The Expression of Forkhead Box P3 T Regulatory Lymphocytes as a Prognostic Factor in Malignant Melanomas.

Since transcription factor Forkhead Box P3 (FoxP3) was identified as a specific regulatory T cell (Treg) marker, researchers have scrutinized its value as a potential novel therapeutic target or a prognostic factor in various types of cancer with inconsistent results. The present analysis was performed to assess the influence of Treg FoxP3 expression on the prognosis of primary melanoma and to evaluate the correlations with various clinicopathological prognostic factors. We analyzed all eligible patients with stage pT3 primary malignant melanomas treated in a tertiary cancer center. Immunohistochemical staining for Treg FoxP3 expression was performed on retrospectively identified paraffin blocks and subsequently correlated with the outcomes of the patients. A total of 81% of the patients presented a positive Treg FoxP3 expression, being correlated with a higher risk of lymph node metastasis, tumor relapse, and death. Moreover, positive expression was statistically associated with a shorter OS. The tumor relapse rate was estimated at 36.7%. A positive expression of Treg FoxP3 and lymph node metastasis were associated with a higher risk of death based on multivariate analysis. Treg FoxP3 expression may be used as an independent prognostic factor in patients with malignant melanoma to evaluate tumor progression and survival.

CT Angiography-Guided Needle Insertion for Interstitial Brachytherapy in Locally Advanced Cervical Cancer.

CT angiography might be a suitable procedure to avoid arterial puncture in combined intracavitary and interstitial brachytherapy for cervical cancer curatively treated with combined chemoradiation and brachytherapy boost. Data in the literature about this technique are scarce. We introduced this method and collected brachytherapy data from patients treated in our department between May 2021 and April 2024. We analyzed the applicator subtype, needle insertion (planned versus implanted), implanted depth and the role of CT angiography in selecting needle trajectories and insertion depths. None of the patients managed through this protocol experienced atrial puncture and consequent hemorrhage. Needle positions were accurately selected with the aid of CT angiography with proper coverage of brachytherapy targets and avoidance of organs at risk. CT angiography is a promising method for guiding needle insertion during interstitial brachytherapy.

The transcriptional landscape of cancer stem-like cell functionality in breast cancer.

BACKGROUND: Cancer stem-like cells (CSCs) have been extensively researched as the primary drivers of therapy resistance and tumor relapse in patients with breast cancer. However, due to lack of specific molecular markers, increased phenotypic plasticity and no clear clinicopathological features, the assessment of CSCs presence and functionality in solid tumors is challenging. While several potential markers, such as CD24/CD44, have been proposed, the extent to which they truly represent the stem cell potential of tumors or merely provide static snapshots is still a subject of controversy. Recent studies have highlighted the crucial role of the tumor microenvironment (TME) in influencing the CSC phenotype in breast cancer. The interplay between the tumor and TME induces significant changes in the cancer cell phenotype, leading to the acquisition of CSC characteristics, therapeutic resistance, and metastatic spread. Simultaneously, CSCs actively shape their microenvironment by evading immune surveillance and attracting stromal cells that support tumor progression. METHODS: In this study, we associated in vitro mammosphere formation assays with bulk tumor microarray profiling and deconvolution algorithms to map CSC functionality and the microenvironmental landscape in a large cohort of 125 breast tumors. RESULTS: We found that the TME score was a significant factor associated with CSC functionality. CSC-rich tumors were characterized by an immune-suppressed TME, while tumors devoid of CSC potential exhibited high immune infiltration and activation of pathways involved in the immune response. Gene expression analysis revealed IFNG, CXCR5, CD40LG, TBX21 and IL2RG to be associated with the CSC phenotype and also displayed prognostic value for patients with breast cancer. CONCLUSION: These results suggest that the characterization of CSCs content and functionality in tumors can be used as an attractive strategy to fine-tune treatments and guide clinical decisions to improve patients therapy response.

