Interaction of Hoechst 33342 with POPC Membranes at Different pH Values.

Hoechst 33342 (H33342) is a fluorescent probe that is commonly used to stain the DNA of living cells. To do so, it needs to interact with and permeate through cell membranes, despite its high overall charge at physiological pH values. In this work, we address the effect of pH in the association of H33342 with lipid bilayers using a combined experimental and computational approach. The partition of H33342 to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid membranes was experimentally quantified using fluorescence spectroscopy and isothermal titration calorimetry (ITC) measurements. Quantum chemical calculations were performed to select the most stable isomer of H33342 for the overall charges 0, +1, and +2, expected to predominate across the 5 < pH < 10 range. The interaction of these isomers with POPC bilayers was then studied by both unrestrained and umbrella sampling molecular dynamics (MD) simulations. Both experimental results and computational free energy profiles indicate that the partition coefficient of H33342 displays a small variation over a wide pH range, not exceeding one order of magnitude. The enthalpy variation upon partition to the membrane suggests efficient hydrogen bonding between the probe and the lipid, namely, for the protonated +2 form, which was confirmed in the MD simulation studies. The relatively high lipophilicity obtained for the charged species contrasts with the decrease in their general hydrophobicity as estimated from octanol/water partition. This highlights the distinction between lipophilicity and hydrophobicity, as well as the importance of considering the association with lipid bilayers when predicting the affinity for biomembranes.

A Preliminary Evaluation of the Pro-Chondrogenic Potential of 3D-Bioprinted Poly(ester Urea) Scaffolds.

Degeneration of articular cartilage (AC) is a common healthcare issue that can result in significantly impaired function and mobility for affected patients. The avascular nature of the tissue strongly burdens its regenerative capacity contributing to the development of more serious conditions such as osteoarthritis. Recent advances in bioprinting have prompted the development of alternative tissue engineering therapies for the generation of AC. Particular interest has been dedicated to scaffold-based strategies where 3D substrates are used to guide cellular function and tissue ingrowth. Despite its extensive use in bioprinting, the application of polycaprolactone (PCL) in AC is, however, restricted by properties that inhibit pro-chondrogenic cell phenotypes. This study proposes the use of a new bioprintable poly(ester urea) (PEU) material as an alternative to PCL for the generation of an in vitro model of early chondrogenesis. The polymer was successfully printed into 3D constructs displaying adequate substrate stiffness and increased hydrophilicity compared to PCL. Human chondrocytes cultured on the scaffolds exhibited higher cell viability and improved chondrogenic phenotype with upregulation of genes associated with type II collagen and aggrecan synthesis. Bioprinted PEU scaffolds could, therefore, provide a potential platform for the fabrication of bespoke, pro-chondrogenic tissue engineering constructs.

Cholinergic Oculomotor Nucleus Activity Is Induced by REM Sleep Deprivation Negatively Impacting on Cognition.

Several efforts have been made to understand the involvement of rapid eye movement (REM) sleep for cognitive processes. Consolidation or retention of recognition memories is severely disrupted by REM sleep deprivation (REMSD). In this regard, pedunculopontine tegmental nucleus (PPT) and other brainstem nuclei, such as pontine nucleus (Pn) and oculomotor nucleus (OCM), appear to be candidates to take part in this REM sleep circuitry with potential involvement in cognition. Therefore, the objective of this study was to investigate a possible association between the performance of Wistar rats in a declarative memory and PPT, Pn, and OCM activities after different periods of REMSD. We examined c-Fos and choline acetyltransferase (ChaT) expressions as indicators of neuronal activity as well as a familiarity-based memory test. The animals were distributed in groups: control, REMSD, and sleep rebound (REB). At the end of the different REMSD (24, 48, 72, and 96 h) and REB (24 h) time points, the rats were immediately tested in the object recognition test and then the brains were collected. Results indicated that OCM neurons presented an increased activity, due to ChaT-labeling associated with REMSD that negatively correlated (r = -0.32) with the cognitive performance. This suggests the existence of a cholinergic compensatory mechanism within the OCM during REMSD. We also showed that 24 h of REMSD impacted similarly in memory, compared to longer periods of REMSD. These data extend the notion that REM sleep is influenced by areas other than PPT, i.e., Pn and OCM, which could be key players in both sleep processes and cognition.

Telomere protein complexes and their role in lymphoid malignancies.

