Substance use disorders (SUDs) commonly co-occur with mental illness. However, the ongoing addiction crisis raises the question of how opioid use disorder (OUD) impacts healthcare utilization relative to other SUDs. This study examines the utilization patterns of patients with major depressive disorder (MDD) and: (1) co-occurring OUD (MDD-OUD); (2) a co-occurring SUD other than OUD (MDD-NOUD); and (3) no co-occurring SUD (MDD-NSUD). We analyzed electronic health records (EHRs) derived from multiple health systems across the New York City (NYC) metropolitan area between January 2008 and December 2017. 11,275 patients aged ≥18 years with a gap of 30–180 days between 2 consecutive MDD diagnoses and an antidepressant prescribed 0–180 days after any MDD diagnosis were selected, and prevalence of any SUD was 24%. Individuals were stratified into comparison groups and matched on age, gender, and select underlying comorbidities. Prevalence rates and encounter frequencies were measured and compared across outpatient, inpatient, and emergency department (ED) settings. Our key findings showed that relative to other co-occurring SUDs, OUD was associated with larger increases in the rates and odds of using substance-use-related services in all settings, as well as services that integrate mental health and substance abuse treatments in inpatient and ED settings. OUD was also associated with larger increases in total encounters across all settings. These findings and our proposed policy recommendations could inform efforts towards targeted OUD interventions, particularly for individuals with underlying mental illness whose treatment and recovery are often more challenging. That same year, more than 4 million US adults with a SUD experienced a major depressive episode, and well over 500,000 of those specifically had an opioid use disorder (OUD)5.In 2018, nearly 20% of US adults experienced mental illness, and nearly 20% of those with mental illness experienced a co-occurring substance use disorder (SUD)9. Furthermore, depression has been documented as a risk factor for misusing opioids , while co-occurring substance use may compromise adherence to and the mood-stabilizing effects of antidepressant medications10.Major depressive disorder (MDD) and SUDs have a bidirectional relationship: symptoms of one disorder increase and reinforce the risk of the other, making this patient population particularly challenging to treat. Left untreated, patients with co-occurring disorders typically exhibit poorer health outcomes—including greater depressive symptomatology, more severe functional impairment, poorer recovery rates, increased suicidal ideation and attempt, and higher rates of healthcare utilization—compared to those with a diagnosis of MDD alone11. In fact, studies have shown that integrated approaches which coordinate mental health and substance use therapies may produce more effective outcomes than parallel or sequential treatments delivered in separate settings17. For example, such interventions for MDD that simultaneously reduce substance use are more likely to improve symptoms of both disorders, reduce relapse rates, and enhance recovery20. However, in 2018, only 11% of adults with co-occurring mental illness and SUDs received such integrated treatments, and over 30% received no treatment at all1.Given the significant overlaps, theories suggest addressing the dual care needs of patients with co-occurring MDD and SUD is vital to improving morbidity and mortality outcomes among this population22. In hospitals, depression has been associated with increased risk of inpatient admissions, length of stay, and risk of 30-day readmission24. Similarly, patients with SUDs typically have increased emergency department (ED) encounters and inpatient hospitalizations25. When combined with MDD, SUDs are associated with increased psychiatric and other medical utilization, including hospitalizations, lengths of stay, and costs (largely attributed to increased psychiatric inpatient encounters)29.On the other hand, patients with MDD or SUD who do ultimately receive treatment are high healthcare utilizers. Primary care patients with MDD typically have more annual healthcare visits, specialist referrals, laboratory tests, and radiologic scans and procedures than patients without MDD31. Patients with OUD are particularly costly, as they are the highest healthcare utilizers among patients with SUDs and are more likely to use crisis- and substance use-related services34. Although OUD shares the same category as other SUDs, several features set OUD apart in terms of specialized management and treatment. For example, withdrawal symptoms from opioids are far more severe than from other substances and can lead to physical dependence in as little as 4–8 weeks35. Second, the supply and access to prescription opioids in medical practice fueled the opioid epidemic and provided a key gateway to non-medical heroin use35. Third, users who relapse on opioids are at a significantly higher risk of overdose and mortality compared to users who relapse on other substances such as cannabis or alcohol36. Finally, whereas treatment for all other SUDs can take place outside the formal healthcare system, successful treatment of OUD demands patients to be on medications , which in turn requires healthcare providers and clinics to be part of this treatment ‘ecosystem’.Such trends are especially intriguing when parsing out the various types of SUDs. In particular, the United States is grappling with an epidemic marked by high rates of addiction treatment admissions, hospitalizations, and overdose deaths related to prescription opioids and heroinWhile co-occurring SUDs have been associated with increased healthcare utilization in individuals with MDD, it remains unclear to what extent co-occurring OUD and non-OUD SUDs increase healthcare utilization among individuals with MDD. This study aims to fill this knowledge gap by using a pharmacotherapy-treated sample of adult patients to examine the patterns of healthcare services utilization in three groups of patients with MDD: (1) those with co-occurring OUD (MDD-OUD); (2) those with a co-occurring SUD other than OUD (MDD-NOUD); and (3) those with no co-occurring SUD (MDD-NSUD).37. Institutional IRB exemption was granted for use of this database for research. Data were extracted from EHRs using the Observational Medical Outcomes Partnership (OMOP) Common Data Model. Figure n = 11,275) from the INSIGHT CRN dataset.Using fully de-identified electronic health record (EHR) data from the PCORI-funded INSIGHT Clinical Research Network (CRN), we identified and analyzed the utilization of patients during the decade from January 2008 to December 2017. The INSIGHT CRN compiles EHRs of 12 million patients from six large medical centers across New York City, including Montefiore Medical Center, New York-Presbyterian Hospital, Mount Sinai Health System, New York University Langone Medical Center, Columbia University Medical Center, and Weill Cornell Medicinen = 2672) and (2) patients with no lifetime co-occurring SUD . To reduce their potentially confounding effects on service use, MDD-SUD was age, gender, and comorbidity-matched to an equally sized sample of MDD-NSUD (n = 2672) using nearest-neighbor propensity score matching39. To isolate OUD from non-OUD SUDs, MDD-SUD was further stratified into two groups using ICD-9 and ICD-10 codes: (1) patients with lifetime co-occurring OUD  and (2) patients with one or more lifetime co-occurring SUDs other than OUD . Similarly, MDD-OUD was age, gender, and comorbidity-matched to an equally sized sample of MDD-NOUD (n = 424) using nearest neighbor propensity score matching39. Overall demographics and health status were evaluated for each of the MDD-OUD, MDD-NOUD, and MDD-NSUD comparison groups, and Fig. First, to evaluate the overall demographic makeup of our study sample, all 11,275 patients in the MDD cohort were classified by age, gender, race, and ethnicity. To assess the overall health status of our study sample, the MDD cohort was evaluated for prevalence of select common chronic conditions, co-occurring mental health disorders, and SUDs using ICD-9 and ICD-10 codes. Next, to establish the patterns and measures of utilization associated with MDD alone, i.e. in the absence of co-occurring OUD or other SUDs, the MDD cohort was stratified into two groups using ICD-9 and ICD-10 codes: (1) patients with one or more lifetime co-occurring SUDs  with 95% confidence intervals (CIs) were also computed to estimate effect sizes. Second, to understand individual-level patterns of utilization, visit frequencies were measured for each individual who had at least one encounter in MDD-OUD, MDD-NOUD, and MDD-NSUD and median, first quartile, and third quartile values were computed for each group. Multiple pairwise comparisons were performed using Mann–Whitney U (Wilcoxon rank-sum) tests to determine between which groups the differences mostly occurred, and a Bonferroni-corrected p < 0.05/3 or 0.0167 was used as the threshold for defining statistical significance. The data met the respective assumptions of all statistical tests performed. R version 3.6.0 was used for all analyses40.First, to understand system-level patterns of utilization, we calculated prevalence rates of healthcare utilization among MDD-OUD, MDD-NOUD, and MDD-NSUD. Differences were compared using chi-squared tests, and According to Table p < 0.001), but OUD was associated with a larger increase . MDD-OUD used substance use-related outpatient services the most (64%) compared to MDD-NOUD (51%) and MDD-NSUD (<1%). Also, OUD and non-OUD SUDs were associated with increased rates of using integrated outpatient services (p < 0.001). MDD-NSUD used integrated outpatient services the least (<1%) compared to MDD-NOUD (55%) and MDD-OUD (60%).Among patients with MDD, both co-occurring OUD and non-OUD SUDs were associated with increased rates of using substance use-related outpatient services (p < 0.001). MDD-NSUD used inpatient services the least (46%) compared to MDD-NOUD (74%) and MDD-OUD (81%). Further analysis of inpatient services stratified by service type showed that OUD and non-OUD SUDs were associated with increased rates of using psychiatric inpatient services . MDD-NOUD used psychiatric inpatient services the most (48%), followed by MDD-OUD (44%) and MDD-NSUD (36%). Also, both OUD and non-OUD SUDs were associated with increased rates of using substance use-related inpatient services (p < 0.001), but OUD was associated with a larger increase . MDD-OUD used substance use-related inpatient services the most (37%) compared to MDD-NOUD (28%) and MDD-NSUD (<1%). Finally, both OUD and non-OUD SUDs were associated with increased rates of using integrated inpatient services (p < 0.001), but OUD was associated with a larger increase . MDD-OUD used integrated inpatient services the most (72%), followed by MDD-NOUD (57%) and MDD-NSUD (<1%).Among patients with MDD, co-occurring OUD and non-OUD SUDs were associated with increased rates of inpatient health services use (p < 0.001). MDD-NSUD used ED services the least (52%) compared to MDD-NOUD (82%) and MDD-OUD (84%). Further analysis of ED services stratified by the service nature showed that OUD and non-OUD SUDs were associated with increased rates of using psychiatric ED services (p < 0.001). MDD-NSUD used psychiatric ED services the least (13%) compared to MDD-NOUD (22%) and MDD-OUD (25%). Also, both OUD and non-OUD SUDs were associated with increased rates of using substance use-related ED services (p < 0.001), but OUD was associated with a larger increase . MDD-OUD used substance use-related ED