Near Infrared Spectroscopy Facilitates Rapid Identification of Both Young and Mature Amazonian Tree Species

Precise identification of plant species requires a high level of knowledge by taxonomists and presence of reproductive material. This represents a major limitation for those working with seedlings and juveniles, which differ morphologically from adults and do not bear reproductive structures. Near-infrared spectroscopy (FT-NIR) has previously been shown to be effective in species discrimination of adult plants, so if young and adults have a similar spectral signature, discriminant functions based on FT-NIR spectra of adults can be used to identify leaves from young plants. We tested this with a sample of 419 plants in 13 Amazonian species from the genera Protium and Crepidospermum (Burseraceae). We obtained 12 spectral readings per plant, from adaxial and abaxial surfaces of dried leaves, and compared the rate of correct predictions of species with discriminant functions for different combinations of readings. We showed that the best models for predicting species in early developmental stages are those containing spectral data from both young and adult plants (98% correct predictions of external samples), but even using only adult spectra it is still possible to attain good levels of identification of young. We obtained an average of 75% correct identifications of young plants by discriminant equations based only on adults, when the most informative wavelengths were selected. Most species were accurately predicted (75–100% correct identifications), and only three had poor predictions (27–60%). These results were obtained despite the fact that spectra of young individuals were distinct from those of adults when species were analyzed individually. We concluded that FT-NIR has a high potential in the identification of species even at different ontogenetic stages, and that young plants can be identified based on spectra of adults with reasonable confidence.     

Intramembrane Proteolysis by Signal Peptide Peptidases: A Comparative Discussion of GXGD-type Aspartyl Proteases

Intramembrane-cleaving proteases are required for reverse signaling and membrane protein degradation. A major class of these proteases is represented by the GXGD-type aspartyl proteases. GXGD describes a novel signature sequence that distinguishes these proteases from conventional aspartyl proteases. Members of the family of the GXGD-type aspartyl proteases are the Alzheimer disease-related γ-secretase, the signal peptide peptidases and their homologs, and the bacterial type IV prepilin peptidases. We will describe the major biochemical and functional properties of the signal peptide peptidases and their relatives. We then compare these properties with those of γ-secretase and discuss common mechanisms but also point out a number of substantial differences.During the last years, a number of intramembrane-cleaving proteases termed I-CLiPs³ have been identified (1). I-CLiPs are generally involved in regulated intramembrane proteolysis (2). Upon shedding of a large part of the ectodomain of membrane proteins, the remaining membrane-retained stub is cleaved by specialized proteases within the hydrophobic lipid membrane. Generally, this cleavage can have two predominant biological functions: first, signaling via the liberated ICD within the substrate-expressing cell (reverse signaling) (2); and second, degradation of membrane-retained stubs, which are not required for any further biological function (3). I-CLiPs of three protease classes, metalloproteases, serine proteases, and aspartyl proteases, have been discovered so far (see accompanying minireview by Wolfe (44)).Intramembrane-cleaving aspartyl proteases are represented by the class of the GXGD-type proteases (4). These are unconventional aspartyl proteases that, like the conventional aspartyl proteases, utilize two critical aspartyl residues for peptide bond cleavage. However, in contrast to the conventional proteases, the critical aspartyl residues are located within two TMDs (Fig. 1A). Moreover, these aspartyl residues are embedded in active-site motifs that are completely different from those of conventional aspartyl proteases. The class of GXGD-type aspartyl proteases is currently represented by three different protease families, the most prominent of which is the PS family, providing the catalytically active subunit of γ-secretase (Fig. 1A) (4). PS/γ-secretase is the I-CLiP that liberates amyloid β-peptide, the major component of senile plaques in Alzheimer disease patients (5). In addition, the bacterial type IV prepilin peptidases also belong to the class of the GXGD-type proteases (6). Besides these two protease families, two additional subfamilies of related proteases that also belong to the GXGD-type aspartyl protease family have been identified. These include SPP as well as the SPP homologs, the SPP-like (SPPL) proteases (Fig. 1A) (7, 8).Open in a separate windowFIGURE 1.A, schematic representation of SPPL2a/b, a member of the SPP/SPPL family, and PS, the catalytic core of theγ-secretase. Note the opposite topology of the active sites (indicated by arrows) of the two proteases and their substrates, APP for PS and TNFα for SPPL2a/b. B, proteolytic processing of APP and TNFα. Shedding releases the extracellular part of APP (APPs) and TNFα (TNFα soluble). In the case of APP, a C-terminal fragment (APP CTF), and in case of TNFα, an N-terminal fragment (TNFα NTF) are produced. These membrane-bound fragments are substrate to intramembrane cleavage by PS or SPPL2a/b, respectively, releasing small peptides to the extracellular space (Aβ and TNFα C-domain, respectively) and to the cytosol (APP intracellular domain (AICD) and TNFα ICD), respectively). TNFα FL, full-length TNFα.We will first describe the biochemical, functional, and structural properties of SPP family members. By comparison of these properties, we will then identify common mechanisms of intramembrane proteolysis by GXGD-type proteases but also point out some fundamental differences.     

