Proteolytic pan-RAS cleavage leads to tumor regression in patient-derived pancreatic cancer xenografts

Molecular cancer therapeutics

2022 Feb 28

Vidimar, V;Park, M;Stubbs, CK;Ingram, NK;Qiang, W;Zhang, S;Gursel, DB;Melnyk, RA;Satchell, KJ;
PMID: 35247912 | DOI: 10.1158/1535-7163.MCT-21-0550

The lack of effective RAS inhibition represents a major unmet medical need in the treatment of pancreatic ductal adenocarcinoma (PDAC). Here, we investigate the anticancer activity of RRSP-DTB, an engineered biologic that cleaves the Switch I of all RAS isoforms, in KRAS-mutant PDAC cell lines and patient-derived xenografts (PDXs). We first demonstrate that RRSP-DTB effectively engages RAS and impacts downstream ERK signaling in multiple KRAS-mutant PDAC cell lines inhibiting cell proliferation at picomolar concentrations. We next tested RRSP-DTB in immunodeficient mice bearing KRAS-mutant PDAC PDXs. Treatment with RRSP-DTB led to 95% tumor regression after 29 days. Residual tumors exhibited disrupted tissue architecture, increased fibrosis and fewer proliferating cells compared to controls. Intratumoral levels of phospho-ERK were also significantly lower, indicating in vivo target engagement. Importantly, tumors that started to regrow without RRSP-DTB shrank when treatment resumed, demonstrating resistance to RRSP-DTB had not developed. Tracking persistence of the toxin activity following intraperitoneal injection showed that RRSP-DTB is active in sera from immunocompetent mice for at least one hour, but absent after 16 hours, justifying use of daily dosing. Overall, we report that RRSP-DTB strongly regresses hard-to-treat KRAS-mutant PDX models of pancreatic cancer, warranting further development of this pan-RAS biologic for the management of RAS-addicted tumors.

Coding and long non-coding gene expression changes in the CNS traumatic injuries

Cellular and molecular life sciences : CMLS

2022 Feb 07

Wu, X;Wei, H;Wu, JQ;
PMID: 35129669 | DOI: 10.1007/s00018-021-04092-2

Traumatic brain injury (TBI) and spinal cord injury (SCI) are two main central nervous system (CNS) traumas, caused by external physical insults. Both injuries have devastating effects on the quality of life, and there is no effective therapy at present. Notably, gene expression profiling using bulk RNA sequencing (RNA-Seq) and single-cell RNA-Seq (scRNA-Seq) have revealed significant changes in many coding and non-coding genes, as well as important pathways in SCI and TBI. Particularly, recent studies have revealed that long non-coding RNAs (lncRNAs) with lengths greater than 200 nucleotides and without protein-coding potential have tissue- and cell type-specific expression pattern and play critical roles in CNS injury by gain- and loss-of-function approaches. LncRNAs have been shown to regulate protein-coding genes or microRNAs (miRNAs) directly or indirectly, participating in processes including inflammation, glial activation, cell apoptosis, and vasculature events. Therefore, lncRNAs could serve as potential targets for the diagnosis, treatment, and prognosis of SCI and TBI. In this review, we highlight the recent progress in transcriptome studies of SCI and TBI and insights into molecular mechanisms.

Considerations for a Reliable In Vitro Model of Chemotherapy-Induced Peripheral Neuropathy

Toxics

2021 Nov 11

Eldridge, S;Scuteri, A;Jones, EMC;Cavaletti, G;Guo, L;Glaze, E;
PMID: 34822690 | DOI: 10.3390/toxics9110300

Chemotherapy-induced peripheral neuropathy (CIPN) is widely recognized as a potentially severe toxicity that often leads to dose reduction or discontinuation of cancer treatment. Symptoms may persist despite discontinuation of chemotherapy and quality of life can be severely compromised. The clinical symptoms of CIPN, and the cellular and molecular targets involved in CIPN, are just as diverse as the wide variety of anticancer agents that cause peripheral neurotoxicity. There is an urgent need for extensive molecular and functional investigations aimed at understanding the mechanisms of CIPN. Furthermore, a reliable human cell culture system that recapitulates the diversity of neuronal modalities found in vivo and the pathophysiological changes that underlie CIPN would serve to advance the understanding of the pathogenesis of CIPN. The demonstration of experimental reproducibility in a human peripheral neuronal cell system will increase confidence that such an in vitro model is clinically useful, ultimately resulting in deeper exploration for the prevention and treatment of CIPN. Herein, we review current in vitro models with a focus on key characteristics and attributes desirable for an ideal human cell culture model relevant for CIPN investigations.

