Probes for INS

Gene-protein assay (GPA), a combination of immunohistochemistry and dual in situ hybridization, allows simultaneous visualization of HER2 protein and gene on a single slide. We aimed to clarify the clinical significance of HER2 intratumoral heterogeneity (ITH) using GPA. We investigated the relationships between various HER2 ITH indicators and clinical course in 102 patients with HER2-positive breast cancer, treated with neoadjuvant trastuzumab and chemotherapy. Five representative microscopic images were captured from each GPA slide of pre-therapeutic biopsy materials. All evaluable cancer cells in the images were individually assessed for HER2 gene copy number and protein expression. Mean and coefficient of variation (CV) of both gene copy number and protein category were calculated, and each was divided into negative, equivocal, and positive. Based on their combined status, cancer cells were classified into nine types. Pathological complete response (pCR) to neoadjuvant treatments showed positive relationships to mean gene copy number (P < 0.001), mean protein category (P < 0.001), and proportion of gene- and protein-positive tumor cells (P < 0.001) and showed negative relationships to the CV of protein category (P < 0.001) and the proportion of gene-amplified but protein-negative tumor cells (P = 0.002). Two diagnostic models, created by combining clinicopathological factors and ITH indicators, showed excellent potential diagnostic ability for pCR (mean gene copy number and protein category CV; AUC = 0.837, proportion of gene- and protein-positive tumor cells; AUC = 0.831). HER2 ITH quantified by GPA is a potential predictive indicator for efficacy of HER2-targeted treatment.

Ticks are important ectoparasites that are capable of transmitting multiple classes of pathogens and are currently linked with many emerging tick-borne diseases worldwide. With increasing occurrences of tick-borne diseases in both humans and veterinary species, there is a continuous need to further our understanding of ticks and the pathogens they transmit. Whole tick histology provides a full scope of the tick internal anatomy, allowing researchers to examine multiple organs of interest in a single section. This is in contrast to other techniques that are more commonly utilized in tick-borne disease research, such as electron microscopy and light microscopy of individual organs. There is a lack of literature describing a practical technique to process whole tick histologic sections. Therefore, the current study aims to provide researchers with a workable protocol to prepare high quality paraffin-embedded whole tick histology sections. Amblyomma americanum adults were used as an example species for this study. After a series of pilot experiments using a combination of various fixatives, softening agents and processing techniques, we elected to compare two common fixatives, 10% neutral-buffered formalin (NBF) and Bouin’s solution for whole ticks. Equal numbers of A. americanum adults (n = 10/fixative) were processed identically and their whole tick histology sections were individually scored. Higher scores were assigned to whole tick sections that contained more internal organs that are crucial for tick-borne disease research (e.g. salivary glands and midgut), high integrity of tissues and exoskeleton on the section, and good fixation and staining quality of the tissues. The mean total scores for Bouin’s-fixed ticks were significantly higher compared to NBF-fixed ticks (p = 0.001). To further assess our preferred technique, we also demonstrated the feasibility of producing high quality whole tick sections for three other common tick species of medical importance (Rhipicephalus sanguineus, Ixodes scapularis, and Dermacentor variabilis) using Bouin’s solution. While this technique may require further optimization for other tick species, we described a feasible protocol that uses commonly available tools, reagents and standard histologic equipment. This should allow any investigator to easily make adjustments to this protocol as needed based on their experimental goals.

Background PIM 1 and PI3K/mTOR pathways are frequently dysregulated in prostate cancer and may lead to decreased survival invasion and metastasis. Moreover, anti-tumour drug resistance has been associated with the interconnection of these pathways. Furthermore, current treatments exhibit issues with toxicity. Hence, these pathways were co-targeted with novel preclinical multikinase PIM/PI3K/mTOR inhibitor- AUM302, PI3K/mTOR inhibitor BEZ235 (Dactolisib) and PIM inhibitor, AZD-1208 in our laboratory using a cohort of cancer explants emanating from our PEOPLE: PatiEnt prOstate samPLes for rEsea ch study and our current SCREEN study. This cohort has a high Gleason grade score of ≥  8. Therefore, this study aims to assess the effect of the combination therapy on the transcriptional landscape of ex vivo prostate cancer models derived from prostate cancer patients. Methods Using the Nanostring GeoMX DSP technology, we aim to analyse the spatial transcriptomic profile of the co-targeted therapy treated ex vivo models to decipher the effects of heterogeneity on the co-targeted therapies' efficacy. Tissue microarrays of co-targeted treated twenty-five ex vivo 3mm cores derived from 4 patients will be analysed. Following RNA Scope analysis, morphology markers, including PAN CK positive and PAN CK negative, will be used to guide the selection of 270 regions of interest (ROI). ROI will be segmented and profiled using immunofluorescence. The morphological markers will define these segments into areas of illumination (AOIs) using a combination of the absence or presence of CD45 and pSTAT3. The AOIs will generate multiple expression profiles for the related ROI. We intend to use this flexible, high-dimensional spatial profiling to identify the spatial transcriptomic signatures and explore phosphorylation sites in cancer-targeted therapies. Results The spatial transcriptomics analysis of this study is in view. Conclusions Our findings will contribute to understanding how the spatial landscape of the tumour microenvironment enhances the efficacy of anti-tumour drugs and what subset of patients are more likely to benefit from such therapy.

