Enzyme-Activatable Near-Infrared Hemicyanines as Modular Scaffolds for in vivo Photodynamic Therapy.

Photodynamic therapy is an anti-cancer treatment that requires illumination of photosensitizers to induce local cell death. Current near-infrared organic photosensitizers are built from large and non-modular structures that cannot be tuned to improve safety and minimize off-target toxicity. This work describes a novel chemical platform to generate enzyme-activatable near-infrared photosensitizers. We optimized the Se-bridged hemicyanine scaffold to include caging groups and biocompatible moieties, and generated cathepsin-triggered photosensitizers for effective ablation of human glioblastoma cells. Furthermore, we demonstrated that enzyme-activatable Se-bridged hemicyanines are effective photosensitizers for the safe ablation of microtumors in vivo, creating new avenues in the chemical design of targeted anti-cancer photodynamic therapy agents.

PTPRH promotes the progression of non-small cell lung cancer via glycolysis mediated by the PI3K/AKT/mTOR signaling pathway.

BACKGROUND: The protein tyrosine phosphatase H receptor (PTPRH) is known to regulate the occurrence and development of pancreatic and colorectal cancer. However, its association with glycolysis in non-small cell lung cancer (NSCLC) is still unclear. In this study, we aimed to investigate the relationship between PTPRH expression and glucose metabolism and the underlying mechanism of action. METHODS: The expression of PTPRH in NSCLC cells was evaluated by IHC staining, qRT‒PCR and Western blotting. The effect of PTPRH on cell biological behavior was evaluated by colony assays, EdU experiments, Transwell assays, wound healing assays and flow cytometry. Changes in F-18-fluorodeoxyglucose (18F-FDG) uptake and glucose metabolite levels after altering PTPRH expression were detected via a gamma counter and lactic acid tests. The expression of glycolysis-related proteins in NSCLC cells was detected by Western blotting after altering PTPRH expression. RESULTS: The results showed that PTPRH was highly expressed in clinical patient tissue samples and closely related to tumor diameter and clinical stage. In addition, PTPRH expression was associated with glycometabolism indexes on 18F-FDG positron emission tomography/computed tomography (PET/CT) imaging, the expression level of Ki67 and the expression levels of glycolysis-related proteins. PTPRH altered cell behavior, inhibited apoptosis, and promoted 18F-FDG uptake, lactate production, and the expression of glycolysis-related proteins. In addition, PTPRH modulated the glycometabolism of NSCLC cells via the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, as assessed using LY294002 and 740Y-P (an inhibitor and agonist of PI3K, respectively). The same results were validated in vivo using a xenograft tumor model in nude mice. Protein expression levels of PTPRH, glycolysis-related proteins, p-PI3K/PI3K and p-AKT/AKT were measured by IHC staining using a subcutaneous xenograft model in nude mice. CONCLUSIONS: In summary, we report that PTPRH promotes glycolysis, proliferation, migration, and invasion via the PI3K/AKT/mTOR signaling pathway in NSCLC and ultimately promotes tumor progression, which can be regulated by LY294002 and 740Y-P. These results suggest that PTPRH is a potential therapeutic target for NSCLC.

LncRNA AP000695.2 promotes glycolysis of lung adenocarcinoma via the miR-335-3p/TEAD1 axis.

AP000695.2 is a novel long non-coding RNA (lncRNA). Its aberrant high expression is remarkably associated with poor prognosis of patients with lung adenocarcinoma (LUAD). However, its role and underlying mechanism in LUAD remains unclear. Previous bioinformatics analysis indicated that AP000695.2 may be closely related to the glycolysis of LUAD. This study aims to verify and explore the mechanism of AP000695.2 in glycolysis of LUAD. Overexpression plasmid and siRNA are used to construct cell models of upregulation and downregulation of AP000695.2, respectively. AP000695.2 is highly expressed in lung cancer cell lines as revealed by qPCR. Western blot analysis, FDG uptake, lactate production assay and ECAR determination results show that high expression of AP000695.2 facilitates glycolysis of LUAD cells. CCK-8, EdU staining, Transwell and wound healing assays show that high expression of AP000695.2 promotes cell growth and migration of LUAD. The relationship between AP000695.2 and miR-335-3p is confirmed by bioinformatics analysis and dual-luciferase reporter assays. Through the dual-luciferase reporter assay, TEA domain transcription factor 1 (TEAD1) is identified as a target gene of miR-335-3p. Rescue experiments are applied to verify the relationship among AP000695.2, miR-335-3p and TEAD1. Our study indicates that AP000695.2 is involved in the mechanism of LUAD through functioning as a ceRNA to competitively sponge miR-335-3p, thereby regulating the expression of TEAD1. In the in vivo models, AP000695.2 depletion restrains tumor growth and glycolysis. AP000695.2 promotes the glycolysis of LUAD by regulating the miR-335-3p/TEAD1 axis, and it may serve as a potential target of anti-tumor energy metabolism therapy.

