Peripheral Blood CD8+ T-Lymphocyte Immune Response in Benign and Subpopulations of Breast Cancer Patients.

Peripheral blood CD8+ T lymphocytes play a crucial role in cell-mediated immunity and tumor-related immune responses in breast cancer. In this study, label-free quantification analysis and gene set enrichment analysis (GSEA) of CD8+ T lymphocytes in the peripheral blood of benign patients and patients with different breast cancer (BC) subtypes, i.e., luminal A, luminal B, and triple-negative breast cancer (TNBC), were performed using nano-UHPLC and Orbitrap mass spectrometry. Differential protein expression in CD8+ T lymphocytes revealed significant downregulation (log2 FC ≥ 0.38 or ≤-0.38, adj. p < 0.05), particularly in proteins involved in cytotoxicity, cytolysis, and proteolysis, such as granzymes (GZMs) and perforin 1 (PRF1). This downregulation was observed in the benign group (GZMH, GZMM, and PRF1) and luminal B (GZMA, GZMH) subtypes, whereas granzyme K (GZMK) was upregulated in TNBC in comparison to healthy controls. The RNA degradation pathway was significantly downregulated (p < 0.05, normalized enrichment score (NES) from -1.47 to -1.80) across all BC subtypes, suggesting a potential mechanism for regulating gene expression during T cell activation. Also, the Sm-like proteins (LSM2, LSM3, and LSM5) were significantly downregulated in the RNA degradation pathway. Proteomic analysis of CD8+ T lymphocytes in peripheral blood across different breast cancer subtypes provides a comprehensive view of the molecular mechanisms of the systemic immune response that can significantly contribute to advancements in the diagnosis, treatment, and prognosis of this disease.

Differential Urinary Proteomic Analysis of High-Risk Cervical Intraepithelial Neoplasia.

Human papillomavirus (HPV)-associated lesions and malignancies exhibit alterations in the composition and functionality of the extracellular matrix (ECM) that represent the complex molecular pathways present between infection and disease. A total of 20 urine samples were used, including from 10 patients with cervical intraepithelial neoplasia grade 3 (CIN3) and 10 healthy controls to perform the label-free quantitative analysis using the nano-HPLC and ESI-MS ion trap mass analyzer and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) fast screening. Among 476 identified/quantified proteins, 48 were significantly changed (log2-fold change ≥1.0 or ≤-1.0, -log10 (bbinominal, p-value ≥ 1.3), of which were 40 proteins (down-regulated) and 8 proteins (up-regulated) in CIN3, in comparison to healthy controls. The biological function and key pathway enrichment of the gene set using gen set enrichment analysis (GSEA) were analyzed. The ECM-receptor interaction pathway (NES = -1.64, p = 0.026) was down-regulated by 13 proteins (HSPG2, COL6A1, COL6A3, SPP1, THBS1, TNC, DAG1, FN1, COMP, GP6, VTN, SDC1, and CD44; log2 FC range from -0.03 to -1.48) for the CIN3 group in the KEGG database. The MALDI-TOF/MS screening showed the difference of protein profiles between the control and CIN3 groups, i.e., using the scatter plot with a well-separated shape, as well as effectively distinguishing both groups (control and CIN3) using genetic algorithms (GA) with cross-validation (51.56%) and recognition capability (95.0%). Decreased levels of ECM-receptor interaction proteins may cause disturbances in the interactions of cells with the ECM and play an important role in the development and progression of cervical cancer.

The Role of ARHGAP1 in Rho GTPase Inactivation during Metastasizing of Breast Cancer Cell Line MCF-7 after Treatment with Doxorubicin.