services the most (26%), followed by MDD-NOUD (15%) and MDD-NSUD (<1%). Furthermore, both OUD and non-OUD SUDs were associated with increased rates of using integrated ED services (p < 0.001), but OUD was associated with a larger increase . MDD-OUD used integrated ED services the most (24%) compared to MDD-NOUD (16%) and MDD-NSUD (<1%). Finally, OUD and non-OUD SUDs were associated with increased rates of using other medical ED services (p < 0.001). MDD-NSUD used other medical ED services the least (43%) compared to MDD-NOUD (72%) and MDD-OUD (73%).Among patients with MDD, co-occurring OUD and non-OUD SUDs were associated with increased rates of ED health services use (p < 0.001), but OUD was associated with a larger increase (p = 0.0027). On average, MDD-OUD had more outpatient visits (89) than MDD-NOUD (68) and MDD-NSUD (44). Further analysis of outpatient visits stratified by the visit type showed that only OUD was associated with increased psychiatric outpatient encounters (p < 0.001). On average, MDD-OUD had more psychiatric outpatient visits (11) than MDD-NOUD (9) and MDD-NSUD (8). Also, only OUD was associated with increased substance use-related outpatient encounters (p = 0.0068). On average, MDD-OUD had more substance use-related outpatient visits (6) than MDD-NOUD (3) and MDD-NSUD (2). Furthermore, only OUD was associated with increased integrated outpatient encounters (p = 0.0082). On average, MDD-OUD had more integrated outpatient visits (5) than MDD-NOUD (3) and MDD-NSUD (2). Finally, OUD and non-OUD SUDs were associated with increased other medical outpatient encounters (p < 0.001). On average, MDD-NSUD had fewer other medical outpatient visits (31) than MDD-NOUD (45) and MDD-OUD (47).Among MDD patients who used any outpatient services, both OUD and non-OUD SUDs were associated with increased outpatient encounters (p < 0.001), but OUD was associated with a larger increase (p < 0.001). On average, MDD-OUD had more inpatient visits (8) than MDD-NOUD (5) and MDD-NSUD (3). Further analysis of inpatient visits stratified by the visit type showed that only OUD was associated with increased psychiatric inpatient encounters (p < 0.001). On average, MDD-OUD had more psychiatric inpatient visits (4) than MDD-NOUD (3) and MDD-NSUD (3). Furthermore, both OUD and non-OUD SUDs were associated with increased integrated inpatient encounters (p < 0.001), but OUD was associated with a larger increase (p < 0.001). On average, MDD-OUD had more integrated inpatient visits (4) than MDD-NOUD (2) and MDD-NSUD (1).Among MDD patients who used any inpatient services, both OUD and non-OUD SUDs were associated with increased inpatient encounters (p < 0.001), but OUD was associated with a larger increase (p < 0.001). On average, MDD-OUD had more ED visits (13) than MDD-NOUD (8) and MDD-NSUD (5). Further analysis of ED visits stratified by the visit type showed that both OUD and non-OUD SUDs were associated with increased other medical ED encounters (p < 0.001), but OUD was associated with a larger increase (p < 0.001). On average, MDD-OUD had more other medical ED visits (6) than MDD-NOUD (4) and MDD-NSUD (3).Among MDD patients who used any ED services, both OUD and non-OUD SUDs were associated with increased ED encounters  compared to patients with other SUDs, patients with OUD were more likely to use inpatient and ED services that integrate mental health and substance abuse treatments, and (2) only OUD was associated with increased psychiatric and integrated encounters in both outpatient and longer term inpatient settings.Conversely, the differential utilization patterns we observed expand on recent findings that could suggest OUD may more strongly reinforce the risk and symptoms of mental illness compared to other SUDs. While little is currently known about OUD in relation to other SUDs among patients with major depression, a recent analysis using a national sample of people with severe mental illness found that those with a co-occurring heroin use disorder were 19 times more likely than those without co-occurring SUDs to have criminal justice system involvement, while those with all other co-occurring SUDs  were only five times more likely. In addition, those with a co-occurring prescription painkiller use disorder were 2.4 times more likely to attempt suicide than those without co-occurring SUDs, while those with all other co-occurring SUDs were 1.8 times more likely48. This underscores the complexity of patients presenting to clinics with multiple co-occurring mental illnesses, and moreover sheds light on the overlapping symptomatologies that cloud differential diagnosis in this population51.Current literature suggests that the etiologies and risk factors for mental health disorders and OUD overlap, and that if either is untreated, both will impact patient outcomes. It is important to note that nearly half of the MDD patients included in this study had other co-occurring mental health disorders (45%), and in particular bipolar disorder (43%). In one study, individuals with bipolar depression had more psychiatric hospitalization, mental health-related outpatient visits, social services visits, and ED visits than individuals with unipolar depressionOur analysis of MDD patients indicates that co-occurring OUD and non-OUD SUDs differentially increase utilization across settings, requiring equally differential treatment planning. Whereas the clinical features and needs that distinguish OUD from other SUDs are helpful for understanding treatment of an individual patient, the utilization patterns observed in this study may help guide selection of trade-offs in policy and other interventions. For example, practitioners should screen for opioid dependence in patients who present with mental illness, especially those with a history of substance use including prescription opioids. In addition, practitioners should initiate treatment for OUD prior to evaluating and treating mental health disorders soon thereafter. This is based on an earlier suggestion that co-occurring OUD may more strongly reinforce the risk and symptoms of mental illness compared to other co-occurring SUDs. Further investigation is needed to ascertain whether temporality of mental health and substance abuse treatment impacts long-term outcomes.52. Limiting the use of parallel treatments in outpatient settings and instead shifting towards integrated treatment models could be more cost-effective and reduce the rate of medical and psychiatric hospitalizations related to a dual diagnosis55. For cases that necessitate hospitalization, we agree with the authors of a recent study of SUD readmissions in NYC hospitals who suggested that hospital settings could be useful venues for substance use-related interventions, and could benefit from close coordination with outpatient providers and more targeted discharge planning56.Furthermore, among MDD patients with co-occurring OUD, the inpatient utilization rate of integrated services was much higher (72%) as compared to non-integrated psychiatric (44%) and substance use-related (37%) treatment services. Conversely, the outpatient utilization rate of integrated services was the lowest (60%) as compared to non-integrated psychiatric (80%) and substance use-related (64%) treatment services. Our broader inpatient and outpatient results resemble those of a national sample of individuals treated for OUD during a similar study periodp < 0.001), although supplemental findings suggest these decreases were recaptured by increased integrated outpatient utilization. A potential future research question could examine whether introducing mental health treatment during the treatment for SUDs improves non-integrated outpatient mental health treatment entry and ambulatory care utilization after hospital discharge in dual diagnosis patients.Interestingly, both OUD and non-OUD SUDs were associated with decreased rates of using psychiatric outpatient services in patients with MDD 58. Finally, the INSIGHT CRN pertains to health centers within the NYC metropolitan area, limiting our ability to accurately control for prescription drug use patterns (in particular prescription opioids) and generalize findings to a national scale. It is also possible that depressed patients captured within, but prescribed antidepressants outside of, the INSIGHT CRN were excluded from the study cohort.The findings from this study should be understood within the context of a few methodological limitations associated with the use of EHR data. First, although it had minimal impact on depression, it is important to consider how the transition from ICD-9-CM to ICD-10-CM impacted coding of mental health conditions59. However, the alarmingly high rate of co-occurring substance use among mentally ill patients makes it important to understand how this impacts relevant service utilization and health outcomes. Our key findings showed that relative to other co-occurring SUDs, OUD was associated with larger increases in the rates and odds of using substance-use-related services in all settings, as well as services that integrate mental health and substance abuse treatments in inpatient and ED settings. OUD was also associated with larger increases in total encounters across all settings. Our analysis expands and motivates further inquiry on recent preliminary evidence that could suggest co-occurring OUD may more strongly reinforce the risk and symptoms of mental illness relative to other co-occurring SUDs. We propose several policy recommendations for better managing co-occurring OUD, including prioritized screening and initiation of treatment for opioid dependence, as well as closer coordination between inpatient, ED, and outpatient care settings. In the midst of an ongoing addiction crisis, recognition among stakeholders  of the heterogeneity among SUDs will inform targeted, evidence-based interventions for OUD. Further investigation is needed to assess the efficacy of our proposed policy recommendations on long-term health outcomes related to the management of co-occurring OUD.The United States continues to make strides in improving access to mental health servicesSupplementary


