Multiple Markers Research Articles

Development of Tumor Markers for Breast Cancer Immunotherapy.

Although breast cancer treatment has been developed remarkably in recent years, it remains the primary cause of death among women. Immune checkpoint blockade therapy has significantly altered the way breast cancer is treated, although not all patients benefit from the changes. At present, the most effective mechanism of immune checkpoint blockade application in malignant tumors is not clear and efficacy may be influenced by many factors, including host, tumor, and tumor microenvironment dynamics. Therefore, there is a pressing need for tumor immunomarkers that can be used to screen patients and help determine which of them would benefit from breast cancer immunotherapy. At present, no single tumor marker can predict treatment efficacy with sufficient accuracy. Multiple markers may be combined to more accurately pinpoint patients who will respond favorably to immune checkpoint blockade medication. In this review, we have examined the breast cancer treatments, developments in research on the role of tumor markers in maximizing the clinical efficacy of immune checkpoint inhibitors, prospects for the identification of novel therapeutic targets, and the creation of individualized treatment plans. We also discuss how tumor markers can provide guidance for clinical practice.

Abstract 1536: Deciphering the crosstalk within the tumor microenvironment of NSCLC by a virtual microdissection approach

Abstract Introduction: Non-small cell lung cancer (NSCLC) is a highly heterogenous disease with the largest number of cancer-related mortality worldwide, one of the reasons for this is the complex and diverse tumor microenvironment (TME) comprising of numerous cell types. Several studies have already highlighted the importance of TME in dictating progression steps and response to therapies; however, a transcriptome-based molecular subtyping of patients in lung adenocarcinomas (LUADs) and lung squamous cell carcinomas (LUSCs) can further determine the distinct tumor immune microenvironment (TiME), which can eventually provide a systematic overview to improve the diagnosis and prognosis of patients. Material and method: To elucidate such nature of interactions between tumor cells and cells comprising the TME, we exploited the transcriptome of 300 early stages (Ib-IIIa) NSCLC recruited in the prospective observational clinical trial PROMOLE. With the help of a clustering approach, initially we performed a molecular-based virtual stratification/dissection on the NSCLC patients. Next, to elucidate the relative cell-type abundance, a deconvolution approach was applied to identify the possibility of tumor infiltrating immune cells within these subgroups. Immunohistochemistry (IHC) was then used to substantiate these predictions on tumor cells. Results and discussion: The resulting subgroups of LUADs and LUSCs are biologically well-characterized by mutational and gene expression profiles. Cell-type abundance approach identified samples which are enriched with tumor infiltrating immune cells like Neutrophils, Tcells, macrophages, etc. These findings were positively confirmed by IHC with multiple cell markers such as MPO, CD4, CD8, CD68, etc. Integrating these two results highlighted the proportion of TiME in the two different sub-populations along with shedding some light on the crosstalk happening between different cancer-/immune- cell lines. Conclusion: The in-silico predictions on bulk RNA data by virtual micro-dissection, distinguished the two distinct NSCLC subtypes, each associated with clinical and molecular features. Furthermore, the immune cells infiltration suggests a possible role of infiltrating tumor immune cells with the prognosis of patients. Our analysis successfully performed an intra-sample and inter-sample comparison, which can unveil new prognostic markers that can provide relevant information for cancer immunotherapy. Citation Format: Sushant Parab, Francesca Napoli, Davide Corà, Gabriella Doronzo, Valentina Communanza, Luisella Righi, Luca Primo, Valentina Monica, Lorenzo Manganaro, Bianco Selene, Paolo Bironzo, Giorgio Scagliotti, Federico Bussolino. Deciphering the crosstalk within the tumor microenvironment of NSCLC by a virtual microdissection approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1536.

Improvement of differential diagnosis of lung cancer by use of multiple protein tumor marker combinations.

Differential diagnosis of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) in hospitalized patients is crucial for appropriate treatment choice. To investigate the relevance of serum tumor markers (STMs) and their combinations for the differentiation of NSCLC and SCLC subtypes. Between 2000 and 2003, 10 established STMs were assessed retrospectively in 311 patients with NSCLC, 128 with SCLC prior systemic first-line therapy and 51 controls with benign lung diseases (BLD), by automatized electrochemiluminescence immunoassay technology. Receiver operating characteristic (ROC) curves and logistic regression analyses were used to evaluate the diagnostic efficacy of both individual and multiple STMs with corresponding sensitivities at 90% specificity. Standards for Reporting of Diagnostic Accuracy (STARD guidelines) were followed. CYFRA 21-1 (cytokeratin-19 fragment), CEA (carcinoembryonic antigen) and NSE (neuron specific enolase) were significantly higher in all lung cancers vs BLD, reaching AUCs of 0.81 (95% CI 0.76-0.87), 0.78 (0.73-0.84), and 0.88 (0.84-0.93), respectively. By the three marker combination, the discrimination between benign and all malignant cases was improved resulting in an AUC of 0.93 (95% CI 0.90-0.96). In NSCLC vs. BLD, CYFRA 21-1, CEA and NSE were best discriminative STMs, with AUCs of 0.86 (95% CI 0.81-0.91), 0.80 (0.74-0.85), and 0.85 (0.79-0.91). The three marker combination also improved the AUC: 0.92; 95% CI 0.89-0.96). In SCLC vs. BLD, ProGRP (pro-gastrin-releasing peptide) and NSE were best discriminative STMs, with AUCs of 0.89 (95% CI 0.84-0.94) and 0.96 (0.93-0.98), respectively, and slightly improved AUC of 0.97 (95% CI 0.95-0.99) when in combination. Finally, discrimination between SCLC and NSCLC was possible by ProGRP (AUC 0.86; 95% CI 0.81-0.91), NSE (AUC 0.83; 0.78-0.88) and CYFRA 21-1 (AUC 0.69; 0.64-0.75) and by the combination of the 3 STMs (AUC 0.93; 0.91-0.96), with a sensitivity of 88% at 90% specificity. The results confirm the power of STM combinations for the differential diagnosis of lung cancer from benign lesions and between histological lung cancer subtypes.

