A Bioinformatic Workflow for InDel Analysis in the Wheat Multi-Copy α-Gliadin Gene Family Engineered with CRISPR/Cas9

The α-gliadins of wheat, along with other gluten components, are responsible for bread viscoelastic properties. However, they are also related to human pathologies as celiac disease or non-celiac wheat sensitivity. CRISPR/Cas was successfully used to knockout α-gliadin genes in bread and durum wheat, therefore, obtaining low gluten wheat lines. Nevertheless, the mutation analysis of these genes is complex as they present multiple and high homology copies arranged in tandem in A, B, and D subgenomes. In this work, we present a bioinformatic pipeline based on NGS amplicon sequencing for the analysis of insertions and deletions (InDels) in α-gliadin genes targeted with two single guides RNA (sgRNA). This approach allows the identification of mutated amplicons and the analysis of InDels through comparison to the most similar wild type parental sequence. TMM normalization was performed for inter-sample comparisons; being able to study the abundance of each InDel throughout generations and observe the effects of the segregation of Cas9 coding sequence in different lines. The usefulness of the workflow is relevant to identify possible genomic rearrangements such as large deletions due to Cas9 cleavage activity. This pipeline enables a fast characterization of mutations in multiple samples for a multi-copy gene family.     

Starch-based coatings: effect on refrigerated strawberry (Fragaria ananassa) quality

Edible coatings can provide an alternative for extending post-harvest life of refrigerated fruit and vegetables. The influence of different starch-based coating formulations on quality attributes of strawberries stored at 0°C and a relative humidity of 84·8% was studied. Starch sources were classified according to the amylose content in starch, with medium amylose content (potato and corn) and high amylose content (amylomaize and amylose-rich product). Quality of fruits was evaluated by weight loss, firmness retention, microbial decay, surface colour development, titratable acidity and sugar content. The effects of starch amylose content and glycerol (plasticiser) concentration on coating properties were also analysed. The coatings reduced the number of infected fruits and extended storage life of strawberries by retarding senescence. The addition of glycerol improved coating performance, with 20 g litre⁻¹ the most effective concentration. The starch source had a significant effect on surface colour development, weight loss and firmness retention. Coated strawberries produced the lowest ratios of chromaticity parameters (a/b, red/yellow) with regard to the control fruits, thus retarding senescence. High amylose content starches reduced weight loss, maintained firmness and reduced decay better than medium amylose content starches. © 1998 SCI.     

Adipsin Serum Concentrations and Adipose Tissue Expression in People with Obesity and Type 2 Diabetes

(1) Adipsin is an adipokine that may link increased fat mass and adipose tissue dysfunction to obesity-related cardiometabolic diseases. Here, we investigated whether adipsin serum concentrations and adipose tissue (AT) adipsin mRNA expression are related to parameters of AT function, obesity and type 2 diabetes (T2D). (2) Methods: A cohort of 637 individuals with a wide range of age and body weight (Age: 18–85 years; BMI: 19–70 kg/m²) with (n = 237) or without (n = 400) T2D was analyzed for serum adipsin concentrations by ELISA and visceral (VAT) and subcutaneous (SAT) adipsin mRNA expression by RT-PCR. (3) Results: Adipsin serum concentrations were significantly higher in patients with T2D compared to normoglycemic individuals. We found significant positive univariate relationships of adipsin serum concentrations with age (r = 0.282, p < 0.001), body weight (r = 0.264, p < 0.001), fasting plasma glucose (r = 0.136, p = 0.006) and leptin serum concentrations (r = 0.362, p < 0.001). Neither VAT nor SAT adipsin mRNA expression correlated with adipsin serum concentrations after adjusting for age, sex and BMI. Independent of T2D status, we found significantly higher adipsin expression in SAT compared to VAT (4) Conclusions: Our data suggest that adipsin serum concentrations are strongly related to obesity and age. However, neither circulating adipsin nor adipsin AT expression reflects parameters of impaired glucose or lipid metabolism in patients with obesity with or without T2D.     