Tryptophan residues in TDP-43 and SOD1 modulate the cross-seeding and toxicity of SOD1.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons. Neuronal superoxide dismutase-1 (SOD1) inclusion bodies are characteristic of familial ALS with SOD1 mutations, while a hallmark of sporadic ALS is inclusions containing aggregated WT TAR DNA-binding protein 43 (TDP-43). We show here that co-expression of mutant or WT TDP-43 with SOD1 leads to misfolding of endogenous SOD1 and aggregation of SOD1 reporter protein SOD1G85R-GFP in human cell cultures and promotes synergistic axonopathy in zebrafish. Intriguingly, this pathological interaction is modulated by natively solvent-exposed tryptophans in SOD1 (tryptophan-32) and TDP-43 RNA-recognition motif RRM1 (tryptophan-172), in concert with natively sequestered TDP-43 N-terminal domain tryptophan-68. TDP-43 RRM1 intrabodies reduce WT SOD1 misfolding in human cell cultures, via blocking tryptophan-172. Tryptophan-68 becomes antibody-accessible in aggregated TDP-43 in sporadic ALS motor neurons and cell culture. 5-fluorouridine inhibits TDP-43-induced G85R-GFP SOD1 aggregation in human cell cultures and ameliorates axonopathy in zebrafish, via its interaction with SOD1 tryptophan-32. Collectively, our results establish a novel and potentially druggable tryptophan-mediated mechanism whereby two principal ALS disease effector proteins might directly interact in disease.

Exploring the Influence of Zinc Ions on the Conformational Stability and Activity of Protein Disulfide Isomerase.

The interplay between metal ion binding and the activity of thiol proteins, particularly within the protein disulfide isomerase family, remains an area of active investigation due to the critical role that these proteins play in many vital processes. This research investigates the interaction between recombinant human PDIA1 and zinc ions, focusing on the subsequent implications for PDIA1's conformational stability and enzymatic activity. Employing isothermal titration calorimetry and differential scanning calorimetry, we systematically compared the zinc binding capabilities of both oxidized and reduced forms of PDIA1 and assessed the structural consequences of this interaction. Our results demonstrate that PDIA1 can bind zinc both in reduced and oxidized states, but with significantly different stoichiometry and more pronounced conformational effects in the reduced form of PDIA1. Furthermore, zinc binding was observed to inhibit the catalytic activity of reduced-PDIA1, likely due to induced alterations in its conformation. These findings unveil a potential regulatory mechanism in PDIA1, wherein metal ion binding under reductive conditions modulates its activity. Our study highlights the potential role of zinc in regulating the catalytic function of PDIA1 through conformational modulation, suggesting a nuanced interplay between metal binding and protein stability in the broader context of cellular redox regulation.

Mammographic assessment of breast density as a tool for predicting the response to neoadjuvant therapy in breast cancer patients.

Background and aims: Breast cancer (BC) is the most frequently diagnosed cancer and the leading cause of cancer-related death among women worldwide. For locally advanced diseases and high-risk tumors, neoadjuvant therapy (NAT) is the treatment of choice. Some studies show that mammographic density (MD) tumor margins and the presence of microcalcifications play a prognostic role in BC patients. Hence, the objective of this retrospective study was to assess if MD could predict the response to NAT among different molecular subtypes of BC patients undergoing NAT at The "Prof. Dr I. Chiricuta" Oncology Institute of Cluj-Napoca, Romania (IOCN). Furthermore, the association between MD, tumor margins and the presence of microcalcifications with clinico-pathological data was analyzed. Methods: Eighty-four breast cancer patients diagnosed and treated at IOCN were included in this study. The morphological characteristics of the tumors were framed according to the BIRADS lexicon. The presence or absence of microcalcifications was also assessed. First, the significance of associations between breast density, margins and microcalcifications and clinico-pathological parameters of the patients were tested with Fisher or Fisher-Freeman-Halton Exact Test. Next, using multinomial logistic regression, we modelled the associations between the pathological response measured by Miller Payne and Residual cancer burden (RCB) systems and the BI-RADS. Variables having significant univariate tests were selected as candidates for the multivariable analysis (adjusted model). Results: Breast densities were significantly associated with the age of the patients (p=0.01), number of positive lymph nodes (p=0.037), margins (p=0.002) and combined categories of Miller-Payne (p=0.034) and RCB pathological response (p=0.021). Margins was significantly associated with ki67 proliferation index (p=0.029), estrogen receptor (ER) (p=0.007), progesterone receptor (PR) (p=0.019), molecular subtype (p<0.001) and the number of clinically observed positive lymph nodes at diagnosis (p=0.019). Conclusions: In our cohort, BC patients with lower MD had higher odds of achieving pCR following NAT, suggesting the role of MD as a clinical prognostic marker. Larger multicenter studies are warranted to validate the prognostic value of MD, which could aid in patients stratification based on their likelihood to respond to NAT.