Chaphamaparvovirus antigen and nucleic acids are not detected in kidney tissues from cats with chronic renal disease or immunocompromised cats

Veterinary pathology

2021 Oct 03

Michel, AO;Donovan, TA;Roediger, B;Lee, Q;Jolly, CJ;Monette, S;
PMID: 34601998 | DOI: 10.1177/03009858211045439

Chronic kidney disease (CKD) is a common cause of morbidity and mortality in domestic cats, but the cause is still largely elusive. While some viruses have been associated with this disease, none have been definitively implicated as causative. Recently, Rodent chaphamaparvovirus 1 was recognized as the cause of murine inclusion body nephropathy, a disease reported for over 40 years in laboratory mice. A novel virus belonging to the same genus, Carnivore chaphamaparvovirus 2, was recently identified in the feces of cats with diarrhea. The goal of this study was to investigate the possible role of chaphamaparvoviruses including members of Rodent chaphamaparvovirus 1 and Carnivore chaphamaparvovirus 2 in the development of feline CKD. The presence of these viruses was retrospectively investigated in formalin-fixed paraffin-embedded feline kidney samples using polymerase chain reaction, in situ hybridization, and immunohistochemistry. Cats were divided into 3 groups: normal (N = 24), CKD (N = 26), and immunocompromised (N = 25). None of the kidney tissues from any of the 75 cats revealed the presence of chaphamaparvovirus DNA, RNA, or antigen. We conclude that viruses belonging to the chaphamaparvovirus genus are unlikely to contribute to the occurrence of feline CKD.

Recovery of Latent HIV-1 from Brain Tissue by Adoptive Cell Transfer in Virally Suppressed Humanized Mice

Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

2021 Sep 15

Su, H;Sravanam, S;Sillman, B;Waight, E;Makarov, E;Mathews, S;Poluektova, LY;Gorantla, S;Gendelman, HE;Dash, PK;
PMID: 34528173 | DOI: 10.1007/s11481-021-10011-w

Defining the latent human immunodeficiency virus type 1 (HIV-1) burden in the human brain during progressive infection is limited by sample access. Human hematopoietic stem cells (hu-HSCs)-reconstituted humanized mice provide an opportunity for this study. The model mimics, in measure, HIV-1 pathophysiology, transmission, treatment, and elimination in an infected human host. However, to date, brain HIV-1 latency in hu-HSC mice during suppressive antiretroviral therapy (ART) was not studied. To address this need, hu-HSC mice were administered long acting (LA) ART 14 days after HIV-1 infection was established. Animals were maintained under suppressive ART for 3 months, at which time HIV-1 infection was detected at low levels in brain tissue by droplet digital polymerase chain reaction (ddPCR) test on DNA. Notably, adoptive transfer of cells acquired from the hu-HSC mouse brains and placed into naive hu-HSC mice demonstrated viral recovery. These proof-of-concept results demonstrate replication-competent HIV-1 reservoir can be established in hu-HSC mouse brains that persists during long-term ART treatment. Hu-HSC mice-based mouse viral outgrowth assay (hu-MVOA) serves as a sensitive tool to interrogate latent HIV-1 brain reservoirs.

FANCI plays an essential role in spermatogenesis and regulates meiotic histone methylation

Cell death & disease

2021 Aug 09

Xu, L;Xu, W;Li, D;Yu, X;Gao, F;Qin, Y;Yang, Y;Zhao, S;
PMID: 34373449 | DOI: 10.1038/s41419-021-04034-7

FANCI is an essential component of Fanconi anemia pathway, which is responsible for the repair of DNA interstrand cross-links (ICLs). As an evolutionarily related partner of FANCD2, FANCI functions together with FANCD2 downstream of FA core complex. Currently, growing evidences showed that the essential role of FA pathway in male fertility. However, the underlying mechanisms for FANCI in regulating spermatogenesis remain unclear. In the present study, we found that the male Fanci-/- mice were sterile and exhibited abnormal spermatogenesis, including massive germ cell apoptosis in seminiferous tubules and dramatically decreased number of sperms in epididymis. Besides, FANCI deletion impaired maintenance of undifferentiated spermatogonia. Further investigation indicated that FANCI was essential for FANCD2 foci formation and regulated H3K4 and H3K9 methylation on meiotic sex chromosomes. These findings elucidate the role and mechanism of FANCI during spermatogenesis in mice and provide new insights into the etiology and molecular basis of nonobstructive azoospermia.