Nanosystems Applied to HIV Infection: Prevention and Treatments

Sexually-transmitted infections (STIs) are a global health concern worldwide as they cause acute diseases, infertility, and significant mortality. Among the bacterial, viral, and parasitic pathogens that can be sexually transmitted, human immunodeficiency virus (HIV) has caused one of the most important pandemic diseases, which is acquired immune deficiency syndrome (AIDS). 32.7 million people have died from AIDS-related illnesses since the start of the epidemic. Moreover, in 2019, 38 million people were living with HIV worldwide. The need to deal with this viral infection becomes more obvious, because it represents not only a problem for public health, but also a substantial economic problem. In this context, it is necessary to focus efforts on developing methods for prevention, detection and treatment of HIV infections that significantly reduce the number of newly infected people and provide a better quality of life for patients. For several decades, biomedical research has been developed allowing quick solutions through the contribution of effective tools. One of them is the use of polymers as vehicles, drug carrier agents, or as macromolecular prodrugs. Moreover, nanosystems (NSs) play an especially important role in the diagnosis, prevention, and therapy against HIV infection. The purpose of this work is to review recent research into diverse NSs as potential candidates for prevention and treatment of HIV infection. Firstly, this review highlights the advantages of using nanosized structures for these medical applications. Furthermore, we provide an overview of different types of NSs used for preventing or combating HIV infection. Then, we briefly evaluate the most recent developments associated with prevention and treatment alternatives. Additionally, the implications of using different NSs are also addressed.     

Olaparib-Based Photoaffinity Probes for PARP-1 Detection in Living Cells

The poly-ADP-ribose polymerase (PARP) is a protein from the family of ADP-ribosyltransferases that catalyzes polyadenosine diphosphate ribose (ADPR) formation in order to attract the DNA repair machinery to sites of DNA damage. The inhibition of PARP activity by olaparib can cause cell death, which is of clinical relevance in some tumor types. This demonstrates that quantification of PARP activity in the context of living cells is of great importance. In this work, we present the design, synthesis and biological evaluation of photo-activatable affinity probes inspired by the olaparib molecule that are equipped with a diazirine for covalent attachment upon activation by UV light and a ligation handle for the addition of a reporter group of choice. SDS-PAGE, western blotting and label-free LC-MS/MS quantification analysis show that the probes target the PARP-1 protein and are selectively outcompeted by olaparib; this suggests that they bind in the same enzymatic pocket. Proteomics data are available via ProteomeXchange with identifier PXD018661.     

Mass transfer kinetics and diffusion coefficient estimation of bioinsecticide terpene ketones in LDPE films obtained by supercritical CO2‐assisted impregnation

Release kinetics of thymoquinone and R ‐(+)‐pulegone impregnated in low‐density polyethylene (LDPE) films into air and the effect of supercritical CO₂‐assisted impregnation process on the diffusional properties of these films were investigated. The incorporation of both ketones into LDPE films was performed under different conditions (pressure, depressurization rate, time, and initial ketone mole fraction). Release experiments were performed under controlled laboratory conditions (24 °C, 60% relative humidity), and the total release profile was determined gravimetrically, while the individual release of each ketone was quantified by Fourier transformed infrared. The experimental data were used to fit a mass transfer model based on the second Fick's law for unsteady‐state diffusion, and the diffusion coefficients of both ketones in LDPE were estimated, ranging from 2.35 × 10^?13 to 5.53 × 10^?13 m² s^?1 (thymoquinone) and from 1.24 × 10^?13 to 4.52 × 10^?13 m² s^?1 (pulegone). Finally, analysis of variance testing indicated that impregnation pressure and depressurization rate (and their combination) have significant effects on the diffusion coefficient values. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134 , 45558.     