Proteasome inhibition triggers tissue-specific immune responses against different pathogens in C. elegans.

Protein quality control pathways play important roles in resistance against pathogen infection. For example, the conserved transcription factor SKN-1/NRF up-regulates proteostasis capacity after blockade of the proteasome and also promotes resistance against bacterial infection in the nematode Caenorhabditis elegans. SKN-1/NRF has 3 isoforms, and the SKN-1A/NRF1 isoform, in particular, regulates proteasomal gene expression upon proteasome dysfunction as part of a conserved bounce-back response. We report here that, in contrast to the previously reported role of SKN-1 in promoting resistance against bacterial infection, loss-of-function mutants in skn-1a and its activating enzymes ddi-1 and png-1 show constitutive expression of immune response programs against natural eukaryotic pathogens of C. elegans. These programs are the oomycete recognition response (ORR), which promotes resistance against oomycetes that infect through the epidermis, and the intracellular pathogen response (IPR), which promotes resistance against intestine-infecting microsporidia. Consequently, skn-1a mutants show increased resistance to both oomycete and microsporidia infections. We also report that almost all ORR/IPR genes induced in common between these programs are regulated by the proteasome and interestingly, specific ORR/IPR genes can be induced in distinct tissues depending on the exact trigger. Furthermore, we show that increasing proteasome function significantly reduces oomycete-mediated induction of multiple ORR markers. Altogether, our findings demonstrate that proteasome regulation keeps innate immune responses in check in a tissue-specific manner against natural eukaryotic pathogens of the C. elegans epidermis and intestine.

Molecular identification, risk factor assessment, and phylogenetic analysis of tick-borne pathogens in symptomatic and asymptomatic cattle from South-Eastern Iran.

Tick-borne pathogens (TBPs) represent a substantial threat to cattle globally, exerting adverse impacts on production, health, and economic viability. This study delves into the prevalence and implications of TTBPs in cattle sourced from resource-limited smallholder livestock farms situated in southeastern Iran, proximate to Afghanistan and Pakistan. Blood and tick specimens were systematically collected from a cohort of 230 cattle, comprising 150 asymptomatic and 80 symptomatic individuals. Genomic DNA isolated from blood samples underwent rigorous examination for the presence of key TBPs, including Anaplasma marginale, A. phagocytophilum, A. bovis, A. centrale, Babesia bigemina, and Theileria annulata, utilizing multiple genetic markers. Nucleotide sequence analysis facilitated the reconstruction of phylogenetic relationships. The study also evaluated various potential risk factors, such as clinical status, gender, age, breed, tick infestation, and management practices, to elucidate their associations with TTBPs. Among the cattle cohort, a staggering 87.8% (202/230) tested positive for at least one pathogen. Prevalence statistics encompassed A. marginale (72.2%), T. annulata (68.3%), A. phagocytophilum/A. platys-like complex (66.1%), A. centrale (16.7%), B. bigemina (10.0%), and A. bovis (6.1%). Remarkably, mixed infections involving two, three, and four pathogens were detected in 23%, 52.1%, and 2.2% of animals, respectively. Notably, all asymptomatic cattle were positive for at least one TBP. Tick infestation was observed in 62.2% (143/230) of cattle, predominantly caused by Hyalomma anatolicum (82.5%), Rhipicephalus (Boophilus) annulatus (13.1%), and R. sanguineus sensu lato (4.4%). Risk factors linked to TBPs encompassed tick infestation, older age, and crossbred animals. Clinical presentations among symptomatic cattle encompassed fever, anemia, weight loss, anorexia, jaundice, and enlarged superficial lymph nodes. This study underscores the pivotal role of asymptomatic carriers in the propagation of TTBPs within endemic regions. Furthermore, it emphasizes the potential for the implementation of molecular diagnostics to unmask subclinical infections, thereby affording the opportunity for targeted interventions aimed at ameliorating the burden of TTBPs in resource-constrained smallholder dairy farms.

