Negative Marker Research Articles

Clinical Significance of Undetectable High-sensitivity Cardiac Troponin I in Thai Individuals with Low Cardiovascular Risk

Background: The long-term prognosis in individuals who have undetectable high-sensitivity cardiac troponin I (hs-TnI) is poorly defined in those with low cardiovascular risk. We aimed to examine whether individuals with undetectable hs-TnI have better cardiovascular outcomes in the low-cardiovascular-risk population. Methods: Data from a low-cardiovascular-risk population (<7.5%) with no established atherosclerotic cardiovascular disease were derived from the Electricity Generating Authority of Thailand 2007–09 survey. Hs-TnI was measured using the Abbott ARCHITECT STAT assay. We categorised hs-TnI levels into undetectable hs-TnI, low hs-TnI and intermediate and higher hs-TnI by sex-specific cut-off. The low-cardiovascular-risk population was classified into extremely low risk (<5%) and low risk (5–7.5%). Data on major adverse cardiovascular events (MACE) and all-cause mortality were collected until 31 December 2019. Survival analysis and subgroup analysis were performed. Results: A total of 3,442 participants were eligible. Mean age was 43.6 years; 65.5% were male. Hs-TnI was detected in 79.8% (median hs-TnI 2.6 ng/l). During a median follow-up of 10.3 years, 52 events (1.5%) of MACE and 60 events (1.7%) of all-cause mortality occurred. After adjusting for conventional risk factors, an increasing level of hs-TnI was significantly associated with the incidence of MACE (HR 1.03; 95% CI [1.02–1.04]; p<0.001). Compared with the intermediate and higher hs-TnI group, the undetectable hs-TnI group and low hs-TnI group, respectively, had 79% and 52% lower incidences of MACE (HR 0.21; 95% CI [0.05–0.79] and HR 0.48; 95% CI [0.23–1.00]), p for trend 0.04. In subgroup analysis, the incidence of MACE remained higher in individuals with either extremely low or low CV risk with detectable hs-TnI compared with those undetectable hs-TnI (P for interaction 0.83). Conclusion: Among individuals with low cardiovascular risk, hs-TnI testing can provide risk prognostication. Undetectable hs-TnI could serve as a negative risk marker for adverse cardiovascular events.

Negative concord by phase: multiple downward agree and the parametrization of edge features

Abstract We divorce Negative Concord (NC) among two or more n-items, which is invariably present in Romance languages, from the mutual exclusion or cooccurrence between clausal negation markers (CNMs) and other n-items. The standard minimalist treatment in terms of Agree with respect to a formal feature [n] (for [negation]) is maintained here. However, an EPP-like principle, conceived as a (parametrized) condition regulating the representation of [n] at phase edges is introduced to deal with CNMs. By divorcing the n-EPP condition from n-Agree, we eliminate Zeijlstra’s Upward Agree in favor of Multiple Downward Agree between a phase head probe endowed with Formal Feature FF and one or more elements endowed with the same interpretable feature FF in the WorkSpace. We argue that these theoretical moves are beneficial from an empirical point of view. Focusing on varieties spoken in Northern Italy, we argue that an exhaustive typology of Romance negative systems can be derived by the parametrized [n]-EPP. We claim that the present approach allows this variation to be accounted for within the syntax – specifically, no recourse is made to semantic variation.

