Design, synthesis, and antiviral properties of 4'-substituted ribonucleosides as inhibitors of hepatitis C virus replication: the discovery of R1479

A series of 4'-substituted ribonucleoside derivatives has been prepared and evaluated for inhibition of hepatitis C virus (HCV) RNA replication in cell culture. The most potent and non-cytotoxic derivative was compound 28 (4'-azidocytidine, R1479) with an IC(50) of 1.28 microM in the HCV replicon system. The triphosphate of compound 28 was prepared and shown to be an inhibitor of RNA synthesis mediated by NS5B (IC(50)=320 nM), the RNA polymerase encoded by HCV. Data on related analogues have been used to generate some preliminary requirements for activity within this series of nucleosides.     

Prophylactic treatment with fms-like tyrosine kinase-3 ligand after burn injury enhances global immune responses to infection

Severely burned patients are susceptible to infections with opportunistic organisms due to altered immune responses and frequent wound contamination. Immunomodulation to enhance systemic and local responses to wound infections may be protective after burn injury. We previously demonstrated that pretreatments with fms-like tyrosine kinase-3 (Flt3) ligand (Flt3L), a dendritic cell growth factor, increase the resistance of mice to a subsequent burn injury and wound infection by a dendritic cell-dependent mechanism. This study was designed to test the hypothesis that Flt3L administration after burn injury decreases susceptibility to wound infections by enhancing global immune cell activation. Mice were treated with Flt3L after burn injury and examined for survival, wound and systemic bacterial clearance, and immune cell activation after wound inoculation with Pseudomonas aeruginosa. To gain insight into the local effects of Flt3L at the burn wound, localization of Langerhans cells was examined. Mice treated with Flt3L had significantly greater numbers of CD25-expressing T cells and CD69-expressing T and B cells, neutrophils, and macrophages after, but not before, infection. Overall leukocyte apoptosis in response to infection was decreased with Flt3L treatment. Survival and local and systemic bacterial clearance were enhanced by Flt3L. Langerhans cells appeared in the dermis of skin bordering the burn wound, and further increased in response to wound infection. Flt3L augmented the appearance of Langerhans cells in response to both injury and infection. These data suggest that dendritic cell enhancement by Flt3L treatments after burn injury protects against opportunistic infections through promotion of local and systemic immune responses to infection.     

Only Akt1 is required for proliferation, while Akt2 promotes cell cycle exit through p21 binding

Protein kinase B (PKB/Akt) is an important modulator of insulin signaling, cell proliferation, and survival. Using small interfering RNA duplexes in nontransformed mammalian cells, we show that only Akt1 is essential for cell proliferation, while Akt2 promotes cell cycle exit. Silencing Akt1 resulted in decreased cyclin A levels and inhibition of S-phase entry, effects not seen with Akt2 knockdown and specifically rescued by microinjection of Akt1, not Akt2. In differentiating myoblasts, Akt2 knockout prevented myoblasts from exiting the cell cycle and showed sustained cyclin A expression. In contrast, overexpression of Akt2 reduced cyclin A and hindered cell cycle progression in M-G1 with increased nuclear p21. p21 is a major target in the differential effects of Akt isoforms, with endogenous Akt2 and not Akt1 binding p21 in the nucleus and increasing its level. Accordingly, Akt2 knockdown cells, and not Akt1 knockdown cells, showed reduced levels of p21. A specific Akt2/p21 interaction can be reproduced in vitro, and the Akt2 binding site on p21 is similar to that in cyclin A spanning T145 to T155, since (i) prior incubation with cyclin A prevents Akt2 binding, (ii) T145 phosphorylation on p21 by Akt1 prevents Akt2 binding, and (iii) binding Akt2 prevents phosphorylation of p21 by Akt1. These data show that specific interaction of the Akt2 isoform with p21 is key to its negative effect on normal cell cycle progression.     

Apoptosis Versus Cell Differentiation: Role of Heat Shock Proteins HSP90, HSP70 and HSP27

Heat shock proteins HSP27, HSP70 and HSP90 are molecular chaperones whose expression is increased after many different types of stress. They have a protective function helping the cell to cope with lethal conditions. The cytoprotective function of HSPs is largely explained by their anti-apoptotic function. HSPs have been shown to interact with different key apoptotic proteins. As a result, HSPs can block essentially all apoptotic pathways, most of them involving the activation of cystein proteases called caspases. Apoptosis and differentiation are physiological processes that share many common features, for instance, chromatin condensation and the activation of caspases are frequently observed. It is, therefore, not surprising that many recent reports imply HSPs in the differentiation process. This review will comment on the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation. HSPs may determine de fate of the cells by orchestrating the decision of apoptosis versus differentiation.Key Words: apoptosis, differentiation, heat shock proteins, chaperones, cancer cells, anticancer drugs     