Fluorogenic Granzyme A Substrates Enable Real-Time Imaging of Adaptive Immune Cell Activity.

Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.

Interleukin-35 suppresses antitumor activity of circulating CD8+ T cells in osteosarcoma patients.

Purpose/Aim of the study: Interleukin (IL)-35 is a newly identified IL-12 cytokine family member and reveals immunosuppressive activity to CD8+ T cells in inflammation, infectious diseases, and cancers. However, little is known regarding IL-35 function in osteosarcoma. Thus, the aim of the current study was to investigate the regulatory function of IL-35 to CD8+ T cells in osteosarcoma. Materials and methods: Thirty-five osteosarcoma patients and 20 healthy individuals were enrolled. Serum CD4+CD25+CD127dim/- regulatory T cells (Tregs) and CD8+ T cells were purified. IL-35 concentration in serum and cultured supernatants was measured by enzyme-linked immunosorbent assay. Osteosarcoma cell line MG-63 cells and CD8+ T cells were stimulated with recombinant IL-35 in vitro, and modulatory function of IL-35 on these cells was assessed by investigation of cellular proliferation, cell cycle, apoptosis, and cytokine production. Results: Serum IL-35 and Treg-secreting IL-35 were significantly elevated in osteosarcoma patients. IL-35 stimulation did not affect proliferation, apoptosis, or cell cycle of MG-63 cells. Purified peripheral CD8+ T cells from osteosarcoma patients revealed dysfunctional property, which presented as decreased mRNA expressions for perforin, granzyme B, and granulysin, as well as reduced cytolytic (direct lysis of target MG-63 cells) and noncytolytic (interferon-γ and tumor necrosis factor-α production) function in coculture systems. Moreover, IL-35 stimulation further diminished cytolytic and noncytolytic activity of CD8+ T cells from osteosarcoma patients. Conclusions: The current data indicated that IL-35 contributed to CD8+ T-cell dysfunction and limited antitumor immune response in osteosarcoma.

Natural product-inspired profluorophores for imaging NQO1 activity in tumour tissues.

Herein we designed a collection of trimethyl-lock quinone profluorophores as activity-based probes for imaging NAD(P)H:quinone oxidoreductase (NQO1) in cancer cells and tumour tissues. Profluorophores were prepared via synthetic routes from naturally-occurring quinones and characterised in vitro using recombinant enzymes, to be further validated in cells and fresh frozen canine tumour tissues as potential new tools for cancer detection and imaging.

The Influence of Grit on Life Satisfaction: Self-Esteem as a Mediator.

Improving people's life satisfaction has become an important goal for many individuals and societies. In this study we investigate how grit influences life satisfaction. We propose that individuals' self-esteem mediates the relationship between grit and life satisfaction. Study 1, with a sample of 243 employees enrolled in a business training course, found that an individual's grit was positively related to life satisfaction and that self-esteem fully mediated this relationship. In Study 2, with 218 full-time employees, self-efficacy, self-control, and self-consciousness were included as mediators, but they did not exceed the power of self-esteem in explaining the relationship between grit and life satisfaction. Implications, limitations and future research directions are discussed.

Extra-articular blocking technique for acetabular bone defect reconstruction.