Breast cancer is the most prevalent cancer type in women worldwide. It proliferates rapidly and can metastasize into farther tissues at any stage due to the gradual invasiveness and motility of the tumor cells. These crucial properties are the outcome of the weakened intercellular adhesion, regulated by small guanosine triphosphatases (GTPases), which hydrolyze to the guanosine diphosphate (GDP)-bound conformation. We investigated the inactivating effect of ARHGAP1 on Rho GTPases involved signaling pathways after treatment with a high dose of doxorubicin. Label-free quantitative proteomic analysis of the proteome isolated from the MCF-7 breast cancer cell line, treated with 1 µM of doxorubicin, identified RAC1, CDC42, and RHOA GTPases that were inactivated by the ARHGAP1 protein. Upregulation of the GTPases involved in the transforming growth factor-beta (TGF-beta) signaling pathway initiated epithelial-mesenchymal transitions. These findings demonstrate a key role of the ARHGAP1 protein in the disruption of the cell adhesion and simultaneously allow for a better understanding of the molecular mechanism of the reduced cell adhesion leading to the subsequent metastasis. The conclusions of this study corroborate the hypothesis that chemotherapy with doxorubicin may increase the risk of metastases in drug-resistant breast cancer cells.

Release of Monomers from Dental Composite Materials into Saliva and the Possibility of Reducing the Toxic Risk for the Patient.

Background and Objectives: The objective of this study was (1) to measure the amount of monomers released into the saliva depending on the time elapsed after the hardening of the composite and on the type of monomer used; and (2) with the prolongation of the light-curing procedure, to publish information on whether it would be possible to influence the level of leached monomers. Materials and Methods: HPLC technique was used to monitor the levels of the unpolymerized monomers Bis-GMA, Bis/EMA, TEGDMA, and UDMA from the four commonly used composite materials, released into the saliva of a volunteer with intact dentition. The levels were monitored in 3 time periods during 24 h after composite hardening. From every composite material, 4 samples were formed and cured with an LED lamp for 10 s, 20 s, 40 s, and 60 s. After the light curing, the same polishing procedure was used and the samples were leached in blank saliva samples. Results: We observed that every monitored composite material eluted monomers into the saliva after its application. The amount of monomers depended on the time elapsed after the curing of the composite and on the type of composite used. A 40 s LED curing procedure can reduce the amount of leached monomers in comparison with the standard 20 s procedure, especially for monomers of higher molecular weight. Conclusions: Our study confirmed the hypothesis that the release of monomers gradually decreases with increasing time after the hardening of the composite filling.

Human peripheral blood mononuclear cells: A review of recent proteomic applications.

Human peripheral blood mononuclear cells (PBMCs) represent a sentinel blood sample which reacts to different pathophysiological stimuli in the form of immunological responses/immunophenotypic changes. The study of molecular content of PBMCs can provide better understanding of immune processes giving the possibility of monitoring the health conditions of the host organism. Proteomic analysis of PBMCs can achieve mentioned goal as important immune-related biomarkers are easily accessible for analysis. PBMCs have been gaining attention in different research areas including preclinical or clinical investigations. In this review, recent applications of proteomic analysis of PBMCs are described and discussed. Approaches are divided based on different proteomic workflows such as in-gel, in-solution and on-filter modes. The effect of various diseases such as autoimmune, cancer, neurodegenerative, viral, metabolic, and various immune stimulations such as radiation, vaccine, corticosteroids over PBMCs proteome, are described with emphasis on promising protein biomarker candidates.

Evaluation of an Integrated Smart Sensor System for Real-Time Characterization and Digitalization of Postoperative Abdominal Drain Output: A Pilot Study.

Background: For centuries, surgeons have relied on surgical drains during postoperative care. Despite all advances in modern medicine and the area of digitalization, as of today, most if not all assessment of abdominal secretions excreted via surgical drains are carried out manually. We here introduce a novel integrated Smart Sensor System (Smart Drain) that allows for real-time characterization and digitalization of postoperative abdominal drain output at the patient's bedside. Methods: A prototype of the Smart Drain was developed using a sophisticated spectrometer for assessment of drain output. The prototype measures 10 × 6 × 6 cm and therefore easily fits at the bedside. At the time of measurement with our Smart Drain, the drain output was additionally sent off to be analyzed in our routine laboratory for typical markers of interest in abdominal surgery such as bilirubin, lipase, amylase, triglycerides, urea, protein, and red blood cells. A total of 45 samples from 19 patients were included. Results: The measurements generated were found to correlate with conventional laboratory measurements for bilirubin (r = .658, P = .000), lipase (r = .490, P = .002), amylase (r = .571, P = .000), triglycerides (r = .803, P = .000), urea (r = .326, P = .033), protein (r = .387, P = .012), and red blood cells (r = .904, P = .000). Conclusions: To our best knowledge, for the first time we describe a device using a sophisticated spectrometer that allows for real-time characterization and digitalization of postoperative abdominal drain output at the patient's bedside.

