Cryptorchidism consists in the failure of one or both testicles to fully descend into the scrotum. The position of the retained testes can be abdominal or inguinal and may occur unilaterally or bilaterally. This retrospective, multi-center study aimed to describe the computed tomography (CT) features of retained testes in dogs. Nineteen CT scans of dogs, with either unilateral or bilateral cryptorchidism, were analyzed with both pre- and post-contrast imaging. The location, size, shape, margins, homogeneous parenchyma, and density calculated were examined with the Hounsfield unit. Statistical analyses were performed to assess the differences between the scrotal (ST) and undescended testes (UT) and to detect any correlation between the features, ages, and size of the dog. CT identified the retained testes and provided enhanced three-dimensional visualization compared to traditional ultrasound. This study revealed that the UT were significantly smaller than ST, with UT measuring around 70% of ST size. Additionally, the UT exhibited increased density in both pre- and post-contrast scans, potentially due to the reduced or absent spermatogenesis or to histological changes occurring in the parenchyma. While CT showed clear advantages, such as three-dimensional spatial resolution and deeper tissue penetration, limitations such as general anesthesia and radiation exposure should also be considered. However, the present study showed that CT could serve as a valuable second step tool in cases where ultrasound fails, particularly in challenging anatomical situations.

CoREST complexes (LSD1, HDAC1/2, and RCoR1/2/3) are pivotal in neurodevelopment and have long been recognized as transcriptional repressors across various cancers. However, distinct roles of RCoR factors remain underexplored. Here, we unveil non-canonical functions of RCoR2 in MYCN-amplified neuroblastoma (NB), underscoring its unique significance compared to its paralogs. This insight shifts the paradigm, highlighting RCoR2 as a key determinant of the NB chromatin landscape. Our findings demonstrate that RCoR2 is a super-enhancer-driven gene, which, unlike RCoR1, acts as a positive regulator of gene expression as a partner of the adrenergic NB core regulatory circuitry (CRC). We propose a model in which RCoR2 facilitates interactions between CRC-bound enhancers and their associated transcription start sites, thereby sustaining the expression of genes critical for NB cell survival. Thus, we identify RCoR2 as a critical vulnerability in high-risk NBs and a promising target for cancer therapeutics.

IntroductionUltrasound imaging (US) is the method of choice to assess the canine prostate gland. Whilst recent studies have documented the role of castration in the development of prostatic neoplasia, little is known about parenchymal and perfusion features of the normal and abnormal prostate in neutered dogs. No data are available concerning prostatic changes after the first 90 days following castration. The present study aimed to acquire data on the long-term ultrasonographic changes occurring to the canine prostate after castration.Materials and methodsTen adult neutered dogs underwent B-mode US and contrast-enhanced ultrasound (CEUS) of the prostate on two occasions: day of the first examination (T0) and six years later (T1). The prostate was evaluated via B-mode US and the volume was calculated using Atalan’s formula. For CEUS examination, an intravenous contrast agent (SonoVue) was administered to assess prostatic perfusion. Videoclips were recorded, and time-intensity curves were obtained to determine contrast parameters: peak enhancement (PPI) and time to peak (TTP). Volumetric and perfusion results were then compared between timepoints.ResultsAt both examinations, the prostate appeared similar on B-mode US in terms of morphology and echotexture, minimally decreasing in volume over time. Prostate perfusion was significantly reduced in all dogs between T0 and T1, with a PPI decrease from 54.9 to 29.6% and an increase in TTP from 26.3 to 47 s.DiscussionThese preliminary data provide baseline information on the B-mode appearance and CEUS measurements of the prostate gland of neutered dogs and suggest that prostatic involution after castration is not a short-term process but continues over several months.