STEM-19. TARGETINGGIHCG LNCRNA IN GLIOBLASTOMA STEM CELLS: A POTENTIAL THERAPEUTIC STRATEGY

Abstract Glioblastoma multiforme (GBM) remains the most lethal and prevalent primary brain tumor worldwide. Despite advances in understanding its biology and multimodal therapy options, the overall prognosis for GBM patients remains bleak, primarily due to the high recurrence rate, which is intimately linked to tumor resistance against standard therapeutic interventions. Glioblastoma stem cells (GSCs) contribute to therapeutic resistance and cellular heterogeneity through their self-renewal properties and ability to adapt. Thus, identifying new molecular targets driving GBM progression is crucial for developing effective therapies. Recent advancements in genome biology have revealed that long non-coding RNAs (lncRNAs) play roles in various biological functions, and the dysregulation of numerous lncRNAs has been shown to be associated with specific cancers. To identify GSC-associated lncRNAs, a comprehensive analysis was performed using single-cell RNA sequencing datasets from GBM tumors, GBM organoids, GSC-enriched GBM tumors, as well as normal human brain samples. A chromatin-enriched lncRNA, GIHCG, was identified as being highly expressed in GSCs compared to non-neoplastic neural stem cells. The GIHCG gene, located on human chromosome 12q14.1, is frequently amplified in glioblastoma and impacts overall survival. Bioinformatic and functional analyses revealed that GIHCG is amplified in 13% of GBM patient samples, and high expression of GIHCG is a negative prognostic marker for GBM patients, with significantly reduced overall survival compared to the low expression group. In addition, the depletion of GIHCG significantly reduced GSC proliferation and migration. Further, the knockdown of GIHCG decreased both mRNA and protein expression levels of critical GSC stemness factors, including SOX2, NESTIN, and OLIG2. Moreover, GIHCG depletion reduced the sphere formation capacity of GSCs, demonstrating the functional importance of GIHCG in the maintenance of GSC stemness. These results indicate that GIHCG is essential for GSC proliferation, migration, and stemness, underscoring its potential therapeutic value in RNA-based therapies for combating glioblastoma malignancy.

EXTH-38. EVALUATION OF STORAGE CONDITIONS ON SERUM-DERIVED EXTRACELLULAR VESICLES IN BRAIN TUMOR SAMPLES

Abstract Tumor-derived Extracellular Vesicles (EVs) are emerging as potential liquid biopsy tool in the field of cancer diagnosis and therapeutic targets. The functionality of EVs depends on its diverse cargo, which is known to alter host-receipient cells. Nevertheless, extracting EVs while preserving its integrity and functionality of its contents is challenging, specifically in Low-middle Income countries (LMIC) countries. Our study focussed on extraction of serum derived EVs analysis from brain-tumor samples encompassing Oligodendrogliomas, Astrocytomas and Glioblastomas. The stability of EVs were examined in serum stored for 2 years at -80°C to understand the effect of preservation and storage in different conditions, that were left exposed to room temperature for variable periods of time before freezing. To explore the optimal suitable conditions, EVs were isolated from serum samples using IZON column size-exclusion based-method, and evaluated for structural integrity and stability through nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) technology. Interestingly, the majority of isolated vesicles exhibited round, intact structure with bilayer membrane of size varying between 30 - 250nm. Moreover, protein content of EVs positive markers (CD63, CD81 and CD9) was also detected with minimal contaminations evidenced by weak bands for negative marker (ApoB), as determined by Western blot analysis. Further, column-based EV-derived RNA demonstrated optimal yield and purity was confirmed by RNA integrity number (RIN) using Qubit. While cryopreservation had minimum detrimental effects on mRNA sample quality, leaving it exposed to room temperature before freezing had a significant effect on EV isolation, as samples were often haemolyzed. This will provide insights in optimizing pre-analytical and analytical procedures for more effective outcomes in the clinical setting

Efficient enzyme-free isolation of brain-derived extracellular vesicles.

Extracellular vesicles (EVs) have gained significant attention as pathology mediators and potential diagnostic tools for neurodegenerative diseases. However, isolation of brain-derived EVs (BDEVs) from tissue remains challenging, often involving enzymatic digestion steps that may compromise the integrity of EV proteins and overall functionality. Here, we describe that collagenase digestion, commonly used for BDEV isolation, produces undesired protein cleavage of EV-associated proteins in brain tissue homogenates and cell-derived EVs. In order to avoid this effect, we studied the possibility of isolating BDEVs with a reduced amount of collagenase or without any protease. Characterization of the isolated BDEVs from mouse and human samples (both female and male) revealed their characteristic morphology and size distribution with both approaches. However, we show that even minor enzymatic digestion induces 'artificial' proteolytic processing in key BDEV markers, such as Flotillin-1, CD81, and the cellular prion protein (PrPC), whereas avoiding enzymatic treatment completely preserves their integrity. We found no major differences in mRNA and protein content between non-enzymatically and enzymatically isolated BDEVs, suggesting that the same BDEV populations are purified with both approaches. Intriguingly, the lack of Golgi marker GM130 signal, often referred to as contamination indicator (or negative marker) in EV preparations, seems to result from enzymatic digestion rather than from its actual absence in BDEV samples. Overall, we show that non-enzymatic isolation of EVs from brain tissue is possible and avoids artificial pruning of proteins while achieving an overall high BDEV yield and purity. This protocol will help to understand the functions of BDEV and their associated proteins in a near-physiological setting, thus opening new research approaches.