Epidermal growth factor receptor (EGFR) is an anticancer drug target for a number of cancers, such as non-small cell lung cancer. However, unsatisfying treatment effects, terrible side-effects, and development of drug resistance are current insurmountable challenges of EGFR targeting treatments for cancers. With the advancement of nanotechnology, an increasing number of inorganic nanomaterials are applied in EGFR-mediated therapy to improve those limitations and further potentiate the efficacy of molecular targeted cancer therapy. Given their facile preparation, easy modification, and biosecurity, inorganic nanoparticles (iNPs) have been extensively explored in cancer treatments to date. This review presents an overview of the application of some typical metal nanoparticles and nonmetallic nanoparticles in EGFR-targeted therapy, then discusses and summarizes the relevant advantages. Moreover, we also highlight future perspectives regarding their remaining issues. We hope these discussions inspire future research on EGFR-targeted iNPs. Cancer is a major public health problem worldwide . The incTumor tissue is mainly composed of parenchyma and stroma. Tumor parenchyma essentially refers to carcinoma cells, which has specificity in relation to tissue. Carcinoma cells are characterized by intense proliferation due to the insensitiveness to apoptotic signal, escaping from apoptosis and so on. The tumor stroma, namely TME, provides survival necessity and conditions for tumor cell growth and metastasis . TME conEpidermal growth factor receptor (EGFR) is a transmembrane protein that widely distributes in epithelial cells, fibroblasts, spongiocytes, and keratinocytes. EGFR plays a crucial role in cell growth metastasis and angiogenesis . FrequenThe use of modern nanoscience and material science has provided new approaches to conquer cancer . Novel nRecent years have witnessed the wide application of oncotherapy of iNPs. Due to their particular physico-chemical properties, metallic NPs (MNPs) have been explored in EGFR-targeted therapy to achieve synergistic treatment, improve therapeutic effects, and delay the development of drug resistance. These multifarious adopted strategies in EGFR-targeted therapy are summarized as follows.111In-labelled EGF-targeted agent (111In-EGF-Au-PEG) , enhance the tumor uptake, and reduce liver uptake compared to the unlabeled 111In- Au-PEG NPs. Au NPs are also qualified for the delivery of EGFR antibodies and cetuximab is the most widely used and reliable EGFR antibody. Groysbeck et al. conjugated water-soluble thiolate-protected Au NPs with cetuximab (AuNP-Cetuximab) against EGFR expressing glioblastoma cells. The obvious inhibition of EGFR autophosphorylation was observed (Gold NPs (Au NPs) are one of the most studied diagnosis and treatment agents for the treatment of cancer due to their ease of functionalization, good stability, low toxicity, and high biocompatibility. Au NPs-based targeted nanomedicine, such as AurImmune™ , has entered clinical trials. Au NPs have been demonstrated as EGFR antibodies and KTIs’ vehicles in molecule targeted therapy because of their large surface area for attachment of targeting drugs. For example, EGF-tagged Au NPs, as a versatile delivery, was bound to indium-111 to form an -Au-PEG) . The outobserved . The conobserved . The impobserved   to achieve cancer inhibition through dual pathways. On one hand, the designed Au10Peptide5-GE11 could target EGFRs in both active and inactive states, and then induce the oxidative stress mediated apoptosis in the active EGFR mediated endocytosis process. On the other hand, the inactive state of EGFR at the membrane of tumor cells could be maintained by binding Au10Peptide5-GE11 to avoid the dimerization and further inhibit the activities of tumor cells. Therefore, employing Au10Peptide5 as EGFR TKIs delivery results in inhibiting cancer cells through dual pathways , Ag/C225 nanocomposites cause irreversible cell growth inhibition. Therefore, Ag/C225 nanocomposites, as a specific radiosensitizer, exhibited better anti-proliferative effects in nasopharyngeal carcinoma cell lines by assistive EGFR-targeting of C225. Cho et al. developed a targeted Au/Ag drug delivery (AuHNS-EGFR-DOX) for near-infrared (NIR) light induced thermo-chemotherapy  have been recognized as useful tools for diagnosis and treatment in anticancer and antimicrobial fields. Ag NPs were found to have potential utility as radiosensitizers to improve the outcomes of cancer radiotherapy. Therefore, the incorporation of the EGFR-specific small molecules (e.g. gefitinib) or humanized McAb targeting EGFR  into Ag NPs is an alternative strategy. For instance, C225-coated Ag NPs (Ag/C225) have been tested for their enhanced radiosensitization in nasopharyngeal carcinoma epithelial cell lines . The antotherapy . The Au/or tumor .In this section, we selectively present various typical nanometallic nanoparticles that have been synthesized and conjugated to EGFR-targeted small molecule drugs for numerous oncotherapies in recent years. The applications in oncotherapy of non-metallic nanomaterials consisting mainly of selenium (Se), iron-based nanomaterials, metallic chalcogenide, metallic oxideare, silicon, and carbon-based materials have been described in the following sections.Se elements with anticancer and immunoregulation properties are indispensable and necessary in humans. Gao’s team found that selenium nanoparticles could inhibit the growth of prostate cancer cells in part through cystepsin mediated apoptosis and Ahmadrezashahverdi’s team demonstrated that Se nanoparticles could significantly induce the immune response of 4T1 breast cancer tumors in mice . ConsequIron is another indispensable microelement in the human body. Iron is required in multitudinous biological processes such as composition of hemoglobin to deliver oxygen around the body. Importantly, iron-based nanomaterials have been approved by the FDA in inorganic nanomedicines . Iron ox3O4 (EGFRmAb-SPIONs) were synthesized and served as a targeted MRI contrast agent for the EGFR-positive detection in vitro and in vivo. The MRI results suggested that the brain glioma cells treated with EGFRmAb-SPIONs exhibited a more significant negative contrast enhancement even at low concentrations than that observed in the tumor cells incubated only with SPIONs   probe for imaging of EGFR positive tumor cells. The outcomes indicated that Au-IONPs nanoprobe provided significant specificity and high sensitivity for both PET and MRI imaging in the human EGFR-expressing tumor cells (3O4/Au nanoparticle was conjugated with a single-chain antibody (scFv) to obtain an EGFR-specific MRI bioprobe (scFv@Fe3O4/Au). The in vivo results showed that scFv@Fe3O4/Au could specifically transfer Fe3O4/Au to detect EGFR-positive non-small cell lung cancer through MRI method. The scFv@Fe3O4/Au immunonanoparticles were detected in the cell cytoplasm of EGFR-overexpressing SPC-A1 cells, while very little scFv@Fe3O4/Au could be detected in EGFR-deficient H69 cells. Therefore, these IONPs-based MRI agents are potential strategies for both selective imaging and cell screening. C225-encapsulated core-shell Fe3O4@Au were fabricated as a therapeutic nano-system (Fe3O4@Au-C225) to conduct targeted magneto-photothermal therapy against glioma cells  was fabricated to realize radiation therapy  was used to be an EGFR-targeted tumor tracer for radiation therapy. The in vitro experimental results revealed that the enhanced inhibition of human nasopharyngeal carcinoma cell combined with X-ray treatment was found. In a word, this multifunctional nanocomposite Fe3O4/Ag/C225 might be a potential EGFR-targeted radiosensitizer for treating human nasopharyngeal carcinoma tumor.A number of EGFR-targeted composite nanoplatforms with IONPs as the main component for targeting MRI and therapy have been studied so far. Cheng’s group developed a hetero-nanostructural trimodality nanoprobe, which was composed of Feu-IONPs) . In thisor cells . Moreovema cells . Fe3O4@A therapy . Herein,in vivo and in vitro results all demonstrated that EGFR-targeted nanosystems could effectively ablate tumor tissue by synergistic treatment of chemotherapy and photothermal therapy  as a carrier to delivery IONPs and doxorubicin (DOX), and further modified this magnetic graphene oxide with cetuximab (MGO-PEG-CET) . This EG therapy . In addi therapy . A two-fIn addition, some metallic sulfides and metallic selenides  have been intensely studied. Herein, some recent reports about zinc sulfide (ZnS), copper sulfide (CuS), silver sulfide (AgS), and cadmium selenide (CdSe) as subjects for EGFR-targeting oncotherapy are summarized in the next section.2+ (PLGA-ZnS) conjugated with cetuximab was used to accomplish targeted imaging and delivery of anti-cancer drugs. As the cell uptake results showed, the uptake of targeted NPs was over 80%, while that of the nontargeted NPs was only 40% (2). Therefore, the designed CuS-Ab NPs fully exploit higher local tumoricidal effect and lower nephric and systemic toxicity by EGFR-targeting with cetuximab -based Ag2S QD (ZEGFR:1907-Ag2S QDs) nanoprobes have been successfully synthesized and used for targeted photoacoustic imaging of EGFR-overexpressed tumor  and Bxpc3 with β-actin (EGFR-negative). The uptake of ZEGFR:1907-Ag2S QDs was up to 80% in A431 cells due to the prominent targeting and specificity endowed by ZEGFR:1907. In one word, the photoacoustic imaging results showed that ZEGFR:1907-Ag2S QDs could detect EGFR-positive tumor cell lines perfectly.For example, poly (lactic-co-glycolic acid) coated ZnS:Mnonly 40% . Gong etonly 40% . The preonly 40% . The cetonly 40% . With thetuximab . Additioetuximab . The resed tumor . The pre2). Apt@GO@Au-His@a-ZnO@DOX NCs was demonstrated as an excellent photothermal nanomaterial with high photothermal conversion efficiency (η = 38%). The resultant Apt@GO@Au-His@a-ZnO@DOX NCs could release antitumor Zn2+ ions in the acidic endosome/lysosome of tumor tissue simultaneously. Importantly, Apt@GO@Au-His@a-ZnO@DOX NCs exhibited much higher targeting efficacy and tumor inhibition to EGFR-positive A549 cells than that of GO@Au-His@a-ZnO@DOX NCs according to the laser scanning confocal microscope (LSCM) images for cellular uptake and in vivo experiments  . For exaeriments .Except for the above mentioned metallic chalcogenide, silica nanoparticles (SNs) exhibit excellent biodegradability and thus were always chosen as the nanocarriers due to their high porosity and surface area . Herein,−1. And the inhibition rate in EGFRAb-SN-pyrrolidine-2 treated group reached 64%  to use as a targeted prodrug delivery for effective lung cancer therapy . Accordiched 64% . Kong etched 64% . Impressched 64% .in vivo models. Furthermore, the nanocomplex showed high accumulation at the tumor site in vivo and exhibited good safety with no damage to major organs. Ji et al. also proved that using MSNs to deliver cetuximab and DOX could accomplish effective management of EGFR-mutant lung cancer and overcome acquired drug resistance (PC9-DR). The resultant MSNs have the specific surface area of 887.9 m2/g and the pore size of 2.5 nm, which is suitable to carry cetuximab, gefitinib, and DOX into cells with high EGFR expression by endocytosis. Significant effect upon inhibiting PC9-DR xenograft tumor growth was observed; the weight of the xenograft tumors decreased to 0.1 g, indicating the potential ability to solve drug resistance  to directly silence the gene expression with complementary messenger RNA (mRNA) sequence, particularly the reversal of drug resistance . The inhsistance . Brinkersistance . They emsistance .3+) is well known as an exogenous contrast agent with strong T1-weighted image signal intensity, however, high doses and indiscriminate accumulation result in terrible side-effects for normal tissues. Therefore, EGFR antibody conjugated SNs were utilized for geometric confinement of small molecule Gd-chelate  is an important auxiliary to implement contrast enhancement. FITC-labeled SNs with the attachment of anti-EGF-receptor-antibodies (Alexa Fluor®555) were built as a novel contrast agent (AF555-EGFR-FITC-SiO2-NPs)  . It couliO2-NPs) . MoreoveiO2-NPs) . The C22in vitro experiments, targeted imaging, and effective apoptosis of cancer cells were caused by the specific internalization of SFN-CDs  also proved that this nanosystem possessed an effective capability for targeted fluorescence imaging of pancreatic cancer cells (The fabrication and bioimaging applications of carbon dots (CQDs) have rapidly advanced in recent years because of their photostablity and chemical stability. Importantly, CQDs contain no heavy metals, suggesting lower biotoxicity than semiconductor QDs . When CQ SFN-CDs . Interes SFN-CDs . The fluer cells . It is win vitro. But there is still room for further improvement so that the nanoparticles can be suitable for clinical trials and applications. Some of the challenges that still need to be addressed include improving the stability of nanoparticles in a variety of complex in vivo environments, designing proper functionalization to improve biocompatibility and reducing cytotoxicity, effective