A molecular index for biological age identified from the metabolome and senescence-associated secretome in humans.

Unlike chronological age, biological age is a strong indicator of health of an individual. However, the molecular fingerprint associated with biological age is ill-defined. To define a high-resolution signature of biological age, we analyzed metabolome, circulating senescence-associated secretome (SASP)/inflammation markers and the interaction between them, from a cohort of healthy and rapid agers. The balance between two fatty acid oxidation mechanisms, β-oxidation and ω-oxidation, associated with the extent of functional aging. Furthermore, a panel of 25 metabolites, Healthy Aging Metabolic (HAM) index, predicted healthy agers regardless of gender and race. HAM index was also validated in an independent cohort. Causal inference with machine learning implied three metabolites, β-cryptoxanthin, prolylhydroxyproline, and eicosenoylcarnitine as putative drivers of biological aging. Multiple SASP markers were also elevated in rapid agers. Together, our findings reveal that a network of metabolic pathways underlie biological aging, and the HAM index could serve as a predictor of phenotypic aging in humans.

Topical antibiotics limit depigmentation in a mouse model ofvitiligo.

Oral neomycin administration impacts the gut microbiome and delays vitiligo development in mice, and topical antibiotics may likewise allow the microbiome to preserve skin health and delay depigmentation. Here, we examined the effects of 6-week topical antibiotic treatment on vitiligo-prone pmel-1 mice. Bacitracin, Neosporin, or Vaseline were applied to one denuded flank, while the contralateral flank was treated with Vaseline in all mice. Ventral depigmentation was quantified weekly. We found that topical Neosporin treatment significantly reduced depigmentation and exhibited effects beyond the treated area, while Bacitracin ointment had no effect. Stool samples collected from four representative mice/group during treatment revealed that Neosporin treatment aligned with reduced abundance of the Alistipes genus in the gut, while relevant changes to the skin microbiome at end point were less apparent. Either antibiotic treatment led to reduced expression of MR1, potentially limiting mucosal-associated invariant T-cell activation, while Neosporin-treated skin selectively revealed significantly reduced CD8+ T-cell abundance. The latter finding aligned with reduced expression of multiple inflammatory markers and markedly increased regulatory T-cell density. Our studies on favorable skin and oral antibiotic treatment share the neomycin compound, and in either case, microbial changes were most apparent in stool samples. Taken together, neomycin-containing antibiotic applications can mediate skin Treg infiltration to limit vitiligo development. Our study highlights the therapeutic potential of short-term antibiotic applications to limit depigmentation vitiligo.

Abstract B082: A pro-tumorigenic, aged microenvironment may aid in worsened disease progression in an HGSOC model

Abstract Over 70% of HGSOC patients are diagnosed after 55 and the average patient succumbs to disease at 70 years old. Despite this, most ovarian cancer research, and cancer studies generally, utilize young mice that are mimetic of human teenagers and/or do not investigate age as a variable despite it being the top cancer risk factor. An improved understanding of how age affects HGSOC progression may enable development of alternative treatment strategies aimed at intercepting metastasis and preventing cancer cells from establishing in the peritoneum. Our preliminary studies show tumor burden and disease severity are significantly higher in aged versus young animals. We developed an epithelial cell line derived from C57Bl/6 oviducts and used CRISPR-Cas9 genome editing to develop a syngeneic tumorigenic line harboring Trp53, Pten, and Brca2 deletions. After intraperitoneal injection into 6-week and 14-month-old immunocompetent C57Bl/6 mice, we observed a stark increase in tumor burden in aged mice. Furthermore, older mice died at a faster rate; all aged mice succumbed to disease within 1-month of inoculation compared to young mice surviving up to 3 months. Total RNA-sequencing of tumors harvested from aged and young mice is ongoing. Characterization of the tumor-naïve peritoneal compartment revealed the aged microenvironment to be highly inflammatory. When assessed via cytokine array, peritoneal fluid of aged mice had higher concentrations of inflammatory markers including soluble TNF receptor-1 (sTNFR-1), L-selectin, and VCAM-1. Flow cytometry of peritoneal lavage indicated an over 2-fold increase in the number CD45+ cells present in the peritoneal cavity, coinciding with cytokine array results. Within this population, there was nearly complete depletion of macrophage and neutrophil populations and a significant increase in B cells and CD4+ and CD8+ T cells. The percentage of PD-1+ CD8+ cells also increased. In addition to increased signs of inflammation, a significantly higher concentration of insulin-like growth factor-1 (IGF-I) and higher concentrations of the associated IGF binding proteins -3 and -6 were found in peritoneal fluid of aged mice. In addition, during tissue collection, organs of the aged mice were found to be much larger than young mice, a common effect of elevated IGF-I; aged omentum weighed 2-4 times that of young. Notably, IGF-I is known to inhibit apoptosis and promote proliferation and increased IGF-I is correlated with increased risk of breast, prostate, and lung cancer. A drastic difference in disease severity occurs when identical cancer cells are injected into 6-week and 14-month-old mice. To identify possible microenvironmental differences in the two populations, a broad panel of cytokines was assessed, and immune populations were quantified in pre-cancerous mice. Overall, multiple pro-inflammatory markers and the number of lymphocytes were upregulated in the aged peritoneal cavity, and markers of immune fatigue were present. Further work is aimed at identifying immune cell state via further cytometry and sequencing. Citation Format: Katherine A. Cummins, Ronny I. Drapkin, Matthew Knarr. A pro-tumorigenic, aged microenvironment may aid in worsened disease progression in an HGSOC model [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B082.