Imaging Methods in Xenopus Cells, Embryos, and Tadpoles

Cold Spring Harbor protocols

2021 Jul 08

Davidson, LA;Lowery, LA;
PMID: 34244350 | DOI: 10.1101/pdb.top105627

Xenopus is an excellent vertebrate model system ideally suited for a wide range of imaging methods designed to investigate cell and developmental biology processes. The individual cells of Xenopus are much larger than those in many other vertebrate model systems, such that both cell behavior and subcellular processes can more easily be observed and resolved. Gene function in Xenopus can be manipulated and visualized using a variety of approaches, and the embryonic fate map is stereotypical, such that microinjections can target specific tissues and cell types during development. Tissues, organotypic explants, and individual cells can also be mounted in stable chambers and cultured easily in simple salt solutions without cumbersome environmental controls. Furthermore, Xenopus embryonic tissues can be microsurgically isolated and shaped to expose cell behaviors and protein dynamics in any regions of the embryo to high-resolution live-cell imaging. The combination of these attributes makes Xenopus a powerful system for understanding cell and developmental processes as well as disease mechanisms, through quantitative analysis of protein dynamics, cell movements, tissue morphogenesis, and regeneration. Here, we introduce various methods, of both fixed and living tissues, for visualizing Xenopus cells, embryos, and tadpoles. Specifically, we highlight protocol updates for whole-mount in situ hybridization and immunofluorescence, as well as robust live imaging approaches including methods for optimizing the time-lapse imaging of whole embryos and explants.

Fluorescent nanoparticle-mediated semiquantitative MYC protein expression analysis in morphologically diffuse large B-cell lymphoma

Pathology international

2021 Jun 25

Takayanagi, N;Momose, S;Kikuchi, J;Tanaka, Y;Anan, T;Yamashita, T;Higashi, M;Tokuhira, M;Kizaki, M;Tamaru, JI;
PMID: 34171161 | DOI: 10.1111/pin.13131

The current World Health Organization (WHO) classification defines a new disease entity of high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, making fluorescence in situ hybridization (FISH) screening for these genes mandatory. In addition, the prognostic significance of MYC expression was reported, with a cut-off value of 40%. However, interobserver discrepancies arise due to the heterogeneous intensity of MYC expression by immunohistochemistry. Moreover, a cut-off value of positivity for MYC protein in diffuse large B-cell lymphoma (DLBCL) varies among studies at present. Here, we applied a high-sensitivity semiquantitative immunohistochemical technique using fluorescent nanoparticles called phosphor-integrated dots (PID) to evaluate the MYC expression in 50 de novo DLBCL cases, and compared it with the conventional diaminobenzidine (DAB)-developing system. The high MYC expression detected by the PID-mediated system predicted poor overall survival in DLBCL patients. However, we found no prognostic value of MYC protein expression for any cut-off value by the DAB-developing system, even if the intensity was considered. These results indicate that the precise evaluation of MYC protein expression can clarify the prognostic values in DLBCL, irrespective of MYC rearrangement.

Amygdala microglia modify neuronal plasticity via complement C1q/C3-CR3 signaling and contribute to visceral pain in a rat model

American journal of physiology. Gastrointestinal and liver physiology

2021 May 05

Yuan, T;Orock, A;Greenwood-Van Meerveld, B;
PMID: 33949202 | DOI: 10.1152/ajpgi.00123.2021

Stress can trigger symptoms in patients with irritable bowel syndrome (IBS). Previously we demonstrated that chronic psychological stress induced microglial remodeling in the central nucleus of amygdala (CeA), and contributed to the development of visceral hypersensitivity via synaptic engulfment. However, the specific signaling mechanisms that microglia depend upon to recognize target neurons to facilitate visceral pain remain unknown. Here we test the hypothesis that the microglia in the CeA contribute to chronic stress-induced visceral hypersensitivity via complement C1q/C3-CR3 signaling-mediated synaptic remodeling. In male and female Fischer-344 rats, micropellets of corticosterone (CORT) or cholesterol (control) were stereotaxically implanted bilaterally onto the CeA. After 7 days, microglial C1q, complement receptor 3 (CR3) expression and microglia-mediated synaptic engulfment were assessed via RNAscope, quantitative PCR and immunofluorescence. The microglial inhibitor minocycline, CR3 antagonist neutrophil inhibitory factor (NIF) or vehicle were daily infused into the CeA following CORT implantations. Visceral sensitivity was assessed via a visceromotor response (VMR) to graded pressures of isobaric colorectal distension (CRD). Our results suggest that chronic exposure to elevated CORT in the CeA induced visceral hypersensitivity and amygdala microglial morphological remodeling. CORT increased microglial C1q and CR3 expression, and increased microglia-mediated synaptic engulfment. Both groups of animals with minocycline or NIF infusions reversed microglia-mediated synaptic remodeling, and attenuated CORT-induced visceral hypersensitivity. Our findings demonstrate that C1q/C3-CR3 signaling is critical for microglia-mediated synaptic remodeling in the CeA and contributes to CORT-induced visceral hypersensitivity.

