of long interspersed nuclear element 1 (LINE-1), HERV-K Gag, and Env genes themselves plus their transcripts and protein products, and HERV-K reverse transcriptase (RT). Therapies effective for HERV-K-associated tumors mostly target invasive autoimmune responses or growth of tumors through suppression of HERV-K Gag or Env protein and RT. To design new therapeutic options, more studies are needed to better understand whether HERV-K and its products (Gag/Env transcripts and HERV-K proteins/RT) are the initiators of tumor formation or just the disorder's developers. Accordingly, this review aims to present evidence that highlights the association between HERV-K and tumorigenicity and introduces some of the available or potential therapies against HERV-K-induced tumors.

Modeling Pkd1 gene-targeted strategies for correction of polycystic kidney disease

Molecular Therapy - Methods & Clinical Development

2023 Apr 01

Kurbegovic, A;Pacis, R;Trudel, M;
| DOI: 10.1016/j.omtm.2023.03.016

Autosomal dominant polycystic kidney disease (ADPKD) causes renal cysts and leads to end-stage-renal-disease in midlife due mainly to PKD1 gene mutations. Virtually no studies have explored gene therapeutic strategies for long-term effective treatment of PKD. Toward this aim, the severely cystic Pkd1 null mouse model was targeted by series of transgene transfer using genomic Pkd1 under its regulatory elements (Pkd1wt), kidney-targeted Pkd1 gene (SBPkd1) or Pkd1Minigene. The introduced Pkd1 wt gene constructs with ∼8-fold overexpression display similar endogenous cellular profile, full complementation of Pkd1-/- phenotype and establish the referral Pkd1 genomic length for proper regulation. SBPkd1 transgene transfers expressing 0.6- or 7-fold Pkd1 endogenous levels are sufficient to correct glomerular and proximal tubular cysts as well as markedly postpone cysts in other tubular segments, showing that the small SB elements appreciably overlap with Pkd1 promoter/5’UTR regulation. Renal targeted Pkd1Minigene at high-copy conveys similar expression level to endogenous Pkd1 gene with widespread and homogeneous weak Pkd1 cellular signal, partially rescuing all cystic tubular segments. These transgene transfers determine that Pkd1 intragenic sequences not only regulate expression levels but also spatiotemporal pattern. Importantly, our study demonstrates that Pkd1 re-expression from hybrid therapeutic constructs can ameliorate, with considerably extended lifespan, or eliminate PKD.

Nucleus Accumbens Local Circuit for Cue-Dependent Aversive Learning

bioRxiv : the preprint server for biology

2023 Feb 07

Belilos, A;Gray, C;Sanders, C;Richie, C;Sengupta, A;Hake, H;Francis, TC;
PMID: 36798245 | DOI: 10.1101/2023.02.06.527338

Response to threatening environmental stimuli requires holistic detection and encoding of important environmental features that dictate threat. Animals need to recognize the likelihood that an environmental stimulus predicts threat and respond to these salient aversive stimuli appropriately. The nucleus accumbens is uniquely positioned to process this salient, aversive information and promote motivated output, through plasticity on the major projection neurons in the brain area. Here, we uncover a nucleus accumbens core local circuit whereby excitatory plasticity facilitates learning and recall of discrete aversive cues. We demonstrate that nucleus accumbens substance P release and long-term excitatory plasticity on dopamine 2 receptor expressing projection neurons is required for learning about aversion-associated cues. Additionally, we found learning and recall were dependent on different projection-neuron subtypes. Our work demonstrates a critical role for Nucleus Accumbens substance P in cue-dependent aversive learning.

Nucleic Acid Pharmaceutical Agents

Haschek and Rousseaux's Handbook of Toxicologic Pathology, Volume 2 : Safety Assessment Environmental Toxicologic Pathology

2023 Feb 27

Kohnken, R;Harbison, C;Klein, S;Engelhardt, J;
| DOI: 10.1016/B978-0-12-821047-5.00017-8

Nucleic acid pharmaceutical (NAP) agents are a relatively recent class of therapeutics that are uniquely capable of inhibiting protein translation through direct interaction with RNA. These classes of pharmaceuticals have demonstrated clinical benefit for diseases previously considered untreatable by small molecules and biologics by their theoretical ability to target any cellular RNA associated with disease. This chapter provides an overview of the major types of NAPs and the types of indications for which they are being developed, with examples of therapeutics on the market and in clinical development. The reader is also provided with a review of the most common delivery systems and chemical modifications that enable the biodistribution and efficacy of these drugs. A brief discussion of the nonclinical safety package is provided, and finally the most common toxicities that have been observed in preclinical species are discussed.