Ultrasound is an important tool in small animal andrology for assessing the integrity of the testes. This study explores ultrasound’s role in assessing the canine testes and understanding the correlation of sonographic parameters to sperm quality. It investigates B-mode and Doppler ultrasound techniques alongside advanced methods like Shear-wave Elastography (SWE) and Contrast-enhanced ultrasound (CEUS). The aim was to standardize a multiparametric ultrasound (MPUS) evaluation protocol using these techniques in normozoospermic dogs. Eight healthy male dogs were assessed. B-mode assessed testicular morphology, while Doppler evaluated testicular artery waveform morphology and velocimetric parameters. SWE measured testicular stiffness, and CEUS assessed testicular perfusion. Seminal parameters were also analyzed. Results revealed normal B-mode sonographic findings and some correlations between Doppler parameters and sperm quality. SWE demonstrated consistent testicular stiffness regardless of the depth of evaluation, with correlations of the stiffness of the head of the epididymis to the ejaculate volume. CEUS identified blood flow differences between testes and some perfusion parameters correlated with sperm features. This comprehensive ultrasound assessment provides valuable insights into canine reproductive health, providing basis for further studies on dogs with abnormal sperm quality and with different fertility statuses.

Guanine quadruplexes (G4s) are non-canonical DNA structures that can trigger micronuclei (MNi). Mechanisms of micronuclei formation by G4s are not fully understood. Here, we show that G4 stabilization can trigger cell-cycle-phase-specific mechanisms of replication fork stalling and DNA synthesis restart dependent on translesion synthesis (TLS) DNA polymerases (Pols). Fork stalling is caused by G-loops and high transcription during early S only. Moreover, while induction of micronuclei is dependent on DNA Pol η throughout S phase, primase and DNA-directed polymerase (PrimPol) is required in late S only. DNA breakage is not an immediate response to stabilized G4s but rather a consequence of persistent G4-mediated replication stress. Thus, different modes of fork stalling and restart, based on genomic context and TLS Pols, avoid immediate DNA breakage at stalled forks but at the expense of a risk of later mitotic chromosomal instability. The insights can lead to the development of more effective therapies for cancer and neurological diseases.

IntroductionEpididymitis in dogs is an underreported but clinically relevant condition, often associated with scrotal pain, systemic inflammation, and infertility. Diagnosis commonly relies on clinical and ultrasonographic findings, even though information concerning the latter are poorly documented. This report aims to describe the ultrasonographic features of epididymitis in a series of clinical canine cases, including clinical and laboratory findings.Materials and methodsClinical cases of dogs affected by epididymitis were retrospectively included. Clinical signs, andrological findings, ultrasonographic abnormalities of the epididymis and testicles, blood analysis and, where available, cytological or histopathological data were recorded. Ultrasonographic features evaluated included epididymal size, capsular integrity, echotexture, vascularization, presence of mineralization and scrotal effusion.ResultsFourteen dogs with epididymitis were included, and the condition was unilateral in 11 dogs (eight left-sided) and bilateral in three. On B-mode ultrasound the epididymal tail was the most frequently affected portion. Other frequent findings included epididymal enlargement, capsular irregularity, and inhomogeneous echotexture. Mineralization, hypoechoic cavities, and peri-epididymal oedema were observed, but not present in all cases. Concurrent testicular and urogenital abnormalities, along with leucocytosis and neutrophilic left shift, were common in acute cases.ConclusionThis is the first case series describing ultrasonographic patterns of epididymitis in dogs. Early recognition via ultrasound may support targeted treatment and fertility preservation.

Sonoelastography is a rapid and non-invasive ultrasound-based technique to assess tissue elasticity. Interest in this technique’s application in veterinary medicine is progressively increasing, with preliminary reports also available to assess canine testicular parenchyma abnormalities. Since elastographic techniques are different and it is of paramount importance to standardize procedures, this work aimed to compare two different elastographic techniques, SE and 2D SWE, on the same testes and compare the data with the current literature. Thirty healthy canine testes were included, and complete B-mode, color and power Doppler examinations were performed on each one. SE was performed qualitatively through an elastogram evaluation and semiquantitatively comparing the parenchyma with peripheral skin. 2D SWE was performed and m/s measurements were taken. Each measurement was collected in the cranial, middle and caudal portions, considering the sections above and below the mediastinum separately. The healthy testes appeared to be intermediate in stiffness with differences in the various portions for SE and expressed a mean value from 1.3 m/s to 1.4 m/s for 2D SWE, consistent with the previous literature. Considering the technical specifics of the software for the two elastography methods, both of them are valuable in assessing canine testis stiffness and are potentially clinically appliable.