Automation of single-cell proteomic sample preparation.

Molecular heterogeneity exists at different spatial scales in biological samples and is an important parameter in the development of pathologies and resistances to therapies. When aiming to reach molecular heterogeneity of cells at extremely low spatial scales, single-cell analysis can be the ultimate choice. Proteomics performed in bulk population of cells (macroproteomics) is prone to mask molecular heterogeneity. Mass spectrometry-based single cell proteomics (SCP-MS) is the right solution to overcome this issue. Three main problems can be identified using SCP-MS: (i) analytical loss during sample preparation, (ii) inefficient microinjection/delivery of proteins/peptides from samples to MS and (iii) low analytical throughput. Technologies for automation of SCP have recently gained attention to improve methods accuracy, sensitivity, throughput and in-depth and low-biased proteome analysis. In this minireview, we therefore overview the state-of-the-art of automation of SCP-MS sample preparation approaches.

Microproteomic sample preparation.

Multiple applications of proteomics in life and health science, pathology and pharmacology, require handling size-limited cell and tissue samples. During proteomic sample preparation, analyte loss in these samples arises when standard procedures are used. Thus, specific considerations have to be taken into account for processing, that are summarised under the term microproteomics (µPs). Microproteomic workflows include: sampling (e.g., flow cytometry, laser capture microdissection), sample preparation (possible disruption of cells or tissue pieces via lysis, protein extraction, digestion in bottom-up approaches, and sample clean-up) and analysis (chromatographic or electrophoretic separation, mass spectrometric measurements and statistical/bioinformatic evaluation). All these steps must be optimised to reach wide protein dynamic ranges and high numbers of identifications. Under optimal conditions, sampling is adapted to the studied sample types and nature, sample preparation isolates and enriches the whole protein content, clean-up removes salts and other interferences such as detergents or chaotropes, and analysis identifies as many analytes as the instrumental throughput and sensitivity allow. In the suggested review, we present and discuss the current state in µP applications for processing of small number of cells (cell µPs) and microscopic tissue regions (tissue µPs).

Invadopodia Structure in 3D Environment Resolved by Near-Infrared Branding Protocol Combining Correlative Confocal and FIB-SEM Microscopy.

Cancer cell invasion through tissue barriers is the intrinsic feature of metastasis, the most life-threatening aspect of cancer. Detailed observation and analysis of cancer cell behaviour in a 3D environment is essential for a full understanding of the mechanisms of cancer cell invasion. The inherent limits of optical microscopy resolution do not allow to for in-depth observation of intracellular structures, such as invadopodia of invading cancer cells. The required resolution can be achieved using electron microscopy techniques such as FIB-SEM. However, visualising cells in a 3D matrix using FIB-SEM is challenging due to difficulties with localisation of a specific cell deep within the resin block. We have developed a new protocol based on the near-infrared branding (NIRB) procedure that extends the pattern from the surface grid deep inside the resin. This 3D burned pattern allows for precise trimming followed by targeted 3D FIB-SEM. Here we present detailed 3D CLEM results combining confocal and FIB-SEM imaging of cancer cell invadopodia that extend deep into the collagen meshwork.

Cell Viability Assessment Using Fluorescence Vital Dyes and Confocal Microscopy in Evaluating Freezing and Thawing Protocols Used in Cryopreservation of Allogeneic Venous Grafts.

