Potential applications of CRISPR/Cas for next-generation biomonitoring of harmful algae blooms: A review.

Research on harmful algal and cyanobacterial blooms (HABs and CHABs) has risen dramatically due to their increasing global distribution, frequency, and intensity. These blooms jeopardize public health, ecosystem function, sustainability and can have negative economic impacts. Numerous monitoring programs have been established using light microscopy, liquid chromatography coupled to mass spectrometry (LC-MS), ELISA, and spectrophotometry to monitor HABs/CHABs outbreaks. Recently, DNA/RNA-based molecular methods have been integrated into these programs to replace or complement traditional methods through analyzing environmental DNA and RNA (eDNA/eRNA) with techniques such as quantitative polymerase chain reaction (qPCR), fluorescent in situ hybridization (FISH), sandwich hybridization assay (SHA), isothermal amplification methods, and microarrays. These have enabled the detection of rare or cryptic species, enhanced sample throughput, and reduced costs and the need for visual taxonomic expertise. However, these methods have limitations, such as the need for high capital investment in equipment or detection uncertainties, including determining whether organisms are viable. In this review, we discuss the potential of newly developed molecular diagnosis technology based on Clustered Regularly Interspaced Short Palindromic Repeats/Cas proteins (CRISPR/Cas), which utilizes the prokaryotic adaptative immune systems of bacteria and archaea. Cas12 and Cas13-based platforms can detect both DNA and RNA with attomolar sensitivity within an hour. CRISPR/Cas diagnostic is a rapid, inexpensive, specific, and ultrasensitive technology that, with some further development, will provide many new platforms that can be used for HABs/CHABs biomonitoring and research.

Strategy for biological co-production of levulinic acid and polyhydroxyalkanoates by using mixed microbial cultures fed with synthetic hemicellulose hydrolysate.

Hemicellulose hydrolysates (HH), which could be an interesting carbon source to feed mixed microbial cultures (MMC) able to accumulate high value-added compounds. This research focused on the evaluation of a culture strategy to achieve the simultaneous biological production of Levulinic Acid (LA) and Polyhydroxyalcanoates (PHA) by MMC fed with a synthetic HH (SHH). The culture strategy involves the use of sequential batch reactors (SBR) to select microorganisms capable of producing LA and PHA. This work proved that the cultivation strategy used allowed the biological production of LA, reaching 37%w/w when the SHH was composed of 85% pentoses. In addition, the simultaneous biological production of LA and PHB was possible when the SHH was enriched with acetate (45% pentoses - 50% acetate). Finally, this study showed that the composition of the SHH impacts directly on the selected microorganism genus and the type and quantity of the value-added compounds obtained.

Integrins and cAMP mediate netrin-induced growth cone collapse.

Growth cones integrate a remarkably complex concert of chemical cues to guide axons to their appropriate destinations. Recent work suggests that integrins contribute to axon guidance by interacting with a wide range of extracellular molecules including axon guidance molecules, by mechanisms that are not fully understood. Here, we describe an interaction between integrins and netrin-1 in growth cones that contributes to growth cone collapse. Our data show that netrin-1 causes growth cone collapse in a substratum-specific manner and is integrin-dependent. Netrin-1 causes collapse of cultured chick dorsal root ganglion (DRG) growth cones extending on high levels of laminin-1 (LN) but not growth cones extending on low levels of LN or on fibronectin. Blocking integrin function significantly decreases netrin-induced growth cone collapse on high LN. Netrin-1 and integrins interact on growth cones; netrin-1 causes integrin activation, a conformational shift to a high ligand-affinity state. Netrin-1 directly binds to integrin α3 and α6 peptides, further suggesting a netrin-integrin interaction. Interestingly, our data reveal that netrin-1 increases growth cone levels of cAMP in a substratum-specific manner and that netrin-induced growth cone collapse requires increased cAMP in combination with integrin activation. Manipulations that either decrease cAMP levels or integrin activation block netrin-induced collapse. These results imply a common mechanism for growth cone collapse and novel interactions between integrins, netrin-1 and cAMP that contribute to growth cone guidance.