Glioblastoma and radiotherapy: A multicenter AI study for Survival Predictions from MRI (GRASP study).

BACKGROUND: The aim was to predict survival of glioblastoma at 8 months after radiotherapy (a period allowing for completing a typical course of adjuvant temozolomide), by applying deep learning to the first brain MRI after radiotherapy completion. METHODS: Retrospective and prospective data were collected from 206 consecutive glioblastoma, isocitrate dehydrogenase -wildtype patients diagnosed between March 2014 and February 2022 across 11 UK centers. Models were trained on 158 retrospective patients from 3 centers. Holdout test sets were retrospective (n = 19; internal validation), and prospective (n = 29; external validation from 8 distinct centers). Neural network branches for T2-weighted and contrast-enhanced T1-weighted inputs were concatenated to predict survival. A nonimaging branch (demographics/MGMT/treatment data) was also combined with the imaging model. We investigated the influence of individual MR sequences; nonimaging features; and weighted dense blocks pretrained for abnormality detection. RESULTS: The imaging model outperformed the nonimaging model in all test sets (area under the receiver-operating characteristic curve, AUC P = .038) and performed similarly to a combined imaging/nonimaging model (P > .05). Imaging, nonimaging, and combined models applied to amalgamated test sets gave AUCs of 0.93, 0.79, and 0.91. Initializing the imaging model with pretrained weights from 10 000s of brain MRIs improved performance considerably (amalgamated test sets without pretraining 0.64; P = .003). CONCLUSIONS: A deep learning model using MRI images after radiotherapy reliably and accurately determined survival of glioblastoma. The model serves as a prognostic biomarker identifying patients who will not survive beyond a typical course of adjuvant temozolomide, thereby stratifying patients into those who might require early second-line or clinical trial treatment.

Usefulness of Saline Sealing in Preventing Pneumothorax after CT-Guided Biopsies of the Lung.

This study aimed to assess the effectiveness of saline sealing in reducing the incidence of pneumothorax after a CT-guided lung biopsy. This was a retrospective case-control study of patients who underwent CT-guided biopsies for lung tumors using 18 G semiautomatic core needles in conjunction with 17 G coaxial needles. The patients were divided into two consecutive groups: a historical Group A (n = 111), who did not receive saline sealing, and Group B (n = 87), who received saline sealing. In Group B, NaCl 0.9% was injected through the coaxial needle upon its removal. The incidence of pneumothorax and chest tube insertion was compared between the two groups. Multivariate logistic regression was performed to verify the contribution of other pneumothorax risk factors. The study included 198 patients, with 111 in Group A and 87 in Group B. There was a significantly (p = 0.02) higher pneumothorax rate in Group A (35.1%, n = 39) compared to Group B (20.7%, n = 18). The difference regarding chest tube insertion was not significant (p = 0.1), despite a tendency towards more insertions in Group A (5.4%, n = 6), compared to Group B (1.1%, n = 1). Among the risk factors for pneumothorax, only the presence of emphysema (OR = 3.5, p = 0.0007) and belonging to Group A (OR = 2.2, p = 0.02) were significant. Saline sealing of the needle tract after a CT-guided lung biopsy can significantly reduce the incidence of pneumothorax. This technique is safe, readily available, and inexpensive, and should be considered as a routine preventive measure during this procedure.

Whole-Tumor ADC Texture Analysis Is Able to Predict Breast Cancer Receptor Status.