The authors present their contribution to the improvement of methods suitable for the detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. They studied the post-thaw viability of cells of the wall of cryopreserved venous grafts (CVG) immediately after thawing and after 24 and 48 h culture at +37 °C in two groups of six CVG selected randomly for slow thawing in the refrigerator and rapid thawing in a water bath at +37 °C. The grafts were collected from multi-organ and tissue brain-dead donors, cryopreserved, and stored in a liquid nitrogen vapor phase for five years. The viability was assessed from tissue slices obtained by perpendicular and longitudinal cuts of the thawed graft samples using in situ staining with fluorescence vital dyes. The mean and median immediate post-thaw viability values above 70% were found in using both thawing protocols and both types of cutting. The statistically significant decline in viability after the 48-h culture was observed only when using the slow thawing protocol and perpendicular cutting. The possible explanation might be the "solution effect damage" during slow thawing, which caused a gentle reduction in the graft cellularity. The possible influence of this phenomenon on the immunogenicity of CVG should be the subject of further investigations.

The unfolded protein response controls endoplasmic reticulum stress-induced apoptosis of MCF-7 cells via a high dose of vitamin C treatment.

Recent in vitro studies have shown that vitamin C (Vit C) with pro-oxidative properties causes cytotoxicity of breast cancer cells by selective oxidative stress. However, the effect of Vit C in itself at different concentration levels on MCF-7 breast cancer cell line after 24 h, has not yet been described. We aimed to examine the effect of Vit C on the viability and signalling response of MCF-7/WT (MCF-7 wild-type) cells that were exposed to various concentrations (0.125-4 mM) of Vit C during 24 h. The cytotoxic effect of Vit C on MCF-7/VitC (MCF-7/WT after added 2 mM Vit C) was observed, resulting in a decrease of cell index after 12 h. Also, the cytotoxicity of Vit C (2 mM) after 24 h was confirmed by flow cytometry, i.e., increase of dead, late apoptotic, and depolarized dead MCF-7/VitC cells compared to MCF-7/WT cells. Moreover, changes in proteomic profile of MCF-7/VitC cells compared to the control group were investigated via label-free quantitative mass spectrometry and post-translational modification. Using bioinformatics assessment (i.e., iPathwayGuide and SPIA R packages), a significantly impacted pathway in MCF-7/VitC was identified, namely the protein processing in endoplasmic reticulum. The semi-quantitative change (log2fold change = 4.5) and autophosphorylation at Thr-446 of protein kinase (PKR) (involved in this pathway) indicates that PKR protein could be responsible for the unfolded protein response and inhibition of the cell translation during endoplasmic reticulum stress, and eventually, for cell apoptosis. These results suggest that increased activity of PKR (Thr-446 autophosphorylation) related to cytotoxic effect of Vit C (2 mM) may cause the MCF-7 cells death.

Detection of FLT3 Mutations in Patients from Eastern Slovakia.

BACKGROUND: The study investigated FLT3 gene mutations in patients from eastern Slovakia using a simple molecular method. PATIENTS AND METHODS: We analyzed 141 patients with primary acute myeloid leukemia (AML) and 8 patients with AML that developed from myelodysplastic syndrome (MDS) who were aged 19-81 years. DNA isolated from peripheral blood and/or bone marrow was analyzed by PCR. FLT3 internal tandem duplication (FLT3-ITD) was detected by amplification of exons 14 and 15. Point mutations in the FLT3 tyrosine kinase domain (FLT3-TKD) were detected by digesting the PCR product of exon 20 with the restriction endonuclease EcoRV. Fragments were separated electrophoretically. PCR products of the positive samples were also analyzed using a microchip device (Bioanalyzer 2100). RESULTS: LT3-ITD and point mutations in the FLT-TKD were detected in 19 and 8% of patients, resp. Two patients (1%) harbored both types of mutations. Patients with and without FLT3 mutations were called FLT+ and FLT-, resp. Most FLT3+ patients had no chromosomal aberrations (59%) or harbored the t (15; 17) translocation in PML-RARA (15%). The mortality rate was 33% among FLT3+ patients and 10% among FLT3-patients. Among FLT3+ patients, the mortality rates of patients with FLT3-ITD and point mutations of the FLT-TKD were almost the same. A 77-year-old female patient with both FLT3-ITD and a point mutation in the FLT3-TKD was in remission. The eight patients who developed AML from MDS were assessed separately. Of these, three patients were FLT3+; two patients displayed FLT3-ITD, and one patient harbored a point mutation in the FLT3-TKD. No other genetic aberrations were detected. FLT3+ patients lived for longer than FLT3-patients. These analyses of FLT3 gene mutations in patients from eastern Slovakia are consistent with published data from other databases. CONCLUSION: The applied PCR method is reliable, relatively fast, and affordable, and can be used for routine monitoring of FLT3 gene mutations. FLT3 mutations can be verified using a microchip as an alternative to capillary electrophoresis. Key words: acute myelogenous leukemia - DNA - PCR - mutation - FLT3-ITD - FLT3-TKD The study was supported by the European Regional Development grant OPVaV-2009/2.2/05- -SORO (ITMS code: 26220220143). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical paper Submitted: 19. 10. 2017 Accepted: 15. 2. 2018.