cost control, and optimization of synthesis protocols. This paper sums up the description and the benefits of different EGFR-loaded nanotechnological platforms based on iNPs.The rapid advance of nanotechnology in recent years has provided progressive strategies for cancer therapy. Various advantages of novel iNPs have been identified as multifunctional nanotherapeutics, especially molecular targeted therapy for cancers. Metal nanoparticles and nonmetallic nanoparticles have unique properties, such as easy accumulation in tumor cells, fluorescence imaging, enhanced Raman scattering, photothermal, and antimicrobial properties. A large number of biomedical applications have been successfully demonstrated As we previously described, EGFR-targeted iNPs have a solid role in imaging diagnosis and cancer therapy. However, these EGFR-targeted antineoplastic agents still have a great distance from nanomedicine in lab to clinical reality and there are many unresolved issues to be elucidated. First of all, regarding the synthesis, the controllable preparation of iNPs with uniform size, high crystallinity, and good morphology is important. Second, combining EGFR-mediated iNPs with the guidance of fluorescence, CT, or MRI imaging has a great perspective to achieve more satisfying therapeutic effect. Therefore, the construction of imaging-guided and EGFR-targeted therapy in one nanoplatform still needs to be further explored to achieve drug delivery monitoring, tumor localization, and treatment effect monitoring. Additionally, whether and why the combination of iNPs with EGFR-KIT or McAb could overcome the drug resistance to a certain extent and suppress the progression of point mutation in the DNA sequence is an urgent issue to understand. Moreover, the pharmacodynamic and pharmacokinetic properties of inorganic nanoparticles are immature. As a consequence, numerous comprehensive studies need to be carried out to scrutinize the pharmacokinetic, biodistribution, and safety profiles of iNPs. Given the interactions between iNPs and immune system in body, the immunotoxicity of iNPs should be further assessed in future.In a word, great efforts are required to meet the challenges in implementing the transformation from nanomedecine to clinical trials. With profound exploration, iNPs will revolutionize molecular targeted cancer therapy in the near future.


Self-immolativepolymers are an emerging class of macromoleculeswith distinct disassembly profiles that set them apart from othergeneral degradable materials. These polymers are programmed to disassemblespontaneously from head to tail, through a domino-like fragmentation,upon response to extremal stimuli. In the time since we first reportedthis unique type of molecule, several groups around the world havedeveloped new, creative molecular structures that perform analogouslyto our pioneering polymers. Self-immolative polymers are now widelyrecognized as an important class of stimuli-responsive materials fora wide range of applications such as signal amplification, biosensing,drug delivery, and materials science. The quinone-methide eliminationwas shown to be an effective tool to achieve rapid domino-like fragmentationof polymeric molecules. Thus, numerous applications of self-immolativepolymers are based on this disassembly chemistry. Although severalother fragmentation reactions achieved the function requested forsequential disassembly, we predominantly focused in this Perspectiveon examples of self-immolative polymers that disassemble through thequinone-methide elimination. Selected examples of self-immolativepolymers that disassembled through other chemistries are briefly described.The growing demand for stimuli-responsive degradable materials withnovel molecular backbones and enhanced properties guarantees the futureinterest of the scientific community in this unique class of polymers. Since we developed this type of macromolecules,the field of self-immolative polymers has grown beyond our expectations.Numerous groups from around the world have reported new, creativemolecular structures, which perform in manners analogous to our originalconcept of self-immolative polymers. Currently, self-immolative polymersare widely recognized as an important emerging class of stimuli-responsivematerials for a wide range of applications.3 Inthis Perspective, we first give a historical overview of the fieldand then focus on selected examples that, in our view, reflect theunique significance of self-immolative polymers. Finally, we describethe recent developments of new molecular structures that cleverlyachieve the distinctive functions demanded of self-immolative polymers.Stimuli-responsivematerials are a promising type of smart molecularcompounds, with properties that can be significantly changed in acontrolled fashion upon response to applied stimuli. About 14 yearsago our group developed a distinct class of such molecular compoundsthat we termed as self-immolative polymers.6 However, there is a fundamental difference betweensuch depolymerization processes and the disassembly pathway of self-immolativepolymers. The disassembly of a self-immolative polymer is initiatedupon removal of a specific masking group at the polymer terminus andoccurs from head to tail through a domino-like manner, while depolymerizationof polymers with low ceiling temperatures is initiated at variouslocations along the polymer backbone by radiation or photoacids. Thus,the level of control on the disassembly of self-immolative polymersis more modular and versatile.Depolymerization reactions of polymers with low ceiling temperatures,such as poly(olefin sulfone) and poly, were describedby Ito and others, long before we reported the concept of self-immolativepolymers.7 In 1981, Katzenellenbogen reported the use ofa novel connector linkage, based on 4-aminobenzyl alcohol, as a self-immolativelinker backbone, the fluorescence of the aniline is almost completely quenched.The polymer was capped with 4-hydroxy-2-butanone as a head trigger,which is designed for removal through a β-elimination reactioncatalyzed by the protein bovine serum albumin (BSA). Incubation ofthis self-immolative polymer with BSA initiates the domino-like disassemblyof the polymer molecule to its aniline monomeric units. As a result,a green fluorescence signal gradually increases in intensity correlatedto the number of monomers released oligomer to develop a strategy for quantifying active enzyme analytesin a paper-based device23 2C. A sel25 Our group reported the developmentof self-immolative poly(benzylcarbonates) that generate a distinctchemiluminescence diagnostic signal16  self-immolative polymers correlatedwith their lengths.Chemiluminescence is a powerful tool for the development of sensitivediagnostic assays.signal16 2D. This 32 Matson and co-workers reported the synthesis of a self-immolativepolymer with a poly(benzylthiourethane) backbone suitable for diagnosticuse33 self-immolative polymer with multiple responsive groups incorporatedat the benzylic positions of each building block15 3A. The p15  backboneto prepare self-immolative brush polymers by grafting azide-terminatedside chains onto an alkyne-bearing scaffold35 3C. In or36  backbone40 (The Liu group preparedamphiphilic block copolymers by linkinga poly(benzylcarbamate) self-immolative polymer with a hydrophilicpolymeric block 4B. Theseckbone40 4C. Them41  was introducedas an end cap via an ester linkage to produce an amphiphilic blockcopolymer. This copolymer was assembled into nanoparticles that werecapable of encapsulating and subsequently releasing a fluorescentdye in aqueous solution through a slow domino-like disassembly process.The group of Gilliesprepared self-immolative polymers that candisassemble through alternating cyclization and quinone-eliminationreactions44 5B. TheseN,N′-dimethylethylenediamineand 2-mercaptoethanol linked by carbamate and thiocarbamate bonds45 (46 (47 As observed for the poly(glyoxylates), these new polymers disassemblefrom head to tail upon exposure to a specific stimulus that removesthe terminal capping group. Interestingly, poly(glyoxylamide) exhibitedthermal properties that differ from those of poly(glyoxylates). Suchproperties may be advantageous for certain polymer applications.Gillies and co-workers have also reported a new type of self-immolativepolymer backbone based on  bonds45 5C. A disds45 (46 5D. The c48 (The Phillips group designed and synthesized a unique class of self-immolativepolymer based on a poly(carboxypyrrole) backbone48 5E. The p49  backbone.52 Theyhave shown that such polymers can thermally decompose via carbonateelimination as the rate-determining step.The Sasaki group was among the first to develop polymerswith apoly(olefin sulfone) backbone, equipped with photobase groups locatedinternally51 6A. Expos54 The resulting poly(olefin sulfone)-SWCNT composites serve as activetransducers in a novel class of γ-ray dosimeters (Swager and co-workers synthesized poly(olefin sulfone)polymersbearing pyrene side groups and used them to wrap single-walled carbonnanotubes (SWCNTs) through aromatic π–π interactions.simeters 6A. The p55 Other groupshave reported similar developments of degradable polymers based onthe poly(disulfide) structure polymer that can disassemble via cyclization reactions upon exposureto base or reductive agents.tructure 6B. The rtructure 6B.1.The56 (dl-dithiothreitol (DTT) as the monomer. Poly(DTT)was produced by solid-state polymerization in a robust and easilyscalable process by mechanically mixing DTT with 2,2′-dithiodipyridineas the end-capping agent  polymer to successfully mask an active site of anenzyme to form an inactive zymogen56 6B.2. Theng agent 6C. These67 In addition to the examples described so far in this Perspective,several other polymer backbones with related disassembly mechanismhave recently been reported.71 Moreover, the progress of the field has inspiredscientists to develop polymers that employ a self-immolative dendriticunit as a focal junction for disassembly. Such polymers usually carrya responsive group on their self-immolative dendritic unit, whichis attached to two polymeric side chains. Removal of the responsivegroup by an analyte of interest initiates the polymer disassembly,through two consecutive quinone-methide elimination reactions. Almutairiand co-workers were the first to report such polymeric nanoparticles.72 They used a polyester backbone attached to aself-immolative dendritic unit, which is equipped with a boronic estertriggering group as a substrate for hydrogen peroxide. The polymerwas formulated in the shape of a capsule that underwent disassemblyupon exposure to reactive oxygen species. Shortly thereafter, theCheng group reported similar polymeric structures that were composedof polycarbamates and polyester backbones attached to self-immolativedendritic units.73 The polymers are ableto trap and release a dye through their disassembly pathway.The majority of examples associated with self-immolativepolymersare based on polybenzylcatbamates, most likely due to the simplicityof the monomer budling blocks and the efficiency of the polymerizationprotocol. Therefore, numerous publications describing various applicationswith such self-immolative polymers have appeared during the past fewyears.74 Upon irradiation with UV light, the polymerbackbone disassembles and the drug molecules are released.In later work, the same group reported a new polymeric materialin which the responsive group of the self-immolative dendritic unitcould be removed by exposure to UV light. The polymer backbone wascomposed of the anticancer drug 10-hydroxycamptothecin and a self-immolativedendritic