The patatin-containing phospholipase A pPLAIIα modulates oxylipin formation and water loss in Arabidopsis thaliana

The patatin-related phospholipase A (pPLA) hydrolyzes membrane glycerolipids to produce monoacyl compounds and free fatty acids. Phospholipids are cleaved by pPLAIIα at the sn-1 and sn-2 positions, and galactolipids, including those containing oxophytodienoic acids, can also serve as substrates. Ablation of pPLAIIα decreased lysophosphatidylcholine and lysophosphatidylethanolamine levels, but increased free linolenic acid. pPLAIIα-deficient plants displayed a higher level of jasmonic acid and methyl jasmonate, as well as the oxylipin-biosynthetic intermediates 13-hydroperoxylinolenic acid and 12-oxophytodienoic acid than wild-type (WT) plants. The expression of genes involved in oxylipin production was also higher in the pPLAIIα-deficient mutant than in WT plants. The mutant plants lost water more quickly than WT plants. The stomata of WT and mutant plants responded similarly to abscisic acid. In response to desiccation, the mutant and WT leaves produced abscisic acid at the same rate, but, after 4?h of desiccation, the jasmonic acid level was much higher in mutant than WT leaves. These results indicate that pPLAIIα negatively regulates oxylipin production and suggest a role in the removal of oxidatively modified fatty acids from membranes.     

Role of primary sensorimotor cortex and supplementary motor area in volitional swallowing: a movement-related cortical potential study

We investigated the role of the cerebral cortex, particularly the face/tongue area of the primary sensorimotor (SMI) cortex (face/tongue) and supplementary motor area (SMA), in volitional swallowing by recording movement-related cortical potentials (MRCPs). MRCPs with swallowing and tongue protrusion were recorded from scalp electrodes in eight normal right-handed subjects and from implanted subdural electrodes in six epilepsy patients. The experiment by scalp EEG in normal subjects revealed that premovement Bereitschaftspotentials (BP) activity for swallowing was largest at the vertex and lateralized to either hemisphere in the central area. The experiment by epicortical EEG in patients confirmed that face/tongue SMI and SMA were commonly involved in swallowing and tongue protrusion with overlapping distribution and interindividual variability. BP amplitude showed no difference between swallowing and tongue movements, either at face/tongue SMI or at SMA, whereas postmovement potential (PMP) was significantly larger in tongue protrusion than in swallowing only at face/tongue SMI. BP occurred earlier in swallowing than in tongue protrusion. Comparison between face/tongue SMI and SMA did not show any difference with regard to BP and PMP amplitude or BP onset time in either task. The preparatory role of the cerebral cortex in swallowing was similar to that in tongue movement, except for earlier activation in swallowing. Postmovement processing of swallowing was lesser than that of tongue movement in face/tongue SMI; probably suggesting that the cerebral cortex does not play a significant role in postmovement processing of swallowing. SMA plays a supplementary role to face/tongue SMI both in swallowing and tongue movements.     

MMP-9, uPAR and Cathepsin B Silencing Downregulate Integrins in Human Glioma Xenograft Cells In Vitro and In Vivo in Nude Mice

Background

Involvement of MMP-9, uPAR and cathepsin B in adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well established. In the present study, MMP-9, uPAR and cathepsin B genes were downregulated in glioma xenograft cells using shRNA plasmid constructs and we evaluated the involvement of integrins and changes in their adhesion, migration and invasive potential.

Methodology/Principal Findings

MMP-9, uPAR and cathepsin B single shRNA plasmid constructs were used to downregulate these molecules in xenograft cells. We also used MMP-9/uPAR and MMP-9/cathepsin B bicistronic constructs to evaluate the cumulative effects. MMP-9, uPAR and cathepsin B downregulation significantly inhibits xenograft cell adhesion to several extracellular matrix proteins. Treatment with MMP-9, uPAR and cathepsin B shRNA of xenografts led to the downregulation of several alpha and beta integrins. In all the assays, we noticed more prominent effects with the bicistronic plasmid constructs when compared to the single plasmid shRNA constructs. FACS analysis demonstrated the expression of αVβ3, α6β1 and α9β1 integrins in xenograft cells. Treatment with bicistronic constructs reduced αVβ3, α6β1 and α9β1 integrin expressions in xenograft injected nude mice. Migration and invasion were also inhibited by MMP-9, uPAR and cathepsin B shRNA treatments as assessed by spheroid migration, wound healing, and Matrigel invasion assays. As expected, bicistronic constructs further inhibited the adhesion, migration and invasive potential of the xenograft cells as compared to individual treatments.