Mitochondrial damage and clearance in retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is a devastating eye disease that causes permanent vision loss in the central part of the retina, known as the macula. Patients with such severe visual loss face a reduced quality of life and are at a 1.5 times greater risk of death compared to the general population. Currently, there is no cure for or effective treatment for dry AMD. There are several mechanisms thought to underlie the disease, for example, ageing-associated chronic oxidative stress, mitochondrial damage, harmful protein aggregation and inflammation. As a way of gaining a better understanding of the molecular mechanisms behind AMD and thus developing new therapies, we have created a peroxisome proliferator-activated receptor gamma coactivator 1-alpha and nuclear factor erythroid 2-related factor 2 (PGC1α/NFE2L2) double-knockout (dKO) mouse model that mimics many of the clinical features of dry AMD, including elevated levels of oxidative stress markers, damaged mitochondria, accumulating lysosomal lipofuscin and extracellular drusen-like structures in retinal pigment epithelial cells (RPE). In addition, a human RPE cell-based model was established to examine the impact of non-functional intracellular clearance systems on inflammasome activation. In this study, we found that there was a disturbance in the autolysosomal machinery responsible for clearing mitochondria in the RPE cells of one-year-old PGC1α/NFE2L2-deficient mice. The confocal immunohistochemical analysis revealed an increase in autophagosome marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) as well as multiple mitophagy markers such as PTE-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase (PARKIN), along with signs of damaged mitochondria. However, no increase in autolysosome formation was detected, nor was there a colocalization of the lysosomal marker LAMP2 or the mitochondrial marker, ATP synthase β. There was an upregulation of late autolysosomal fusion Ras-related protein (Rab7) in the perinuclear space of RPE cells, together with autofluorescent aggregates. Additionally, we observed an increase in the numbers of Toll-like receptors 3 and 9, while those of NOD-like receptor 3 were decreased in PGC1α/NFE2L2 dKO retinal specimens compared to wild-type animals. There was a trend towards increased complement component C5a and increased involvement of the serine protease enzyme, thrombin, in enhancing the terminal pathway producing C5a, independent of C3. The levels of primary acute phase C-reactive protein and receptor for advanced glycation end products were also increased in the PGC1α/NFE2L2 dKO retina. Furthermore, selective proteasome inhibition with epoxomicin promoted both nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondrial-mediated oxidative stress, leading to the release of mitochondrial DNA to the cytosol, resulting in potassium efflux-dependent activation of the absent in melanoma 2 (AIM2) inflammasome and the subsequent secretion of interleukin-1β in ARPE-19 cells. In conclusion, the data suggest that there is at least a relative decrease in mitophagy, increases in the amounts of C5 and thrombin and decreased C3 levels in this dry AMD-like model. Moreover, selective proteasome inhibition evoked mitochondrial damage and AIM2 inflammasome activation in ARPE-19 cells.

Magnetic photoelectrochemical sensor array utilizing addressing sensing strategy for simultaneous detection of amyloid-β 42 and microtubule-associated protein tau

The accurate diagnosis of diseases can be improved by detecting multiple biomarkers simultaneously. This study presents the development of a magnetic photoelectrochemical (PEC) immunosensor array for the simultaneous detection of amyloid-β 42 (Aβ) and microtubule-associated protein (Tau), which are markers for neurodegenerative disorders. A metal-organic framework (MOF) derivative, Fe2O3@FeS2 magnetic composites with exceptional photoelectric and ferromagnetic properties was synthesized while preserving the original structure and advantages. Thus, the immunoassembly process of the sensor can be carried out in homogeneous solution and recovered by magnetic separation. For simultaneous detection, a chip is divided into multiple independent sensing sites, which have the same preparation and detection environment, allowing for the implementation of a self-calibration method. The sensor array demonstrates considerable detection ranges of 0.01–100 ng mL−1 for Aβ and 0.05–100 ng mL−1 for Tau, with low detection limits of 2.1 pg mL−1 for Aβ and 7.9 pg mL−1 for Tau. The PEC sensor array proposed in this study exhibits exceptional stability, selectivity, and reproducibility, providing a new method for detecting multiple markers.

A FACS-based novel isolation technique identifies heterogeneous CTCs in oral squamous cell carcinoma.