Sintered metallic foams for biodegradable bone replacement materials

The iron open cell foams were synthesized by the replication method based on powder metallurgical technologies as biodegradable bone replacement material. Open cell metal foams provide extraordinary combinations of the properties. Samples containing carbon nanotubes (CNTs) and Mg were produced with the aim to affect degradation rate and enhance the biocompatibility. The microstructure, corrosion behaviour and in vitro biocompatibilities were investigated by scanning electron microscopy, immersion tests in Hank’s solution during the time of 8 weeks, cytotoxicity and haemolysis tests. The addition of CNTs and Mg induced the higher surface inhomogeneity and roughness, which was more pronounced for Fe–Mg sample. The homogenous degradation on the whole surface was registered for bare iron sample, while progressive local corrosion sites were found on the surface of samples with CNTs and Mg. Highest corrosion rate was determined for Fe–Mg sample which came to be disintegrated after 3 weeks of immersion. The slowest degradation rate was detected for Fe–CNTs sample. The fast dieback of fibroblast cells was registered under static conditions in monolayer cell culture. However, all the experimental samples were found to be highly haemocompatible. Additionally, the haemolysis percentage value of about 2 % determined for Fe–Mg sample proved its good performance for blood vessel related cellular application.     

A two step chemo-biotechnological conversion of polystyrene to a biodegradable thermoplastic

A novel approach to the recycling of polystyrene is reported here; polystyrene is converted to a biodegradable plastic, namely polyhydroxyalkanoate (PHA). This unique combinatorial approach involves the pyrolysis of polystyrene to styrene oil, followed by the bacterial conversion of the styrene oil to PHA by Pseudomonas putida CA-3 (NCIMB 41162). The pyrolysis (520 degrees C) of polystyrene in a fluidized bed reactor (Quartz sand (0.3-0.5 mm)) resulted in the generation of an oil composed of styrene (82.8% w/w) and low levels of other aromatic compounds. This styrene oil, when supplied as the sole source of carbon and energy allowed for the growth of P. putida CA-3 and PHA accumulation in shake flask experiments. Styrene oil (1 g) was converted to 62.5 mg of PHA and 250 mg of bacterial biomass in shake flasks. A 1.6-fold improvement in the yield of PHA from styrene oil was achieved by growing P. putida CA-3 in a 7.5 liter stirred tank reactor. The medium chain length PHA accumulated was comprised of monomers 6, 8, and 10 carbons in length in a molar ratio of 0.046:0.436:1.126, respectively. A single pyrolysis run and four fermentation runs resulted in the conversion of 64 g of polystyrene to 6.4 g of PHA.     

Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies

Bispecific single-chain diabodies (scDb) consist of the variableheavy and light chain domains of two antibodies connected bythree linkers. The structure of an scDb in the V_H–V_L orientationis V_HA–linkerA–V_LB–linkerM–V_HB–linkerB–V_LA,with linkers A and B routinely chosen to be 5–6 residuesand linker M 15–20 residues. Here, we applied displayof scDb on filamentous phage to analyse the composition of optimallinker sequences. The three linkers were randomized in lengthand sequence using degenerated triplets coding for only sixhydrophilic or aliphatic amino acids (Thr, Ser, Asp, Asn, Gly,Ala). Antigen-binding clones were then isolated by one to tworounds of selection on the two different antigens recognizedby the bispecific scDb. Using an scDb directed against carcinoembryonicantigen (CEA) and ß-galactosidase (Gal), we foundthat monomeric scDb had a preferred length of 15 or more aminoacid residues for the middle linker M and of 3–6 residuesfor the linkers A and B. No obvious bias towards a preferredlinker sequence was observed. Reduction of the middle linkerbelow 13 residues led to the formation of dimeric scDb, whichmost likely results from interchain pairing between all theV_H and V_L domains. Dimeric scDb were also formed by fragmentspossessing a long linker M and linkers A and B of 0 or 1 residue.We assume that these dimeric scDb are formed by intrachain pairingof the central variable domains and interchain pairing of theflanking variable domains. Thus, the latter molecules representa novel format of bispecific and tetravalent molecules. Thedescribed strategy allows for the isolation of both optimizedand minimal linker sequences for the assembly of monomeric ordimeric single-chain diabodies.     

Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: Its purification and characterization

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5 kDa according to SDS–PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40 °C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe²⁺ > Hg²⁺ > Mn²⁺ > K⁺ > Mg²⁺ > Li⁺ > Cu²⁺). Trypsin activity decreased continuously as NaCl concentration increased (0–30%). K_m and k_cat values were 0.13 mM and 1.46 s⁻¹, respectively. Results suggest the enzyme have a potential application where room processing temperatures (25–35 °C) or high salt (30%) concentration are needed, such as in fish sauce production.