A phenomap of TTR amyloidosis to aid diagnostic screening.

Cardiac amyloidosis due to transthyretin (ATTR) remains an underdiagnosed cause of cardiomyopathy. As awareness of the disease grows and referrals for ATTR increase, clinicians are likely to encounter more atypical forms of the condition in clinical practice. Therefore, physicians and treating cardiologists should be aware of the full phenotypic spectrum of ATTR. The phenotypic manifestation of ATTR varies depending on the stage of the disease, the presence and type of TTR mutation and the patient's comorbidities. ATTR findings can be grouped into four major categories: clinical profile and cardiac phenotype, extra-cardiac findings, electrocardiogram and imaging findings, which cumulatively form the full phenomap of ATTR. Results from any diagnostic test for ATTR should be interpreted in light of the pre-test probability for the disease. Findings that suggest negative markers for ATTR can point towards other forms of amyloidosis (such as AL amyloidosis) or alternate causes of left ventricular hypertrophy, including hypertrophic cardiomyopathy or Fabry disease. The rising number of referrals for ATTR cardiomyopathy presents a challenge in daily clinical practice. To prevent an increase in false-positive diagnostic test results, an ATTR phenomap can serve as a valuable tool for guiding diagnostic assessments, interpreting test outcomes and prioritizing appropriate referrals for ATTR screening.

Low chronotropic response in post-myocardial infarction exercise test predicts worse prognosis in patients with preserved or mildly reduced left ventricular ejection fraction

Abstract Background Chronotropic incompetence is common in post-myocardial infarction (MI) patients and is associated with reduced exercise capacity. However, its prognostic significance and a determination of threshold values for prognosis remain unclear. Methods & Results We assessed 96 post-MI patients 4 weeks after their initial event with left ventricular ejection fraction (LVEF) ≥ 40%. All patients underwent combined stress echocardiography and cardiopulmonary exercise tests. The chronotropic response was measured as a percent of the maximal predicted heart rate at peak exercise (%MPHR). Exclusion criteria included patients with exercise limitations for pulmonary or peripheral reasons and those who declined to complete the study. Follow-up was conducted in 86 patients. The primary outcome measured was a composite of all-cause mortality or hospitalization for ischemic heart disease events (acute MI, unstable angina, or revascularization procedures) or heart failure exacerbation. The median follow-up duration was 4.0 (IQR 2, 5.6) years. The median patient age was 60 (IQR 53, 65) years, 64% of patients were males, and 84% were on beta-blockers. Median LVEF was 57 (IQR 51, 62)%, and median peak VO2 was 19 (IQR 15, 22) mL/kg/min. There were 15 composite end-point events, including 3 deaths, during the follow-up period. The %MPHR threshold of 67% had the best predictive value with an area under the curve (AUC) of 77% for predicting the primary outcome (PPV 31%, NPV 97%, sensitivity 93%, specificity 55%). A significant difference (p = 0.0022) in the occurrence of the composite end-point was noted between patients with a %MPHR below versus above this threshold (Figure. Kaplan-Meier curves for survival free of primary outcome). In the multivariate Cox regression analysis adjusting for demographic, clinical variables, and beta-blocker doses, only chronotropic response and current smoking correlated with the primary end-point (Table. The Cox proportional hazards regression model for prediction of the primary outcome). Conclusions A low chronotropic response to exercise is a negative prognostic marker in post-myocardial infarction patients with either preserved or mildly reduced left ventricular ejection fraction.Figure.Table.