The acetabular bone defect reconstruction is of great challenge in total hip arthroplasty (THA). Although several solutions such as autologous bone grafting, trabecular metal augment, or compromising techniques such as the medial protrusion, high inclination angle, and elevated hip center have been raised, their efficacy and reliability have not been fully substantiated. Traditional reconstruction methods may lead to bone resorption, aggravation of bone defects, unequal length of lower limbs, unbalance of hip-spine relationship, increased costs, and so on. Our team proved a new technique named extra-articular blocking to resolve this problem. The extra-articular blocking technique was a simple, economic and effective acetabular reconstructive method to resolve the massive acetabular bone defect in congenital (especially for developmental dysplasia of the hip, DDH), inflammatory, and osteolytic pathologies. This article organized as surgical technique, aims to report the surgical principle, indication, and procedure of using extra-articular blocking technique. With this technique, we have successfully solved the difficult problem of acetabular bone defect reconstruction. We found after 3 months of the surgery, there were fluoroscopic healing and remodeling. And there were no bone loss or graft absorption until the last follow-up as evidenced by radiographic observation. The survival rate of the acetabular component was 100%, no radiolucent line, changes in inclination and anteversion of the shell, as well as migration of the rotation center were identified.

18 F-FAPI-42 PET/CT Findings in a Patient With Left Ventricular Mural Thrombus.

ABSTRACT: 18 F-FAPI-42 PET/CT is a novel imaging tool targeting fibroblast activation protein (FAP). We describe the 18 F-FAPI-42 PET/CT findings of a left ventricular mural thrombus in a 50-year-old man who had chest tightness. The 18 F-FAPI-42 PET/CT showed annular uptake at the apex of the left ventricle, but there was no uptake of 18 F-FDG. This case showed that abnormal 18 F-FAPI-42 uptake in the heart may be associated with mural thrombus and should be evaluated clinically.

Invariant Content Representation for Generalizable Medical Image Segmentation.

Domain generalization (DG) for medical image segmentation due to privacy preservation prefers learning from a single-source domain and expects good robustness on unseen target domains. To achieve this goal, previous methods mainly use data augmentation to expand the distribution of samples and learn invariant content from them. However, most of these methods commonly perform global augmentation, leading to limited augmented sample diversity. In addition, the style of the augmented image is more scattered than the source domain, which may cause the model to overfit the style of the source domain. To address the above issues, we propose an invariant content representation network (ICRN) to enhance the learning of invariant content and suppress the learning of variability styles. Specifically, we first design a gamma correction-based local style augmentation (LSA) to expand the distribution of samples by augmenting foreground and background styles, respectively. Then, based on the augmented samples, we introduce invariant content learning (ICL) to learn generalizable invariant content from both augmented and source-domain samples. Finally, we design domain-specific batch normalization (DSBN) based style adversarial learning (SAL) to suppress the learning of preferences for source-domain styles. Experimental results show that our proposed method improves by 8.74% and 11.33% in overall dice coefficient (Dice) and reduces 15.88 mm and 3.87 mm in overall average surface distance (ASD) on two publicly available cross-domain datasets, Fundus and Prostate, compared to the state-of-the-art DG methods. The code is available at https://github.com/ZMC-IIIM/ICRN-DG .

[Efficacy and Safety of Bortezomib or Thalidomide Combined with rhEPO in the Treatment of Multiple Myeloma].

