Neonatal Antibiotic Treatment Can Affect Stool Pattern and Oral Tolerance in Preterm Infants.

Preterm neonates are at high risk of infectious and inflammatory diseases which require antibiotic treatment. Antibiotics influence neonatal gut microbiome development, and intestinal dysbiosis has been associated with delayed gastrointestinal transit. Neonates who take less time to pass meconium have a better tolerance to enteral feeding. We analyzed the effect of neonatal antibiotic treatment on the stool pattern and oral tolerance in 106 preterm infants < 33 weeks gestational age. Neonates were classified in 3 groups according to neonatal antibiotic (ABT) treatment days: no antibiotics, 3−7 d ABT, and ≥8 d ABT. Preterm infants from the ≥8 d ABT group took longer to pass meconium and to start green and yellow stools, took longer to reach 100 and 150 mL/kg/day, and reached reduced volumes in enteral feeds at day of life 14 and 28 than infants from no ABT and 3−7 d ABT groups. Multiple linear regression models showed that neonatal antibiotic treatment, birth weight, invasive mechanical ventilation, surfactant, enteral feeding start day, neonatal parenteral nutrition, and neonatal fasting days are associated with the stool pattern and oral tolerance in preterm infants.

Purification and biophysical analysis of microtubule-severing enzymes in vitro.

Microtubule-severing enzymes are a novel class of microtubule regulators. They are enzymes from the ATPases associated with various cellular activities family (AAA+) that utilize ATP to cut microtubules into smaller filaments. Discovered over 20 years ago, there are still many open questions about severing enzymes. Both cellular and biochemical studies need to be pursued to fully understand how these enzymes function mechanistically in the cell. Here, we present methods to express, purify, and test the biophysical nature of these proteins in vitro to begin to address the biochemical and biophysical mechanisms of this important and novel group of microtubule destabilizers.

Drosophila katanin-60 depolymerizes and severs at microtubule defects.

Microtubule (MT) length and location is tightly controlled in cells. One novel family of MT-associated proteins that regulates MT dynamics is the MT-severing enzymes. In this work, we investigate how katanin (p60), believed to be the first discovered severing enzyme, binds and severs MTs via single molecule total internal reflection fluorescence microscopy. We find that severing activity depends on katanin concentration. We also find that katanin can remove tubulin dimers from the ends of MTs, appearing to depolymerize MTs. Strikingly, katanin localizes and severs at the interface of GMPCPP-tubulin and GDP-tubulin suggesting that it targets to protofilament-shift defects. Finally, we observe that binding duration, mobility, and oligomerization are ATP dependent.

Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration.

Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. Here we show that the Drosophila katanin Dm-Kat60 functions to generate a dynamic cortical-microtubule interface in interphase cells. Dm-Kat60 concentrates at the cell cortex of S2 Drosophila cells during interphase, where it suppresses the polymerization of microtubule plus-ends, thereby preventing the formation of aberrantly dense cortical arrays. Dm-Kat60 also localizes at the leading edge of migratory D17 Drosophila cells and negatively regulates multiple parameters of their motility. Finally, in vitro, Dm-Kat60 severs and depolymerizes microtubules from their ends. On the basis of these data, we propose that Dm-Kat60 removes tubulin from microtubule lattice or microtubule ends that contact specific cortical sites to prevent stable and/or lateral attachments. The asymmetric distribution of such an activity could help generate regional variations in microtubule behaviours involved in cell migration.

Novel structural and functional findings of the ehFLN protein from Entamoeba histolytica.

The ehFLN protein (previously known as EhABP-120) is the first filamin to be identified in the parasitic protozoan Entamoeba histolytica. Filamins are a family of cross-linking actin-binding proteins that organize filamentous actin in networks and stress fibers. It has been reported that filamins of different organisms directly interact with more than 30 cellular proteins and some PPIs. The biochemical consequences of such interactions may have either positive or negative effects on the cross-linking function. Besides, filamins form a link between cytoskeleton and plasma membrane. In this work, the ehFLN protein was biochemically characterized; amoebae filamin was found to associate with both PA and PI(3)P in vitro, new lipid targets for a member of the filamins. By molecular modeling analysis and protein-lipid overlay assays, K-609, 709, and 710 were determined to be essential for the PA-ehFLN1 complex stability. Also, the integrity of the 4th repeat of ehFLN is essential to keep interaction with the PI(3)P. Transfected trophozoites that overexpressed the d100, d50NH(2), and d50COOH regions of ehFLN1 displayed both increased motility and chemotactic response to TYI-S-33 media. Together, these results suggest that short regions of ehFLN are involved in signaling events that, in cooperation with phosphatidic acid, EhPLD2 and EhPI3K, could promote cell motility.

EhPAK2, a novel p21-activated kinase, is required for collagen invasion and capping in Entamoeba histolytica.

p21-activated kinases (PAKs) are a highly conserved family of enzymes that are activated by Rho GTPases. All PAKs contain an N-terminal Cdc42/Rac interacting binding (CRIB) domain, which confers binding to these GTPases, and a C-terminal kinase domain. In addition, some PAKs such as Cla4p, Skm1p and Pak2p contain an N-terminal pleckstrin homology (PH) domain and form a distinct group of PAK proteins involved in cell morphology, cell-cycle and gene transcription. Here, we describe a novel p21-activated kinase, denominated EhPAK2, on the parasitic protozoan Entamoeba histolytica. This is the first reported Entamoeba PAK member that contains a N-terminal PH domain and a highly conserved CRIB domain. EhPAK2 CRIB domain shares 29% of amino acid identity and 53% of amino acid homology with these of DdPAKC from Dictyostelium discoideum and Cla4p from Saccharomyces cerevisiae and binds in vitro and in vivo to EhRacA GTPase. This domain also possesses the conserved residues His123, Phe134 and Trp141, which are important for the interaction with the effector loop and strand beta2 of the GTPase; and the residues Met121 and Phe145, which are specific for the interaction of EhPAK2 with EhRacA. Functional studies of EhPAK2 showed that its C-terminal kinase domain had activity toward myelin basic protein. Cellular studies showed that Entamoeba trophozoites transfected with the vector pExEhNeo/kinase-myc, had a 90% decrease in the ability to invade a collagen matrix as well as severe defects in capping, suggesting the involvement of EhPAK2 in these cellular processes.

The ABP-120 C-end region from Entamoeba histolytica interacts with sulfatide, a new lipid target.

EhABP-120 is the first filamin identified in the parasitic protozoan Entamoeba histolytica. Filamins are a family of cross-linking actin-binding proteins that promote a dynamic orthogonal web. They have been reported to interact directly with more than 30 cellular proteins and some phosphoinositides. The biochemical consequences of these interactions may have either positive or negative effects on the cross-linking function and also form a link between the cytoskeleton and plasma membrane. In this study, the EhABP-120 carboxy-terminal domain (END) was biochemically characterized. This domain was able to associate to 3-sulfate galactosyl ceramide, a new lipid target for a member of the filamin family. Also, the END domain was able to dimerize "in vitro." Molecular modeling analysis showed that the dimeric region is stabilized by a disulfide bond. Electrostatic and docking studies suggest that an electropositive concave pocket at the dimeric END domain interacts simultaneously with several sulfogalactose moieties of the sulfatide.