Isolating circulating tumour cells (CTCs) from the blood is challenging due to their low abundance and heterogeneity. Limitations of conventional CTC detection methods highlight the need for improved strategies to detect and isolate CTCs. Currently, the Food and Drug Administration (FDA)-approved CellSearch™ and other RUO techniques are not available in India. Therefore, we wanted to develop a flexible CTC detection/isolation technique that addresses the limitation(s) of currently available techniques and is suitable for various downstream applications. We developed a novel, efficient, user-friendly CTC isolation strategy combining density gradient centrifugation and immuno-magnetic hematogenous cell depletion with fluorescence-activated cell sorting (FACS)-based positive selection using multiple CTC-specific cell-surface markers. For FACS, a stringent gating strategy was optimised to exclude debris and doublets by side scatter/forward scatter (SSC/FSC) discriminator, remove dead cells by 4',6-diamidino-2-phenylindole (DAPI) staining, and eliminate non-specific fluorescence using a "dump" channel. APC-labelled anti-CD45mAB was used to gate remaining hematogenous cells, while multiple epithelial markers (EpCAM, EGFR, and Pan-Cytokeratin) and an epithelial-mesenchymal transition (EMT) marker (Vimentin) labelled with fluorescein isothiocyanate (FITC) were used to sort cancer cells. The technique was initially developed by spiking Cal 27 cancer cells into the blood of healthy donors and then validated in 95 biopsy-proven oral squamous cell carcinoma (OSCC) patients. CTCs isolated from patients were reconfirmed by Giemsa staining, immuno-staining, and whole transcriptome amplification (WTA), followed by qRT-PCR. In vitro culture and RNA sequencing (RNA-Seq) were also performed to confirm their suitability for various downstream applications. The mean detection efficiency for the Cal 27 tongue cancer cells spiked in the whole blood of healthy donors was 32.82% ± 12.71%. While ~75% of our patients (71/95) had detectable CTCs, the CTC positivity was independent of the TNM staging. The isolated potential cancer cells from OSCC patients were heterogeneous in size. They expressed different CTC-specific markers in various combinations as identified by qRT-PCR after WTA in different patients. Isolated CTCs were also found to be suitable for downstream applications like short-term CTC culture and RNA-Seq. We developed a sensitive, specific, flexible, and affordable CTC detection/isolation technique, which is scalable to larger patient cohorts, provides a snapshot of CTC heterogeneity, isolates live CTCs ready for downstream molecular analysis, and, most importantly, is suitable for developing countries.

Integration of Augmented Reality and Building Information Modeling for Enhanced Construction Inspection—A Case Study

This research addresses a significant challenge in the construction industry: the traditional reliance on CAD drawings and manual methods for building inspection and monitoring. This article presents a transformative proposal utilizing Augmented Reality (AR) to revolutionize these processes. The proposed model is based on an innovative integration of AR technology with Building Information Modeling (BIM), aiming to enhance data life-cycle management and improve the efficiency of construction management practices. The main goal of the article is to demonstrate the practical feasibility and benefits of this AR-BIM integration in construction management, particularly in the realms of inspection and monitoring. This involves developing and testing an accessible, user-friendly, and affordable AR application prototype for mobile devices, employing multiple markers, as a potential replacement for traditional methods. The research methodology comprises five phases, starting with the conversion of 2D CAD drawings into 3D BIM, followed by the simulation of AR-BIM integration at a construction site using a commercial AR application. This is succeeded by assessing the applicability of the commercial AR app and developing a multiple-marker AR application prototype suitable for general platforms. The final phase encompasses the testing and evaluation of this prototype. The findings suggest that the integration of 3D BIM with AR technology is not only feasible, but also beneficial in replacing paper-based processes, thereby enhancing information sharing, communication, and overall project execution efficiency. However, the accuracy of the superimposition between virtual and actual objects needs improvement to reduce discrepancies between as-built and as-planned scenarios. These results hold significant potential for transforming construction project execution by replacing traditional methods with more efficient digital solutions. Future research directions include extensive testing in various construction scenarios, improving model complexity handling, exploring the application of machine learning algorithms for data analysis, and expanding the study to encompass other stages of the construction lifecycle.

Phylogeography and demographic history of macaques, fascicularis species group, in East Asia: Inferred from multiple genomic markers

Climate changes at larger scales have influenced dispersal and range shifts of many taxa in East Asia. The fascicularis species group of macaques is composed of four species and is widely distributed in Southeast and East Asia. However, its phylogeography and demographic histories are currently poorly understood. Herein, we assembled autosomal, mitogenome, and Y-chromosome data for 106 individuals, and combined them with 174 mtDNA dloop haplotypes of this species group, with particular focus on the demographic histories and dispersal routes of Macaca fuscata, M. cyclopis, and M. mulatta. The results showed: (1) three monophyletic clades for M. fuscata, M. cyclopis, and M. mulatta based on the multiple genomics analyses; (2) the disparate demographic trajectories of the three species after their split ∼1.0 Ma revealed that M. cyclopis and M. fuscata were derived from an ancestral M. mulatta population; (3) the speciation time of M. cyclopis was later than that of M. fuscata, and their divergence time occurred at the beginning of “Ryukyu Coral Sea Stage” (1.0–0.2 Ma) when the East China Sea land bridge was completely submerged by the sea level rose; and (4) the three parallel rivers (Nujiang, Lancangjiang, and Jinshajiang) of Southwestern China divided M. mulatta into Indian and Chinese genetic populations ∼200 kya. These results shed light on understanding not only the evolutionary history of the fascicularis species group but also the formation mechanism of faunal diversity in East Asia during the Pleistocene.

