Histone Deacetylase 6 (HDAC6) Promotes the Pro-survival Activity of 14-3-3ζ via Deacetylation of Lysines within the 14-3-3ζ Binding Pocket

The phospho-binding protein 14-3-3ζ acts as a signaling hub controlling a network of interacting partners and oncogenic pathways. We show here that lysines within the 14-3-3ζ binding pocket and protein-protein interface can be modified by acetylation. The positive charge on two of these lysines, Lys⁴⁹ and Lys¹²⁰, is critical for coordinating 14-3-3ζ-phosphoprotein interactions. Through screening, we identified HDAC6 as the Lys⁴⁹/Lys¹²⁰ deacetylase. Inhibition of HDAC6 blocks 14-3-3ζ interactions with two well described interacting partners, Bad and AS160, which triggers their dephosphorylation at Ser¹¹² and Thr⁶⁴², respectively. Expression of an acetylation-refractory K49R/K120R mutant of 14-3-3ζ rescues both the HDAC6 inhibitor-induced loss of interaction and Ser¹¹²/Thr⁶⁴² phosphorylation. Furthermore, expression of the K49R/K120R mutant of 14-3-3ζ inhibits the cytotoxicity of HDAC6 inhibition. These data demonstrate a novel role for HDAC6 in controlling 14-3-3ζ binding activity.     

Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor

Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP), can arise through mutation of FECV to FIP virus (FIPV). The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro) with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for further development for important coronaviruses in animals and humans.     

A simple fluid-structure coupling algorithm for the study of the anastomotic mechanics of vascular grafts

Vascular anastomoses constitute a main factor in poor graft performance due to mismatches in distensibility between the host artery and the graft. This work aims at computational fluid-structure investigations of proximal and distal anastomoses of vein grafts and synthetic grafts. Finite element and finite volume models were developed and coupled with a user-defined algorithm. Emphasis was placed on the simplicity of the coupling algorithm. An artery and vein graft showed a larger dilation mismatch than an artery and synthetic graft. The vein graft distended nearly twice as much as the artery while the synthetic graft displayed only approximately half the arterial dilation. For the vein graft, luminal mismatching was aggravated by development of an anastomotic pseudo-stenosis. While this study focused on end-to-end anastomoses as a vehicle for developing the coupling algorithm, it may serve as useful point of departure for further investigations such as other anastomotic configurations, refined modelling of sutures and fully transient behaviour.     

Fungal-bacterial biofilms: their development for novel biotechnological applications

The attachment of microbes on biotic or abiotic surfaces to form biofilm structures has a great impact on biodegradation and biosynthesis in nature. Various interactions in such biofilms and their extracellular polymeric substances (EPS) layer make them considerably different in physiology and action, compared to that of their individual microbes in planktonic (free swimming) mode of growth. Expression of new genes is up-regulated in the biofilm cells, due in part to the cellular interactions, compared with the planktonic cells. Formation of fungal-bacterial biofilms (FBB) by bacterial colonization on biotic fungal surface gives the biofilm enhanced metabolic activities compared to monocultures, and perhaps multi-species bacterial or fungal biofilms on abiotic surfaces. Incorporation of a N₂-fixing rhizobial strain to the FBB to form fungal-rhizobial biofilms (FRB) has been shown to improve potential biofilm applications in N-deficient settings and in the production of biofilmed inocula for biofertilizers and biocontrol in plants. Their applications in agricultural and environmental settings, enzyme technology, drug discovery studies and energy research are being investigated. Thus, it has already been shown that the use of the FBB is a promising technology for many applications. This review deals with the different areas in which FBB/FRB have been seen to be applied with successful results as well as the numerous emerging avenues in which they show promising potential.     