OBJECTIVE: To explore the efficacy and safety of bortezomib or thalidomide combined with recombinant human erythropoietin (rhEPO) in the treatment of multiple myeloma (MM). METHODS: A total of 80 patients with MM who were treated in the Second People's Hospital of Wuhu from January 2013 to December 2018 were selected as the research subjects, and they were divided into bortezomib group (n=40) and thalidomide group (n=40) by the simple randomization method. The bortezomib group received bortezomib regimen combined with rhEPO therapy, and the thalidomide group was given thalidomide regimen combined with rhEPO therapy, and all patients were treated for 3 courses with every 3 weeks as a course of treatment. The clinical efficacy after 3 courses of treatment, and tumor-related biochemical indicators [lactate dehydrogenase (LDH), ß2-microglobulin (ß2-MG), vascular endothelial growth factor (VEGF), apoptosis inhibitory protein Survivin], bone marrow-related indicators [serum M-protein, bone marrow plasma cells, hemoglobin (Hb)] and coagulation function indicators [activated partial thromboplastin time (APTT), prothrombin time (PT), plasminogen activator inhibitor (PAI), total circulating microparticles (TMPs)] before treatment and after 3 courses of treatment were compared between the two groups of patients. The occurrence of adverse reactions during the treatment in the two groups of patients was recorded. RESULTS: After 3 courses of treatment, the ORR rate of 92.5% in bortezomib group was higher than 90.0% in thalidomide group, but the difference was not statistically significant (P >0.05). The levels of LDH, ß2-MG, VEGF, Survivin, serum M-protein, bone marrow plasma cells, APTT, PT, PAI and TMPs in the two groups after 3 courses of treatment were significantly lower or shorter than those before treatment, and the above indicators in bortezomib group were significantly lower or shorter than those in thalidomide group (P <0.05). After 3 courses of treatment, the expression level of Hb in the two groups was significantly higher than that before treatment, and the Hb level in bortezomib group was significantly higher than that in thalidomide group (P <0.05). During the treatment process, the incidence rates of adverse reactions in bortezomib group were significantly lower than those in thalidomide group (P <0.05). CONCLUSION: Thalidomide regimen or bortezomib regimen combined with rhEPO has similar clinical efficacy on MM, but bortezomib regimen combined with rhEPO is more prominent and safer on improving tumor-related biochemical indicators, bone marrow-related indicators and coagulation status in patients with MM.

Multi-scale consistent self-training network for semi-supervised orbital tumor segmentation.

PURPOSE: Segmentation of orbital tumors in CT images is of great significance for orbital tumor diagnosis, which is one of the most prevalent diseases of the eye. However, the large variety of tumor sizes and shapes makes the segmentation task very challenging, especially when the available annotation data is limited. METHODS: To this end, in this paper, we propose a multi-scale consistent self-training network (MSCINet) for semi-supervised orbital tumor segmentation. Specifically, we exploit the semantic-invariance features by enforcing the consistency between the predictions of different scales of the same image to make the model more robust to size variation. Moreover, we incorporate a new self-training strategy, which adopts iterative training with an uncertainty filtering mechanism to filter the pseudo-labels generated by the model, to eliminate the accumulation of pseudo-label error predictions and increase the generalization of the model. RESULTS: For evaluation, we have built two datasets, the orbital tumor binary segmentation dataset (Orbtum-B) and the orbital multi-organ segmentation dataset (Orbtum-M). Experimental results on these two datasets show that our proposed method can both achieve state-of-the-art performance. In our datasets, there are a total of 55 patients containing 602 2D images. CONCLUSION: In this paper, we develop a new semi-supervised segmentation method for orbital tumors, which is designed for the characteristics of orbital tumors and exhibits excellent performance compared to previous semi-supervised algorithms.

Changing impact of COVID-19 on life expectancy 2019-2023 and its decomposition: Findings from 27 countries.

Background: The World Health Organization declared COVID-19 no longer a global health emergency on 5th May 2023; however, the impact of COVID-19 on life expectancy throughout the pandemic period is not clear. This study aimed to quantify and decompose the changes in life expectancy during 2019-2023 and corresponding age and gender disparities in 27 countries. Methods: Data were sourced from the Human Mortality Database, the World Population Prospects 2022 and the United Kingdom's Office for National Statistics. Life expectancy was estimated using the abridged life table method, while differentials of life expectancies were decomposed using the age-decomposition algorithm. Results: There was an overall reduction in life expectancy at age 5 among the 27 countries in 2020. Life expectancy rebounded in Western, Northern and Southern Europe in 2021 but further decreased in the United States, Chile and Eastern Europe in the same year. In 2022 and after, lost life expectancy years in the United States, Chile and Eastern Europe were slowly regained; however, as of 7th May 2023, life expectancy in 22 of the 27 countries had not fully recovered to its pre-pandemic level. The reduced life expectancy in 2020 was mainly driven by reduced life expectancy at age 65+, while that in subsequent years was mainly driven by reduced life expectancy at age 45-74. Women experienced a lower reduction in life expectancy at most ages but a greater reduction at age 85+. Conclusions: The pandemic has caused substantial short-term mortality variations during 2019-2023 in the 27 countries studied. Although most of the 27 countries experienced increased life expectancy after 2022, life expectancy in 22 countries still has not entirely regained its pre-pandemic level by May 2023. Threats of COVID-19 are more prominent for older adults and men, but special attention is needed for women aged 85+ years.

