Analysis of self-organizing maps and explainable artificial intelligence to identify hydrochemical factors that drive drinking water quality in Haor region.

Water contamination undermines human survival and economic growth. Water resource protection and management require knowledge of water hydrochemistry and drinking water quality characteristics, mechanisms, and factors. Self-organizing maps (SOM) have been developed using quantization and topographic error approaches to cluster hydrochemistry datasets. The Piper diagram, saturation index (SI), and cation exchange method were used to determine the driving mechanism of hydrochemistry in both surface and groundwater, while the Gibbs diagram was used for surface water. In addition, redundancy analysis (RDA) and a generalized linear model (GLM) were used to determine the key drinking water quality parameters in the study area. Additionally, the study aimed to utilize Explainable Artificial Intelligence (XAI) techniques to gain insights into the relative importance and impact of different parameters on the entropy water quality index (EWQI). The SOM results showed that thirty neurons generated the hydrochemical properties of water and were organized into four clusters. The Piper diagram showed that the primary hydrochemical facies were HCO3--Ca2+ (cluster 4), Cl---Na+ (all clusters), and mixed (clusters 1 and 4). Results from SI and cation exchange show that demineralization and ion exchange are the driving mechanisms of water hydrochemistry. About 45 % of the studied samples are classified as "medium quality"," that could be suitable as drinking water with further refinement. Cl- may pose increased non-carcinogenic risk to adults, with children at double risk. Cluster 4 water is low-risk, supporting EWQI findings. The RDA and GLM observations agree in that Ca2+, Mg2+, Na+, Cl- and HCO3- all have a positive and significant effect on EWQI, with the exception of K+. TDS, EC, Na+, and Ca2+ have been identified as influencing factors based on bagging-based XAI analysis at global and local levels. The analysis also addressed the importance of SO4, HCO3, Cl, Mg2+, K+, and pH at specific locations.

Difference in presentation, outcomes, and hospital epidemiologic trend of COVID-19 among first, second, and third waves: a review of hospital records and prospective cohort study.

This study aimed to examine the differences in epidemiologic and disease aspects among patients with coronavirus disease-19 (COVID-19). Methods: The authors reviewed the hospital records between April 2020 and September 2021 and followed up on the patients for post-COVID complications. Findings: Older adult patients were predominantly affected during the third wave, and middle-aged patients were predominantly affected during the first and second waves. Men were predominantly admitted, considering the three waves, although more women were admitted in the second wave. Cough was more common in the second and third waves than in the first wave 522 (59.7%). Respiratory distress was the most common in the third wave, 251(67.1%), and least common in the first wave, 403 (46.1%). Anosmia was more common in the third wave 116 (31.2%). In the third wave, patients presenting in a critical state 23 (6.2%) and with severe disease 152 (40.8%) were more common. The hospital admission median (IQR) was longer in the first wave, 12 (8-20), than in other waves. More patients were admitted in the first wave (52%) than in the other waves, and patients received more oxygen in the third wave (75%) than in the other waves. Death occurred more commonly in the first wave (51%) than in the other waves. The positivity rate was higher in the third wave (22.8%) than in the other waves. In the third wave, the positivity rate was higher in women (24.3%) than in men. Post-COVID cough increased in the second wave, and fatigue was higher in the third wave than in the other waves. Tiredness and memory loss were greater during the second wave than in other waves. Conclusion: The authors found differences in the presentation, outcomes, and hospital epidemiologic trend of COVID-19 among the three waves.

Dynamic antibody response in SARS-CoV-2 infected patients and COVID-19 vaccine recipients alongside vaccine effectiveness in comorbid and multimorbid groups.