TDP1 removes transcription-blocking topoisomerase I cleavage complexes (TOP1ccs), and its inactivating H493R mutation causes the neurodegenerative syndrome SCAN1. However, the molecular mechanism underlying the SCAN1 phenotype is unclear. Here, we generate human SCAN1 cell models using CRISPR-Cas9 and show that they accumulate TOP1ccs along with changes in gene expression and genomic distribution of R-loops. SCAN1 cells also accumulate transcriptional DNA double-strand breaks (DSBs) specifically in the G1 cell population due to increased DSB formation and lack of repair, both resulting from abortive removal of transcription-blocking TOP1ccs. Deficient TDP1 activity causes increased DSB production, and the presence of mutated TDP1 protein hampers DSB repair by a TDP2-dependent backup pathway. This study provides powerful models to study TDP1 functions under physiological and pathological conditions and unravels that a gain of function of the mutated TDP1 protein, which prevents DSB repair, rather than a loss of TDP1 activity itself, could contribute to SCAN1 pathogenesis.

Ovarian torsion (OT) is a condition that can affect both humans and animals, although it is less common in the latter, with very few cases documented in the literature. To our knowledge, no previous reports have documented the occurrence of this condition in rabbits. In this study, we present the first documented case of spontaneous OT in a 2-year-old female intact rabbit. The patient was brought to the clinic for a routine check-up, during which a firm, large abdominal mass was palpated. Subsequent ultrasound examination of the abdomen revealed the presence of a large, hypoechoic, non-vascularized mass occupying the majority of the caudal abdomen. A computed tomography (CT) scan further confirmed the presence of a heterogeneous mass exhibiting the Whirlpool sign, which is characteristic of organ torsion. Upon laparotomy, an enlargement of the right ovary was observed, characterized by twisting of the ovarian pedicle, consistent with the mass detected via ultrasound and CT scan. Ovariohysterovaginectomy was performed, and the mass was subsequently analyzed. Microscopic analysis of the mass revealed predominantly necrotic tissue, with only a few ovarian epithelial cells present. The underlying cause of the OT described in this study remains unclear. However, it is plausible that a previous neoplastic condition or ovarian necrosis led to an increase in the size and weight of the mass, ultimately resulting in the twisting of the supporting structures.

DNA topoisomerase I can contribute to cancer genome instability. During catalytic activity, topoisomerase I forms a transient intermediate, topoisomerase I-DNA cleavage complex (Top1cc) to allow strand rotation and duplex relaxation, which can lead to elevated levels of DNA-RNA hybrids and micronuclei. To comprehend the underlying mechanisms, we have integrated genomic data of Top1cc-triggered hybrids and DNA double-strand breaks (DSBs) shortly after Top1cc induction, revealing that Top1ccs increase hybrid levels with different mechanisms. DSBs are at highly transcribed genes in early replicating initiation zones and overlap with hybrids downstream of accumulated RNA polymerase II (RNAPII) at gene 5 '-ends. A transcription factor IIS mutant impairing transcription elongation further increased RNAPII accumulation likely due to backtracking. Moreover, Top1ccs can trigger micronuclei when occurring during late G1 or early/mid S, but not during late S. As micronuclei and transcription-replication conflicts are attenuated by transcription factor IIS, our results support a role of RNAPII arrest in Top1cc-induced transcription-replication conflicts leading to DSBs and micronuclei.

