The protective effect of low-dose minocycline on brain microvascular ultrastructure in a rodent model of subarachnoid hemorrhage.

The multifaceted nature of subarachnoid hemorrhage (SAH) pathogenesis is poorly understood. To date, no pharmacological agent has been found to be efficacious for the prevention of brain injury when used for acute SAH intervention. This study was undertaken to evaluate the beneficial effects of low-dose neuroprotective agent minocycline on brain microvascular ultrastructures that have not been studied in detail. We studied SAH brain injury using an in vivo prechiasmatic subarachnoid hemorrhage rodent model. We analyzed the qualitative and quantitative ultrastructural morphology of capillaries and surrounding neuropil in the rodent brains with SAH and/or minocycline administration. Here, we report that low-dose minocycline (1 mg/kg) displayed protective effects on capillaries and surrounding cells from significant SAH-induced changes. Ultrastructural morphology analysis revealed also that minocycline stopped endothelial cells from abnormal production of vacuoles and vesicles that compromise blood-brain barrier (BBB) transcellular transport. The reported ultrastructural abnormalities as well as neuroprotective effects of minocycline during SAH were not directly mediated by inhibition of MMP-2, MMP-9, or EMMPRIN. However, SAH brain tissue treated with minocycline was protected from development of other morphological features associated with oxidative stress and the presence of immune cells in the perivascular space. These data advance the knowledge on the effect of SAH on brain tissue ultrastructure in an SAH rodent model and the neuroprotective effect of minocycline when administered in low doses.

Presence of tubuloreticular inclusions in ultrastructural studies of renal biopsies in children with lupus nephropathy - one-center preliminary study.

Introduction: Juvenile systemic lupus erythematosus (jSLE) is an autoimmune disease that develops as a result of multi-level immune dysregulation, including the interferon pathway. Nephropathy develops at an early stage and eventually affects 90% of patients. A renal biopsy allows one to classify lupus nephritis and determine the proper treatment. Biopsy assessment should be done not only in a light microscope but also in a transmission electron microscope (TEM). Its usage may reveal the presence of intracellular tubuloreticular inclusions (TRIs), considered as a morphological marker of interferon hyperactivity. Material and methods: Renal biopsies of 10 children with jSLE and nephropathy were analyzed in TEM. The location, structure, and size of TRIs were assessed. Demographic data, nephropathy manifestation, non-renal symptoms, and serological activity of lupus were analyzed. Results: All the patients were female with an average onset at 12.7 years of age and met SLE criteria. Nephropathy manifested with proteinuria (n = 10) and hematuria (n = 6). Glomerular filtration rate (GFR) was normal in all patients. In three children with early disease onset, it manifested with hematological disorders. TRIs were revealed in 7 biopsies, with the highest expression in the youngest children, with peripheral cytopenia, membranous glomerulonephritis, and lupus nephritis. Conclusions: Demonstration of TRIs in renal biopsies of children with juvenile systemic lupus may confirm the diagnosis of lupus nephritis and is a sign of involvement of the interferon pathway at the early stage of the disease.

Notch signalling pathway as an oncogenic factor involved in cancer development.

Notch signalling is an evolutionarily conserved signalling pathway, which plays a significant role in a wide array of cellular processes including proliferation, differentiation, and apoptosis. Nevertheless, it must be noted that Notch is a binary cell fate determinant, and its overexpression has been described as oncogenic in a broad range of human malignancies. This finding led to interest in therapeutically targeting this pathway especially by the use of GSIs, which block the cleavage of Notch at the cell membrane and inhibit release of the transcriptionally active NotchIC subunit. Preclinical cancer models have clearly demonstrated that GSIs suppress the growth of such malignancies as pancreatic, breast, and lung cancer; however, GSI treatment in vivo is associated with side effects, especially those within the gastrointestinal tract. Although intensive studies are associated with the role of γ-secretase in pathological states, it should be pointed out that this complex impacts on proteolytic cleavages of around 55 membrane proteins. Therefore, it is clear that GSIs are highly non-specific and additional drugs must be designed, which will more specifically target components of the Notch signalling.

Electron microscopy of human fascia lata: focus on telocytes.