Objectives: Underlying medical conditions are critical risk factors for COVID-19 susceptibility and its rapid clinical manifestation. Therefore, the preexisting burden of non-communicable diseases (NCDs) makes the preparedness for COVID-19 more challenging for low- and middle-income countries (LMICs). These countries have relied on vaccination campaigns as an effective measure to tackle COVID-19. In this study, we investigated the impact of comorbidities on humoral antibody responses against the specific receptor-binding domain (RBD) of SARS-CoV2. Methods: A total of 1005 patients were selected for the SARS-CoV-2 specific immunoglobulin G (IgG1, IgG2, IgG3, and IgG4 subclasses) and total antibody (TAb) tests (IgG and IgM), of which 912 serum samples were ultimately selected based on the specimen cutoff analyte value. Patients with multimorbidity (N = 60) were recruited for follow-up studies from the initial cohort, and their immune response (IgG and TAb) was measured at multiple time points after the second dose of vaccination. Siemens Dimension Vista SARS-CoV-2 IgG (CV2G) and SARS-CoV-2 TAb assay (CV2T) were used to carry out the serology test. Results: Out of a total of 912 participants, vaccinated individuals (N = 711) had detectable antibody responses up to 7-8 months. The synergistic effect of natural infection and vaccine response was also studied. Participants with breakthrough infections (N = 49) mounted a greater antibody response compared to individuals with normal vaccination response (N = 397) and those who were naturally infected before receiving the second dose of vaccine (N = 132). Investigation of the impact of comorbidities revealed that diabetes mellitus (DM) (N = 117) and kidney disease (N = 50) had a significant negative impact on the decline of the humoral antibody response against SARS-CoV-2. IgG and TAb declined more rapidly in diabetic and kidney disease patients compared to the other four comorbid groups. Follow-up studies demonstrated that antibody response rapidly declined within 4 months after receiving the second dose. Conclusion: The generalized immunization schedule for COVID-19 needs to be adjusted for high-risk comorbid groups, and a booster dose must be administered early within 4 months after receiving the second dose.

Structure of Human Mitochondrial Translation Initiation Factor 3 Bound to the Small Ribosomal Subunit.

The human mitochondrial translational initiation factor 3 (IF3mt) carries mitochondrial-specific amino acid extensions at both its N and C termini (N- and C-terminal extensions [NTE and CTE, respectively]), when compared with its eubacterial counterpart. Here we present 3.3- to 3.5-Å-resolution cryoelectron microscopic structures of the mammalian 28S mitoribosomal subunit in complex with human IF3mt. Unique contacts observed between the 28S subunit and N-terminal domain of IF3mt explain its unusually high affinity for the 28S subunit, whereas the position of the mito-specific NTE suggests NTE's role in binding of initiator tRNA to the 28S subunit. The location of the C-terminal domain (CTD) clarifies its anti-association activity, whereas the orientation of the mito-specific CTE provides a mechanistic explanation for its role in destabilizing initiator tRNA in the absence of mRNA. Furthermore, our structure hints at a possible role of the CTD in recruiting leaderless mRNAs for translation initiation. Our findings highlight unique features of IF3mt in mitochondrial translation initiation.

Characterization of a wheat pathogenesis-related protein, TaBWPR-1.2, in seminal roots in response to waterlogging stress.

We examined the role of pathogenesis-related protein TaBWPR-1.2 in the context of molecular and physiological responses of wheat (Triticum aestivum) seminal roots under waterlogging stress. Two cDNAs corresponding to the TaBWPR-1.2 gene, TaBWPR-1.2#2 and TaBWPR-1.2#13 were cloned from seminal roots. These cDNAs were predicted to encode proteins of 173 and 172 amino acids, respectively. In a time-course experiment, TaBWPR-1.2 gene expression was highest in whole seminal roots after 1 day of waterlogging treatment and higher than the control for at least 10 days; significantly increased protein abundance was observed after 7 days of waterlogging. Drought, another abiotic stress, did not influence TaBWPR-1.2 gene expression in wheat seminal roots at 5-d-old seedlings. Tissue-specific studies revealed that the highest TaBWPR-1.2 gene expression and protein levels were in the aerenchymatous root zone. TaBWPR-1.2 expression in seminal roots was also increased by the signalling molecules 1-aminocyclopropane-1-carboxylic acid (ACC; an ethylene precursor), H2O2, jasmonic acid (JA), and nitric oxide (NO); however, treatment with abscisic acid (ABA), salicylic acid (SA), and ethanol did not alter its expression. Interestingly, aerenchyma formation in the seminal root cortex was induced only by ACC and H2O2. Taken together, these results indicate that TaBWPR-1.2 is a waterlogging-responsive gene that might be associated with root cortex tissue alteration in wheat plants through ACC and/or H2O2 regulatory mechanisms.