Mutations in cytoplasmic dynein lead to a Huntington's disease-like defect in energy metabolism of brown and white adipose tissues

The molecular motor dynein is regulated by the huntingtin protein, and Huntington's disease (HD) mutations of huntingtin disrupt dynein motor activity. Besides abnormalities in the central nervous system, HD animal models develop prominent peripheral pathology, with defective brown tissue thermogenesis and dysfunctional white adipocytes, but whether this peripheral phenotype is recapitulated by dynein dysfunction is unknown. Here, we observed prominently increased adiposity in mice harboring the legs at odd angles (Loa/+) or the Cramping mutations (Cra/+) in the dynein heavy chain gene. In Cra/+ mice, hyperadiposity occurred in the absence of energy imbalance and was the result of impaired norepinephrine-stimulated lipolysis. A similar phenotype was observed in 3T3L1 adipocytes upon chemical inhibition of dynein showing that loss of functional dynein leads to impairment of lipolysis. Ex vivo, dynein mutant adipose tissue displayed increased reactive oxygen species production that was, at least partially, responsible for the decreased cellular responses to norepinephrine and subsequent defect in stimulated lipolysis. Dynein mutation also affected norepinephrine efficacy to elicit a thermogenic response and led to morphological abnormalities in brown adipose tissue and cold intolerance in dynein mutant mice. Interestingly, protein levels of huntingtin were decreased in dynein mutant adipose tissue. Collectively, our results provide genetic evidence that dynein plays a key role in lipid metabolism and thermogenesis through a modulation of oxidative stress elicited by norepinephrine. This peripheral phenotype of dynein mutant mice is similar to that observed in various animal models of HD, lending further support for a functional link between huntingtin and dynein.     

Fouling and its control in heat exchangers in the dairy industry

Fouling of heat transfer equipment poses a major problem in the food and especially the dairy industry. A review of recent progress made in the study and understanding of fouling and cleaning mechanisms in relation to exchangers and heat exchange surfaces in contact with milk components is given.

The first part describes the different materials and methods used in the study of fouling and cleaning, from laboratory to pilot‐plant and industrial scale.

The second part concerns the different fouling dynamics observed with milk and dairy products, and gives an interpretation of the different behaviours which occur, on the basis of the most recent results on the composition and structure of the deposits and the biochemical mechanisms of fouling.

The last part is a complete review of cleaning of milk deposits, taking into account the chemical reaction approach to cleaning as a mass transfer phenomenon.     

Pulmonary vascular response to thrombin: Effects of thromboxane synthetase inhibition with OKY-046 and OKY-1581

We examined the effects of thromboxane synthetase inhibition with OKY-1581 and OKY-046 on pulmonary hemodynamics and lung fluid balance after thrombin-induced intravascular coagulation. Studies were made in anesthetized sheep prepared with lyng lymph fistulas. Pulmonary intravascular coagulation was induced by i.v. infusion of α-thrombin over a 15 min period. Thrombin infusion in control sheep resulted in immediate increases in pulmonary artery pressure (P ) and pulmonary vascular resistance (PVR), which associated with rapid 3-fold increase in pulmonary lymph flow (Q̇lym) and a delayed increase in lymph-to-plasma protein concentration (L/P) ratio, indicating an increase in the pulmonary microvascular permeability to proteins. Thrombin-induced intravascular coagulation alos increased arterial thromboxane B₂ (a metabolite of thromboxane A₂) and 6-keto-PGF_1α concentrations (a metabolite of prostacyclin). Both OKY-1581 and OKY-046 prevented thromboxane B₂ and 6-keto-PGF_1α generation. The initial increments in P and PVR were attenuated in both treated groups. The increases in Q̇lym were gradual in the treated groups but attained the same levels as in control group. However, the increases in Q̇lym were associated with decreases in L/P ratio. In both treated groups, the leukocyte count decreased after thrombin infusion but then increased steadily above the baseline value, whereas the leukocyte count remained depressed in the control group after thrombin. These studies indicate that a part of the initial pulmonary vasoconstrictor response to thrombin-induced intravascular coagulation is mediated by thromboxane generation. In addition, thromboxane may also contribute to the increase in lung vascular permeability to proteins that occurs after intravascular coagulation and this effect may be mediated by a thromboxane-neutrophil interaction.     