Impact of sarcopenia and fat distribution on outcomes in penile cancer

Sarcopenia, defined as age-associated loss of skeletal muscle function and muscle mass, is a negative prognostic marker for survival in several tumor entities. However, data evaluating the impact of sarcopenia and fat distribution on penile cancer are rarely described. We performed a retrospective study including 38 patients who were diagnosed with squamous cell carcinoma of the penis. By measuring skeletal muscle mass and fat distribution at axial abdominal computed tomography images at the third lumbar vertebra several body composition parameters including skeletal muscle index (SMI), psoas muscle index (PMI), visceral obesity and visceral-to-subcutaneous fat ratio were determined. Among 38 patients, 26% (n = 10) of the patients with penile cancer were identified as sarcopenic. SMI, age, lymph node metastases, distant metastases and penile cancer of the shaft were identified as significant risk factors for overall survival. PMI and distant metastases were significantly associated with cancer specific survival. None of the analysed adipose tissue parameters could be identified as risk factors for survival in this study. We showed that sarcopenia occurs in a relevant part of patients with penile cancer and is a significant risk factor for overall survival (p = 0.032) and cancer specific survival (p = 0.034) for patients with penile cancer. Regarding fat distribution further studies are needed to evaluate its impact on sarcopenia and survival.

A formal approach to reanalysis and the Early Semantic Stability Hypothesis: exploring the test case of the negative counterfactual marker ʾilmale in Hebrew and Aramaic

Abstract This study delves into the intricate process of reanalysis, wherein linguistic expressions undergo grammatical or semantic changes, or sometimes both. The primary objective of this study is to explore the theoretical aspects surrounding historical changes of this nature. To facilitate a comprehensive understanding of the topic, we provide a formal description of reanalysis as an analytical tool. Our formal description allows for the differentiation of various change scenarios, enabling us to identify distinct types of shifts from one analysis to another. This approach not only focuses on what has been reanalyzed, be it the morphology, syntax, or the semantics, but also emphasizes the interplay between all three linguistic modules (Form, Grammar, and Meaning) and their relationships. This holistic perspective enables a systematic examination of the significance of what remains constant at both points in time during the reanalysis process. The key insight arising from this analysis leads us to propose and substantiate the Early Semantic Stability Hypothesis. This hypothesis posits that the truth-conditional semantics of the original proposition remain unchanged throughout reanalysis, either in all contexts or in specific “bridging contexts” where the reanalysis occurs. To demonstrate these phenomena, we present a compelling test case, focusing on the development of the counterfactual conditional marker ʾilmale in Hebrew and Aramaic. Through a detailed examination of the syntactic and semantic reanalyses it underwent, we observe the emergence of unique semantic features. By adopting a formal semantic perspective, we address fundamental questions such as the level of ambiguity required for reanalysis to take place, the potential existence of universal constraints on reanalysis, and potential motivations driving these linguistic changes. This investigation provides valuable insights into the intricate mechanisms at play during reanalysis and contributes to the broader understanding of linguistic evolution and development.

A case of complete response to radiotherapy combined with durvalumab and tremelimumab in a patient with unknown primary hepatocellular carcinoma arising in the lumbar spine.

A 58-year-old man visited an orthopedic clinic complaining of pain in his right lower back and numbness in his lower limbs for one month. Imaging tests revealed a tumorous lesion from the left side of the second lumbar vertebra to the paraspinal muscles. CT-guided biopsy of the tumor was performed, and immunostaining results diagnosed hepatocellular carcinoma (HCC). Although the liver showed signs of chronic liver damage, no primary tumor was found within the liver or in other organs. Blood tests showed negative hepatitis virus markers for both HBV and HCV. The tumor markers AFP, AFP-L3, and DCP were high. Because he developed spinal cord compression syndrome due to a lumbar tumor, radiation therapy and denosumab administration were performed. Subsequently, systemic therapy with durvalumab plus tremelimumab was started. In the year following the start of treatment, the tumor has shrunk, and no new lesions have been observed. Tumor markers have also decreased. We have experienced a case of HCC in the lumbar spine without a primary tumor in the liver. This is a very rare case, and the combination therapy with durvalumab and tremelimumab resulted in a complete response, which we consider to be a valuable case.