Conclusions/Significance

Downregulation of MMP-9, uPAR and cathespin B alone and in combination inhibits adhesion, migration and invasive potential of glioma xenografts by downregulating integrins and associated signaling molecules. Considering the existence of integrin inhibitor-resistant cancer cells, our study provides a novel and effective approach to inhibiting integrins by downregulating MMP-9, uPAR and cathepsin B in the treatment of glioma.     

Mesenchymal Stem Cells from Rat Bone Marrow Downregulate Caspase-3-mediated Apoptotic Pathway After Spinal Cord Injury in Rats

Mesenchymal stem cells have been intensively studied for their potential use in reparative strategies for neurodegenerative diseases and traumatic injuries. We used mesenchymal stem cells (rMSC) from rat bone marrow to evaluate the therapeutic potential after spinal cord injury (SCI). Immunohistochemistry confirmed a large number of apoptotic neurons and oligodendrocytes in caudal segments 2 mm away from the lesion site. Expression of caspase-3 on both neurons and oligodendrocytes after SCI was significantly downregulated by rMSC. Caspase-3 downregulation by rMSC involves increased expression of FLIP and XIAP in the cytosol and inhibition of PARP cleavage in the nucleus. Animals treated with rMSC had higher Basso, Beattie, Bresnahan (BBB) locomotor scoring and better recovery of hind limb sensitivity. Treatment with rMSC had a positive effect on behavioral outcome and histopathological assessment after SCI. The ability of rMSC to incorporate into the spinal cord, differentiate and to improve locomotor recovery hold promise for a potential cure after SCI. Special issue in honor of Naren Banik.     

Impact of different pH control agents on biopesticidal activity of<Emphasis Type="Italic"> Bacillus thuringiensis</Emphasis> during the fermentation of starch industry wastewater

Different pH control agents (NaOH/H₂SO₄—SodSulp, NaOH/CH₃COOH—SodAcet, NH₄OH/CH₃COOH—AmmoAcet and NH₄OH/H₂SO₄—AmmoSulp) were used to investigate their effects on growth, enzyme production (alkaline protease and amylase), and entomotoxicity of Bacillus thuringiensis var. kurstaki HD-1 (Btk) against eastern spruce budworm larvae (Choristoneura fumiferana) using starch industry wastewater (SIW) as a raw material in a 15-l fermentor. AmmoSulp and SodSulp were found to be the best pH control agents for alkaline protease and amylase production, respectively; whereas, the fermented broth obtained by using SodAcet as pH control agents recorded the highest delta-endotoxin production of 1043.0 mg/l and entomotoxicity value 18.4 × 10⁹ SBU/l. Entomotoxicity of re-suspended centrifuged pellet in one-tenth of original volume in case of SodAcet as pH control agents was 26.7 × 10⁹ SBU/l and was the highest value compared to three other pH control agents.     