Identification and characterization of CHCHD1, AURKAIP1, and CRIF1 as new members of the mammalian mitochondrial ribosome.

Defects in mitochondrial ribosomal proteins (MRPs) cause various diseases in humans. Because of the essential role of MRPs in synthesizing the essential subunits of oxidative phosphorylation (OXPHOS) complexes, identifying all of the protein components involved in the mitochondrial translational machinery is critical. Initially, we identified 79 MRPs; however, identifying MRPs with no clear homologs in bacteria and yeast mitochondria was challenging, due to limited availability of expressed sequence tags (ESTs) in the databases available at that time. With the improvement in genome sequencing and increased sensitivity of mass spectrometry (MS)-based technologies, we have established four previously known proteins as MRPs and have confirmed the identification of ICT1 (MRP58) as a ribosomal protein. The newly identified MRPs are MRPS37 (Coiled-coil-helix-coiled-coil-helix domain containing protein 1-CHCHD1), MRPS38 (Aurora kinase A interacting protein1, AURKAIP1), MRPS39 (Pentatricopeptide repeat-containing protein 3, PTCD3), in the small subunit and MRPL59 (CR-6 interacting factor 1, CRIF1) in the large subunit. Furthermore, we have demonstrated the essential roles of CHCHD1, AURKAIP1, and CRIF1in mitochondrial protein synthesis by siRNA knock-down studies, which had significant effects on the expression of mitochondrially encoded proteins.

Contacts between mammalian mitochondrial translational initiation factor 3 and ribosomal proteins in the small subunit.

Mammalian mitochondrial translational initiation factor 3 (IF3(mt)) binds to the small subunit of the ribosome displacing the large subunit during the initiation of protein biosynthesis. About half of the proteins in mitochondrial ribosomes have homologs in bacteria while the remainder are unique to the mitochondrion. To obtain information on the ribosomal proteins located near the IF3(mt) binding site, cross-linking studies were carried out followed by identification of the cross-linked proteins by mass spectrometry. IF3(mt) cross-links to mammalian mitochondrial homologs of the bacterial ribosomal proteins S5, S9, S10, and S18-2 and to unique mitochondrial ribosomal proteins MRPS29, MRPS32, MRPS36 and PTCD3 (Pet309) which has now been identified as a small subunit ribosomal protein. IF3(mt) has extensions on both the N- and C-termini compared to the bacterial factors. Cross-linking of a truncated derivative lacking these extensions gives the same hits as the full length IF3(mt) except that no cross-links were observed to MRPS36. IF3 consists of two domains separated by a flexible linker. Cross-linking of the isolated N- and C-domains was observed to a range of ribosomal proteins particularly with the C-domain carrying the linker which showed significant cross-linking to several ribosomal proteins not found in prokaryotes.

Hemagglutinin fusion peptide mutants in model membranes: structural properties, membrane physical properties, and PEG-mediated fusion.

While the importance of viral fusion peptides (e.g., hemagglutinin (HA) and gp41) in virus-cell membrane fusion is established, it is unclear how these peptides enhance membrane fusion, especially at low peptide/lipid ratios for which the peptides are not lytic. We assayed wild-type HA fusion peptide and two mutants, G1E and G13L, for their effects on the bilayer structure of 1,2-dioleoyl-3-sn-phosphatidylcholine/1,2-dioleoyl-3-sn-phosphatidylethanolamine/Sphingomyelin/Cholesterol (35:30:15:20) membranes, their structures in the lipid bilayer, and their effects on membrane fusion. All peptides bound to highly curved vesicles, but fusion was triggered only in the presence of poly(ethylene glycol). At low (1:200) peptide/lipid ratios, wild-type peptide enhanced remarkably the extent of content mixing and leakage along with the rate constants for these processes, and significantly enhanced the bilayer interior packing and filled the membrane free volume. The mutants caused no change in contents mixing or interior packing. Circular dichroism, polarized-attenuated total-internal-reflection Fourier-transform infrared spectroscopy measurements, and membrane perturbation measurements all conform to the inverted-V model for the structure of wild-type HA peptide. Similar measurements suggest that the G13L mutant adopts a less helical conformation in which the N-terminus moves closer to the bilayer interface, thus disrupting the V-structure. The G1E peptide barely perturbs the bilayer and may locate slightly above the interface. Fusion measurements suggest that the wild-type peptide promotes conversion of the stalk to an expanded trans-membrane contact intermediate through its ability to occupy hydrophobic space in a trans-membrane contact structure. While wild-type peptide increases the rate of initial intermediate and final pore formation, our results do not speak to the mechanisms for these effects, but they do leave open the possibility that it stabilizes the transition states for these events.