unit, attached each to other in an alternating order.75 The polymers undergo splitdisassembly into two equally sized fragments on exposure to a specificstimulus. Interestingly, when the polymer was formulated into a thinfilm, the quinone-methide-based disassembly occurred in the solidphase.Amirand co-workers designed photoactivated self-immolative junctionsthat enables the transformation of nonresponsive polymers into photocleavablepolymers.The inspiration for the developmentof the disassembly chemistry,used to achieve the function of self-immolative polymers, first originatedfrom the quinone-methide elimination reaction. This unique reactionenabled scientists to design and synthesize spacer molecules thatare known as self-immolative linkers. Such molecules, usually basedon 4-aminobenzyl alcohol or 4-hydroxybenzyl alcohol, can undergo afragmentation process, which is initiated at one terminus site, torelease a target molecule from the other terminus. Incorporation of4-aminobenzyl alcohol as a building block unit in a polymer backboneresults in a macromolecular linear structure that can disassemblefrom head to tail. Capping of the polymer head with a responsive groupseals the polymer with a trigger, which can be activated upon exposureto specific extrenal stimuli.The distinct disassembly profileof self-immolative polymers setsthem apart from other degradable materials. Our original report ofself-immolative polymers in 2008 has opened the door for a new classof stimuli-responsive disassembled materials. After this report, severalgroups from around the world developed new molecular building blocksthat can be used to compose self-immolative polymers. Our group hasmainly focused on the use of self-immolative polymers as tools toachieve effective signal amplification and controlled-release functions.Other groups have also developed applications for materials scienceand for biomedicine.The growing interest in self-immolativepolymers is clearly reflectedby the increasing number of publications in this field. Remarkably,new examples of structures and applications of self-immolative polymerskeep emerging even while these lines are being read. Ultimately, thedevelopment of new molecular backbones for self-immolative polymerswith enhanced properties and disassembly behavior still remains ageneral challenge. The continuous demand for stimuli-responsive degradablematerials guarantees the future interest of the scientific communityin this unique class of polymers.


Ultrasonographic indices of the inferior vena cava are useful for predicting right heart filling pressures in people.To determine whether ultrasonographic indices of caudal vena cava (CVC) differ between dogs with right-sided CHF (R-CHF), left-sided CHF (L-CHF), and noncardiac causes of cavitary effusion (NC).max and CVCmin), CVCmax indexed to aortic dimension (CVC:Ao), and CVC collapsibility index (CVC-CI). Variables were compared between study groups using Kruskal-Wallis and Dunn’s-Bonferroni testing, and receiver operating characteristics curves were used to assess sensitivity and specificity.113 dogs diagnosed with R-CHF (n = 51), L-CHF (30), or NC effusion (32) were enrolled. Seventeen of the R-CHF dogs had pericardial effusion and tamponade. Focused ultrasound was performed prospectively to obtain 2-dimensional and M-mode subxiphoid measures of CVC maximal and minimal size (CVCP < 0.001). Variables demonstrating the highest diagnostic accuracy for discriminating R-CHF versus NC were CVC-CI <33% in 2D (91% sensitive and 96% specific) and presence of hepatic venous distension (84% sensitive and 90% specific). L-CHF dogs had higher CVC:Ao and lower CVC-CI compared to NC dogs  but increased CVC-CI compared to the R-CHF group (P < 0.001).All sonographic CVC indices were significantly different between R-CHF and NC dogs (Ultrasonographic indices of CVC size and collapsibility differed between dogs with R-CHF compared to NC causes of cavitary effusions. Dogs with L-CHF have CVC measurements intermediate between R-CHF and NC dogs. Ultrasonography of the inferior vena cava (IVC) is commonly utilized as a noninvasive estimate of right heart filling pressures in people –3. Maximmax), CVCmax to aortic ratio (CVCmax:Ao), CVC collapsibility index (CVC-CI), hepatic vein diameter, and gallbladder wall edema in healthy dogs [max and CVCmin measurements above normal reference range [In veterinary medicine, previous studies have reported reference ranges for maximum caudal vena cava diameter  and North Carolina State University (protocol number 17-063-O). Informed owner consent was obtained for each patient. A power calculation determined that a population of at least 20 subjects in each group would be needed to detect a difference in sonographic markers of 10% with a statistical power of 0.90 and α = 0.05.Client-owned dogs presented to the Iowa State University Lloyd Veterinary Medical Center and North Carolina State Veterinary Hospital were prospectively recruited for this study between October 27, 2018, and December 31, 2019. To be included in this study, dogs were required to have one or both of the following as assessed by the attending clinician: (1) cavitary effusion  of at least moderate volume identified sonographically (defined as fluid accumulation >2 cm such that the attending clinician deemed the volume sufficient for diagnostic centesis), or (2) cardiogenic pulmonary edema with clinical signs (increased respiratory rate or effort) warranting diuretic therapy. Exclusion criteria included lack of owner consent or instability of patient precluding focused ultrasound. Cases were also excluded if subsequent fluid analysis identified the effusion as hemorrhagic (packed cell volume >10%), septic exudate (neutrophilic inflammation with intracellular bacteria), or urine (fluid creatinine higher than serum creatinine).The following data were collected from the patient’s physical examination on hospital presentation: patient signalment, body weight, temperature, heart rate, respiratory rate, presence and description of heart murmur, presence and description of arrhythmias, Doppler systolic blood pressure, and sedation protocol if applicable. Fluid location(s) , presence or absence of cardiac tamponade, and centesis information  were also recorded.Focused ultrasound examinations were performed by ACVIM-boarded cardiologists or supervised cardiology residents using platform ultrasound units  coupled to phased-array transducers . Dogs were stabilized prior to focused ultrasound  as deemed appropriate by the attending clinician. Images were obtained in all dogs according to a standard protocol as follows. Dogs were scanned in right lateral recumbency on a specialized echocardiography table with cutout allowing cardiac imaging from below. Starting at the right parasternal location, ultrasound cine-loops of the cardiac long-axis 4-chamber view and the short-axis view at the level of the LV were obtained using 2-dimensional (2D) echocardiography for measurement of the ratio of right ventricular to left ventricular dimension (RV:LV) in long-axis and short-axis . Subxiphmax and CVCmin) were measured in both 2D and M-mode by identifying the maximum and minimum visible diameters of the CVC [max was also indexed to aortic diameter from the right parasternal short-axis heart base view (CVC:Ao). Gallbladder wall thickness was measured from cross-sectional view from leading edge to leading edge. Presence of gallbladder wall edema was defined as a hypoechoic layer within the hyperechoic gallbladder wall [Archived cine-loop and still images of sonographic right-sided cardiac markers were evaluated off-line by investigators blinded to patient identity. Five cardiac cycles in sinus rhythm for each measured cardiac parameter were averaged and used for further analysis. Maximal right ventricular end-diastolic dimension and maximal left ventricular end-diastolic dimension were obtained at the level of the right and left ventricular papillary muscles in long-axis 4-chamber view and short-axis 2-chamber view, respectively, and RV:LV was calculated in both long-axis and short-axis . Ratio o the CVC , 14. CVCder wall , 27. Preder wall , 28, 29.Final clinical diagnosis of the cause of cavitary effusions was determined by retrospective review of each patient’s entire medical record by a single independent investigator at each study location blinded to focused ultrasound results. The final diagnosis incorporated physical examination findings and diagnostic test results other than the above right-sided sonographic markers, including clinicopathologic and cytologic analysis of cavitary effusions, labwork , advanced imaging , and response to therapy. Dogs were assigned to one of three study groups based on the location and etiology of their cavitary effusions. The R-CHF group was defined as dogs with cavitary effusions and severe right heart disease identified on complete echocardiogram. Dogs with pericardial effusion and cardiac tamponade were included in the R-CHF group but also subanalyzed as separate group (PCEFF). The L-CHF group was defined as dogs with radiographic evidence of cardiogenic pulmonary edema and severe left heart disease identified on complete echocardiogram. Dogs with cavitary effusions of noncardiac etiology were categorized as the NC group. Dogs with severe cardiac disease who manifested both pulmonary edema and cavitary effusions (biventricular CHF) were categorized as R-CHF or L-CHF based on the predominant fluid location, considering clinical severity of each fluid accumulation and need for therapeutic centesis. Dyspneic dogs with pulmonary edema that also had trace to mild cavitary effusions not requiring centesis were categorized as L-CHF. Dogs requiring therapeutic abdominocentesis or thoracocentesis were classified as R-CHF, even if they also had mild pulmonary edema.P-values < 0.05 considered statistically significant). Univariate logistic regression was performed to evaluate clinical and ultrasound variables associated with diagnosis group. Final multivariable models were created with a forward inclusion threshold of alpha = 0.05 using a backwards stepwise procedure. Receiver operating characteristic curves were developed to assess diagnostic accuracy for prediction of diagnosis group, and sensitivity and specificity were calculated for each index.Statistical analyses were performed using commercial software . Normality of data was assessed using the Shapiro-Wilk test. Parametric data are reported as mean ± standard deviation, nonparametric data are reported as median (interquartile range), and categorical data are reported as count (percent). Outliers were included in all analyses; missing data were omitted. Variables were compared between study groups using Kruskal-Wallis rank-sum testing and post hoc pairwise Dunn’s testing with Bonferroni correction , and included 5 intact females, 52 spayed females, 14 intact males, and 42 castrated males. Various breeds were represented , with the most common breeds being mixed breed (n = 25), Yorkshire terrier (9), Cavalier King Charles spaniel (5), Chihuahua (5), German shepherd (5), golden retriever (4), and Labrador retriever (4). Other clinical variables describing the study population are shown in The final study population of 113 dogs comprised 88 dogs from Iowa State University and 25 dogs from North Carolina State University  dogs were categorized as having R-CHF, 30 (27%) as L-CHF, and 32 (28%) as NC causes of effusion. Seventeen dogs with R-CHF had pericardial effusion with tamponade (PCEFF subgroup), representing 33% of dogs with R-CHF and 15% of the total study sample. Underlying causes of R-CHF included