Design and Dosimetric Analysis of an Exposure Facility for Investigating Possible Effects of 2.45 GHz Wi-Fi Signals on Human Sleep

A new head exposure system for double-blind provocation studies investigating possible effects of 2.45 GHz Wi-Fi exposure on human sleep was developed and dosimetrically analyzed. The exposure system includes six simultaneously radiating directional antennas arranged along a circle (radius 0.6 m) around the test subject's head, and enables a virtually uniform head exposure, i.e. without any preferred direction of incidence, during sleep. The system is fully computer-controlled and applies a real wireless local area network (WLAN) signal representing different transmission patterns as expected in real WLAN scenarios, i.e. phases of “beacon only” as well as phases of different data transmission rates. Sham and verum are applied in a double-blind crossover study design and all relevant exposure data, i.e. forward and reverse power at all six antenna inputs, are continuously recorded for quality control. For a total antenna input power (sum of all antennas) of 220 mW, typical specific absorption rate (SAR) in cortical brain regions is approximately 1–2 mW/kg (mass average SAR over respective brain region), which can be seen as a realistic worst-case exposure level in real WLAN scenarios. Taking into account variations of head positions during the experiments, the resulting exposure of different brain regions may deviate from the given average SAR levels up to 10 dB. Peak spatial 10 g average SAR in all brain and all head tissues is between 1.5–3.5 and 10.4–25 mW/kg, respectively. Bioelectromagnetics. © 2020 Bioelectromagnetics Society.     

BCL-XL is crucial for progression through the adenoma-to-carcinoma sequence of colorectal cancer

Evasion of apoptosis is a hallmark of cancer, which is frequently mediated by upregulation of the antiapoptotic BCL-2 family proteins. In colorectal cancer (CRC), previous work has highlighted differential antiapoptotic protein dependencies determined by the stage of the disease. While intestinal stem cells (ISCs) require BCL-2 for adenoma outgrowth and survival during transformation, ISC-specific MCL1 deletion results in disturbed intestinal homeostasis, eventually contributing to tumorigenesis. Colon cancer stem cells (CSCs), however, no longer require BCL-2 and depend mainly on BCL-XL for their survival. We therefore hypothesized that a shift in antiapoptotic protein reliance occurs in ISCs as the disease progresses from normal to adenoma to carcinoma. By targeting antiapoptotic proteins with specific BH3 mimetics in organoid models of CRC progression, we found that BCL-2 is essential only during ISC transformation while MCL1 inhibition did not affect adenoma outgrowth. BCL-XL, on the other hand, was crucial for stem cell survival throughout the adenoma-to-carcinoma sequence. Furthermore, we identified that the limited window of BCL-2 reliance is a result of its downregulation by miR-17-5p, a microRNA that is upregulated upon APC-mutation driven transformation. Here we show that BCL-XL inhibition effectively impairs adenoma outgrowth in vivo and enhances the efficacy of chemotherapy. In line with this dependency, expression of BCL-XL, but not BCL-2 or MCL1, directly correlated to the outcome of chemotherapy-treated CRC patients. Our results provide insights to enable the rational use of BH3 mimetics in CRC management, particularly underlining the therapeutic potential of BCL-XL targeting mimetics in both early and late-stage disease.Subject terms: Cancer models, Cancer stem cells, Cell biology     

Encapsulated mesenchymal stromal cells for in vivo transplantation

Immunomodulatory human mesenchymal stromal cells (hMSC) have been incorporated into therapeutic protocols to treat secondary inflammatory responses post-spinal cord injury (SCI) in animal models. However, limitations with direct hMSC implantation approaches may prevent effective translation for therapeutic development of hMSC infusion into post-SCI treatment protocols. To circumvent these limitations, we investigated the efficacy of alginate microencapsulation in developing an implantable vehicle for hMSC delivery. Viability and secretory function were maintained within the encapsulated hMSC population, and hMSC secreted anti-inflammatory cytokines upon induction with the pro-inflammatory factors, TNF-α and IFN-γ. Furthermore, encapsulated hMSC modulated inflammatory macrophage function both in vitro and in vivo, even in the absence of direct hMSC-macrophage cell contact and promoted the alternative M2 macrophage phenotype. In vitro, this was evident by a reduction in macrophage iNOS expression with a concomitant increase in CD206, a marker for M2 macrophages. Finally, Sprague-Dawley rat spinal cords were injured at vertebra T10 via a weight drop model (NYU model) and encapsulated hMSC were administered via lumbar puncture 24 h post-injury. Encapsulated hMSC localized primarily in the cauda equina of the spinal cord. Histological assessment of spinal cord tissue 7 days post-SCI indicated that as few as 5 × 10(4) encapsulated hMSC yielded increased numbers of CD206-expressing macrophages, consistent with our in vitro studies. The combined findings support the inclusion of immobilized hMSC in post-CNS trauma tissue protective therapy, and suggest that conversion of macrophages to the M2 subset is responsible, at least in part, for tissue protection.