There are different breast cancer molecular subtypes with differences in incidence, treatment response and outcome. They are roughly divided into estrogen and progesterone receptor (ER and PR) negative and positive cancers. In this retrospective study, we included 185 patients augmented with 25 SMOTE patients and divided them into two groups: the training group consisted of 150 patients and the validation cohort consisted of 60 patients. Tumors were manually delineated and whole-volume tumor segmentation was used to extract first-order radiomic features. The ADC-based radiomics model reached an AUC of 0.81 in the training cohort and was confirmed in the validation set, which yielded an AUC of 0.93, in differentiating ER/PR positive from ER/PR negative status. We also tested a combined model using radiomics data together with ki67% proliferation index and histological grade, and obtained a higher AUC of 0.93, which was also confirmed in the validation group. In conclusion, whole-volume ADC texture analysis is able to predict hormonal status in breast cancer masses.

Texture Analysis in Uterine Cervix Carcinoma: Primary Tumour and Lymph Node Assessment.

The conventional magnetic resonance imaging (MRI) evaluation and staging of cervical cancer encounters several pitfalls, partially due to subjective evaluations of medical images. Fifty-six patients with histologically proven cervical malignancies (squamous cell carcinomas, n = 42; adenocarcinomas, n = 14) who underwent pre-treatment MRI examinations were retrospectively included. The lymph node status (non-metastatic lymph nodes, n = 39; metastatic lymph nodes, n = 17) was assessed using pathological and imaging findings. The texture analysis of primary tumours and lymph nodes was performed on T2-weighted images. Texture parameters with the highest ability to discriminate between the two histological types of primary tumours and metastatic and non-metastatic lymph nodes were selected based on Fisher coefficients (cut-off value > 3). The parameters' discriminative ability was tested using an k nearest neighbour (KNN) classifier, and by comparing their absolute values through an univariate and receiver operating characteristic analysis. Results: The KNN classified metastatic and non-metastatic lymph nodes with 93.75% accuracy. Ten entropy variations were able to identify metastatic lymph nodes (sensitivity: 79.17-88%; specificity: 93.48-97.83%). No parameters exceeded the cut-off value when differentiating between histopathological entities. In conclusion, texture analysis can offer a superior non-invasive characterization of lymph node status, which can improve the staging accuracy of cervical cancers.

The Role of miR-375-3p, miR-210-3p and Let-7e-5p in the Pathological Response of Breast Cancer Patients to Neoadjuvant Therapy.

Background and Objectives: Prediction of response to therapy remains a continuing challenge in treating breast cancer, especially for identifying molecular tissue markers that best characterize resistant tumours. Microribonucleic acids (miRNA), known as master modulators of tumour phenotype, could be helpful candidates for predicting drug resistance. We aimed to assess the association of miR-375-3p, miR-210-3p and let-7e-5p in breast cancer tissues with pathological response to neoadjuvant therapy (NAT) and clinicopathological data. Material and methods: Sixty female patients diagnosed with invasive breast cancer at The Oncology Institute "Ion Chiricuța", Cluj-Napoca, Romania (IOCN) were included in this study. Before patients received any treatment, fresh breast tissue biopsies were collected through core biopsy under echographic guidance and processed for total RNA extraction and miRNA quantification. The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) database was used as an independent external validation cohort. Results: miR-375-3p expression was associated with more differentiated tumours, hormone receptor presence and lymphatic invasion. According to the Miller-Payne system, a higher miR-375-3p expression was calculated for patients that presented with intermediate versus (vs.) no pathological response. Higher miR-210-3p expression was associated with an improved response to NAT in both Miller-Payne and RCB evaluation systems. Several druggable mRNA targets were correlated with miR-375-3p and miR-210-3p expression, with upstream analysis using the IPA knowledge base revealing a list of possible chemical and biological targeting drugs. Regarding let-7e-5p, no significant association was noticed with any of the analysed clinicopathological data. Conclusions: Our results suggest that tumours with higher levels of miR-375-3p are more sensitive to neoadjuvant therapy compared to resistant tumours and that higher miR-210-3p expression in responsive tumours could indicate an excellent pathological response.

Circulating Small EVs miRNAs as Predictors of Pathological Response to Neo-Adjuvant Therapy in Breast Cancer Patients.