pulmonary hypertension (n = 10), congenital heart diseases including pulmonic stenosis or tricuspid valve dysplasia (6), dilated cardiomyopathy (4), degenerative mitral valve disease (DMVD) or degenerative tricuspid valve disease (4), bradyarrhythmia (4), inflow obstruction due to compressive neoplasia (3), arrhythmogenic right ventricular cardiomyopathy (2), and iatrogenic circulatory overload (1). Causes of PCEFF were right atrial or auricular mass (n = 7), heart base tumor (3), idiopathic pericarditis (3), left atrial rupture (2), mesothelioma (1), and septic pericarditis (1). Causes of L-CHF were DMVD (n = 22), dilated cardiomyopathy (6), patent ductus arteriosus (1), and bradyarrhythmia (1).Extracardiac neoplasia was the most common cause of NC effusion (n = 11), with lymphoma and hepatic masses being diagnosed most frequently (n = 3 each). Other diagnosed NC diseases included hypoalbuminemia (n = 9), chylothorax (3), pancreatitis (2), hepatopathy (2), and 1 dog with intra-abdominal cysts. Cause of effusion was ultimately unknown in 4 cases with incomplete diagnostic workup; these cases were assigned to the NC group based on complete echocardiography showing no evidence of structural cardiac disease.Table 1). Heart rate differed between the L-CHF and NC group, with L-CHF dogs having higher heart rate compared to dogs with NC disease (P = 0.036). Respiratory rate was significantly higher in the L-CHF group compared to NC (P < 0.001) and R-CHF (P = 0.004) groups. Murmur incidence was higher in L-CHF dogs compared to NC (P < 0.0001) or R-CHF (P = 0.021) dogs, as well as in R-CHF dogs compared to NC (P = 0.0008). Systolic blood pressure was lower in L-CHF dogs compared to dogs with NC effusion . Location of fluid accumulation  is also presented in  these data were not statistically compared between groups since fluid location contributed to the definition and classification of disease groups.No significant differences were detected between study groups in terms of age, sex, body weight, rectal temperature, presence of arrhythmia, or incidence of sedation  and M-mode . CVCmin measurements were also higher in dogs with R-CHF compared to NC or L-CHF dogs in both 2D and M-mode . The CVC:Ao ratio was higher in the R-CHF and L-CHF groups compared to the NC group in both 2D  and M-mode . CVC-CI measurements in both 2D and M-mode were significantly lower in the R-CHF group compared to NC or L-CHF groups . CVC-CI in 2D was also lower in the L-CHF group compared to the NC group (P = 0.043). The RV:LV ratio was lower in the L-CHF group compared to R-CHF and NC groups in both long-axis  and short-axis . Gallbladder wall thickness was higher in the R-CHF group compared to NC dogs (P = 0.0036), and hepatic venous distension was more common in the R-CHF group compared to either the L-CHF or NC groups . Fig 1 displays results for select sonographic indices by disease group. Fig 2 provides example ultrasound images for CVC indices comparing representative dogs from the R-CHF versus NC groups.Performance of focused ultrasound was technically feasible in all dogs. Imaging was performed by a total of 2 cardiologists and 5 cardiology residents. Sonographic indices obtained from the three study groups are summarized in Table 3). The only difference in clinical variables between these subgroups was lower incidence of heart murmur in PCEFF dogs (P = 0.0004), and the only difference in focused ultrasound variables was higher long-axis RV:LV ratio in dogs with other causes of R-CHF .Clinical and focused ultrasound variables were also subanalyzed within the R-CHF group, separating dogs with PCEFF from dogs with other causes of R-CHF (see Table 4. Table 5 depicts sensitivity and specificity of bodyweight-independent focused sonographic indices for the differentiation of R-CHF (including PCEFF) versus NC causes of effusion, representing the clinical scenario wherein focused ultrasound may provide the most useful diagnostically relevant information.Receiver operating characteristic curves were performed to identify clinical and sonographic indices that were predictive of different disease groups. Variables found to be independently predictive in multivariate regression are listed in As hypothesized, the focused ultrasound indices investigated in this study differed between disease groups and were diagnostically useful for prediction of R-CHF as a cause of cavitary effusions in dogs. Variables with the highest sensitivity and specificity for detection of R-CHF included CVC-CI, CVC:Ao, and presence of hepatic venous distension. These findings are logical since elevated central venous hydrostatic pressure in patients with R-CHF is transmitted to the CVC, resulting in increased size and decreased distensibility of this vessel and the hepatic veins.This study represents the first investigation of focused ultrasound parameters for the diagnosis of R-CHF in veterinary medicine. Diagnostic accuracy of CVC-CI reported in our study  was similar or higher than inferior vena cava collapsibility index (IVC-CI) accuracy in multiple human studies , 9. In psystolic pressure, few would be expected to have elevated right ventricular diastolic  pressure. Changes to CVC size or distensibility would only be expected in dogs with advanced right heart disease leading to elevated central venous pressure and R-CHF. In our study, only dogs diagnosed with overt R-CHF (cavitary effusion) secondary to their right heart disease were included in the R-CHF group.Predictive performances of focused ultrasound for detection of R-CHF versus NC disease in our analysis was higher than in a previous study investigating the utility of similar sonographic indices to predict pulmonary hypertension in dogs, which reported only 11% sensitivity and 88% specificity . These vIn addition to differentiating R-CHF and NC effusions, several parameters evaluated in this study also performed well in discriminating other disease groups. Indices that best differentiated L-CHF from other groups included respiratory rate and RV:LV ratio in long-axis, explained by the enlargement of left-sided cardiac chambers and presence of pulmonary edema in dogs with L-CHF. The L-CHF group was also distinguishable from the NC group using CVC-CI, CVC:Ao, and hepatic venous distension; L-CHF dogs had values for these parameters that were clinically and statistically intermediate between the R-CHF and NC groups. This is consistent with findings from a previous study in dogs with DMVD, which reported higher CVC:Ao and decreased CVC:CI in dogs with stage C or D (previous or current L-CHF) compared to dogs in earlier stages of disease . These rDogs with PCEFF and cardiac tamponade were included in the R-CHF group in this study because the pathophysiology of other cavitary effusions in cardiac tamponade  is the same as other causes of R-CHF; therefore, similar changes to the CVC and hepatic vasculature would be expected. However, because PCEFF represents a distinct clinical scenario (obstruction to right heart filling rather than right heart pressure or volume overload), subanalyses were also performed comparing dogs with PCEFF to other causes of R-CHF. Not surprisingly, the only variables that differed between these subgroups were incidence of heart murmur and RV:LV ratio. This reflects the fact that dogs with PCEFF and tamponade typically have structurally normal hearts, while dogs with other causes of R-CHF have severe right heart remodeling.Interestingly, results of this study revealed a much larger spread of CVC-CI measurements for the L-CHF group compared to other disease groups. We speculate that this disparity occurred because the change in the size of the CVC depends on the change in intrathoracic pressure during respiration. Respiratory rate and degree of respiratory distress were higher and more variable within the L-CHF group compared to the other disease groups, wherein most dogs had normal respiratory rate and effort. This variation in respiratory effort and thus intrathoracic pressure during respiration in dyspneic dogs could cause more variability in magnitude of CVC collapse, or more translational motion affecting ability to image the center of the CVC. The influence of diaphragmatic movement on IVC-CI has been demonstrated in humans , 34. HowThere was no statistically significant difference in gallbladder wall edema between the study groups, suggesting that presence or absence of gallbladder wall edema is a poor screening test for R-CHF . This is not necessarily surprising, as a wide variety of noncardiac causes for gallbladder wall edema have been identified, including anaphylaxis , hepatitexcluding dogs with PCEFF and cardiac tamponade), ascites was present in all 34 (100%) dogs, trace PCEFF was present in 9 (27%) dogs, and pleural effusion was only present in 5/34 (15%) cases; isolated pleural effusion occurred exclusively in the NC group. In a previous study of dogs with right-sided manifestations of CHF secondary to dilated cardiomyopathy or DMVD, 40/60 (67%) of dogs with R-CHF had ascites, 26/60 (43%) had pleural effusion, 13/60 (22%) had PCEFF, and similarly no dogs had pleural effusion only [Among dogs with R-CHF  were similar to measurements reported from the subxiphoid view in similarly-sized healthy dogs  or intravenous fluids had been administered before ultrasound was performed. Operators were not blinded to the presumptive diagnosis of the patient at the time of focused ultrasound, which may have led to bias during image acquisition. It is possible that sedative drugs may have affected central venous pressure (and thus CVC measurements) in some dogs, although use of sedation did not differ between disease groups. Additionally, a minority of dogs with CHF had evidence of both pulmonary edema and cavitary effusions, and this pathophysiologic overlap may have obscured some differences between R-CHF and L-CHF groups.max, but not for CVCmin or CVC-CI [A further limitation is that focused ultrasound examinations in this study were performed by cardiologists or cardiology residents utilizing a platform cardiac ultrasound machine and specialized echocardiography table. Personnel and equipment would be different in a primary care setting or emergency department, which is the intended setting for application of these techniques as a focused ultrasonographic screening tool. Differences in the operators, ultrasound model, probes, and software can lead to disparities in imaging quality and results; future studies may assess diagnostic accuracy of these measurements performed by non-specialists in a point-of-care setting. Additionally, interobserver reliability was not assessed in this study. Previous studies show good to excellent inter-rater agreement of CVC measurements performed at the hepatic, paralumbar, and spleno-renal views , 16, 18.r CVC-CI . TherefoIn conclusion, the present study demonstrated that focused ultrasound of the CVC is a feasible and accurate diagnostic test for the detection of R-CHF in dogs with cavitary effusion. Dogs with NC effusion had CVC size and collapsibility similar to normal dogs, while dogs with R-CHF had large non-distensible CVC and hepatic venous distension, and dogs with L-CHF had CVC measurements in between these extremes. CVC-CI in either 2D or M-mode was the best ultrasonographic index to differentiate between study groups, and a cutoff value of less than approximately 30% was more than 90% sensitive and specific for diagnosis of R-CHF versus NC effusion. Focused ultrasound of the CVC could have diagnostic utility to increase or decrease index of suspicion for R-CHF as the cause of cavitary effusions.S1 File(XLSX)Click here for additional data file.