A pilot study for monitoring changes in the microbiological component of metalworking fluids as a function of time and use in the system.

This article describes the results of a pilot study to examine changes in the biological component of metalworking fluids (MWF) as a function of use. Fluid samples were taken from two newly charged systems, designated BT-7415 and BT-7707, at 1-week intervals for 8 weeks and characterized with respect to the kinds and numbers of bacteria present and presence of soluble protein in cell-free supernatants. In addition, lipid extracts of pelleted cells from fluids in BT-7415 were examined by gas chromatography/mass spectroscopy for the kinds and relative amounts of phospholipid fatty acids (PLFA) present. A total of 19 different bacterial species was cultured and identified, more than half (12/19) of which were gram-negative. Total colony-forming units (CFU) reached levels of 2.2 x 10(3)/mL in BT-7415 and 2.4 x 10(5)/mL in BT-7707. The most common genus isolated was Pseudomonas. Estimations of cell numbers based on total biomass from PLFA in samples from BT-7415 indicated 1.1 x 10(7)/mL after 8 weeks of use. Both the numbers of PLFA identified and the amounts of each detected in BT-7415 increased as the fluids were used. The chromatograms were dominated by two fatty acids, the amounts of which increased with time. These fatty acids, 18:2 omega 6 and 18:1 omega 9c, are not commonly associated with pseudomonads. This suggests that there is an important component of the biological consortium in MWF is not being detected by currently used culture techniques. There was no soluble protein detected in any of the samples from either system.

Differences in intracellular transport of a fluorescent phosphatidylcholine analog in established cell lines.

The transport and metabolism of a fluorescent phosphatidylcholine analog, 1-palmitoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)- aminocaproyl-phosphatidylcholine [palmitoyl, C6-NBD)-PC), in BHK, CHO-K1, CHO-15B, MDCK, VA-2, Vero, V79 and WI-38 cells has been investigated. When liposomes containing (palmitoyl, C6-NBD)-PC were incubated with cells at 2 degrees C, spontaneous transfer of the fluorescent lipid from the liposomes to the cells' plasma membranes occurred. Most of the lipid transferred to the cells could be removed by incubating the cells in the presence of nonfluorescent liposomes or media containing 10% serum, suggesting that the fluorescent probe resided exclusively in the outer leaflet of the plasma membrane at 2 degrees C. After insertion into the plasma membrane, internalization of (palmitoyl, C6-NBD)-PC occurred when the cells were warmed to 37 degrees C. This resulted in four different labeling patterns: (1) little or no internalization of (palmitoyl, C6-NBD)-PC into punctate vesicles was observed in Vero cells. (2) Transport of (palmitoyl, C6-NBD)-PC to the region of the Golgi apparatus and to a small number of intracellular vesicles was observed in both V79 and CHO-K1 cell lines. (3) A large number of fluorescently labeled intracellular vesicles with little or no labeling in the region of the Golgi apparatus appeared after the internalization of (palmitoyl, C6-NBD)-PC in BHK, CHO-15B, MDCK and WI-38 cell lines. (4) Accumulation of (palmitoyl, C6-NBD)-PC in small vesicles, mitochondria and the nuclear envelope was observed in VA-2 cells. In addition, cells having a defect in glycoprotein processing and those transformed with simian virus 40 (SV40) internalized the fluorescent lipid probe differently compared with parental lines. Neither differences in rates of endocytosis nor rates of (palmitoyl, C6-NBD)-PC degradation between cell types appears to cause the observed dissimilarities in intracellular lipid transport. We suggest that these different cell types may have dissimilar pathways of intracellular lipid trafficking or differential regulation of a common transport pathway, and that the predominant pathway of lipid translocation can be altered in cells by changing the composition of their glycoproteins or by viral transformation.