From the histological point of view, fascia lata is a dense connective tissue. Although extracellular matrix is certainly the most predominant fascia's feature, there are also several cell populations encountered within this structure. The aim of this study was to describe the existence and characteristics of fascia lata cell populations viewed through a transmission electron microscope. Special emphasis was placed on telocytes as a particular interstitial cell type, recently discovered in a wide variety of tissues and organs such as the heart, skeletal muscles, skin, gastrointestinal tract, uterus and urinary system. The conducted study confirmed the existence of a telocyte population in fascia lata samples. Those cells fulfil main morphological criteria of telocytes, namely, the presence of very long, thin cell processes (telopodes) extending from a relatively small cell body. Aside from telocytes, we have found fibroblasts, mast cells and cells with features of myofibroblastic differentiation. This is the first time it has been shown that telocytes exist in human fascia. Currently, the exact role of those cells within the fascia is unknown and definitely deserves further attention. One can speculate that fascia lata telocytes likewise telocytes in other organs may be involved in regeneration, homeostasis and intracellular signalling.

Role of Notch signaling pathway in gastric cancer pathogenesis.

Notch signaling pathway is activated dynamically during evolution playing significant role in cell fate determination and differentiation. It has been known that alterations of this pathway may lead to human malignancies, including gastric cancer. Despite a decline in the overall incidence, this disease still remains an important global health problem. Therefore, a better understanding of the molecular alterations underlying gastric cancer may contribute to the development of rationally designed molecular targeted therapies. It has been reported that Notch1 receptor could become a prognostic marker of gastric cancer and novel target for gastric cancer therapy. Among the novel and targeted approaches for the treatment of gastric cancer is also the process of Notch receptors regulation by specific microRNA. γ-secretase inhibitors are also taken into consideration.

Switch them off or not: selective rRNA gene repression in grasses.

Nucleolar dominance (ND) is selective epigenetic silencing of 35-48S rDNA loci. In allopolyploids, it is frequently manifested at the cytogenetic level by the inactivation of nucleolar organiser region(s) (NORs) inherited from one or several evolutionary ancestors. Grasses are ecologically and economically one of the most important land plant groups, which have frequently evolved through hybridisation and polyploidisation events. Here we review common and unique features of ND phenomena in this monocot family from cytogenetic, molecular, and genomic perspectives. We highlight recent advances achieved by using an allotetraploid model grass, Brachypodium hybridum, where ND commonly occurs at a population level, and we cover modern genomic approaches that decipher structural features of core arrays of NORs.

Heat shock protein A2 is a novel extracellular vesicle-associated protein.

70-kDa Heat Shock Proteins (HSPA/HSP70) are chaperones playing a central role in the proteostasis control mechanisms. Their basal expression can be highly elevated as an adaptive response to environmental and pathophysiological stress conditions. HSPA2, one of poorly characterised chaperones of the HSPA/HSP70 family, has recently emerged as epithelial cells differentiation-related factor. It is also commonly expressed in cancer cells, where its functional significance remains unclear. Previously, we have found that proteotoxic stress provokes a decrease in HSPA2 levels in cancer cells. In the present study we found that proteasome inhibition-related loss of HSPA2 from cancer cells neither is related to a block in the gene transcription nor does it relate to increased autophagy-mediated disposals of the protein. Proteotoxic stress stimulated extracellular release of HSPA2 in extracellular vesicles (EVs). Interestingly, EVs containing HSPA2 are also released by non-stressed cancer and normal cells. In human urinary EVs levels of HSPA2 were correlated with the levels of TSG101, one of the main EVs markers. We conclude that HSPA2 may constitute basic components of EVs. Nevertheless, its specific role in EVs and cell-to-cell communication requires further investigation.

Molecular Composition of Serum Exosomes Could Discriminate Rectal Cancer Patients with Different Responses to Neoadjuvant Radiotherapy.