Abstract During human Topoisomerase I (Top1) catalytic activity, a transient Top1-DNA cleavage complex (Top1cc) forms to allow DNA relaxation. Top1ccs can be stabilized by antitumor Top1 poisons, such as clinically-effective camptothecin analogs. Camptothecin-stabilized Top1ccs can lead to R-loop-dependent transcription-replication conflicts (TRCs), single-end DNA double-strand breaks (seDSBs) and genome instability in cancer cells. To define the genomic location of TRCs and the underlying mechanisms, we integrated genomic maps of Top1cc-induced DNA cleavage by END-seq and DNA:RNA hybrids by DRIP, determined the cell-cycle specificity of micronuclei formation and the effects of elongation transcription factor IIS (TFIIS). We have defined the genomic sites of both seDSBs and increased R-loop levels (gains) induced by Top1ccs. seDSBs are highly enriched at regions with R-loop gains at highly-transcribed genes in early replicating zones. Kinetics analyses of R-loops and genomic ChIP data of RNA polymerase II (RNAPII) revealed that R-loop gains can either be upstream or downstream of RNAPII. TFIIS effects showed that downstream R-loop gains can follow RNAPII backtracking and arrest. seDSB strandness showed that co-directional TRCs occur mainly at downstream hybrid-associated RNAPII, whereas head-on TRCs occur mainly at upstream hybrid-associated RNAPII. Therefore, Top1ccs can increase two structurally-different R-loops and R-loop-associated backtracked RNAPII causes co-directional TRCs at higher rate than head-on collisions. Moreover, Top1ccs trigger micronuclei formation at G1/early S, but not late S/G2, which are attenuated by TFIIS. Thus, our findings establish a role of RNAPII backtracking at highly-transcribed genes in Top1cc-induced co-directional TRCs and micronuclei formation. The findings can accelerate the development of new effective strategies to treat cancer patients. (Supported by Associazione Italiana per la Ricerca sul Cancro, Milan, Italy - IG 2019 - ID. 23032 to C.G. and by the European Union - NextGenerationEU through the Italian Ministry of University and Research under PNRR - M4C2-I1.3 Project PE_00000019 "HEAL ITALIA" to G.C., CUP J33C22002920006). Citation Format: Renée C. Duardo, Jessica Marinello, Marco Russo, Sara Morelli, Simona Pepe, Federico Guerra, Belen Gomez-González, Andrés Aguilera, Giovanni Capranico. R-loop type determines the orientation of transcription-replication conflicts attenuated by transcription factor IIS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3367.

There is growing evidence by the literature that the unbalance between androgens and estrogens is a relevant condition associated with a common canine reproductive disorder known as cryptorchidism. The role of estrogens in regulating testicular cell function and reproductive events is supposedly due to the wide expression of two nuclear estrogen receptors (ERs), ER-alpha and ER-beta and a trans-membrane G protein-coupled estrogen receptor (GPER) in the testis. In this study, immunohistochemistry, Western blotting and qRT-PCR were used to assess the distribution and expression of GPER in the testis–epididymal complex in the normal and cryptorchid dog. ER-alpha and ER-beta were also evaluated to better characterize the relative abundances of all three receptors. In addition, in these tissues, the expression level of two proteins as SOD1 and Nrf2 normally associated with oxidative stress was investigated to evaluate a possible relationship with ERs. Our data revealed changes in the distribution and expression of the GPER between the normal and cryptorchid dog. In particular, dogs affected by cryptorchidism showed an upregulation of GPER at level of the examined reproductive tract. Also considering the obtained result of a modulation of SOD1 and Nrf2 expression, we could hypothesize the involvement of GPER in the cryptorchid condition. Further studies are, however, necessary to characterize the role of GPER and its specific signaling mechanisms.

Lung cancer has a significant incidence among the population and, unfortunately, has an unfavourable prognosis in most cases. The World Health Organization (WHO) classifies lung tumours into two subtypes based on their phenotype: the Non-Small Cell Lung Cancer (NSCLC) and the Small Cell Lung Cancer (SCLC). SCLC treatment, despite advances in chemotherapy and radiotherapy, is often unsuccessful for cancer recurrence highlighting the need to develop novel therapeutic strategies. In this review, we describe the genetic landscape and tumour microenvironment that characterize the pathological processes of SCLC and how they are responsible for tumour immune evasion. The immunosuppressive mechanisms engaged in SCLC are critical factors to understand the failure of immunotherapy in SCLC and, conversely, suggest that new signalling pathways, such as cGAS/STING, should be investigated as possible targets to stimulate an innate immune response in this subtype of lung cancer. The full comprehension of the innate immunity of cancer cells is thus crucial to open new challenges for successful immunotherapy in treating SCLC and improving patient outcomes.