Insertion domain within mammalian mitochondrial translation initiation factor 2 serves the role of eubacterial initiation factor 1.

Mitochondria have their own translational machineries for the synthesis of thirteen polypeptide chains that are components of the complexes that participate in the process of oxidative phosphorylation (or ATP generation). Translation initiation in mammalian mitochondria requires two initiation factors, IF2(mt) and IF3(mt), instead of the three that are present in eubacteria. The mammalian IF2(mt) possesses a unique 37 amino acid insertion domain, which is known to be important for the formation of the translation initiation complex. We have obtained a three-dimensional cryoelectron microscopic map of the mammalian IF2(mt) in complex with initiator fMet-tRNA(iMet) and the eubacterial ribosome. We find that the 37 amino acid insertion domain interacts with the same binding site on the ribosome that would be occupied by the eubacterial initiation factor IF1, which is absent in mitochondria. Our finding suggests that the insertion domain of IF2(mt) mimics the function of eubacterial IF1, by blocking the ribosomal aminoacyl-tRNA binding site (A site) at the initiation step.

Analysis of proteins in aerenchymatous seminal roots of wheat grown in hypoxic soils under waterlogged conditions.

Hypoxia caused by waterlogging results in a severe loss of crop production. At the primary stage of wheat development, the seminal roots have strategies to survive under hypoxia through alternative metabolism coupling root anatomical modification. The present study used a model system of lysigenous aerenchymatous seminal roots from a representative seedling stage of wheat to elucidate the root physiology in response to soil hypoxia. Seminal roots characteristic with lysigenous aerenchyma tissues were developed in pot cultures for 7 days under two hypoxic conditions, water depths of 15 cm below and 3 cm above the soil surface. Proteins from the roots were separated using two-dimensional polyacrylamide gel electrophoresis and identified using mass spectrometry. The results showed that approximately 345 distinct protein spots were detected by 2-DE, 29 spots changed in the expression levels between the control and two hypoxic plants, and 10 spots exhibited a reproducible up- or down regulated fluctuation. The up-regulated proteins were thought to be involved in alteration in energy and redox status, defense responses and cell wall turnover. These results suggest the effects of soil hypoxia on the activity of the identified up-regulated proteins and their roles in alternative respiration and cell degeneration in wheat in order to gain metabolic adjustment under hypoxia stress.

Identification of protein-protein and protein-ribosome interacting regions of the C-terminal tail of human mitochondrial inner membrane protein Oxa1L.

The mammalian mitochondrial inner membrane protein Oxa1L is involved in the insertion of a number of mitochondrial translation products into the inner membrane. During this process, the C-terminal tail of Oxa1L (Oxa1L-CTT) binds mitochondrial ribosomes and is believed to coordinate the synthesis and membrane insertion of the nascent chains into the membrane. The C-terminal tail of Oxa1L does not contain any Cys residues. Four variants of this protein with a specifically placed Cys residue at position 4, 39, 67, or 94 of Oxa1L-CTT have been prepared. These Cys residues have been derivatized with a fluorescent probe, tetramethylrhodamine-5-maleimide, for biophysical studies. Oxa1L-CTT forms oligomers cooperatively with a binding constant in the submicromolar range. Fluorescence anisotropy and fluorescence lifetime measurements indicate that contacts near a long helix close to position 39 of Oxa1L-CTT occur during oligomer formation. Fluorescence correlation spectroscopy measurements demonstrate that all of the Oxa1L-CTT derivatives bind to mammalian mitochondrial ribosomes. Steady-state fluorescence quenching and fluorescence lifetime data indicate that there are extensive contacts between Oxa1L-CTT and the ribosome-encompassing regions around positions 39, 67, and 94. The results of this study suggest that Oxa1L-CTT undergoes conformational changes and induced oligomer formation when it binds to the ribosome.