Circulating endothelial-derived apoptotic microparticles and insulin resistance in non-diabetic patients with chronic heart failure.

BACKGROUND: The objective of this study was to assess the relationship between insulin resistance and apoptotic endothelial-derived microparticles (EMPs) in patients with chronic heart failure (CHF). METHODS: The study involved 300 CHF patients (186 males) aged 48-62 years with angiographically proven coronary artery disease and/or previously defined myocardial infarction. Insulin resistance was assessed by the homeostasis model assessment for insulin resistance (HOMA-IR). EMPs phenotype was determined by flow cytofluorometry. RESULTS: Depending on HOMA-IR cut-off point (over and <2.77 mmol/L×µU/mL) all patients were divided into two cohorts with (n=171) or without (n=129) IR, respectively. Circulating EMPs were higher in CHF patients with IR than in patients without IR. Interestingly, EMPs were directly related to NYHA functional class of CHF, HOMA-IR, NT-pro-BNP, hs-CRP and BMI. There was a significant association between the level of EMPs and HbA1c, gender (r=0.318, p<0.001 for male), age and smoking. On univariate and multivariate regression analysis we found that the NYHA class of CHF,NT-pro-BNP, hs-CRP, and left ventricular ejection fraction (LVEF) appeared to be independent predictors of increased circulatory apoptotic EMPs. The addition of HOMA-IR to the standard model (NYHA class CHF) improved the relative IDI by 19.9% for increased EMPs. For category-free NRI, 10% of events and 24% of non-events were correctly reclassified by the addition of HOMA-IR to the standard model for increased circulating EMPs. CONCLUSIONS: IR may be a contributing factor increasing circulating levels of apoptotic EMPs in non-diabetic CHF patients.

Genotype/allelic combinations as potential predictors of myocardial infarction.

In order to find new informative predictors of myocardial infarction, we performed an analysis of genotype frequencies of polymorphic markers of SELE (rs2076059, 3832T > C), SELP (rs6131, S290 N), SELL (rs1131498, F206L), ICAM1 (rs5498, K469E), VCAM1 (rs3917010, c.928 + 420A > C), PECAM1 (rs668, V125L), VEGFA (rs35569394, -2549(18)I/D), CCL2 (rs1024611, -2518A > G), NOS3 (rs1799983, E298D), and DDAH1 (rs669173, c.303 + 30998A > G) genes in the group of Russian men with myocardial infarction (N = 315) and the control group of corresponding ethnicity, gender, and age (N = 286). Using Markov chain Monte-Carlo method (APSampler), we found genotype combinations associated with increased and decreased risk of myocardial infarction. The most significant associations were detected for PECAM1*V/V + DDAH1*C (OR = 4.17 CI 1.56-11.15 Pperm = 0.005) SELE*C + VEGFA*I + CCL2*G + VCAM1*A + NOS3*D (OR = 2.74 CI 1.66-4.52 Pperm = 2.09 × 10(-5)), and VEGFA*D/D + CCL2*A + DDAH1*C (OR = 0.44 CI 0.28-0.7 Pperm = 7.89 × 10(-5)) genotype combinations.

Delayed bradykinin postconditioning modulates intrinsic neuroprotective enzyme expression in the rat CA1 region after cerebral ischemia: a proteomic study.