Residential mobility restrictions and adverse mental health outcomes during the COVID-19 pandemic in the UK.

During the COVID-19 pandemic, several governments tried to contain the spread of SARS-CoV-2, the virus that causes COVID-19, with lockdowns that prohibited leaving one's residence unless carrying out a few essential services. We investigate the relationship between limitations to mobility and mental health in the UK during the first year and a half of the pandemic using a unique combination of high-frequency mobility data from Google and monthly longitudinal data collected through the Understanding Society survey. We find a strong and statistically robust correlation between mobility data and mental health survey data and show that increased residential stationarity is associated with the deterioration of mental wellbeing even when regional COVID-19 prevalence and lockdown stringency are controlled for. The relationship is heterogeneous, as higher levels of distress are seen in young, healthy people living alone; and in women, especially if they have young children.

Selective Detection of Active Extracellular Granzyme A by Using a Novel Fluorescent Immunoprobe with Application to Inflammatory Diseases.

Granzymes (Gzms), a family of serine proteases, expressed by immune and nonimmune cells, present perforin-dependent and independent intracellular and extracellular functions. When released in the extracellular space, GzmA, with trypsin-like activity, is involved in the pathophysiology of different inflammatory diseases. However, there are no validated specific systems to detect active forms of extracellular GzmA, making it difficult to assess its biological relevance and potential use as a biomarker. Here, we have developed fluorescence-energy resonance-transfer (FRET)-based peptide probes (FAM-peptide-DABCYL) to specifically detect GzmA activity in tissue samples and biological fluids in both mouse and human samples during inflammatory diseases. An initial probe was developed and incubated with GzmA and different proteases like GzmB and others with similar cleavage specificity as GzmA like GzmK, thrombin, trypsin, kallikrein, or plasmin. After measuring fluorescence, the probe showed very good specificity and sensitivity for human and mouse GzmA when compared to GzmB, its closest homologue GzmK, and with thrombin. The specificity of this probe was further refined by incubating the samples in a coated plate with a GzmA-specific antibody before adding the probe. The results show a high specific detection of soluble GzmA even when compared with other soluble proteases with very similar cleavage specificity like thrombin, GzmK, trypsin, kallikrein, or plasmin, which shows nearly no fluorescence signal. The high specific detection of GzmA was validated, showing that using pure proteins and serum and tissue samples from GzmA-deficient mice presented a significant reduction in the signal compared with WT mice. The utility of this system in humans was confirmed, showing that GzmA activity was significantly higher in serum samples from septic patients in comparison with healthy donors. Our results present a new immunoprobe with utility to detect extracellular GzmA activity in different biological fluids, confirming the presence of active forms of the soluble protease in vivo during inflammatory and infectious diseases.

Efficient CsPbBr3 Perovskite Light-Emitting Diodes via Novel Multi-Step Ligand Exchange Strategy Based on Zwitterionic Molecules.

Perovskite nanocrystals have absorbed increasing interest, especially in the field of optoelectronics, owing to their unique characteristics, including their tunable luminescence range, robust solution processability, facile synthesis, and so on. However, in practice, due to the inherent instability of the traditional long-chain insulating ligands surrounding perovskite quantum dots (PeQDs), the performance of the as-fabricated QLED is relatively disappointing. Herein, the zwitterion 3-(decyldimethylammonio)propanesulfonate (DLPS) with the capability of double passivating perovskite quantum dots could effectively replace the original long-chain ligand simply through a multistep post-treatment strategy to finally inhibit the formation of defects. It was indicated from theexperimental results that the DLPS, as one type of ligand with the bimolecular ion, was very adavntageous in replacing long-chain ligands and further suppressing the formation of defects. Finally, the perovskite quantum dots with greatly enhanced PLQY as high as 98% were effectively achieved. Additionally, the colloidal stability of the corresponding PeQDs has been significantly enhanced, and a transparent colloidal solution was obtained after 45 days under ambient conditions. Finally, the as-fabricated QLEDs based on the ligand-exchanged PeQDs exhibited a maximum brightness of 9464 cd/m2 and an EQE of 12.17%.