Identification of biomarkers that could be used for the prediction of the response to neoadjuvant radiotherapy (neo-RT) in locally advanced rectal cancer remains a challenge addressed by different experimental approaches. Exosomes and other classes of extracellular vesicles circulating in patients' blood represent a novel type of liquid biopsy and a source of cancer biomarkers. Here, we used a combined proteomic and metabolomic approach based on mass spectrometry techniques for studying the molecular components of exosomes isolated from the serum of rectal cancer patients with different responses to neo-RT. This allowed revealing several proteins and metabolites associated with common pathways relevant for the response of rectal cancer patients to neo-RT, including immune system response, complement activation cascade, platelet functions, metabolism of lipids, metabolism of glucose, and cancer-related signaling pathways. Moreover, the composition of serum-derived exosomes and a whole serum was analyzed in parallel to compare the biomarker potential of both specimens. Among proteins that the most properly discriminated good and poor responders were GPLD1 (AUC = 0.85, accuracy of 74%) identified in plasma as well as C8G (AUC = 0.91, accuracy 81%), SERPINF2 (AUC = 0.91, accuracy 79%) and CFHR3 (AUC = 0.90, accuracy 81%) identified in exosomes. We found that the proteome component of serum-derived exosomes has the highest capacity to discriminate samples of patients with different responses to neo-RT when compared to the whole plasma proteome and metabolome. We concluded that the molecular components of exosomes are associated with the response of rectal cancer patients to neo-RT and could be used for the prediction of such response.

New insights into the pathogenesis of cardiac papillary fibroelastomas.

INTRODUCTION: Cardiac papillary fibroelastomas (CPFs) are rare benign cardiac tumors typically found on the heart valves. The previously published data on the CPF focused on its clinical presentation, optimal management, and prognosis. However, histogenesis of these lesions remains controversial. Accordingly, the aim of this study was to establish the role of endocardial endothelium (EE) in CPF formation. MATERIALS AND METHODS: Four CPF tumors removed from the right atrioventricular valves were analyzed using hematoxylin & eosin, orcein, and Masson trichrome staining together with immunochemistry for CD-34, CD-68, vimentin, vWF and a-SMA. Moreover, conventional transmission electron microscopy was used for morphological analysis and a-SMA presence confirmation. RESULTS: Ultrastructural morphology, immunohisto- and immunocytochemical analyses indicated that cells covering collagenous core have an endothelial origin. Some endocardial endothelium cells have the potential to undergo a transition to mesenchymal cells. Moreover, the abundant presence of extracellular vesicles may indicate an active intercellular communication. Within the intermediate translucent zone, amorphous substances with monocytes/macrophage-like cells and fibroblastic cells were found. Finally, within collagenous core activated (myo)fibroblasts were observed. CONCLUSIONS: Our study demonstrated that the endocardial endothelium of the CPF was "double-sided", i.e., it presented both endothelial and mesenchymal cell characteristics. Another finding was the presence of monocytes, and macrophages which were integrated into CPF core and displayed features of a fibroblast that have been shown to contribute to extracellular matrix production. This could be interpreted as being attributed to the CPF histogenesis.

The Lipid Composition of Serum-Derived Small Extracellular Vesicles in Participants of a Lung Cancer Screening Study.

Molecular components of exosomes and other classes of small extracellular vesicles (sEV) present in human biofluids are potential biomarkers with possible applicability in the early detection of lung cancer. Here, we compared the lipid profiles of serum-derived sEV from three groups of lung cancer screening participants: individuals without pulmonary alterations, individuals with benign lung nodules, and patients with screening-detected lung cancer (81 individuals in each group). Extracellular vesicles and particles were purified from serum by size-exclusion chromatography, and a fraction enriched in sEV and depleted of low-density lipoproteins (LDLs) was selected (similar sized vesicles was observed in all groups: 70-100 nm). The targeted mass-spectrometry-based approach enabled the detection of 352 lipids, including 201 compounds used in quantitative analyses. A few compounds, exemplified by Cer(42:1), i.e., a ceramide whose increased plasma/serum level was reported in different pathological conditions, were upregulated in vesicles from cancer patients. On the other hand, the contribution of phosphatidylcholines with poly-unsaturated acyl chains was reduced in vesicles from lung cancer patients. Cancer-related features detected in serum-derived sEV were different than those of the corresponding whole serum. A high heterogeneity of lipid profiles of sEV was observed, which markedly impaired the performance of classification models based on specific compounds (the three-state classifiers showed an average AUC = 0.65 and 0.58 in the training and test subsets, respectively).