Pyramidal cells in the CA1 brain region exhibit an ischemic tolerance after delayed postconditioning; therefore, this approach seems to be a promising neuroprotective procedure in cerebral postischemic injury improvement. However, little is known about the effect of postconditioning on protein expression patterns in the brain, especially in the affected hippocampal neurons after global cerebral ischemia. This study is focused on the examination of the ischemia-vulnerable CA1 neuronal layer and on the acquisition of protection from delayed neuronal death after ischemia. Ischemic-reperfusion injury was induced in Wistar rats and bradykinin was applied 2 days after the ischemic insult in an attempt to overcome delayed cell death. Analysis of complex peptide CA1 samples was performed by automated two dimensional liquid chromatography (2D-LC) fractionation coupled to tandem matrix assisted laser desorption/ionization time-of-flight (MALDI TOF/TOF) mass spectrometry instrumentation. We devoted our attention to differences in protein expression mapping in ischemic injured CA1 neurons in comparison with equally affected neurons, but with bradykinin application. Proteomic analysis identified several proteins occurring only after postconditioning and control, which could have a potentially neuroprotective influence on ischemic injured neurons. Among them, the prominent position occupies a regulator of glutamate level aspartate transaminase AATC, a scavenger of glutamate in brain neuroprotection after ischemia-reperfusion. We identified this enzyme in controls and after postconditioning, but AATC presence was not detected in the ischemic injured CA1 region. This finding was confirmed by two-dimensional differential electrophoresis followed by MALDI-TOF/TOF MS identification. Results suggest that bradykinin as delayed postconditioning may be associated with modulation of protein expression after ischemic injury and thus this procedure can be involved in neuroprotective metabolic pathways.

Unique Presentation of Hematometrocolpos Mimicking Cauda Equina Syndrome: Severe Back Pain and Urinary Incontinence in an Adolescent Girl.

BACKGROUND: Imperforate hymen with hematometrocolpos in adolescent females is a rare pediatric condition. Classical presentation includes abdominal pain or a pelvic mass in female patients with primary amenorrhea. Atypical complaints and reluctance among emergency physicians to perform genital examination in the emergency department or the pediatric emergency department (PED) may delay correct diagnosis. CASE REPORT: We report a unique, cauda equina syndrome-like presentation of hematometrocolpos secondary to imperforate hymen in a 13-year old, previously healthy girl with primary amenorrhea. In the PED, the unusual clinical presentation of severe back pain and urinary incontinence initially mimicked cauda equina syndrome and led to delayed correct diagnosis. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: The novelty of this case is a cauda equina-like presentation of imperforate hymen secondary to hematocolpos. This report illustrates the highly variable clinical presentation of this rare gynecological pediatric entity. It underlines the importance of considering this rare condition in the differential diagnosis of severe upper or lower back pain alongside voiding abnormalities including urinary retention and incontinence in adolescent females with primary amenorrhea. Above all, the importance of performing a thorough history and genital examination in this subgroup early in the investigation process in the PED emerges from this case. Essentially, excellent clinical judgment and genital examination by the emergency physician may minimize unnecessary radiological investigations and ultimately, accelerate correct diagnosis and expedite appropriate surgical treatment. However, not only pediatric and adult emergency physicians, but also pediatricians and general practitioners should be aware of this entity and its diverse clinical presentation.

Impact of platelet phenotype on myocardial infarction.

In acute myocardial infarction patients the injured vascular wall triggers thrombus formation in the damage site. Fibrin fibers and blood cellular elements are the major components of thrombus formed in acute occlusion of coronary arteries. It has been established that the initial thrombus is primarily composed of activated platelets rapidly stabilized by fibrin fibers. This review highlights the role of platelet membrane phenotype in pathophysiology of myocardial infarction. Here, we regard platelet phenotype as quantitative and qualitative parameters of the plasma membrane outer surface, which are crucial for platelet participation in blood coagulation, development of local inflammation and tissue repair.

Regional differences of vasodilatation and vasomotion response to local heating in human cutaneous microcirculation.