Properties of the C-terminal tail of human mitochondrial inner membrane protein Oxa1L and its interactions with mammalian mitochondrial ribosomes.

In humans the mitochondrial inner membrane protein Oxa1L is involved in the biogenesis of membrane proteins and facilitates the insertion of both mitochondrial- and nuclear-encoded proteins from the mitochondrial matrix into the inner membrane. The C-terminal approximately 100-amino acid tail of Oxa1L (Oxa1L-CTT) binds to mitochondrial ribosomes and plays a role in the co-translational insertion of mitochondria-synthesized proteins into the inner membrane. Contrary to suggestions made for yeast Oxa1p, our results indicate that the C-terminal tail of human Oxa1L does not form a coiled-coil helical structure in solution. The Oxa1L-CTT exists primarily as a monomer in solution but forms dimers and tetramers at high salt concentrations. The binding of Oxa1L-CTT to mitochondrial ribosomes is an enthalpy-driven process with a K(d) of 0.3-0.8 microM and a stoichiometry of 2. Oxa1L-CTT cross-links to mammalian mitochondrial homologs of the bacterial ribosomal proteins L13, L20, and L28 and to mammalian mitochondrial specific ribosomal proteins MRPL48, MRPL49, and MRPL51. Oxa1L-CTT does not cross-link to proteins decorating the conventional exit tunnel of the bacterial large ribosomal subunit (L22, L23, L24, and L29).

ROS resistance in Pisum sativum cv. Alaska: the involvement of nucleoside diphosphate kinase in oxidative stress responses via the regulation of antioxidants.

This study investigated the reactive oxygen species (ROS) tolerance mechanism of a paraquat-resistant Pisum sativum line (R3-1) compared with the wild type (WT). Physiological and biochemical analyses showed significant differences in the phenotypes, such as delayed leaf and floral development, superior branching, and greater biomass and yields in the R3-1 line, as well as an increased level of antioxidant pigments and a lower rate of cellular lipid peroxidation in the resistant R3-1. Additionally, the phosphorylation of crude proteins showed distinguishable differences in band mobility and intensity between the R3-1 and WT plants. cDNA cloning and sequence analysis of NDPKs, which were candidate phosphorylated proteins, revealed that two of the deduced amino acids in NDPK2 (IL12L and Glu205Lys) and one in NDPK3 (P45S) were mutated in R3-1. Using glutathione S-transferase-NDPK fusion constructs, we found that the precursor recombinant R3-1 NDPK2 showed an increased level of activity and autophosphorylation in R3-1 plants compared to WT plants. Native PAGE analysis of the crude proteins revealed that NDPK and catalase (CAT) activity co-existed in the same area of the gel. In a yeast two-hybrid assay, the N-terminal region of NDPK2 showed an interaction with the full-length CAT1 protein. Furthermore, we found that WT showed a decreased level of CAT activity compared with R3-1 under illumination and/or on media containing ROS-releasing reagents. Taken together, these results suggest that there is a strong interaction between NDPK2 and CAT1 in R3-1 plants, which possibly plays a vital role in the antioxidant defense against ROS.

The effect of spermine on the initiation of mitochondrial protein synthesis.

Polyamines are important in both prokaryotic and eukaryotic translational systems. Spermine is a quaternary aliphatic amine that is cationic under physiological conditions. In this paper, we demonstrate that spermine stimulates fMet-tRNA binding to mammalian mitochondrial 55S ribosomes. The stimulatory effect of spermine is independent of the identity of the mRNA. The degree of stimulation of spermine is the same at all concentrations of mitochondrial initiation factor 2 (IF2(mt)) and mitochondrial initiation factor 3 (IF3(mt)). This observation indicates that IF2(mt) and IF3(mt), while essential for initiation, are not the primary components of the translation initiation system affected by spermine. IF3(mt) dissociates 55S ribosomes detectably in the absence of spermine, but this effect is strongly inhibited in the presence of spermine. This observation indicates that the positive effect of spermine on initiation is not due to an increase in the availability of the small subunits for initiation. Spermine also promotes fMet-tRNA binding to small subunits of the mitochondrial ribosome in the presence of IF2(mt). The major effect of spermine in promoting initiation complex formation thus appears to be on the interaction of fMet-tRNA with the ribosome.