Outcomes for children with disability in out-of-home care: Evidence from the pathways of care longitudinal study in Australia.

BACKGROUND: Children with disability are over-represented in out-of-home care (OOHC) in Australia and internationally. Yet we know little about their circumstances, placement types, support needs, and the outcomes of their trajectories and wellbeing through care. OBJECTIVE: We examine the wellbeing and outcomes of children with and without disabilities in OOHC. PARTICIPANTS AND SETTING: We use panel data from waves 1-4 of the Pathways of Care Longitudinal Study (POCLS) collected between June 2011 and November 2018 by the New South Wales (NSW) Department of Communities and Justice (DCJ), Australia. The POCLS sampling framework covers all children aged 0-17 years who entered OOHC in NSW for the first time between May 2010 and October 2011 (n = 4126). A subset of these children (n = 2828) had final Children's Court orders by 30 April 2013. Among these, caregivers of 1789 children agreed to participate in the interview component of the POCLS. METHODS: We employ a random effects estimator to analyse the panel data. This is standard practice to exploit a panel database when some of the key explanatory variables are time invariant. RESULTS: Children with disability have poorer wellbeing than children without disability across the three domains of physical health, socio-emotional wellbeing, and cognitive ability. However, children with disability have fewer difficulties at school and better school bonding. The type of placements - namely relative/kinship care, restoration/adoption/guardianship, foster care and residential care - have little or limited association with wellbeing of children with disability. CONCLUSIONS: Children with disability tend to have lower levels of wellbeing in OOHC than children without disability, and this is driven mainly by their disability status rather than care factors.

Fluorescent Probes for Imaging in Humans: Where Are We Now?

Optical imaging has become an indispensable technology in the clinic. The molecular design of cell-targeted and highly sensitive materials, the validation of specific disease biomarkers, and the rapid growth of clinically compatible instrumentation have altogether revolutionized the way we use optical imaging in clinical settings. One prime example is the application of cancer-targeted molecular imaging agents in both trials and routine clinical use to define the margins of tumors and to detect lesions that are "invisible" to the surgeons, leading to improved resection of malignant tissues without compromising viable structures. In this Perspective, we summarize some of the key research advances in chemistry, biology, and engineering that have accelerated the translation of optical imaging technologies for use in human patients. Finally, our paper comments on several research areas where further work will likely render the next generation of technologies for translational optical imaging.

FAPI PET/CT in Diagnostic and Treatment Management of Colorectal Cancer: Review of Current Research Status.

FAPI PET/CT is a novel imaging tool targeting fibroblast activation protein (FAP), with high tumor uptake rate and low background noise. Therefore, the appearance of FAPI PET/CT provides a good tumor-to-background ratio between tumor and non-tumor tissues, which is beneficial to staging, tumor description and detection. Colorectal cancer has the biological characteristics of high expression of FAP, which provides the foundation for targeted FAP imaging. FAPI PET/CT may have a potential role in changing the staging and re-staging of colorectal cancer, monitoring recurrence and treatment management, and improving the prognosis of patients. This review will summarize the application status of FAPI PET/CT in colorectal cancer and provide directions for further application research.

Asymptomatic Prostate Cancer Metastasis in Rectal Mucosa Revealed by 18 F-PSMA-1007 PET/CT.

ABSTRACT: Prostate cancer metastasis to the rectal mucosa, a relatively rare metastatic site, leads to a higher clinical stage and poorer prognosis. A 65-year-old man with prostate cancer underwent 18 F-prostate-specific membrane antigen (PSMA) PET/CT for staging. Intense 18 F-PSMA uptake occurred at the primary lesion, bladder, adjacent seminal vesicle, and rectum. PET/CT imaging revealed increased homogeneous round activity of the rectal wall. The final diagnosis was prostate cancer metastasis to the rectal mucosa. This case suggested that 18 F-PSMA PET/CT may assist in locating rare metastases of prostate cancer, with potential value for early staging.