Reagents for Mass Cytometry

Chemical reviews

2023 Jan 25

Arnett, LP;Rana, R;Chung, WW;Li, X;Abtahi, M;Majonis, D;Bassan, J;Nitz, M;Winnik, MA;
PMID: 36696538 | DOI: 10.1021/acs.chemrev.2c00350

Mass cytometry (cytometry by time-of-flight detection [CyTOF]) is a bioanalytical technique that enables the identification and quantification of diverse features of cellular systems with single-cell resolution. In suspension mass cytometry, cells are stained with stable heavy-atom isotope-tagged reagents, and then the cells are nebulized into an inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS) instrument. In imaging mass cytometry, a pulsed laser is used to ablate ca. 1 μm2 spots of a tissue section. The plume is then transferred to the CyTOF, generating an image of biomarker expression. Similar measurements are possible with multiplexed ion bean imaging (MIBI). The unit mass resolution of the ICP-TOF-MS detector allows for multiparametric analysis of (in principle) up to 130 different parameters. Currently available reagents, however, allow simultaneous measurement of up to 50 biomarkers. As new reagents are developed, the scope of information that can be obtained by mass cytometry continues to increase, particularly due to the development of new small molecule reagents which enable monitoring of active biochemistry at the cellular level. This review summarizes the history and current state of mass cytometry reagent development and elaborates on areas where there is a need for new reagents. Additionally, this review provides guidelines on how new reagents should be tested and how the data should be presented to make them most meaningful to the mass cytometry user community.

Progenitor-derived endothelin controls dermal sheath contraction for hair follicle regression

Nature cell biology

2023 Jan 30

Martino, P;Sunkara, R;Heitman, N;Rangl, M;Brown, A;Saxena, N;Grisanti, L;Kohan, D;Yanagisawa, M;Rendl, M;
PMID: 36717629 | DOI: 10.1038/s41556-022-01065-w

Substantial follicle remodelling during the regression phase of the hair growth cycle is coordinated by the contraction of the dermal sheath smooth muscle, but how dermal-sheath-generated forces are regulated is unclear. Here, we identify spatiotemporally controlled endothelin signalling-a potent vasoconstriction-regulating pathway-as the key activating mechanism of dermal sheath contraction. Pharmacological blocking or genetic ablation of both endothelin receptors, ETA and ETB, impedes dermal sheath contraction and halts follicle regression. Epithelial progenitors at the club hair-epithelial strand bottleneck produce the endothelin ligand ET-1, which is required for follicle regression. ET signalling in dermal sheath cells and downstream contraction is dynamically regulated by cytoplasmic Ca2+ levels through cell membrane and sarcoplasmic reticulum calcium channels. Together, these findings illuminate an epithelial-mesenchymal interaction paradigm in which progenitors-destined to undergo programmed cell death-control the contraction of the surrounding sheath smooth muscle to orchestrate homeostatic tissue regression and reorganization for the next stem cell activation and regeneration cycle.

Targeting Peripheral μ-opioid Receptors or μ-opioid Receptor-Expressing Neurons Does not Prevent Morphine-induced Mechanical Allodynia and Anti-allodynic Tolerance

Neuroscience bulletin

2023 Jan 09

Du, F;Yin, G;Han, L;Liu, X;Dong, D;Duan, K;Huo, J;Sun, Y;Cheng, L;
PMID: 36622575 | DOI: 10.1007/s12264-022-01009-2

The chronic use of morphine and other opioids is associated with opioid-induced hypersensitivity (OIH) and analgesic tolerance. Among the different forms of OIH and tolerance, the opioid receptors and cell types mediating opioid-induced mechanical allodynia and anti-allodynic tolerance remain unresolved. Here we demonstrated that the loss of peripheral μ-opioid receptors (MORs) or MOR-expressing neurons attenuated thermal tolerance, but did not affect the expression and maintenance of morphine-induced mechanical allodynia and anti-allodynic tolerance. To confirm this result, we made dorsal root ganglia-dorsal roots-sagittal spinal cord slice preparations and recorded low-threshold Aβ-fiber stimulation-evoked inputs and outputs in superficial dorsal horn neurons. Consistent with the behavioral results, peripheral MOR loss did not prevent the opening of Aβ mechanical allodynia pathways in the spinal dorsal horn. Therefore, the peripheral MOR signaling pathway may not be an optimal target for preventing mechanical OIH and analgesic tolerance. Future studies should focus more on central mechanisms.

Leveraging circuits to understand addiction

Neurocircuitry of Addiction

2023 Jan 19

Salling, M;
| DOI: 10.1016/B978-0-12-823453-2.00002-3

Advancements in neuroscientific methods often drive new waves of insight into our understanding of addiction. While addiction research questions persist, technical improvements can augment our observational sensitivity, allowing us to update and extend existing addiction models through method development, creative application, and scientific discovery. As a result of this iterative process, we have reached the point where neuroscientists can now readily identify, monitor, and control specific neural circuits during behavior, thereby opening new windows of inquiry into the neurobiology of addiction. The objective of this chapter is to familiarize the reader with standard and emerging techniques used to observe and interrogate neural circuitry that are prevalent in contemporary clinical and preclinical addiction neuroscience labs and that are presented throughout the book. This chapter will further discuss the historical context, benefits, and limitations of these techniques with a look forward into how they can be applied to questions of addiction neurocircuitry.

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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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