Biofilm prevalence and microbial characterisation in chronic wounds in a Sri Lankan cohort

Biofilms have been associated with chronic wound infections in diabetic patients. The study assessed the occurrence of biofilms in chronic diabetic wounds (CDWs) in a Sri Lankan cohort. Tissue specimens collected during surgical debridement were analysed by quantitative differential viable counting, scanning electron microscopy (SEM), fluorescence insitu hybridization (FISH) and light microscopy with Gram and Haematoxylin-Eosin staining. All specimens harboured >5·0 log₁₀ CFU per g bacteria and 2–9 distinct species per specimen were recovered from twenty wounds by culture. The most frequently isolated bacterium was Pseudomonas spp. (12/20;60%). Strict anaerobes were isolated from 10/20 specimens. Gram and Haematoxylin-Eosin staining showed aggregated micro-colonies, embedded in the wound tissue bed (20/20) but the exopolymer matrix was not visible in all samples (13/20). Fluorescence microscopy using a eubacteria-specific FISH probe indicated the presence of bacterial aggregates within the deep layers of the wound tissues (20/20). SEM revealed the presumptive architecture of matrix-embedded microbial clusters (20/20). The approximate diameter of bacterial aggregates in tissues ranged between 12 and 400 µm. Bacterial infiltration into the internal portions of the tissues was apparent using FISH, Gram, and Haematoxylin-Eosin staining. All CDWs carried biofilm-specific morphological features. FISH was more specific than SEM and indicated the presence of microcolonies within deeper tissues.     

Metabolic-Stress-Induced Rearrangement of the 14-3-3ζ Interactome Promotes Autophagy via a ULK1- and AMPK-Regulated 14-3-3ζ Interaction with Phosphorylated Atg9

14-3-3ζ promotes cell survival via dynamic interactions with a vast network of binding partners, many of which are involved in stress regulation. We show here that hypoxia (low glucose and oxygen) triggers a rearrangement of the 14-3-3ζ interactome to favor an interaction with the core autophagy regulator Atg9A. Our data suggest that the localization of mammalian Atg9A to autophagosomes requires phosphorylation on the C terminus of Atg9A at S761, which creates a 14-3-3ζ docking site. Under basal conditions, this phosphorylation is maintained at a low level and is dependent on both ULK1 and AMPK. However, upon induction of hypoxic stress, activated AMPK bypasses the requirement for ULK1 and mediates S761 phosphorylation directly, resulting in an increase in 14-3-3ζ interactions, recruitment of Atg9A to LC3-positive autophagosomes, and enhanced autophagosome production. These data suggest a novel mechanism whereby the level of autophagy induction can be modulated by AMPK/ULK1-mediated phosphorylation of mammalian Atg9A.     

Social mobilisation, drug coverage and compliance and adverse reactions in a Mass Drug Administration (MDA) Programme for the Elimination of Lymphatic Filariasis in Sri Lanka

Background

In Sri Lanka filariasis is endemic in Southern, Western and North Western provinces covering eight districts designated as implementation units in the Programme for the Elimination of Lymphatic Filariasis (PELF). Despite control activities over sixty years including multidose diethylcarbamazine, 6 mg/kg treatment microfilaria rates had persisted at low levels. Following systematic social mobilisation the first MDA with DEC albendazole combination was conducted in 2002.

Methods

We investigated the extent social mobilisation had reached the people, their drug compliance and adverse reactions. Three localities were selected from each district to pick target population samples for pre-tested questionnaire. Three teams each with six people visited one district each day. One team worked from three starting points in one locality. A member applied eight part questionnaire to one family member totalling 150–160 people from one locality. Questions included social mobilisation, drug compliance and adverse reactions.

Results

Information was disseminated by television, radio, banners and leaflets, to a lesser extent by people. Information reached more people in the periphery than in Colombo. 35.2% from Colombo municipality were unaware of the MDA. Drug coverage was 79.6%, home delivery 71.7% and delivery centres 7.9%. 35.6% in Colombo district and 53.4% from Colombo municipality did not receive drugs. Drugs were consumed by 71.4%. 28.6% who did not comply included 20.4% who did not receive them. 91.4% showed no adverse reactions, 7.5% were mild, 1.1% recovered with home remedies.

Conclusion

Drug compliance showed significant positive correlation with awareness of the MDA. Door to door delivery was more successful than delivery from centres. More delivery centres conveniently located would have rectified this disparity. Poor awareness and compliance in Colombo and urban areas could be rectified with separate strategy for urban areas. More time for MDA and trained adequate manpower would ensure coverage to achieve elimination.