Inducible gene modification in the gastric epithelium of Tff1‐CreERT2, Tff2‐rtTA,Tff3‐luc mice

Temporal and spatial regulation of genes mediated by tissue‐specific promoters and conditional gene expression systems provide a powerful tool to study gene function in health, disease, and during development. Although transgenic mice expressing the Cre recombinase in the gastric epithelium have been reported, there is a lack of models that allow inducible and reversible gene modification in the stomach. Here, we exploited the gastrointestinal epithelium‐specific expression pattern of the three trefoil factor (Tff) genes and bacterial artificial chromosome transgenesis to generate a novel mouse strain that expresses the CreERT2 recombinase and the reverse tetracycline transactivator (rtTA). The Tg(Tff1‐CreERT2;Tff2‐rtTA;Tff3‐Luc) strain confers tamoxifen‐inducible irreversible somatic recombination and allows simultaneous doxycycline‐dependent reversible gene activation in the gastric epithelium of developing and adult mice. This strain also confers luciferase activity to the intestinal epithelium to enable in vivo bioluminescence imaging. Using fluorescent reporters as conditional alleles, we show Tff1‐CreERT2 and Tff2‐rtTA transgene activity in a partially overlapping subset of long‐term regenerating gastric stem/progenitor cells. Therefore, the Tg(Tff1‐CreERT2;Tff2‐rtTA;Tff3‐Luc) strain can confer intermittent transgene expression to gastric epithelial cells that have undergone previous gene modification, and may be suitable to genetically model therapeutic intervention during development, tumorigenesis, and other genetically tractable diseases. Birth Defects Research (Part A) 106:626–635, 2016. © 2016 Wiley Periodicals, Inc.     

Semimature stage: a checkpoint in a dendritic cell maturation program that allows for functional reversion after signal-regulatory protein-alpha ligation and maturation signals

CD47 on live cells actively engages signal-regulatory protein-alpha (SIRP-alpha) on phagocytes and delivers a negative signal that prevents their elimination. We evaluated the biological consequences of SIRP-alpha ligation on the dendritic cell (DC) response to maturation signals and the potential interplay with the IL-10/IL-10R inhibitory pathway. At first, CD47/SIRP-alpha allowed the generation of mature migratory DCs not producing IL-12, IFN-gamma-inducible protein-10, and CCL19. Rather, they secreted neutrophils attracting chemokine CXCL5 and IL-1beta, reflecting a partial block in functional DC maturation. Afterward, semimature DCs functionally regressed in an IL-10-independent fashion toward cells that retrieved the cardinal features of immature DCs: re-expression of CCR5, loss of DC-lysosome-associated membrane protein, high endocytosis, and impaired allostimulatory functions. The global gene expression profile of IL-10 and SIRP-alpha-ligated DC demonstrated two distinct molecular pathways. IL-10R and SIRP-alpha expression were reciprocally down-regulated by CD47 and IL-10, respectively. These results emphasize that the SIRP-alpha pathway might be part of the molecular machinery used by the DC to dampen or resolve an inflammatory response in an IL-10-independent manner.     

Heterosubtypic immunity to influenza A virus infection requires a properly diversified antibody repertoire

Heterosubtypic immunity (HSI) is defined as cross-protection to infection with an influenza A virus serotype other than the one used for primary infection. Although HSI has been thought to be mediated by serotype cross-reactive cytotoxic T lymphocytes (CTL) that recognize conserved epitopes of structural proteins, recent studies suggest that antibodies (Abs) may make a significant contribution. In this study, we provide further evidence for the role of Abs in HSI using transgenic mice lacking terminal deoxyribonucleotidyltransferase (TdT), which adds N nucleotides to V-D and D-J junctions of the complementary determining region 3 (CDR3) (TdT(-/-)) and mice with altered Ab repertoires due to replacement of the complete locus of heavy chain diversity segments (D(H)) with an altered D(H) segment (namely, Delta D-iD). Both types of mice failed to generate complete HSI, although they were able to mount protective immunity to a homologous challenge. Lower levels of virus-specific antibodies along with more severely impaired HSI were observed in TdT(-/-) mice compared to those in Delta D-iD mice, while CTL activity remained unchanged in both types of mice. These findings indicate that a properly diversified antibody repertoire is required for HSI and that N addition by TdT is a more effective mechanism in the induction of a properly diversified antibody repertoire and, therefore, complete HSI. The results suggest that the diversity of the antibody repertoire as determined by the composition of the D region of HCDR3 and by N addition are among the mechanisms selected for in evolution to create a favorable environment to resolve infections with mutated viruses.