Neo-adjuvant therapy (NAT) is increasingly used in the clinic for the treatment of breast cancer (BC). Pathological response to NAT has been associated with improved patients' survival; however, the current techniques employed for assessing the tumor response have significant limitations. Small EVs (sEVs)-encapsulated miRNAs have emerged as promising new biomarkers for diagnosis and prediction. Therefore, our study aims to explore the predictive value of these miRNAs for the pathological response to NAT in BC. By employing bioinformatic tools, we selected a set of miRNAs and evaluated their expression in plasma sEVs and BC biopsies. Twelve miRNAs were identified in sEVs, of which, miR-21-5p, 221-3p, 146a-5p and 26a-5p were significantly associated with the Miller-Payne (MP) pathological response to NAT. Moreover, miR-21-5p, 146a-5p, 26a-5p and miR-24-3p were independent as predictors of MP response to NAT. However, the expression of these miRNAs showed no correlation between sEVs and tissue samples, indicating that the mechanisms of miRNA sorting into sEVs still needs to be elucidated. Functional analysis of miRNA target genes and drug interactions revealed that candidate miRNAs and their targets, can be regulated by different NAT regimens. This evidence supports their role in governing the patients' therapy response and highlights their potential use as prediction biomarkers.

Rational Generation of Monoclonal Antibodies Selective for Pathogenic Forms of Alpha-Synuclein.

Misfolded toxic forms of alpha-synuclein (α-Syn) have been implicated in the pathogenesis of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). The α-Syn oligomers and soluble fibrils have been shown to mediate neurotoxicity and cell-to-cell propagation of pathology. To generate antibodies capable of selectively targeting pathogenic forms of α-Syn, computational modeling was used to predict conformational epitopes likely to become exposed on oligomers and small soluble fibrils, but not on monomers or fully formed insoluble fibrils. Cyclic peptide scaffolds reproducing these conformational epitopes exhibited neurotoxicity and seeding activity, indicating their biological relevance. Immunization with the conformational epitopes gave rise to monoclonal antibodies (mAbs) with the desired binding profile showing selectivity for toxic α-Syn oligomers and soluble fibrils, with little or no reactivity with monomers, physiologic tetramers, or Lewy bodies. Recognition of naturally occurring soluble α-Syn aggregates in brain extracts from DLB and MSA patients was confirmed by surface plasmon resonance (SPR). In addition, the mAbs inhibited the seeding activity of sonicated pre-formed fibrils (PFFs) in a thioflavin-T fluorescence-based aggregation assay. In neuronal cultures, the mAbs protected primary rat neurons from toxic α-Syn oligomers, reduced the uptake of PFFs, and inhibited the induction of pathogenic phosphorylated aggregates of endogenous α-Syn. Protective antibodies selective for pathogenic species of α-Syn, as opposed to pan α-Syn reactivity, are expected to provide enhanced safety and therapeutic potency by preserving normal α-Syn function and minimizing the diversion of active antibody from the target by the more abundant non-toxic forms of α-Syn in the circulation and central nervous system.

Optimizing Epitope Conformational Ensembles Using α-Synuclein Cyclic Peptide "Glycindel" Scaffolds: A Customized Immunogen Method for Generating Oligomer-Selective Antibodies for Parkinson's Disease.

Effectively presenting epitopes on immunogens, in order to raise conformationally selective antibodies through active immunization, is a central problem in treating protein misfolding diseases, particularly neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease. We seek to selectively target conformations enriched in toxic, oligomeric propagating species while sparing the healthy forms of the protein that are often more abundant. To this end, we computationally modeled scaffolded epitopes in cyclic peptides by inserting/deleting a variable number of flanking glycines ("glycindels") to best mimic a misfolding-specific conformation of an epitope of α-synuclein enriched in the oligomer ensemble, as characterized by a region most readily disordered and solvent-exposed in a stressed, partially denatured protofibril. We screen and rank the cyclic peptide scaffolds of α-synuclein in silico based on their ensemble overlap properties with the fibril, oligomer-model and isolated monomer ensembles. We present experimental data of seeded aggregation that support nucleation rates consistent with computationally predicted cyclic peptide conformational similarity. We also introduce a method for screening against structured off-pathway targets in the human proteome by selecting scaffolds with minimal conformational similarity between their epitope and the same solvent-exposed primary sequence in structured human proteins. Different cyclic peptide scaffolds with variable numbers of glycines are predicted computationally to have markedly different conformational ensembles. Ensemble comparison and overlap were quantified by the Jensen-Shannon divergence and a new measure introduced here, the embedding depth, which determines the extent to which a given ensemble is subsumed by another ensemble and which may be a more useful measure in developing immunogens that confer conformational selectivity to an antibody.