Burned-out testicular cancer - case report

Introduction and Purpose Germ cell tumors of the testis are the most common malignant tumors in young men aged 15-40, although their incidence in the male population is about 1-1.5%. 90-98% of all testicular tumors originate from germ cells. There has been an increase in the incidence of testicular tumors in recent years, suggesting the influence of both genetic and environmental factors. Testicular cancer, which accounts for about 5% of all urological cancers and mainly affects young men. Most of these cancers originate from germ cells, and cases of spontaneous tumor regression are extremely rare. Aim of the study We present the case of a 36-year-old man who was found to have a left testicular tumor with negative tumor markers during physical examination and testicular ultrasound. The tumor was surgically removed, and the patient was discharged home. Histopathological examination showed that the tumor had completely necrosed, and a CT scan showed no metastasis. Results and Future directions The phenomenon of testicular tumor “burnout” has been described since 1927, but it was only in 2016 that the WHO recognized it as a separate disease entity. Despite the rarity of this phenomenon, more and more cases are being reported in the literature, allowing for a better understanding of the underlying mechanisms. It is important to remember that the tumor can give a variable clinical picture and its symptoms are not necessarily related to the location of the tumor, but to its metastasis. Physical and ultrasound examinations are key to making a correct diagnosis. Histologic features such as scar formation, intravesicular calcification, lymphoplasmocytic infiltration and testicular atrophy can help diagnose germ cell tumor. The findings of this case demonstrate the need for diagnostic vigilance, especially in patients with equivocal tumor marker results but with clinical signs of testicular tumor.

Navigating the HER2 Frontier: Advancing Gastrointestinal Adenocarcinoma Diagnosis through Updated NCCN Guidelines

Abstract Introduction/Objective HER2-status is routinely tested in breast and upper-gastrointestinal (GI) cancers. Studies have shown the importance of anti-HER2 therapy with HER2 being a negative predictive marker in advanced colorectal cancer (CRC). Thus, recent NCCN-guidelines recommend screening for HER2-status in such cases. To increase compliance with the NCCN recommendations, we performed a quality assurance project to screen for HER2 status by immunohistochemistry (IHC) in GI adenocarcinomas. Methods/Case Report We conducted a validation of HER2 IHC for GI adenocarcinomas. Surgical pathologists were informed via email discussing the criteria and significance of HER2 IHC in GI adenocarcinomas. A total of 50 GI adenocarcinoma cases were evaluated in our surgical-pathology service, from June to December 2023. Pathologists were periodically reminded, either through email or in-person meetings, to ensure HER2 IHC assessment, especially for cases initially missed. Results (if a Case Study enter NA) Out of 50 GI adenocarcinomas, HER2 IHC was performed on 27 out of 50 cases (54%) without any reminder. The pathologists were reminded to perform HER2 IHC on the remaining 23 cases, resulting in the IHC performance on an additional 22 cases, giving a yield of 98%. Of 49 cases, HER2 was negative in 46 cases and was positive (3+) in one case, and equivocal (2+) in two cases. Subsequent HER2 FISH analysis of equivocal cases showed HER2 amplification in one case. Thus, HER2 expression/amplification was present in 2 out of 50 cases (4%) including one case each in upper and lower GI tracts. Conclusion While we have long recognized importance of HER2 Status in upper GI adenocarcinomas, it’s now clear that understanding HER2 status is crucial in CRC too. New evidence highlights how anti-HER2 therapy can make a difference, especially for advanced cases. By screening for HER2 early, clinicians can enroll such patients in appropriate clinical trials and also guide them on the appropriate treatment options, offering hope and targeted care.