CT-guided high dose rate brachytherapy can induce multiple systemic proteins of proliferation and angiogenesis predicting outcome in HCC

Background and purposeTo determine the potential prognostic value of proliferation and angiogenesis plasma proteins following CT-guided high dose rate brachytherapy (HDR-BT) of hepatocellular carcinoma (HCC). Materials and methodsFor this prospective study, HDR-BT (1 × 15 Gy) was administered to 24 HCC patients. Plasma was obtained and analyzed using an Olink proteomics Target-96 immuno-oncology-panel that included multiple markers of angiogenesis and proliferation. Fold-change (FC) ratios were calculated by comparing baseline and 48 h post HDR-BT paired samples. Patients were classified as responders (n = 12) if they had no local progression within 6 months or systemic progression within 2 years. Non-responders (n = 12) had recurrence within 6 months and/or tumor progression or extrahepatic disease within 2 years. ResultsProliferation marker EGF was significantly elevated in non-responders compared to responders (p = 0.0410) while FGF-2, HGF, and PlGF showed no significant differences. Angiogenesis markers Angiopoietin-1 and PDGF-B were likewise significantly elevated in non-responders compared to responders (p = 0.0171, p = 0.0462, respectively) while Angiopoietin-2, VEGF-A, and VEGFR-2 did not differ significantly. Kaplan-Meier analyses demonstrated significantly shorter time to systemic progression in patients with increased EGF and Angiopoietin-1 (p = 0.0185, both), but not in patients with one of the remaining proteins elevated (all p > 0.1). Pooled analysis for these 9 proteins showed significantly shorter time to systemic progression for FC ≥1.3 and ≥1.5 for at least 3 proteins elevated (p = 0.0415, p = 0.0193, respectively). ConclusionIncreased plasma levels of EGF and Angiopoietin-1 after HDR-BT for HCC are associated with poor response and may therefore function as predictive biomarkers of outcome.

Chronic malaria exposure is associated with inhibitory markers on T cells that correlate with atypical memory and marginal zone-like B cells.

Chronic immune activation from persistent malaria infections can induce immunophenotypic changes associated with T-cell exhaustion. However, associations between T and B cells during chronic exposure remain undefined. We analyzed peripheral blood mononuclear cells from malaria-exposed pregnant women from Papua New Guinea and Spanish malaria-naïve individuals using flow cytometry to profile T-cell exhaustion markers phenotypically. T-cell lineage (CD3, CD4, and CD8), inhibitory (PD1, TIM3, LAG3, CTLA4, and 2B4), and senescence (CD28-) markers were assessed. Dimensionality reduction methods revealed increased PD1, TIM3, and LAG3 expression in malaria-exposed individuals. Manual gating confirmed significantly higher frequencies of PD1+CD4+ and CD4+, CD8+, and double-negative (DN) T cells expressing TIM3 in malaria-exposed individuals. Increased frequencies of T cells co-expressing multiple markers were also found in malaria-exposed individuals. T-cell data were analyzed with B-cell populations from a previous study where we reported an alteration of B-cell subsets, including increased frequencies of atypical memory B cells (aMBC) and reduction in marginal zone (MZ-like) B cells during malaria exposure. Frequencies of aMBC subsets and MZ-like B cells expressing CD95+ had significant positive correlations with CD28+PD1+TIM3+CD4+ and DN T cells and CD28+TIM3+2B4+CD8+ T cells. Frequencies of aMBC, known to associate with malaria anemia, were inversely correlated with hemoglobin levels in malaria-exposed women. Similarly, inverse correlations with hemoglobin levels were found for TIM3+CD8+ and CD28+PD1+TIM3+CD4+ T cells. Our findings provide further insights into the effects of chronic malaria exposure on circulating B- and T-cell populations, which could impact immunity and responses to vaccination.

Multi-locus sequence analysis of ‘Candidatus Mycoplasma haematomacacae’ in free-ranging macaques from Thailand suggestive of a closer relationship to hemotropic mycoplasmas in capuchins and potential origin from bats