Ileal transposition helps to regulate plasma hepatokine levels in obese Zucker (Crl:ZUC(ORL)-Leprfa) rats.

We studied the long-term effect of ileal transposition (IT) metabolic surgery on the hepatokines: retinol-binding protein-4 (RBP4), α-2-HS-glycoprotein (aHSG/fetuin-A), and fibroblast growth factor 21 (FGF21), C-reactive protein (CRP) plasma levels, glucose metabolism, body weight, liver histology, as well as total lipids concentration in muscle, liver, and fat tissue of obese Zucker (Crl:ZUC(ORL)-Leprfa) rats. 14 adult males were randomly submitted either to IT or SHAM (control) surgery. Pre-operative hepatokines plasma levels were not significantly different in rats submitted to IT or SHAM protocol. Three months after the procedures the plasma levels of RBP4, aHSG, FGF21, and CRP were significantly lower in IT-operated animals when compared to SHAM-operated group. Three and 12 weeks after the IT and SHAM surgery, the AUCOGTT were significantly lower than AUCOGTT before the surgery. HOMA-IR was lower in rats after IT surgery in comparison to the SHAM-operated rats. Muscle and liver total lipids concentration was reduced after the IT procedure when compared to pre-IT conditions. IT had a significant reductive impact on the body weight in comparison to SHAM surgery in the 4th, 6th, 8th, and 10th week after the surgery. We conclude that IT reduces hepatokines' plasma concentrations, muscle and liver total lipids concentration but not the inflammatory processes in the liver of Zucker (Crl:ZUC(ORL)-Leprfa) rats.

4-n-Butylresorcinol-Based Linear and Graft Polymethacrylates for Arbutin and Vitamins Delivery by Micellar Systems.

A novel initiator, bromoester modified 4-n-butylresorcinol (4nBREBr2), was prepared and utilized in controlled atom transfer radical polymerization (ATRP) to obtain three series of amphiphilic copolymers. The V-shaped copolymers of methyl methacrylate (MMA), 2-hydroxyethyl methacrylate (HEMA), and poly(ethylene glycol) methyl ether methacrylate (MPEGMA), abbreviated to P(HEMA-co-MMA), P(HEMA-co-MPEGMA), and P(MMA-co-MPEGMA), were synthesized. Moreover, P((HEMA-graft-PEG)-co-MMA) graft copolymers were prepared by combining the pre-polymerization modification of HEMA and a "click" reaction using a "grafting onto" approach. All copolymers could form micelles with encapsulated active substances (vitamin C (VitC), vitamin E (VitE), arbutin (ARB)), which are used in cosmetology. In vitro studies carried out in a PBS solution (pH 7.4) demonstrates that in most cases the maximum release of active substance was after 1 h. The polymeric systems presenting satisfactory encapsulation characteristics and release profiles are attractive micellar carriers of cosmetic substances, which show a positive effect on the skin condition.

Synthesis and in vitro cytotoxicity evaluation of star-shaped polymethacrylic conjugates with methotrexate or acitretin as potential antipsoriatic prodrugs.

Water-soluble polymer-drug conjugates were obtained and analyzed towards their potential use as prodrugs for two hydrophobic antipsoriatic agents, including methotrexate (MTX) and acitretin (AC). The conjugation efficacy of MTX decreased with a decreasing molar ratio of N,N-dimethylaminoethyl methacrylate (DMAEMA) repeating units in the polymethacrylic chains. Cytotoxicity of positively charged (from +5 to +10 mV) nano- and microparticles (3-1500 nm in DMEM at 37 °C) were estimated by in vitro MTT and Annexin-V apoptosis assays on Me45, NHDF, HaCaT and BEAS-2B cell lines. Further, cell cycle analysis revealed arrest in G0/G1 phase in melanoma cells, while neither apoptosis induction nor cell cycle arrest occurred in normal epidermal and epithelial cells. Tested conjugates displayed a novel cytostatic effect in Me45 cells and a pro-apoptotic effect in HaCaT cells. Epithelial BEAS-2B cells were the most sensitive to the tested conjugates and responded via induction of necrosis. Cell line models allowed for characterization of the biologically relevant potential action of pro-drugs. Additionally, a skin in vitro evaluation assay provided the first known evidence of side-effect reduction with pro-drug use. Histological examinations confirmed the lack of negative effects of conjugates on the skin and showed no irritating properties.