Evaluation of mrkD, pgaC and wcaJ as biomarkers for rapid identification of K. pneumoniae biofilm infections from endotracheal aspirates and bronchoalveolar lavage

Klebsiella pneumoniae has been identified as one of the most important opportunistic pathogens responsible for nosocomial infections. Antibiotic resistance and the ability to form biofilms are the two main factors involved in the persistence of infections. Conventional detection methods involve culture isolation and identification followed by biofilm assay that takes 48–72 h. Timely detection of biofilm-forming resistant pathogens is essential to appropriately treat the infection with the right dose and combinations. The present study focuses on evaluating an RT-PCR panel using mrkD, pgaC, and wcaJ genes to screen for biofilm-forming K. pneumoniae from ETA/BAL specimens. The assay accurately identified K. pneumoniae harboring samples with a limit of detection of 1 ng/µl total RNA. Representative culture-negative-PCR-positive samples were subjected to metagenomics which identified K. pneumoniae reads in these samples confirming the specificity of RT-PCR. mrkD and pgaC act as K. pneumoniae specific identification whereas wcaJ acts as a negative marker for biofilm-forming K. pneumoniae. In addition, RT-PCR results correlated well with the phenotypic biofilm-forming assay. This RT-PCR assay is the first of its kind for rapid identification of biofilm-forming K. pneumoniae. The result of this study highlights that the rapid detection of K. pneumoniae biofilms based on the RT-PCR results coupled with clinical conditions would be appropriate to treat emerging infections or to prevent re-infections in clinical settings.

Presence of cholestasis and its impact on survival in SARS-CoV-2 associated acute respiratory distress syndrome.

Data on cholestasis and biliary injury in patients with COVID-19 are scarce. The primary aim of this study was to evaluate the prevalence of cholestasis and factors associated with its development and outcome in critically ill patients with COVID-19 associated acute respiratory distress syndrome (ARDS). In this retrospective exploratory study, COVID-19 patients with ARDS admitted to an intensive care unit (ICU) at the Medical University of Vienna were evaluated for the development of cholestasis defined as an alkaline phosphatase level of 1.67x upper limit of normal for at least three consecutive days. Simple and multiple logistic regression analysis was used to evaluate parameters associated with development of cholestasis and survival. Of 225 included patients 119 (53%) developed cholestasis during ICU stay. Patients with cholestasis had higher peak levels of alkaline phosphatase, gamma-glutamyl transferase, bilirubin and inflammation parameters. Factors independently associated with cholestasis were extracorporeal membrane oxygenation support, ketamine use, high levels of inflammation parameters and disease severity. Presence of cholestasis and peak ALP levels were independently associated with worse ICU and 6-month survival. Development of cholestasis is a common complication in critically ill COVID-19 patients and represents a negative prognostic marker for survival. It is associated with disease severity and specific treatment modalities of intensive care.

Overexpression of TPD52L2 in HNSCC: prognostic significance and correlation with immune infiltrates.

Evidence has been presented that the tumor protein D52 (TPD52) family plays a critical role in tumor development and progression. As a member of the TPD52 family, the changes in TPD52L2 gene status are instrumental in kinds of cancer development. However, its effects on patient prognosis and immune infiltration in Head and Neck Squamous Carcinoma (HNSCC) are still poorly understood. The Tumor Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and c-BioPortal database was used to explore the expression pattern, prognostic value, and variation of gene status in HNSCC. The LinkedOmics database was used to obtain the co-expression genes of TPD52L2 and identify the diagnostic value of TPD52L2 in HNSCC. The correlations between TPD52L2 expression and six main types of immune cell infiltrations and immune signatures were explored using Tumor Immune Estimation Resource (TIMER). The correlation between TPD52L2 expression and immune checkpoint genes (ICGs) was analyzed by TCGA database. Immunohistochemistry (IHC) was performed to validate the expression of three ICGs (PDL1, PDL2, EGFR) and TPD52L2 using 5 paired HNSCC and normal head and neck tissues. Polymerase Chain Reaction (PCR) and Western Blot (WB) of HNSCC and normal head and neck cell lines were performed to verify the high level of TPD52L2 mRNA and protein expression. protein expression of TPD52L2 in pan-cancer was also validated using UALCAN. TPD52L2 was overexpressed in tumor tissues, and it predicted worse survival status in HNSCC. ROC analysis suggested that TPD52L2 had a diagnostic value. Multivariate Cox analysis identified TPD52L2 as an independent negative prognostic marker of overall survival. Functional network analysis suggested that TPD52L2 was associated with immune-related signaling pathways, cell migration pathways, and cancer-related pathways. High expression of TPD52L2 was associated with a more mutant frequency of TP53. Notably, we found that the expression of TPD52L2 was closely negatively correlated with the infiltration levels of 15 types of immune cells and positively correlated with several immune markers. PCR, WB experiments, and UALCAN database verified the high level of TPD52L2 mRNA and protein expression. TPD52L2 is upregulated in HNSCC, which is an independent factor for adverse prognosis prediction. It probably plays a role in the negative regulation of immune cell infiltration. TPD52L2 might be a promising prognostic biomarker and therapeutic target in HNSCC.