Prognostic factors for in-transit metastasis in patients with malignant melanoma.

Background and aims: Malignant melanoma represents an aggressive and unpredictable malignancy, with high locoregional recurrence rates, regardless of tumor stage and therapeutic management. This study aims to identify the main histopathological prognostic factors involved in the development of in-transit metastasis in patients with malignant melanoma. Methods: The study includes only patients that were diagnosed with malignant melanoma and with histologically confirmed in-transit metastasis who were treated in a comprehensive cancer center between 2010-2021. Histopathological parameters were investigated, univariate and multivariate analysis was performed. Results: A total of 26 patients were included in the analysis. On univariate and multivariate analysis, only primary cutaneous melanomas located on the thorax correlated with the risk of developing in-transit metastasis, whereas clinicopathological factors such as an increased Breslow thickness and Clark level, the presence of ulceration, positive lymph nodes, a non-brisk TIL density, a high mitotic rate, a nodular subtype, and age >50 years may represent risk factors, even though we could not find any correlations. Conclusions: Primary cutaneous melanomas that arise on the thorax present a high risk for the occurrence of locoregional disease, whereas other clinicopathological characteristics could not be used to predict local recurrence. However, prospective and more extensive cohort studies are needed in order to validate these important prognostic factors.

Signet ring cell gastric carcinoma with breast and leptomeningeal metastases: a case report.

Gastric cancer is the 5th most common malignancy worldwide. Signet ring cell histology represents an aggressive subtype of gastric cancer, presenting at a younger age. Both breast and leptomeningeal metastases are rare locations of tumor dissemination, requiring correct and immediate diagnosis and treatment. We present a case of a 45-year old female with signet ring cell gastric carcinoma who developed both left breast and leptomeningeal metastases, requiring multiple chemotherapy lines. As far as we know, this is the first published case in literature following multiple lines of treatment for both breast and leptomeningeal metastases from signet ring cell gastric carcinoma.

Imaging Biomarkers of Glioblastoma Treatment Response: A Systematic Review and Meta-Analysis of Recent Machine Learning Studies.

OBJECTIVE: Monitoring biomarkers using machine learning (ML) may determine glioblastoma treatment response. We systematically reviewed quality and performance accuracy of recently published studies. METHODS: Following Preferred Reporting Items for Systematic Reviews and Meta-Analysis: Diagnostic Test Accuracy, we extracted articles from MEDLINE, EMBASE and Cochrane Register between 09/2018-01/2021. Included study participants were adults with glioblastoma having undergone standard treatment (maximal resection, radiotherapy with concomitant and adjuvant temozolomide), and follow-up imaging to determine treatment response status (specifically, distinguishing progression/recurrence from progression/recurrence mimics, the target condition). Using Quality Assessment of Diagnostic Accuracy Studies Two/Checklist for Artificial Intelligence in Medical Imaging, we assessed bias risk and applicability concerns. We determined test set performance accuracy (sensitivity, specificity, precision, F1-score, balanced accuracy). We used a bivariate random-effect model to determine pooled sensitivity, specificity, area-under the receiver operator characteristic curve (ROC-AUC). Pooled measures of balanced accuracy, positive/negative likelihood ratios (PLR/NLR) and diagnostic odds ratio (DOR) were calculated. PROSPERO registered (CRD42021261965). RESULTS: Eighteen studies were included (1335/384 patients for training/testing respectively). Small patient numbers, high bias risk, applicability concerns (particularly confounding in reference standard and patient selection) and low level of evidence, allow limited conclusions from studies. Ten studies (10/18, 56%) included in meta-analysis gave 0.769 (0.649-0.858) sensitivity [pooled (95% CI)]; 0.648 (0.749-0.532) specificity; 0.706 (0.623-0.779) balanced accuracy; 2.220 (1.560-3.140) PLR; 0.366 (0.213-0.572) NLR; 6.670 (2.800-13.500) DOR; 0.765 ROC-AUC. CONCLUSION: ML models using MRI features to distinguish between progression and mimics appear to demonstrate good diagnostic performance. However, study quality and design require improvement.