Canine prostatic serum esterase (CPSE) is considered a useful tool to identify prostate disorders in dogs, with increasing interest in ultrasound (US)-based sonoelastography to non-invasively detect prostate disorders. Since no report is available about a possible correlation between these diagnostic tools, we aimed to investigate a possible correlation between strain elastography (SE) and 2D-shear wave elastography (SWE) and CPSE. Twenty-one dogs were included and, on each animal, CPSE was evaluated followed by a complete US examination and SE and 2D-SWE application. Healthy dogs were identified based on the CPSE results. All the dogs included were characterized by normal CPSE values (<52.3ng/mL) and normal US prostate appearance. The prostate was characterized by intermediate stiffness with SE (pattern III - 84.7% for the left lobe and 79.27% for the right lobe) and softer than the abdominal wall (SR 0.6 for the left lobe and 0.56 for the right lobe), with low values for both m/s and kilopascals (kPa) for 2D-SWE, pointing that the healthy tissue is not hard. 2D-SWE results were, respectively, 13.515.55kPa and 2.31 +/- 0.42m/s for the left lobe and 18.05 +/- 6.47kPa and 2.39 +/- 0.43m/s for the right lobe. The significant difference between the right and left measurements expressed with kPa, not evidenced with m/s, can be considered indicative of m/s as the most reliable measurement to be considered regarding the prostate parenchyma. Even though no linear correlation was detected between CPSE and elastography values, these preliminary results evidence that the healthy prostates were characterized by a similar elastographic pattern, thus pointing that these techniques can be potentially useful to be applied in case of prostatic disorders to improve the accuracy of the final diagnosis in a non-invasive way.

Small-cell lung cancer (SCLC) is an aggressive cancer characterized by immunosuppressive features leading to poor responses to current immunotherapies. Activation of transposable elements (TE) can trigger an innate immune response, which can synergize with immunotherapeutic protocols in patients. However, TE activity in relation to immune gene response is not fully known in human SCLC. Here, we compared TE expression in 104 human SCLC and 24 normal tissues and established their involvement in innate immune responses. We observed that different intergenic TEs, mainly endogenous retroviral (ERV) families, are deregulated in SCLC. Similarly to other cancers, we detected a subset of LTRs that correlate with innate immune gene signatures and cytosolic RNA sensors, such as RIG-I. These LTRs are downregulated in SCLC tumors vs. normal tissues, and are mainly located at transcriptional repressed regions, marked with H3K4me2 in different cell lines. Analyses of different genomic datasets show that chromatin repression is likely due to de-methylase LSD1 activity. Moreover, high expression levels of ERV LTRs predict a better survival upon chemotherapy of SCLC patients. The findings reveal a specific pattern of TE-mediated activation of innate immune genes in SCLC, which can be exploited to establish more effective immunotherapeutic combinations.

Neuroblastoma (NB) is one of the primary causes of death for pediatric malignancies. Given the high heterogeneity in NB's mutation landscape, optimizing individualized therapies is still challenging. In the context of genomic alterations, MYCN amplification is the most correlated event with poor outcomes. MYCN is involved in the regulation of several cellular mechanisms, including cell cycle. Thus, studying the influence of MYCN overexpression in the G1/S transition checkpoint of the cell cycle may unveil novel druggable targets for the development of personalized therapeutical approaches. Here, we show that high expression of E2F3 and MYCN correlate with poor prognosis in NB despite the RB1 mRNA levels. Moreover, we demonstrate through luciferase reporter assays that MYCN bypasses RB function by incrementing E2F3-responsive promoter activity. We showed that MYCN overexpression leads to RB inactivation by inducing RB hyperphosphorylation during the G1 phase through cell cycle synchronization experiments. Moreover, we generated two MYCN-amplified NB cell lines conditionally knockdown (cKD) for the RB1 gene through a CRISPRi approach. Indeed, RB KD did not affect cell proliferation, whereas cell proliferation was strongly influenced when a non-phosphorylatable RB mutant was expressed. This finding revealed the dispensable role of RB in regulating MYCN-amplified NB's cell cycle. The described genetic interaction between MYCN and RB1 provides the rationale for using cyclin/CDK complexes inhibitors in NBs carrying MYCN amplification and relatively high levels of RB1 expression.