Roles of the N- and C-terminal domains of mammalian mitochondrial initiation factor 3 in protein biosynthesis.

Bacterial initiation factor 3 (IF3) is organized into N- and C-domains separated by a linker. Mitochondrial IF3 (IF3(mt)) has a similar domain organization, although both domains have extensions not found in the bacterial factors. Constructs of the N- and C-domains of IF3(mt) with and without the connecting linker were prepared. The K(d) values for the binding of full-length IF3(mt) and its C-domain with and without the linker to mitochondrial 28S subunits are 30, 60, and 95 nM, respectively, indicating that much of the ribosome binding interactions are mediated by the C-domain. However, the N-domain binds to 28S subunits with only a 10-fold lower affinity than full-length IF3(mt). This observation indicates that the N-domain of IF3(mt) has significant contacts with the protein-rich small subunit of mammalian mitochondrial ribosomes. The linker also plays a role in modulating the interactions between the 28S subunit and the factor; it is not just a physical connector between the two domains. The presence of the two domains and the linker may optimize the overall affinity of IF3(mt) for the ribosome. These results are in sharp contrast to observations with Escherichia coli IF3. Removal of the N-domain drastically reduces the activity of IF3(mt) in the dissociation of mitochondrial 55S ribosomes, although the C-domain itself retains some activity. This residual activity depends significantly on the linker region. The N-domain alone has no effect on the dissociation of ribosomes. Full-length IF3(mt) reduces the binding of fMet-tRNA to the 28S subunit in the absence of mRNA. Both the C-terminal extension and the linker are required for this effect. IF3(mt) promotes the formation of a binary complex between IF2(mt) and fMet-tRNA that may play an important role in mitochondrial protein synthesis. Both domains play a role promoting the formation of this complex.

The interaction of mammalian mitochondrial translational initiation factor 3 with ribosomes: evolution of terminal extensions in IF3mt.

Mammalian mitochondrial initiation factor 3 (IF3(mt)) has a central region with homology to bacterial IF3. This homology region is preceded by an N-terminal extension and followed by a C-terminal extension. The role of these extensions on the binding of IF3(mt) to mitochondrial small ribosomal subunits (28S) was studied using derivatives in which the extensions had been deleted. The K(d) for the binding of IF3(mt) to 28S subunits is approximately 30 nM. Removal of either the N- or C-terminal extension has almost no effect on this value. IF3(mt) has very weak interactions with the large subunit of the mitochondrial ribosome (39S) (K(d) = 1.5 muM). However, deletion of the extensions results in derivatives with significant affinity for 39S subunits (K(d) = 0.12-0.25 muM). IF3(mt) does not bind 55S monosomes, while the deletion derivative binds slightly to these particles. IF3(mt) is very effective in dissociating 55S ribosomes. Removal of the N-terminal extension has little effect on this activity. However, removal of the C-terminal extension leads to a complex dissociation pattern due to the high affinity of this derivative for 39S subunits. These data suggest that the extensions have evolved to ensure the proper dissociation of IF3(mt) from the 28S subunits upon 39S subunit joining.

Properties and structures of the influenza and HIV fusion peptides on lipid membranes: implications for a role in fusion.

The fusion peptides of HIV and influenza virus are crucial for viral entry into a host cell. We report the membrane-perturbing and structural properties of fusion peptides from the HA fusion protein of influenza virus and the gp41 fusion protein of HIV. Our goals were to determine: 1), how fusion peptides alter structure within the bilayers of fusogenic and nonfusogenic lipid vesicles and 2), how fusion peptide structure is related to the ability to promote fusion. Fluorescent probes revealed that neither peptide had a significant effect on bilayer packing at the water-membrane interface, but both increased acyl chain order in both fusogenic and nonfusogenic vesicles. Both also reduced free volume within the bilayer as indicated by partitioning of a lipophilic fluorophore into membranes. These membrane ordering effects were smaller for the gp41 peptide than for the HA peptide at low peptide/lipid ratio, suggesting that the two peptides assume different structures on membranes. The influenza peptide was predominantly helical, and the gp41 peptide was predominantly antiparallel beta-sheet when membrane bound, however, the depths of penetration of Trps of both peptides into neutral membranes were similar and independent of membrane composition. We previously demonstrated: 1), the abilities of both peptides to promote fusion but not initial intermediate formation during PEG-mediated fusion and 2), the ability of hexadecane to compete with this effect of the fusion peptides. Taken together, our current and past results suggest a hypothesis for a common mechanism by which these two viral fusion peptides promote fusion.