MicroRNA Profile of Exosomes and Parental Cells is Differently Affected by Ionizing Radiation.

Exosomes are key mediators of cell-to-cell communication involved in different aspects of the response to ionizing radiation. The functional role of exosomes depends on their molecular cargo, including protein and miRNA content. In this work, we compared the miRNA profile of cells exposed to a high-dose of radiation and the exosomes released by those cells. FaDu cells (derived from human head and neck cancer) were exposed to 2 and 8 Gy doses, exosomes were purified from culture media at 36 h postirradiation using a combination of differential centrifugation, ultrafiltration and precipitation, then microRNA was analyzed using the RNA-seq approach. There were 439 miRNA species quantified, and significant differences in their relative abundance were observed between the cells and exosomes; several low-abundance miRNAs were over-represented while high-abundance miRNA were under-represented in exosomes. There were a few miRNA species markedly affected in irradiated cells and in exosomes released by these cells. However, markedly different radiation-induced effects were observed in both miRNA sets, which could be exemplified by miR-3168 significantly downregulated in cells and upregulated in exosomes. On the other hand, both 2 and 8 Gy radiation doses induced similar effects. Radiation-affected miRNA species present in exosomes are linked to genes involved in the DNA damage and cytokine-mediated response, which may suggest their hypothetical role in the exosome-mediated radiation-induced bystander effect reported elsewhere.

Choline supported poly(ionic liquid) graft copolymers as novel delivery systems of anionic pharmaceuticals for anti-inflammatory and anti-coagulant therapy.

New type of carriers based on grafted poly(ionic liquid)s was designed for delivery of ionically attached salicylates (Sal). Choline derived ionic liquid monomeric units were successfully introduced with various content in the side chains by the controlled radical polymerization. Properly high amounts of ionic pharmaceutics in the polymer systems were achieved by the well-fitted length and grafting degree of the side chains. In aqueous solution the graft copolymers were self-assembled into the spherical superstructures with sizes up to 73 nm. Delivery studies showed "burst" release within 4 h, after that it was slower yielding ~70% of released drug within 80 h. Proposed nanocarriers supported low toxicity against human cells (NHDF and BEAS-2B), anti-inflammation activity evaluated with the use of pro-inflammatory interleukins (IL-6 and IL-8) and antibacterial activities towards E. coli. Adjustment of ionic drug content by structural parameters of graft copolymers, including grafting degree and graft length, are advantageous to tailor nanocarriers with self-assembly properties in aqueous media. Effective release process by ionic exchange and biological activity with low toxicity are promising for further development of this type of drug delivery (DDS).

Proteome Profiling of Exosomes Purified from a Small Amount of Human Serum: The Problem of Co-Purified Serum Components.

Untargeted proteomics analysis of extracellular vesicles (EVs) isolated from human serum or plasma remains a technical challenge due to the contamination of these vesicles with lipoproteins and other abundant serum components. Here we aimed to test a simple method of EV isolation from a small amount of human serum (<1 mL) using the size-exclusion chromatography (SEC) standalone for the discovery of vesicle-specific proteins by the untargeted LC-MS/MS shotgun approach. We selected the SEC fraction containing vesicles with the size of about 100 nm and enriched with exosome markers CD63 and CD81 (but not CD9 and TSG101) and analyzed it in a parallel to the subsequent SEC fraction enriched in the lipoprotein vesicles. In general, there were 267 proteins identified by LC-MS/MS in exosome-containing fraction (after exclusion of immunoglobulins), yet 94 of them might be considered as serum proteins. Hence, 173 exosome-related proteins were analyzed, including 92 proteins absent in lipoprotein-enriched fraction. In this set of exosome-related proteins, there were 45 species associated with the GO cellular compartment term "extracellular exosome". Moreover, there were 31 proteins associated with different immune-related functions in this set, which putatively reflected the major role of exosomes released by immune cells present in the blood. We concluded that identified set of proteins included a bona fide exosomes components, yet the coverage of exosome proteome was low due to co-purified high abundant serum proteins. Nevertheless, the approach proposed in current work outperformed other comparable protocols regarding untargeted identification of exosome proteins and could be recommended for pilot exploratory studies when a small amount of a serum/plasma specimen is available.