This is the first report investigating the involvement of TRIM28-interacting KRAB-ZNFs in kidney cancer progression. We demonstrate a significant negative association between KRAB-ZNFs and cancer stemness followed by an attenuated immune-suppressive response and reveal the prognostic role for several KRAB-ZNFs. Our findings may help better understand the molecular basis of kidney cancer and ultimately pave the way to more appropriate prognostic tools and novel therapeutic strategies directly eradicating the dedifferentiated compartment of the tumor.Krüppel-associated box zinc finger (KRAB-ZNF) proteins are known to regulate diverse biological processes, such as embryonic development, tissue-specific gene expression, and cancer progression. However, their involvement in the regulation of cancer stemness-like phenotype acquisition and maintenance is scarcely explored across solid tumor types, and to date, there are no data for kidney renal clear cell cancer (KIRC). We have harnessed The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database transcriptomic data and used several bioinformatic tools  to verify the relation between the expression and genomic alterations in KRAB-ZNFs and kidney cancer, focusing primarily on tumor dedifferentiation status and antitumor immune response. Our results demonstrate a significant negative correlation between KRAB-ZNFs and kidney cancer dedifferentiation status followed by an attenuated immune-suppressive response. The transcriptomic profiles of high KRAB-ZNF-expressing kidney tumors are significantly enriched with stem cell markers and show a depletion of several inflammatory pathways known for favoring cancer stemness. Moreover, we show for the first time the prognostic role for several KRAB-ZNFs in kidney cancer. Our results provide new insight into the role of selected KRAB-ZNF proteins in kidney cancer development. We believe that our findings may help better understand the molecular basis of KIRC. KRAB-ZN (TIF1β) . TRIM28 Generally, KRAB-ZNFs are sequence-specific DNA binding proteins that bind TRIM28, further recruiting complex epigenetic machinery, resulting in reversible repression of target gene transcription . HoweverKRAB-ZNFs are known to regulate diverse biological processes, such as embryonic development, tissue-specific gene expression, and cancer progression, although their role in kidney cancer development remains largely unknown ,3,5,6. KIt is postulated that cancer cells, at least partially, may experience phases of transition between stem-like and non-stem-like states, and epigenetic dysregulation might contribute to tumorigenicity via facilitating this phenomenon. Stem cell-associated molecular features of cancer cells are indisputably necessary for disease development and progression, endowing cancers with intrinsic resistance to standard therapies and providing tumor relapse after treatment ,9. Recent artificial intelligence-supported approaches have led to the development of so-called stemness indices that quantify tumor dedifferentiation status with great effectiveness. These molecular signatures represent an essential step in designing novel therapeutic schemes ,11,12,13The involvement of KRAB-ZNFs in the regulation of cancer stemness-like phenotype acquisition and maintenance is scarcely explored across solid tumor types, and to date, there are no data for kidney renal clear cell cancer. Therefore, we have harnessed The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database transcriptomic data to verify the relation between the expression and genomic alterations in KRAB-ZNFs and kidney cancer patients’ survival, focusing primarily on tumor dedifferentiation status. Using the Compartmentalized Protein–Protein Interaction Database (ComPPI)  and PathUsing the GSCALite platform , we demoFurthermore, the expression of at least 20 KRAB-ZNFs was significantly associated with tumor dedifferentiation status (tumor grade and disease stage), mostly negatively, which was further confirmed with cancer stemness scores. Moreover, the gene set enrichment analysis (GSEA)  of gene Next, we constructed a potential prognostic KRAB-ZNF-based signature and confirmed its significance in predicting the disease course of patients with KIRC. The gene expression profiles of high-risk KIRC patients were significantly enriched with cancer stemness markers and immune infiltration-associated gene sets. Accordingly, using a CIBERSORT algorithm , we demoThis is the first report that investigates the expression of TRIM28-interacting KRAB-ZNFs in kidney cancer. Our results demonstrate a significant negative correlation between KRAB-ZNFs and kidney cancer dedifferentiation status followed by an attenuated immune-suppressive response. Moreover, we show for the first time the prognostic role for several KRAB-ZNFs in kidney cancer. We believe that our findings may help better understand the molecular basis of KIRC and ultimately pave the way to more appropriate prognostic tools for KIRC and facilitate the development of new therapeutic strategies directly targeting the dedifferentiated compartment of the tumor.In the current study, we used the transcriptomic data of kidney renal clear cell cancer (KIRC) from the TCGA database  and tranhttps://comppi.linkgroup.hu/protein_search, accessed on 12 July 2021), which provides qualitative information on the interactions, proteins, and their localizations integrated from multiple databases [https://www.pathwaycommons.org/, accessed on 12 July 2021), an integrated resource of publicly available information about biological pathways, including biochemical reactions; assembly of biomolecular complexes; transport and catalysis events; and physical interactions involving proteins, DNA, RNA, and small molecules  [To search for TRIM28-interacting KRAB-ZNFs, we used (i) the Compartmentalized Protein–Protein Interaction Database  . KRAB-ZNhttp://bioinfo.life.hust.edu.cn/GSCA/#/, accessed on 13 September 2021) [t-test.The expression of selected KRAB-ZNF family members in tumor tissues and normal adjacent tissues in KIRC tumors was analyzed using the Expression module of the GSCALite (er 2021) . The modhttp://gepia2.cancer-pku.cn/#index, accessed on 15 July 2021) [The association between KRAB-ZNF family members’ expression and patients’ overall survival (OS) in TCGA KIRC tumor patients was analyzed with the Survival_Analysis panel of the GEPIA2 database (ly 2021) . Similarwww.cbioportal.org, accessed on 16 July 2021) database [Genomic data for TCGA KIRC tumors were directly downloaded from the cBioPortal  were cut off at p-value < 0.05 and FDR < 0.05.The RNA sequencing-based mRNA expression data were directly downloaded from the cBioPortal. RNASeq V2 from TCGA is processed and normalized using RSEM . SpecifiThe mRNA-SI stemness score  and othehttp://cis.hku.hk/TISIDB/index.php, accessed on 20 July 2021) [The association between KRAB-ZNF family members’ expression and the histologic tumor grade or stage was assessed using the TISIDB portal (ly 2021) . The corhttp://www.broad.mit.edu/gsea/index.html, accessed on 29 July 2021) [http://www.broad.mit.edu/gsea/.msigdb/msigdb_index.html, accessed on 29 July 2021). All significantly correlated genes  were imported to GSEA. The GSEA was run according to the default parameters: each probe set was collapsed into a single gene vector (identified by its HUGO gene symbol), permutation number = 1000, and permutation type = “gene-sets.” The FDR < 0.01 was used to correct for multiple comparisons and gene set sizes.The Gene Set Enrichment Analysis   was usedPreselected KRAB-ZNFs were fitted in a univariate and multivariate Cox proportional hazards regression analysis . Risk scThe leukocyte fraction and the lymphocyte infiltration signature (LIS) scores were estimated based on the transcriptome profiles of each sample, as previously reported . InfiltrStatistical analyses were carried out with GraphPad Prism 8.0 software . Multiple comparisons were performed with the ANOVA test. The correlation between two variables was assessed with Spearman’s rank correlation coefficient (r).Firstly, using the ComPPI database  and Pathn = 79, GSE11024; Using the GSCALite database , we analNext, using the median expression as a cut-off, we observed that 18 KRAB-ZNFs are significantly associated with better patient survival in KIRC  in contrWe further analyzed the association between KRAB-ZNF expression and tumor dedifferentiation status. Specifically, we looked at the level of preselected KRAB-ZNFs in KIRC regarding tumor stage and tumor grade and observed that most KRAB-ZNFs are negatively associated with both clinicopathological features A,B. SpecAs dedifferentiated tumors clearly exhibit stemness characteristics, we analyzed the association between KRAB-ZNF expression and previously defined transcriptome-based stemness scores, namely mRNA-SI , Ben-Porp < 0.05, FDR < 1%) with each of the tested KRAB-ZNFs in TCGA KIRC data , further confirming a negative association between KRAB-ZNF expression and cancer stemness  were omitted from further prognosis evaluation. The 516 TCGA KIRC patients were randomly divided into a discovery set (n = 258) and a validation set (n = 258). Based on the expression level of seven KRAB-ZNFs and multivariate Cox regression coefficients for a discovery set, we built a risk score formula , suggesting that high-risk tumors exhibit dedifferentiated phenotype.Next, we compared the gene expression profiles of high- and low-risk KIRC patients  and obsep = 0.0009) (p = 0.0014) (p = 0.0371) (p = 0.0054) when compared to low-risk KIRC cohort. Taken together, these results indicate that high-risk KIRC tumors exhibit a more stem cell-like phenotype with accumulated immune-suppressive cells and signaling.Next, we compared the level of transcriptome-based immune-related scores, namely the leukocyte fraction and the leukocyte infiltration signature (LIS) scores , with th 0.0009) E and fol 0.0014) F followe 0.0371) G in highn = 22 and n = 16, respectively). Specifically, the abundances of regulatory T cells (Tregs), follicular helper T cells (Tfh), and