A systematic review relating to bladder cancer epidemiology, risk factors and occupational hazards was conducted. This was to identify the bladder cancer epidemiology and risk factors in muscle invasive disease. The search strategy aimed to identify all references related to bladder cancer AND screening. Search terms used were as follows: (Bladder cancer) AND (Immune checkpoint inhibitors). The following databases were screened from 1989 to June 2020:

Human papillomavirus (HPV)-mediated squamous cell carcinomas of the oropharynx are common, however only rare cases of HPV-mediated oropharyngeal adenocarcinoma have been reported to date. In this report, we describe a 50 year old nonsmoking male who originally presented with an enlarging neck mass. Fine needle aspiration cytology confirmed an HPV-mediated adenocarcinoma. Subsequent surgery identified a 0.7 cm base of tongue primary HPV-mediated carcinoma with focal glandular differentiation and a 4.0 cm cystic lymph node metastasis demonstrating entirely glandular differentiation. Next generation sequencing of the metastasis detected a pathogenic NOTCH1 mutation.

Single-nucleus RNA sequencing allows the profiling of gene expression in isolated nuclei. Here, we describe a step-by-step protocol optimized for adult mouse skeletal muscles. This protocol provides two main advantages compared to the widely used single-cell protocol. First, it allows us to sequence the myonuclei of the multinucleated myofibers. Second, it circumvents the cell-dissociation-induced transcriptional modifications. For complete details on the use and execution of this protocol, please refer to Dos Santos et al. (2020) and Machado, Geara et al. (2021).

Interventions to disrupt memory reconsolidation have held promise for the treatment of stress- and anxiety-related disorders. In the present study, we tested whether an intervention based on these principles, called memory updating, could be adapted for reward-seeking behaviors. Non-treatment seeking tobacco smokers were exposed to smoking cues and/or stress, two stimuli known to trigger smoking. It was predicted that exposure to a stress task would enhance the cues’ motivational salience and yield greater susceptibility to the memory updating procedure.

The supplement telomerase activator TA-65 (purified from Astragalus membranaceus) has been shown to retard cellular senescence, boost the aging immune system, and retard age-related symptoms. Lengthened telomeres retard aging, but because cancers often maintain longevity by lengthening telomeres, dietary telomerase activator might possibly increase tumorigenesis. This study investigated whether oral TA-65 effects the timing of onset and/or the incidence of skin cancers induced by UVB-irradiation and whether that possible effect is different if the oral supplementation is begun only after tumors are first detected clinically or if supplementation is begun before initiation of tumors as well as during and after the inciting UVB exposure. Three groups of ten Skh:1 hairless, nonpigmented mice exposed to UVB for twenty weeks were given (1) no supplementation, (2) TA-65 supplementation starting when the first UV-induced skin cancers were clinically observed, after which the UV exposure was terminated, and (3) TA-65 supplementation before, during, and after UV exposure (as more tumors subsequently appeared). Except for two time points when Group 3 had borderline or statistically more tumors ≥ 2mm per mouse, overall, there was no statistically significant difference in the time of onset, the incidence, or the tumor load of skin cancers with TA-65 with either timing, confirming the safety of this anti-aging supplement in this model of the most frequent human malignancy.

Neuropeptide Y (NPY) in the arcuate nucleus (ARC) is known as one of the most critical regulators of feeding. However, how NPY promotes feeding under obese conditions is unclear. Here, we show that positive energy balance, induced by high-fat diet (HFD) or in genetically obese leptin-receptor-deficient mice, leads to elevated Npy2r expression especially on proopiomelanocortin (POMC) neurons, which also alters leptin responsiveness. Circuit mapping identified a subset of ARC agouti-related peptide (Agrp)-negative NPY neurons that control these Npy2r expressing POMC neurons. Chemogenetic activation of this newly discovered circuitry strongly drives feeding, while optogenetic inhibition reduces feeding. Consistent with that, lack of Npy2r on POMC neurons leads to reduced food intake and fat mass. This suggests that under energy surplus conditions, when ARC NPY levels generally drop, high-affinity NPY2R on POMC neurons is still able to drive food intake and enhance obesity development via NPY released predominantly from Agrp-negative NPY neurons.

The suprachiasmatic nucleus (SCN) can generate robust circadian behaviors in mammals under different environments, but the underlying neural mechanisms remained unclear. Here, we showed that the activities of cholecystokinin (CCK) neurons in the mouse SCN preceded the onset of behavioral activities under different photoperiods. CCK-neuron-deficient mice displayed shortened free-running periods, failed to compress their activities under a long photoperiod, and developed rapid splitting or became arrhythmic under constant light. Furthermore, unlike vasoactive intestinal polypeptide (VIP) neurons, CCK neurons are not directly light sensitive, but their activation can elicit phase advance and counter light-induced phase delay mediated by VIP neurons. Under long photoperiods, the impact of CCK neurons on SCN dominates over that of VIP neurons. Finally, we found that the slow-responding CCK neurons control the rate of recovery during jet lag. Together, our results demonstrated that SCN CCK neurons are crucial for the robustness and plasticity of the mammalian circadian clock.

Ischemia reperfusion injury (IRI), often related to surgical procedures, is one of the important causes of acute kidney injury (AKI). To decipher the dynamic process of AKI caused by IRI (with prolonged ischemia phase), we performed single-cell RNA sequencing (scRNA-seq) of clinically relevant IRI murine model with different ischemic intervals. We discovered that Slc5a2hi proximal tubular cells were susceptible to AKI and highly expressed neutral amino acid transporter gene Slc6a19, which was dramatically decreased over the time course. With the usage of mass spectrometry-based metabolomic analysis, we detected that the level of neutral amino acid isoleucine dropped off in AKI mouse plasma metabolites. And the reduction of plasma isoleucine was also verified in patients with cardiac surgery-associated acute kidney injury (CSA-AKI). The findings advanced the understanding of dynamic process of AKI and introduced reduction of isoleucine as a potential biomarker for CSA-AKI.