BACKGROUND: The aim of this study was to evaluate the vasodilatation and vasomotion response to local heating in the cutaneous microcirculation of the ankle, dorsum of foot and forearm. Recently, it has been suggested that this response differs between the forearm and the leg. PROBANDS AND METHODS: Twenty-nine young healthy adults were recruited. They underwent measurement by laser Doppler flowmetry (LDF) in three sites of the body (ankle, dorsum of foot, forearm). Percentage change of the median flow of the skin before and after provocation and normalised perfusion flow to maximal dilation (cutaneous vascular conductance--CVC % Max) during short provocation test were monitored. Spectral analysis of laser Doppler flowmetry signals was performed using the fast Fourier transform algorithm. RESULTS: Significant differences were found in CVC % Max between ankle/dorsum (45.18±6.38% Max vs. 51.24±6.87% Max, respectively; p<0.05) and between ankle/forearm (45.18±6.38% Max vs. 54.49±5.37% Max, respectively; p<0.05). Percentage change of flux after provocation has revealed significant differences between ankle/dorsum (394.1±204.5% vs. 577.4±273.5%, respectively; p<0.05) and ankle/forearm (394.1±204.5% vs. 637.1±324.7%, respectively; p<0.05). Total spectral activity of vasomotion has differed between ankle/dorsum and ankle/forearm: 69.59 [49.58-96.04] vs. 93.01 [73.15-121.8] (p<0.05) and 69.59 [49.58-96.04] vs. 107.5 [80.55-155.8] (p<0.05), respectively. CONCLUSIONS: Cutaneous microcirculation exhibits regional differences. Significant variability of function between ankle and dorsum of foot suggests that leg microcirculation is not uniform.

Human peripheral blood mononuclear cells as a valuable source of disease-related biomarkers: Evidence from comparative proteomics studies.

PURPOSE: The discovery of specific and sensitive disease-associated biomarkers for early diagnostic purposes of many diseases is still highly challenging due to various complex molecular mechanisms triggered, high variability of disease-related interactions, and an overlap of manifestations among diseases. Human peripheral blood mononuclear cells (PBMCs) contain protein signatures corresponding to essential immunological interplay. Certain diseases stimulate PBMCs and contribute towards modulation of their proteome which can be effectively identified and evaluated via the comparative proteomics approach. EXPERIMENTAL DESIGN: In this review, we made a detailed survey of the PBMCS-derived protein biomarker candidates for a variety of diseases, published in the last 15 years. Articles were preselected to include only comparative proteomics studies. RESULTS: PBMC-derived biomarkers were investigated for cancer, glomerular, neurodegenerative/neurodevelopmental, psychiatric, chronic inflammatory, autoimmune, endocrinal, infectious, and other diseases. A detailed review of these studies encompassed the proteomics platforms, proposed candidate biomarkers, their immune cell type specificity, and potential clinical application. CONCLUSIONS: Overall, PBMCs have shown a solid potential in giving early diagnostic and prognostic biomarkers for many diseases. The future of PBMC biomarker research should reveal its full potential through well-designed comparative studies and extensive testing of the most promising protein biomarkers identified so far.

CKAP5 enables formation of persistent actin bundles templated by dynamically instable microtubules.

Cytoskeletal rearrangements and crosstalk between microtubules and actin filaments are vital for living organisms. Recently, an abundantly present microtubule polymerase, CKAP5 (XMAP215 homolog), has been reported to play a role in mediating crosstalk between microtubules and actin filaments in the neuronal growth cones. However, the molecular mechanism of this process is unknown. Here, we demonstrate, in a reconstituted system, that CKAP5 enables the formation of persistent actin bundles templated by dynamically instable microtubules. We explain the templating by the difference in CKAP5 binding to microtubules and actin filaments. Binding to the microtubule lattice with higher affinity, CKAP5 enables the formation of actin bundles exclusively on the microtubule lattice, at CKAP5 concentrations insufficient to support any actin bundling in the absence of microtubules. Strikingly, when the microtubules depolymerize, actin bundles prevail at the positions predetermined by the microtubules. We propose that the local abundance of available CKAP5-binding sites in actin bundles allows the retention of CKAP5, resulting in persisting actin bundles. In line with our observations, we found that reducing CKAP5 levels in vivo results in a decrease in actin-microtubule co-localization in growth cones and specifically decreases actin intensity at microtubule plus ends. This readily suggests a mechanism explaining how exploratory microtubules set the positions of actin bundles, for example, in cytoskeleton-rich neuronal growth cones.