Although ‘Candidatus Mycoplasma haematomacacae’ (formerly known as ‘Candidatus Mycoplasma haemomacaque’) has been reported on extensively in macaques from Thailand, the USA, Japan, and Brazil, its genetic characterization has primarily been restricted to the 16S rRNA sequences with no exploration on multi-locus sequence analysis. The primary goal of this study was to characterize ‘Ca. M. haematomacacae’ among Thai macaques based on multiple genetic markers. Between April 2018 and November 2021, blood samples were taken from 580 free-ranging macaques (560 Macaca fascicularis and 20 Macaca nemestrina) in 15 locations encompassing 10 provinces throughout Thailand. Using the conventional PCR assay targeting the 16S ribosomal RNA (16S rRNA) gene, 338 out of 580 macaques (58.27 %) tested hemoplasma-positive. Of these, 40 positive samples were further subjected to DNA sequencing, and all were identified as ‘Ca. M. haematomacacae’. Subsequently, the partial nucleotide sequences of 23S ribosomal RNA (23S rRNA) and RNase P RNA (rnpB) genes of this particular hemoplasma species were amplified through nested PCR assay. The analysis of multi-locus genetic markers revealed that the 23S rRNA and rnpB sequences exhibited higher levels of genetic diversity than the 16S rRNA sequences. Furthermore, the 16S rRNA analyses demonstrated that ‘Ca. M. haematomacacae’ infecting Old World monkeys (Macaca spp.) was most closely related to hemotropic Mycoplasma spp. in black-capped capuchins (Sapajus apella) and Marcgrave's capuchins (Sapajus flavius) from Brazil, as well as establishing a common ancestor clade with hemotropic Mycoplasma spp. from the Neotropical bats in Belize and Peru and an Old World bat in Spain. The 23S rRNA analyses likewise evidenced that ‘Ca. M. haematomacacae’ formed a sister clade with hemotropic Mycoplasma spp. in Neotropical bats from Belize and Panama. Thus, the present findings, based on multi-locus sequence analysis, suggest a potential origin of ‘Ca. M. haematomacacae’ from Neotropical and Old World bats. To the best of the authors’ knowledge, this study provided the largest dataset so far of multi-locus genetic sequences of ‘Ca. M. haematomacacae’ isolated from Thai macaques and enhanced the accuracy of phylogenetic analyses, providing insights into their origins among hemotropic Mycoplasma spp. discovered worldwide.

Genetic insights of H9N2 avian influenza viruses circulating in Mali and phylogeographic patterns in Northern and Western Africa.

Avian influenza viruses (AIVs) of the H9N2 subtype have become widespread in Western Africa since their first detection in 2017 in Burkina Faso. However, the genetic characteristics and diffusion patterns of the H9N2 virus remain poorly understood in Western Africa, mainly due to limited surveillance activities. In addition, Mali, a country considered to play an important role in the epidemiology of AIVs in the region, lacks more comprehensive data on the genetic characteristics of these viruses, especially the H9N2 subtype. To better understand the genetic characteristics and spatio-temporal dynamics of H9N2 virus within this region, we carried out a comprehensive genetic characterization of H9N2 viruses collected through active surveillance in live bird markets in Mali between 2021 and 2022. We also performed a continuous phylogeographic analysis to unravel the dispersal history of H9N2 lineages between Northern and Western Africa. The identified Malian H9N2 virus belonged to the G1 lineage, similar to viruses circulating in both Western and Northern Africa, and possessed multiple molecular markers associated with an increased potential for zoonotic transmission and virulence. Notably, some Malian strains carried the R-S-N-R motif at their cleavage site, mainly observed in H9N2 strains in Asia. Our continuous phylogeographic analysis revealed a single and significant long-distance lineage dispersal event of the H9N2 virus to Western Africa, likely to have originated from Morocco in 2015, shaping the westward diffusion of the H9N2 virus. Our study highlights the need for long-term surveillance of H9N2 viruses in poultry populations in Western Africa, which is crucial for a better understanding of virus evolution and effective management against potential zoonotic AIV strain emergence.

Immuno-Diagnostic Interest in Monitoring CD16+CD56+ (Natural Killer) Cells and CD19+CD45+ (B Lymphocytes) in Individuals Newly Diagnosed with HIV in a Tertiary Care Center.

Monitoring multiple cellular markers of immune cells may provide a more accurate evaluation of the immune status of people living with human immunodeficiency virus (PLHIV). This study assessed the value of CD16+CD56+ cells (NK cells) and CD19+ lymphocytes (B cells) phenotyping in indicating viral load, AIDS status, and treatment efficacy. A retrospective, laboratory-based study was conducted at the Diagnostic immunology division of a referral tertiary hospital. It involved 82 newly diagnosed HIV patients treated between 2009-2016. We explored three objectives: (1) the paired change in CD16+CD56+ and CD19+CD45+ cells counts and percentages from baseline to 2-to-6 months after treatment; (2) the association of these phenotypes with 5 gradual categories of viral load; and (3) the accuracy of CD16+CD56+ and CD19+CD45+ cells counts in indicating AIDS stage defined as CD4+ < 200 cells/mm3. The second and third objectives were tested using a pooled analysis (N = 300-373). The median CD19+CD45+ and CD16+CD56+ counts increased by 1.9-fold and 1.3-fold after treatment respectively (p < 0.001). A negative correlation of viral load with both CD16+CD56+ (ρ = -0.29, p < 0.001) and CD19+CD45+ (ρ = -0.34, p < 0.001) counts was observed. CD16+CD56+ count < 73 cells/mm3 and CD19+CD45+ count < 166.5 were indicative for AIDS with 95.5% and 63.6% sensitivity respectively. Findings advocate for the usefulness of CD16+CD56+ and CD19+CD45+ phenotyping in characterizing the severity of HIV infection and its impact on both the humoral and cellular immunity, as well as monitoring the effectiveness of treatment.