gamma/delta T cells (γδ T cells) are negatively correlated with the levels of n = 20, n = 16, and n = 17 KRAB-ZNFs, respectively . As for other immune cell subtypes, we observed both positive and negative associations, depending on the KRAB-ZNF. Lastly, we analyzed the association between KRAB-ZNF expression and the immune landscape of KIRC tumors using previously estimated immune cells fractions. As presented in ectively B. In conn = 14), IFN-α (n = 15) and IFN-γ response (n = 14), as well as IL2-STAT5 (n = 12), IL6-JAK-STAT3 (n = 13), and TNF-α signaling (n = 10), in a substantial number of KRAB-ZNF-associated transcriptome profiles followed by significant enrichment of the TGF-β-mediated signaling pathway (n = 13 KRAB-ZNF-associated transcriptome profiles). We also looked at the activation of immune-associated biological processes in KRAB-ZNF-related transcriptome profiles using the GSEA. As presented in To date, the role of KRAB-ZNFs in clear cell kidney carcinoma development and progression is largely unknown. Here, we demonstrate for the first time a significant association between the upregulation of specific KRAB-ZNFs with the better survival of KIRC patients, which strongly corresponds to tumor dedifferentiation status (mostly negatively) and the level of immune cell infiltration. We also revealed a potential prognostic role for the expression of several KRAB-ZNF and uncovered the association with cancer stemness-like phenotype. The role of most KRAB-ZNF transcription factors in solid tumors’ development and progression remains largely unknown, with only several (out of ~700 proteins) being well studied and documented as either tumor suppressors or oncogenes or harboring both tumor-promoting and -suppressing roles ,5. MoreoThe canonical function of KRAB-ZNFs is to epigenetically repress specific regions within the chromatin, significantly impairing gene expression . To exerRecently, we have demonstrated that KAP1/TRIM28 is significantly associated with cancer stemness across distinct types of solid tumors ,28,29. HAs for other KRAB-ZNFs preselected in our study, several reports regarding tumor development and progression might be found, although with no relevance to clear cell kidney tumors. Briefly, ZFP57—the embryonic stem cell-specific transcription factor—is involved in colorectal cancer spreading , and itsZNF10 promotes the carcinogenesis and progression of breast invasive ductal carcinoma via the Wnt/β-catenin signaling pathway  and receZNF195 is one of the most significant survival-associated genes in bladder cancer  and has Among the tested KRAB-ZNFs, numerous studies demonstrate significant engagement of ZNF224 in the regulation of cancer development and progression, especially in melanoma , breast ZNF250 overexpression significantly predicted reduced survival of TCGA breast cancer patients . The expAn increasing number of reports reveal the role of ZNF350 (ZBRK1) in suppressing the progression of distinct types of solid tumors, including cervical ,47, breaZNF382 was firstly identified as a direct repressor for several oncogenes  across distinct types of cancers, including lung, esophageal, colon, stomach, breast, and cervical tumors . FurtherHigh expression of ZNF420, also known as APAK, is correlated with a poor prognosis of colorectal cancer patients . SimilarZNF689, also known as TIPUH1, promotes the progression of hepatocellular carcinoma by suppressing the apoptotic signaling, and a high ZNF689 level indicates a poor prognosis of hepatocellular carcinoma ,60. ReceHIGH tumors. As cancer stemness is significantly negatively associated with anticancer immunity , we alsoNotably, we observed the negative correlation between the expression of most KRAB-ZNFs and the abundance of regulatory T cells (Tregs), follicular helper T cells (Tfh), and gamma/delta T (γδ T) cells. In cancer, the presence of Tregs prevents the development of effective antitumor immunity in tumor-bearing patients . A recenThe γδ T cells might promote or suppress tumor progression (either directly or through indirect effects), and their actions are robustly influenced by the cytokines present in the tumor microenvironment. However, the role of γδ T cells in the regulation of cancer stemness is still unknown. Recent in vitro studies suggest that the cancer stem cell population exhibits strong resistance to γδ T cell-mediated killing. However, further experimental confirmation is needed to prove this phenomenon in vivo . On the other hand, we observed a significant positive association between the expression of most KRAB-ZNFs and the abundance of resting NK cells or resting mast cells and observed a significant positive correlation with both naïve and memory CD4+ T cells for almost half of the tested KRAB-ZNFs. An increasing number of data demonstrate that NK cells can selectively identify and kill the population of dedifferentiated cancer cells that possess the characteristics of stem cells ,67. As fMoreover, within the KRAB-ZNF-associated transcriptome profiles, we observed significant downregulation of genes expressed in response to interferons α and γ and a substantial depletion of signaling mediated by other proinflammatory cytokines: IL2, IL6, and TNF-α. IFNs participate in tumor immunology as a “double-edged sword”, exhibiting both pro- and antitumorigenic activities , and recHIGH KIRC tumors might directly link to robust depletion of cancer stem cell-like traits and ultimately result in better patient outcomes. However, the exact role of KRAB-ZNFs in this scenario remains to be elucidated. As KRAB-ZNFs are potent transcriptional suppressors, it might be speculated that at least several of them directly target inflammatory-responsive genes and keep them repressed in kidney cancer in the absence of tumor-infiltrating lymphocytes. The abovementioned inflammatory pathways can enhance tumor growth and immune escape, especially by favoring cancer stemness. Therefore, a significant attenuation of this signaling in KRAB-ZNFHIGH KIRC tumors is surprising and requires further confirmation to delineate the impact on tumor dedifferentiation status. Lastly, we observed significant enhancement of the TGF-β mediated signaling pathway in high KRAB-ZNF-expressing tumors that do not exhibit cancer stemness phenotype. The TGF-β pathway is an important way to induce cancer stem cell formation in the epithelial cells of the lung , breast Using our KRAB-ZNF-based gene signature, we were able to filter out high-risk KIRC patients that exhibit stemness-high phenotype accompanied with an enriched immune-suppressive microenvironment that collectively leads to worse patient outcomes. Our results provide new insight into the role of TRIM28-interacting KRAB-ZNF proteins in kidney cancer development.To date, our report is the first that investigates the role of TRIM28-interacting KRAB-ZNFs in kidney cancer progression. Our results demonstrate a robust negative association between KRAB-ZNFs and kidney cancer dedifferentiation status followed by attenuated proinflammatory signaling within the tumor. However, the exact mechanism mediating this stemness-low phenotype of KRAB-ZNF-overexpressing KIRC tumors should be confirmed experimentally. Moreover, we demonstrate for the first time the prognostic role for several KRAB-ZNFs in kidney cancer and believe that our findings may help better understand the molecular basis of KIRC. Ultimately, this approach may pave the way to more suitable prognostic tools for KIRC and promote the development of novel therapeutic strategies directly eradicating the dedifferentiated compartment of the tumor.


We describe a patient in Argentina with severe acute respiratory syndrome coronavirus 2 infection and hantavirus pulmonary syndrome (HPS). Although both coronavirus disease and HPS can be fatal when not diagnosed and treated promptly, HPS is much more lethal. This case report may contribute to improved detection of co-infections in HPS-endemic regions. The current coronavirus disease (COVID‐19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), has resulted in substantial illness and death rates worldwide. Orthohantaviruses are zoonotic viruses responsible for another severe respiratory infectious disease in the Americas, hantavirus pulmonary syndrome (HPS). Although humans generally become infected with HPS through inhaling excreta generated by infected rodents, person-to-person transmission has been well documented in Argentina and Chile  and medium (611-bp) segments, and we submitted the sequences obtained to GenBank (accession nos. OL840325 and OL840326). The highest nucleotide identities matched previous published sequences corresponding to Plata genotype of Andes virus (GenBank accession nos. EU564720 [96% identity] and AY101185 [97.8 identity]). This viral genotype is one of the prevalent pathogenic orthohantaviruses circulating in central Argentina and Uruguay (Because the incubation period for HPS is longer than that for COVID-19, we might speculate that hantavirus infection occurred before coronavirus infection. The respiratory distress syndrome appeared 5 days after the onset of fever, which coincided with the characteristic prodromal period described for HPS. This condition, during the incubation period of HPS, could have induced a higher susceptibility to COVID-19. Because HPS can evolve rapidly to respiratory failure in most patients with severe disease, resulting in high case-fatality rates, alerting health-care workers from HPS-endemic areas is warranted to detect co-infections in the context of the COVID-19 pandemic. In particular, at least 2 genotypes of Andes virus can be transmitted person-to-person, and these species are prevalent in 2 of the 3 hantavirus-endemic regions of Argentina (In conclusion, we detected co-infection with SARS-CoV-2 and Andes virus causing HPS in a patient from a hantavirus-endemic area. Clinicians should be aware of the possibility of co-infection for patients originating, residing, or traveling in hantavirus-endemic areas.Additional information about hantavirus pulmonary syndrome in a COVID-19 patient, Argentina, 2020.