CyCadas: accelerating interactive annotation and analysis of clustered cytometry data.

Single cell profiling by cytometry has emerged as a key technology in biology, immunology and clinical-translational medicine. The correct annotation, which refers to the identification of clusters as specific cell populations based on their marker expression, of clustered high-dimensional cytometry data, is a critical step of the analysis. Its accuracy determines the correct interpretation of the biological data. Despite the progress in various clustering algorithms, the annotation of clustered data still remains a manual, time consuming and error-prone task. We developed a user-friendly cluster annotation and differential abundance detection tool that can be applied on data generated with Self Organizing Map clustering algorithms, thus simplifying the annotation process of datasets that consist of hundreds or thousands of clusters. We present Cytometry Cluster Annotation and Differential Abundance Suite (CyCadas), a semi-automated software tool that facilitates cluster annotation in cytometry data by offering both visual and computational guidance. CyCadas addresses the critical need for efficient and accurate annotation of high-resolution clustered cytometry data, significantly reducing the time needed to perform the analysis compared to both manual gating approaches and manual annotation of clustered data. The tool features a user-friendly interface, visual tools enabling data exploration and automated threshold estimation to separate negative and positive marker expression. It facilitates the definition and annotation of cell phenotypes among multiple clusters in a tree-based data structure. Finally, it calculates the abundance of various cell populations across the conditions with statistical interpretation. It is an ideal resource for researchers aiming to streamline their cytometry workflow. CyCadas is available as open source at: https://github.com/DII-LIH-Luxembourg/cycadas.

Whole transcription analysis identified the regulation of hypoxia-inducible factors in monocytes with immune suppression: implications for clinical outcomes.

Sepsis progression is marked by a complex immune response, where the involvement of hypoxia-inducible factors (HIF) plays an uncertain role. The study aims to elucidate the involvement of HIF-1α in monocyte function during sepsis and its potential as a prognostic indicator. Transcriptomic data from healthy individuals and septic patients in datasets GSE54514, GSE167363, and GSE46955 were analyzed. Additionally, human monocytes were employed to elucidate how HIF regulates immune responses in the context of sepsis. Septic non-survivors exhibited sustained upregulation of HIF-1α expression alongside compromised inflammatory response and antigen presentation, with downregulation of NF-κB and HLADRB1 genes associated with poor sepsis prognosis. Conversely, septic survivors displayed an increased proportion of classical monocytes and enhanced inflammation and expression of antigen presentation-related genes. During the recovery phase of sepsis, monocytes continued to demonstrate elevated HIF-1α expression. In cultured THP1 cells and septic CD14+ monocytes, HIF hindered inflammatory responses and antigen presentation, while also suppressing the proportion of classical monocytes after LPS stimulation. Mechanistically, HIF significantly attenuated LPS-induced immune responses in monocytes by inhibiting the phosphorylation of IKK. HIF in monocytes acts as a suppressor of immune-inflammatory responses and antigen presentation, and may serve as a negative molecular marker for sepsis development.