Telomeres are highly regulated and dynamic complexes that protect the genomic DNA and prevent the end of linear chromosomes from being misrecognized as a broken DNA. Due to the end replication problem, telomeres of somatic cells shorten with each cell division, inducing cell senescence. Telomerase is a reverse transcriptase capable of compensating telomere attrition by adding telomere repeats to the ends of chromosomes. Human telomeres are associated with the shelterin complex which consists of six telomere-associated proteins that specifically bind to telomeric DNA. Alterations or removal of individual shelterin components would lead to telomere uncapping and telomere dysfunction, resulting in cellular senescence and transformation to a malignant state. Another complex of multifunctional proteins, named non-shelterin complex, is thought to prevent telomere degradation and facilitate telomerase-based telomere elongation. As telomerase is highly expressed in most human tumor cells, it is considered an attractive target for new therapeutic strategies. In this review, we will summarize the characteristics of telomeres and telomerase in lymphoid malignancies and discuss the role of telomere-associated proteins in these entities.

Infection by Henneguya sp. (Myxozoa) in the bone tissue of the gill filaments of the Amazonian catfish Hypophthalmus marginatus (Siluriformes).

This study describes aspects of the infection caused by the myxosporean genus Henneguya, which forms cysts in the bony portion of the gill filaments of Hypophthalmusmarginatus. Specimens of this catfish were acquired dead from artisanal fishermen near the town of Cametá, state of Pará, northern Brazil, between July 2011 and May 2012. They were transported in refrigerated containers to the Carlos Azevedo Research Laboratory at the Federal Rural University of Amazonia, in Belém, where analyses were performed. After confirmation of parasitism by the genus Henneguya, observation were made using optical and differential interference contrast (DIC) microscopy. The histological technique of embedment in paraffin was used. Ziehl-Neelsen staining was applied to the histological sections. Necropsy analyses on specimens of H. marginatus showed that 80% of them (40/50) had cysts of whitish coloration inside the bony portion of the gill filaments, filled with Henneguya spores. The present study found inflammatory infiltrate in the vicinity of the cysts. Furthermore, the special Ziehl-Neelsen staining technique made it possible to mark the Henneguya sp. cysts in the bone tissue and in spore isolates in the gill tissue structure. The descriptions of these histopathological findings show that this parasite is very invasive and causes damage to its host tissues.

The critical illness polyneuropathy in septic patients with prolonged weaning from mechanical ventilation: is the diaphragm also affected? A pilot study.

BACKGROUND: Critical illness myopathy and/or neuropathy (CRIMYNE) is a common alteration seen in the ICU. The currently available bedside methods of measuring respiratory and peripheral muscle function in critically ill patients are somewhat inadequate. The objective of this study was to evaluate the presence of diaphragmatic and peripheral CRIMYNE in septic patients with prolonged weaning from mechanical ventilation (MV). METHODS: Cohort prospective study with an entry period of 6 months. In 2 Brazilian medical-surgical ICUs, septic patients ≥ 18 years of age, dependent on MV ≥ 14 days, requiring prolonged weaning from MV, awake (Richmond Agitation Sedation Scale ≥ -2), and with no previous history of polyneuropathy or myopathy were included. Electrophysiological studies of the limbs and also of the respiratory system by phrenic nerve conduction and needle electromyography of the diaphragm were performed in all subjects. RESULTS: Twelve subjects were enrolled during 6 months of study. The electrophysiological signs of peripheral CRIMYNE occurred in 9 subjects, 7 of whom died in the ICU. Three subjects developed critical illness polyneuropathy, 4 critical illness myopathy, and 2 both. Only one subject who developed peripheral CRIMYNE did not present diaphragmatic involvement, whereas no subject developed diaphragm involvement alone. Thus, electrophysiological signs of diaphragmatic CRIMYNE occurred in 8 of the 9 subjects with peripheral CRIMYNE. Upon clinical examination, 8 subjects were not able to moves their limbs against gravity, and these findings were related to the presence of peripheral and diaphragmatic dysfunction. CONCLUSIONS: Our pilot findings suggested that CRIMYNE is common in septic patients with prolonged weaning from MV (MV ≥ 14 d). The inability to move limbs against gravity is frequently associated with peripheral and diaphragmatic CRIMYNE, and the findings of CRIMYNE in peripheral electrophysiological tests are associated with diaphragmatic involvement.