Role of interleukins in heart failure with reduced ejection fraction.

Heart failure (HF) is the leading cause of morbidity and mortality in developed countries, and it is the primary cause of mortality in the elderly worldwide. The processes of inflammatory response activation, production and release of pro-inflammatory cytokines, activation of the complement system, synthesis of autoantibodies, and overexpression of Class II major histocompatibility complex molecules contribute to the HF development and progression. High levels of circulating cytokines correlate with the severity of HF, measured with the use of New York Heart Association's classification, and prognosis of the disease. In HF, there is an imbalance between pro-inflammatory and anti-inflammatory cytokines. Concentrations of several interleukins are increased in HF, including IL-1ß, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, and IL-18, whereas the levels of IL-5, IL-7, or IL-33 are down-regulated. Concentrations of inflammatory mediators are associated with cardiac function and can be HF markers and predictors of adverse outcomes or mortality. This review presents the role of interleukins, which contribute to the HF initiation and progression, the importance of their pathways in transition from myocardial injury to HF, and the role of interleukins as markers of disease severity and outcome predictors.

Self-assembling water-soluble polymethacrylate-MTX conjugates: The significance of macromolecules architecture on drug conjugation efficiency, the final shape of particles, and drug release.

Water-soluble polymer-methotrexate (MTX) conjugates were obtained via efficient carbodiimide-mediated amidation (E = 17-100%). Binding abilities between water-soluble V-shaped or star-shaped copolymers and MTX were studied by isothermal titration calorimetry spectroscopic (UV-vis, NMR) and microscopic (scanning electron microscopy and transmission electron microscopy) techniques. The efficiency of the amidation reaction has depended on the amount of pendant amino alcohol groups and zeta potential (ZP) values of polymeric carries. The sizes of aggregates formed by polymer-drug conjugates in water increased with the number of copolymer arms (202-774 nm at 37°C). Moreover, the conjugates with the high amount of bounded MTX molecules (nMTX > 78) exhibited negative ZP values. The drug release experiments revealed that the amount of the released MTX depends on pH and can be controlled via shape, topology, and composition of polymeric carrier. Preliminary cytotoxicity studies of V-shaped-MTX conjugate on human immortalized nontumorigenic keratinocyte (HaCaT) cells indicated cytocompatibility of the compound in a wide range of concentrations. The results of our studies have shown that physicochemical and drug release properties of obtained polymer-MTX prodrugs can be tailored via the structure and the topology of the polymeric carrier. Thus conjugates might find the application in a different type of treatment (cancer or psoriasis therapy) and administration (intravenous, dermal, or pulmonary). © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2476-2487, 2019.

Apoptosis in Primary Hyperparathyroidism.

Primary hyperparathyroidism (PHPT) is defined by inappropriate elevation of parathormone, caused by parathyroid hyperplasia, also known as multi-gland disease (MGD), parathyroid adenoma (PA), or parathyroid carcinoma (PC). Although several studies have already been conducted, there is a lack of a definite diagnostic marker, which could unambiguously distinguish MGD from PA or PC. The accurate and prompt diagnosis has the key meaning for effective treatment and follow-up. This review paper presents the role of apoptosis in PHPT. The comparison of the expression of Fas, TRAIL, BCL-2 family members, p53 in MGD, PA, and PC, among others, was described. The expression of described factors varies among proliferative lesions of parathyroid gland; therefore, these could serve as additional markers to assist in the diagnosis.

A different ultrastructural face of ribbon synapses in the rat retina.

Ribbon synapses located exclusively within retinal, cochlear and vestibular connections belong to the most interesting cellular structures but their molecular nature and functions had remained unclear. The study has provided a descriptive morphological analysis of rat eye ribbon synapses using high-resolution transmission electron microscopy (TEM). An original collection of untypical, rarely present in the literature sagittal or tangential sections through the single RIBEYE domain of the particular ribbon have been delivered.