Chemical profiling and quality evaluation of Liuweizhiji Gegen-Sangshen oral liquid by UPLC-Q-TOF-MS and HPLC-diode array detector fingerprinting.

Liuweizhiji Gegen-Sangshen (LGS) oral liquid is a Chinese patent medicine that is widely used for the prevention and treatment of alcoholic liver disease in clinical practice. However, the chemical complexity of LGS has not yet been investigated. The aim of this study was to rapidly identify chemical constituents of LGS and establish a quality control method based on fingerprint and quantitative analysis. A comprehensive strategy was used by combining qualitative analysis by ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and fingerprint analysis by high-performance liquid chromatography with diode array detection (HPLC-DAD). A total of 162 chemical components in LGS, including 91 flavonoids, 31 organic acids, and 20 phenolic compounds, were identified or preliminarily characterized in both positive and negative ion modes based on the UPLC-Q-TOF-MS results. Of these, 37 were confirmed with the reference standards. In fingerprint analysis, 23 peaks were chosen as common peaks and used to evaluate the similarity of different batches of LGS. Subsequently, a rapid quantification method was optimized and validated for the simultaneous determination of multiple chemical markers in LGS. The validated quantitative method was successfully used to analyze different batches of LGS samples. The proposed comprehensive strategy combining HPLC-DAD fingerprinting and multi-component quantification demonstrated satisfactory results with high efficiency, accuracy, and reliability. This can be used as a reference for the overall quality consistency evaluation of Chinese patent medicines.

Multiplex electrochemical sensing platforms for the detection of breast cancer biomarkers.

Herein, advancements in electroanalytical devices for the simultaneous detection of diverse breast cancer (BC) markers are demonstrated. This article identifies several important areas of exploration for electrochemical diagnostics and highlights important factors that are pivotal for the successful deployment of novel bioanalytical devices. We have highlighted that the limits of detection (LOD) reported for the multiplex electrochemical biosensor can surpass the sensitivity displayed by current clinical standards such as ELISA, FISH, and PCR. HER-2; a breast cancer marker characterised by increased metastatic potential, more aggressive development, and poor clinical outcomes; can be sensed with a LOD of 0.5 ng/ml using electrochemical multiplex platforms, which falls within the range of that measured by ELISA (from picogram/ml to nanogram/ml). Electrochemical multiplex biosensors are reported with detection limits of 0.53 ng/ml and 0.21 U/ml for MUC-1 and CA 15-3, respectively, or 5.8 × 10-3 U/ml for CA 15-3 alone. The sensitivity of electrochemical assays is improved when compared to conventional analysis of MUC-1 protein which is detected at 11-12 ng/ml, and ≤30 U/ml for CA 15-3 in the current clinical blood tests. The LOD for micro-ribonucleic acid (miRNA) biomarkers analyzed by electrochemical multiplex assays were all notedly superior at 9.79 × 10-16 M, 3.58 × 10-15 M, and 2.54 × 10-16 M for miRNA-155, miRNA-21, and miRNA-16, respectively. The dogma in miRNA testing is the qRT-PCR method, which reports ranges in the ng/ml level for the same miRNAs. Breast cancer exosomes, which are being explored as a new frontier of biosensing, have been detected electrochemically with an LOD of 103-108 particles/mL and can exceed detection limits seen by the tracking and analysis of nanoparticles (∼ 107 particles/ml), flow cytometry, Western blotting and ELISA, etc. A range of concentration at 78-5,000 pg/ml for RANKL and 16-1,000 pg/ml for TNF is reported for ELISA assay while LOD values of 2.6 and 3.0 pg/ml for RANKL and TNF, respectively, are demonstrated by the electrochemical dual immunoassay platform. Finally, EGFR and VEGF markers can be quantified at much lower concentrations (0.01 and 0.005 pg/ml for EGFR and VEGF, respectively) as compared to their ELISA assays (EGRF at 0.31-20 ng/ml and VEGF at 31.3-2,000 pg/ml). In this study we hope to answer several questions: (1) Are the limits of detection (LODs) reported for multiplex electrochemical biosensors of clinical relevance and how do they compare to well-established methods like ELISA, FISH, or PCR? (2) Can a single sensor electrode be used for the detection of multiple markers from one blood drop? (3) What mechanism of electrochemical biosensing is the most promising, and what technological advancements are needed to utilize these devices for multiplex POC detection? (4) Can nanotechnology advance the sensitive and selective diagnostics of multiple BC biomarkers? (5) Are there preferred receptors (antibody, nucleic acid or their combinations) and preferred biosensor designs (complementary methods, sandwich-type protocols, antibody/aptamer concept, label-free protocol)? (6) Why are we still without FDA-approved electrochemical multiplex devices for BC screening?