SERS-based DNA methylation profiling allows the differential diagnosis of malignant lymphadenopathy.

This study highlights the potential of surface-enhanced Raman scattering (SERS) to differentiate between B-cell lymphoma (BCL), T-cell lymphoma (TCL), lymph node metastasis of melanoma (Met) and control (Ctr) samples based on the specific SERS signal of DNA extracted from lymph node tissue biopsy. Differences in the methylation profiles as well as the specific interaction of malignant and non-malignant DNA with the metal nanostructure are captured in specific variations of the band at 1005 cm-1, attributed to 5-methylcytosine and the band at 730 cm-1, attributed to adenine. Thus, using the area ratio of these two SERS marker bands as input for univariate classification, an area under the curve (AUC) of 0.70 was achieved in differentiating between malignant and non-malignant DNA. In addition, DNA from the BCL and TCL groups exhibited differences in the area of the SERS band at 730 cm-1, yielding an AUC of 0.84 in differentiating between these two lymphadenopathies. Lastly, using multivariate data analysis techniques, an overall accuracy of 94.7% was achieved in the differential diagnosis between the BCL, TCL, Met and Ctr groups. These results pave the way towards the implementation of SERS as a novel tool in the clinical setting for improving the diagnosis of malignant lymphadenopathy.

Gamma-Synuclein Dysfunction Causes Autoantibody Formation in Glaucoma Patients and Dysregulation of Intraocular Pressure in Mice.

Dysregulation of intraocular pressure (IOP) is one of the main risk factors for glaucoma. γ-synuclein is a member of the synuclein family of widely expressed synaptic proteins within the central nervous system that are implicated in certain types of neurodegeneration. γ-synuclein expression and localization changes in the retina and optic nerve of patients with glaucoma. However, the mechanisms by which γ-synuclein could contribute to glaucoma are poorly understood. We assessed the presence of autoantibodies to γ-synuclein in the blood serum of patients with primary open-angle glaucoma (POAG) by immunoblotting. A positive reaction was detected for five out of 25 patients (20%) with POAG. Autoantibodies to γ-synuclein were not detected in a group of patients without glaucoma. We studied the dynamics of IOP in response to IOP regulators in knockout mice (γ-KO) to understand a possible link between γ-synuclein dysfunction and glaucoma-related pathophysiological changes. The most prominent decrease of IOP in γ-KO mice was observed after the instillation of 1% phenylephrine and 10% dopamine. The total protein concentration in tear fluid of γ-KO mice was approximately two times higher than that of wild-type mice, and the activity of neurodegeneration-linked protein α2-macroglobulin was reduced. Therefore, γ-synuclein dysfunction contributes to pathological processes in glaucoma, including dysregulation of IOP.

Mechanism of Zn2+ and Ca2+ Binding to Human S100A1.

S100A1 is a member of the S100 family of small ubiquitous Ca2+-binding proteins, which participates in the regulation of cell differentiation, motility, and survival. It exists as homo- or heterodimers. S100A1 has also been shown to bind Zn2+, but the molecular mechanisms of this binding are not yet known. In this work, using ESI-MS and ITC, we demonstrate that S100A1 can coordinate 4 zinc ions per monomer, with two high affinity (KD~4 and 770 nm) and two low affinity sites. Using competitive binding experiments between Ca2+ and Zn2+ and QM/MM molecular modeling we conclude that Zn2+ high affinity sites are located in the EF-hand motifs of S100A1. In addition, two lower affinity sites can bind Zn2+ even when the EF-hands are saturated by Ca2+, resulting in a 2Ca2+:S100A1:2Zn2+ conformer. Finally, we show that, in contrast to calcium, an excess of Zn2+ produces a destabilizing effect on S100A1 structure and leads to its aggregation. We also determined a higher affinity to Ca2+ (KD~0.16 and 24 µm) than was previously reported for S100A1, which would allow this protein to function as a Ca2+/Zn2+-sensor both inside and outside cells, participating in diverse signaling pathways under normal and pathological conditions.