Inactivation of Drosophila DJ-1 leads to impairments of oxidative stress response and phosphatidylinositol 3-kinase/Akt signaling.

Parkinson's disease (PD) is the most common movement disorder characterized by dopaminergic dysfunction and degeneration. The cause of most PD cases is unknown, although postmortem studies have implicated the involvement of oxidative stress. The identification of familial PD-associated genes offers the opportunity to study mechanisms of PD pathogenesis in model organisms. Here, we show that DJ-1A, a Drosophila homologue of the familial PD-associated gene DJ-1, plays an essential role in oxidative stress response and neuronal maintenance. Inhibition of DJ-1A function through RNA interference (RNAi) results in cellular accumulation of reactive oxygen species, organismal hypersensitivity to oxidative stress, and dysfunction and degeneration of dopaminergic and photoreceptor neurons. To identify other genes that may interact with DJ-1A in regulating cell survival, we performed genetic interaction studies and identified components of the phosphatidylinositol 3-kinase (PI3K)/Akt-signaling pathway as specific modulators of DJ-1A RNAi-induced neurodegeneration. PI3K signaling suppresses DJ-1A RNAi phenotypes at least in part by reducing cellular reactive oxygen species levels. Consistent with the genetic interaction results, we also found reduced phosphorylation of Akt in DJ-1A RNAi animals, indicating an impairment of PI3K/Akt signaling by DJ-1A down-regulation. Together with recent findings in mammalian systems, these results implicate impairments of PI3K/Akt signaling and oxidative stress response in DJ-1-associated disease pathogenesis. We also observed impairment of PI3K/Akt signaling in the fly parkin model of PD, hinting at a common molecular event in the pathogenesis of PD. Manipulation of PI3K/Akt signaling may therefore offer therapeutic benefits for the treatment of PD.

Roles of curvature and hydrophobic interstice energy in fusion: studies of lipid perturbant effects.

We have examined the effects of small amounts (1-4 mol %) of lipids of different molecular shapes, long chain lipids, and hydrocarbon on the kinetics of PEG-mediated fusion of 1,2-dioleoyl-3-sn-phosphatidylcholine/1,2-dioleoyl-3-sn-phosphatidylethanolamine/sphingomyelin/cholesterol (DOPC/DOPE/SM/CH, 35:30:15:30) sonicated vesicles. The effects of these lipid perturbants were different for different steps in the fusion process and varied with the ratio of the cross-sectional areas of headgroup to acyl chain moieties. For lipids with a ratio <1 (negative intrinsic curvature), a decrease in this ratio led to a dramatic increase in the initial rate of vesicle contents mixing but left the initial rate of lipid mixing roughly unchanged. For lipids with ratios >1 (positive intrinsic curvature), the initial rates of both lipid and contents mixing decreased mildly with increasing ratio. In the context of the "stalk model" for fusion, lipid mixing reflects mainly formation of the initial fusion intermediate (stalk), while contents mixing reflects conversion of this intermediate either to a second intermediate or to a fusion pore. Results with positively curved lipids (ganglioside, GM1; lysophosphatidylcholine, LPCs) and negatively curved lipids (dioleoylglycerol, DOG, and 1,2-diphytanoyl-sn-glyvero-3-phosphatidylcholine, DPhPC) can be taken as supportive of the usual interpretation of the stalk model in terms of bending energy, but enhancement of fusion in the presence of long-chain phospholipids, hexadecane, as well as a mixture of GM1 plus hexadecane could not be explained by their curvature alone. We propose that the ability of a lipid perturbant to compensate for lipid packing mismatch, that is, to lower "void" energy, must be taken into account, along with intrinsic curvature, to explain the ability of lipid perturbants to promote pore formation.