G-quadruplex (G4) binders have been investigated to discover new anticancer drugs worldwide in past decades. As these ligands are generally not highly cytotoxic, the discovery rational was mainly based on increasing the cell-killing potency. Nevertheless, no G4 binder has been shown yet to be effective in cancer patients. Here, G4 binder activity at low dosages will be discussed as a critical feature to discover ligands with therapeutic effects in cancer patients. Specific effects of G4 binders al low doses have been reported to occur in cancer and normal cells. Among them, genome instability and the stimulation of cytoplasmic processes related to autophagy and innate immune response open to the use of G4 binders as immune-stimulating agents. Thus, we propose a new rational of drug discovery, which is not based on cytotoxic potency but rather on immune gene activation at non-cytotoxic dosage.

Background Current immunotherapy strategies have contrasting clinical results in human lung cancer patients as small-cell lung cancers (SCLC) often show features of immunological cold tumours. Topoisomerase 1 (TOP1) poisons are effective antitumor drugs with good efficacy against lung cancers. Methods We used molecular, genetic and bioinformatic approaches to determine the mechanism of micronuclei formation induced by two TOP1 poisons in different human cancer cells, including SCLC cell lines. Results TOP1 poisons stimulate similar levels of micronuclei in all tested cell lines but downstream effects can vary markedly. TOP1 poisons increase micronuclei levels with a mechanism involving R-loops as overexpression of RNaseH1 markedly reduces or abolishes both H2AX phosphorylation and micronuclei formation. TOP1 poison-induced micronuclei activate the cGAS/STING pathway leading to increased expression of immune genes in HeLa cells, but not in human SCLC cell lines, mainly due to lack of STING and/or cGAS expression. Moreover, the expression of STING and antigen-presenting machinery genes is generally downregulated in patient tumours of human lung cancer datasets. Conclusions Altogether, our data reveal an immune signalling mechanism activated by TOP1 poisons, which is often impaired in human SCLC tumours.

The reduced oocyte competence recorded during the non-breading season (NBS) is one of the key factors affecting the profitability of buffalo farming and limits the IVEP efficiency. The purpose of this experiment was to evaluate whether season influences the lipid content within the ovarian follicle in the Italian Mediterranean buffalo. Abattoir-derived ovaries were collected during the breeding season (BS) and the NBS, and different matrices (follicular fluid, oocytes, cumulus and follicular cells) were recovered. After the extraction of the apolar fraction, all samples were analyzed by H1 nuclear magnetic resonance and FF samples by gas chromatography–mass spectrometry. Seasonal differences in lipid composition were observed in all matrices. In particular, during the NBS, the triglyceride content was higher in the follicular fluid and in the oocytes but reduced in the follicular cells. Both cholesterol and phospholipids were reduced in the follicular fluid and follicular cells during the NBS. Furthermore, the total amount of non-esterified fatty acids was significantly increased in the follicular fluid. The seasonal variation in lipid profile of the follicle may, in part, account for the reduced buffalo oocyte competence during the NBS, due to the critical role played by lipids in regulating ovarian functions.

Diagnostic imaging plays an essential role in the diagnosis and management of reproductive neoplasia in dogs and cats. The initial diagnosis, staging, and planning of surgical and radiation treatment and the response to therapy all involve imaging to varying degrees. Routine radiographs, ultrasound, nuclear medicine, and cross-sectional imaging in the form of computed tomography (CT) and magnetic resonance imaging (MRI) are routinely used in canine reproductive disorders. The choice of imaging modality depends on many factors, including the level of referral and the pathological information required. The biological behaviour of the tumour also guides the choice of imaging in cancer staging, and imaging may play an important role in guiding serial tumour biopsy during the course of therapy. The sophistication of imaging modalities is increasing exponentially. Each modality has advantages and disadvantages in terms of cost, availability, sensitivity, specificity, and qualities of anatomic versus functional imaging.