Primary Cutaneous Monomorphic Post-transplant Lymphoproliferative Disorder Mimicking Squamous Cell Carcinoma In Situ

The American Journal of dermatopathology

2021 Apr 21

Craddock, AP;Gru, AA;Mannschreck, D;Wilson, BB;Raghavan, SS;
PMID: 33899771 | DOI: 10.1097/DAD.0000000000001950

Post-transplant lymphoproliferative disorder (PTLD) is a term used to describe a range of lymphoproliferative disorders that occur after solid organ transplant. Although the clinical presentation is variable, primary cutaneous PTLD typically presents as isolated nodules that appear as dermal-based proliferations. We present a case of a 70-year-old woman with a history of a kidney transplant who presented with a 2-month history of an asymptomatic, erythematous plaque on the right shin, clinically suspected to be squamous cell carcinoma in situ. Histomorphology demonstrated a dermal proliferation of atypical plasma cells with dense chromatin, variable nucleoli, and irregular nuclear borders. The atypical plasma cells were positive for Epstein-Barr virus by in situ hybridization and markedly kappa-restricted by RNAscope in situ hybridization. A diagnosis of cutaneous monomorphic PTLD, plasma cell neoplasm variant, was rendered, a rare diagnosis in the skin. Treatment for PTLD typically involves reduction of immunosuppression, although our patient progressed and developed new lesions despite this intervention. In this study, we present an atypical presentation of cutaneous PTLD, plasma cell neoplasm variant, presenting as squamous cell carcinoma in situ.

Paraventricular Calcitonin Receptor Expressing Neurons Modulate Energy Homeostasis in Male Mice

Endocrinology

2021 Apr 09

Gonzalez, IE;Ramirez-Matias, J;Lu, C;Pan, W;Zhu, A;Myers, MG;Olson, DP;
PMID: 33834205 | DOI: 10.1210/endocr/bqab072

The paraventricular nucleus of the hypothalamus (PVH) is a heterogeneous collection of neurons that play important roles in modulating feeding and energy expenditure. Abnormal development or ablation of the PVH results in hyperphagic obesity and defects in energy expenditure whereas selective activation of defined PVH neuronal populations can suppress feeding and may promote energy expenditure. Here, we characterize the contribution of calcitonin receptor-expressing PVH neurons (CalcR PVH) to energy balance control. We used Cre-dependent viral tools delivered stereotaxically to the PVH of CalcR 2Acre mice to activate, silence and trace CalcR PVH neurons and determine their contribution to body weight regulation. Immunohistochemistry of fluorescently-labelled CalcR PVH neurons demonstrates that CalcR PVH neurons are largely distinct from several PVH neuronal populations involved in energy homeostasis; these neurons project to regions of the hindbrain that are implicated in energy balance control, including the nucleus of the solitary tract and the parabrachial nucleus. Acute activation of CalcR PVH neurons suppresses feeding without appreciably augmenting energy expenditure, whereas their silencing leads to obesity that may be due in part due to loss of PVH melanocortin-4 receptor (MC4R) signaling. These data show that CalcR PVH neurons are an essential component of energy balance neurocircuitry and their function is important for body weight maintenance. A thorough understanding of the mechanisms by which CalcR PVH neurons modulate energy balance might identify novel therapeutic targets for the treatment and prevention of obesity.

High-risk human papillomavirus and ZEB1 in ocular adnexal sebaceous carcinoma

Journal of cutaneous pathology

2021 Mar 21

Moore, RF;Zhang, XR;Allison, DB;Rooper, LM;Campbell, AA;Eberhart, CG;
PMID: 33745190 | DOI: 10.1111/cup.13987

Ocular adnexal (OA) sebaceous carcinoma is an aggressive malignancy. Oncologic drivers of ocular sebaceous carcinoma are incompletely understood. A retrospective search of our pathology archives for OA sebaceous carcinoma identified 18 primary resection specimens. Immunohistochemistry for p16 and ZEB1 and RNA in situ hybridization for high-risk human papillomavirus (HPV) subtypes were performed. High-risk HPV was demonstrated in 2/11 (18%) cases. p16 overexpression was observed in 10/11 (91%). No association between gender, age at presentation, tumor location, intraepithelial spread, tumor size, and T stage was observed between HPV-driven and nonviral cases. High expression of ZEB1 was observed in the intraepithelial component of 4/14 (28%) cases and in the subepithelial component of 1/13 (7%) cases. ZEB1 overexpression was not associated with HPV-status, T stage, or tumor size. As previously described by others, our findings suggest that a subset of OA sebaceous carcinomas may arise via an HPV-dependent pathway. However, unlike high-risk HPV-driven carcinomas of the oropharynx, we did not identify an association between HPV-status and prognostic features. Furthermore, p16 expression was not a useful surrogate marker for HPV-driven disease. ZEB1 overexpression is not associated with HPV in our cohort of ocular sebaceous carcinoma.

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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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