Ethanol production from biodiesel-derived crude glycerol by newly isolated Kluyvera cryocrescens

The rapidly expanding market for biodiesel has increased the supply and reduced the cost of glycerol, making it an attractive sustainable feed stock for the fuel and chemical industry. Glycerol-based biorefinery is the microbial fermentation of crude glycerol to produce fuels and chemicals. A major challenge is to obtain microbes tolerant to inhibitors such as salts and organic solvents present in crude glycerol. Microbial screening was attempted to isolate novel strain capable of growing on crude glycerol as a sole carbon source. The newly isolated bacteria, identified as nonpathogenic Kluyvera cryocrescens S26 could convert biodiesel-derived crude glycerol to ethanol with high yield and productivity. The supplementation of nutrients such as yeast extract resulted in distinguished enhancement in cell growth as well as ethanol productivity under anaerobic condition. When glycerol fermentation is performed under microaerobic condition, there is also a remarkable improvement in cell growth, ethanol productivity and yield, compared with those under strict anaerobic condition. In batch fermentation under microaerobic condition, K. cryocrescens S26 produced 27 g/l of ethanol from crude glycerol with high molar yield of 80% and productivity of 0.61 g/l/h.     

Bactericidal activity of LFchimera is stronger and less sensitive to ionic strength than its constituent lactoferricin and lactoferrampin peptides

The innate immunity factor lactoferrin harbours two antimicrobial moieties, lactoferricin and lactoferrampin, situated in close proximity in the N1 domain of the molecule. Most likely they cooperate in many of the beneficial activities of lactoferrin. To investigate whether chimerization of both peptides forms a functional unit we designed a chimerical structure containing lactoferricin amino acids 17-30 and lactoferrampin amino acids 265-284. The bactericidal activity of this LFchimera was found to be drastically stronger than that of the constituent peptides, as was demonstrated by the need for lower dose, shorter incubation time and less ionic strength dependency. Likewise, strongly enhanced interaction with negatively charged model membranes was found for the LFchimera relative to the constituent peptides. Thus, chimerization of the two antimicrobial peptides resembling their structural orientation in the native molecule strikingly improves their biological activity.     

Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5′-scaffold moiety and variable 3′-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.     

Neuromuscular action of venom from the South American colubrid snake Philodryas patagoniensis

Snakes of the opisthoglyphous genus Philodryas are widespread in South America and cause most bites by colubrids in this region. In this study, we examined the neurotoxic and myotoxic effects of venom from Philodryas patagoniensis in biventer cervicis and phrenic nerve-diaphragm preparations and we compared the biochemical activities of venoms from P. patagoniensis and Philodryas olfersii. Philodryas patagoniensis venom (40 microg/mL) had no effect on mouse phrenic nerve-diaphragm preparations but caused time-dependent neuromuscular blockade of chick biventer cervicis preparations. This blockade was not reversed by washing. The highest concentration of venom tested (40 microg/mL) significantly reduced (p<0.05) the contractures to exogenous acetylcholine (55 microM and 110 microM) and K(+) (13.4 mM) after 120 min; lower concentrations of venom had no consistent or significant effect on these responses. Venom caused a concentration- and time-dependent release of creatine kinase (CK) from biventer cervicis preparations. Histological analysis showed contracted muscle fibers at low venom concentrations and myonecrosis at high concentrations. Philodryas venoms had low esterase and phospholipase A(2) but high proteolytic activities compared to the pitviper Bothrops jararaca. SDS-PAGE showed that the Philodryas venoms had similar electrophoretic profiles, with most proteins having a molecular mass of 25-80 kDa. Both of the Philodryas venoms cross-reacted with bothropic antivenom in ELISA, indicating the presence of proteins immunologically related to Bothrops venoms. RP-HPLC of P. patagoniensis venom yielded four major peaks, each of which contained several proteins, as shown by SDS-PAGE. These results indicate that P. patagoniensis venom has neurotoxic and myotoxic components that may contribute to the effects of envenoming by this species.     