Syntactic and semantic variations in negative interrogatives: Contracted vs. uncontracted forms

This study investigates the forms and uses of negative interrogatives with auxiliary verb contractions in English, focusing on syntactic and semantic differences. Data was drawn from conversations in an English class at a university in Lhokseumawe, Indonesia, between a lecturer and students and students and students. The aim was to examine three types of negative interrogatives: Yes-No questions, negative rhetorical questions, and both marked (n’t) and unmarked (not) forms. The research employs a descriptive method with a synchronic time frame, utilizing distributional analysis, including immediate constituent analysis and mark reading techniques, to categorize and analyze the data. The findings reveal that negative interrogatives involving auxiliary verbs exhibit positive and negative presuppositions, with adjectival negative interrogatives showing variations between contracted and uncontracted forms. Adverbial negative interrogatives were found across all three types, indicating their versatility in forming questions. The study highlights that the choice between ‘not’ and ‘n’t’ affects the tone and emphasis of negative interrogatives, influencing the interpretation of responses. Contracted forms often carry a rhetorical or presumptive tone, while uncontracted forms are more formal and explicit. These variations show the importance of auxiliary verbs and negative markers in shaping the structure and meaning of negative interrogatives. This research contributes to the understanding of how negative interrogatives function in English, revealing the nuanced ways in which they impact communication and interpretation.

Discourse markers of rejection in three varieties of Spanish

In the study of discourse markers in Spanish, a field with great vitality in the last few decades, it is common to overlook the expressions used to accept or reject a proposal, suggestion or petition, often included within the broader functions of agreement and disagreement. These answers, used in an informal oral interactive context, exhibit a rich dialectal variety. Due to the fleeting nature of their contexts of use, they are difficult to formally register for further study, especially when looking at them from a panhispanic perspective. For this reason, an anonymous survey was designed in order to ask native speakers about their familiarity with a series of discourse markers used to answer “no” to a proposal. The questionnaire was disseminated in the countries of Spain, Mexico and Colombia, specifically around the capital areas. Participants (N=223) evaluated from 1 (I do not know this expression) to 5 (I use this expression frequently) a series of possible responses, and they were offered the option to provide some other answers not included in the original list. Results show that, while some discourse markers are used to a certain degree in the three varieties, others are prototypical from one of these dialects. The opportunity to offer new expressions was very productive, especially in the case of participants from Colombia and Mexico. While this study focuses on three particular regions, it opens the door to a comparative analysis of discourse markers of negation throughout the Spanish speaking world.

Paclitaxel, Ifosfamide, and Cisplatin as Initial Salvage Chemotherapy for Germ Cell Tumors: Long-Term Follow-Up and Outcomes for Favorable- and Unfavorable-Risk Disease.

Paclitaxel, ifosfamide, and cisplatin (TIP) is an established salvage regimen for germ cell tumors (GCT) on the basis of a phase II trial, but efficacy on a large patient cohort including patients with unfavorable risk features and long-term outcomes has not been reported. Herein, we report updated treatment efficacy and long-term follow-up with TIP. Patients with GCT who received TIP after cisplatin-based chemotherapy were eligible. Favorable response (complete response or partial response with negative tumor markers), overall survival (OS) and progression-free survival (PFS) rates, relapse, and toxicity were determined. Disease was reclassified according to the International Prognostic Factor Study Group (IPFSG) score. Of the 104 patients, 87 had favorable risk factors and 17 had at least one unfavorable factor by Memorial Sloan Kettering Cancer Center (MSKCC) criteria. Ten patients were treated for a second gonadal primary GCT. With a median follow-up of 8.9 years, the 5-year PFS and OS rates were 66% (95% CI, 55 to 74) and 69% (95% CI, 59 to 77), respectively. Among 87 patients with favorable-risk disease, 69 (79%) achieved a favorable response with 5-year PFS and OS rates of 67% (95% CI, 56 to 76) and 72% (95% CI, 61 to 80), respectively. Among 17 patients with MSKCC unfavorable-risk disease, 13 (76%) achieved a favorable response with 5-year PFS and OS rates of 59% (95% CI, 33 to 78) and 56% (95% CI, 28 to 76), respectively. After IPFSG reclassification, 5-year PFS and OS rates for patients with ≤intermediate-risk disease were 75% (95% CI, 50 to 89) and 73% (95% CI, 55 to 85), respectively. TIP is an effective second-line regimen for patients with GCT. Similar outcomes were observed in patients with favorable- and unfavorable-risk disease. The randomized TIGER trial (ClinicalTrials.gov identifier: NCT02375204) comparing TIP with high-dose chemotherapy will determine the optimal second-line treatment approach.