Single Nucleotide Polymorphisms in IL17A and IL6 Are Associated with Decreased Risk for Pulmonary Tuberculosis in Southern Brazilian Population

In Mycobacterium tuberculosis (MTB) infection, the complex interaction of host immune system and the mycobacteria is associated with levels of cytokines production that play a major role in determining the outcome of the disease. Several single-nucleotide polymorphisms (SNPs) in cytokine genes have been associated with tuberculosis (TB) outcome. The aim of this study was to evaluate the association between previously reported SNPs IL2–330 T>G (rs2069762); IL4–590 C>T (rs2243250); IL6–174 G>C (rs1800795); IL10–592 A>C (rs1800872); IL10–1082 G>A (rs1800896); IL17A -692 C>T (rs8193036); IL17A -197 G>A (rs2275913); TNF -238 G>A (rs361525); TNF -308 G>A (rs1800629) and IFNG +874 T>A (rs2430561) and pulmonary TB (PTB) susceptibility. We conducted a case-control study in individuals from Southern Brazil who were recruited between February 2012 and October 2013 in a high incidence TB city. We performed a multiplex genotyping assay in 191 patients with PTB and 175 healthy subjects. Our results suggest a decreased risk for PTB development associated with the IL17A -197A allele (OR = 0.29; p = 0.04), AA genotype (OR = 0.12; p = 0.04) and A carrier (AG/AA) (OR = 0.29; p = 0.004) and IL6 -174C carrier (CC/CG) (OR = 0.46; p = 0.04). We could not properly analyze IL17A -692 C>T (rs8193036) and IFNG +874T>A due to genotypic inconsistencies and found no evidence of association for the IL2, IL4, IL10 and TNF polymorphisms and PTB. In conclusion, our results show a protective effect of IL17 and IL6 polymorphisms on PTB outcome in Southern Brazilian population.     

Nitrochalcones: Potential in vivo insulin secretagogues

In this study, the in vivo and in vitro anti-hyperglycemic activity of chalcone derivatives of 3,4-methylenedioxy, with a substituent electron-acceptor nitro group in the A or B ring, was investigated. As expected, the second generation sulfonylurea glipizide stimulated insulin secretion and reduced glycemia over the study period. Also, it was demonstrated for the first time that chalcones are able to increase insulin secretion and this event was coincident with serum glucose-lowering in the oral glucose tolerance test. Additionally, the chalcones studied had a similar effect on insulin secretion and serum glucose-lowering as glipizide. The effect of chalcones in terms of inducing insulin secretion was greater than that of glipizide after 30 min. Moreover, chalcones were not able to stimulate glucose uptake in soleus muscle, either in the presence of insulin or in the absence of this hormone. In addition, the oral treatment with chalcones did not alter glycemia in diabetic rats. These reports indicate that the effect of chalcones on serum glucose-lowering in hyperglycemic-normal rats is mainly a consequence of insulin secretion, highlighting these chalcones as novel compounds with strong anti-hyperglycemic properties.     

Effects of Bothrops asper snake venom on the expression of cyclooxygenases and production of prostaglandins by peritoneal leukocytes in vivo,and by isolated neutrophils and macrophages in vitro

In this study, the ability of Bothrops asper snake venom (BaV) to increase the production of prostaglandins PGE₂ and PGD₂ was assessed in a mouse model in vivo and in inflammatory cells in vitro. In addition, the expressions of COX-1 and COX-2 were assessed. BaV induced an increment in the in vivo synthesis of PGE₂ and PGD₂, together with an enhanced expression of COX-2, but not of COX-1. However, enzymatic activities of COX-1 and COX-2 were increased. Incubation of isolated macrophages and neutrophils with a sub-cytotoxic concentration of BaV in vitro resulted in increased release of PGE₂ and PGD₂ by macrophages and PGE₂ by neutrophils, concomitantly with an increment in the expression of COX-2, but not of COX-1 by both cell types. Our results demonstrate the ability of BaV to promote the expression of COX-2 and to induce the synthesis of proinflammatory prostaglandins. Macrophages and neutrophils may be important targets for this venom under in vivo situation.