The histogenesis of somaclones from tomato (Lycopersicon esculentum Mill.) cotyledons

Summary A histological study ofin vitro cultured cotyledonary expiants of tomato (Lycopersicon esculentum) was performed in order to determine the site (differentiated tissue or developing callus) and the mode of plant regeneration.Results have shown that callus develops at the excision sites of cotyledonary expiants and that shoots are formed exclusively within the unorganized callus: excision areas are the only morphogenetic sites and the proximal excision is the preferred site for plant regeneration.Shoots differentiate by organogenesis within the superficial region of the callus. Few neocambial cells cooperate in the neoformation. Origin from a single cell is highly unlikely since rarely observed single activated cells never developed into shoots.Regenerated plants may be chimeras if invitro culture induces genetic diversity in the initial cells.Abbreviations IAA Indole-3-acetic acid - c callus - d vegetative dome - s shoot - ad adaxial - ab abaxial - t tracheid - p parenchyma - S sieve tube     

Truck Drivers Sleep-Wake Time Arrangements

Irregular working hours, including night work, change sleep-wake time arrangements which in turn might affect the ability to drive safely. This study aims to compare the effects of an irregular and a fixed day shift system on the sleep-wake cycle of truck drivers. The investigation of sleep-wake cycle was carried-out with 37 truck drivers working on two transportation plants: 24 working on irregular working hours and 13 on fixed day shift. The truck drivers filled out sleep logs and wore actigraphs for 10 consecutive days to identify activity and rest episodes. The group working in irregular hours showed more sleep episodes per 24 h and they were shorter compared to the fixed shift group (p < 0.05). No differences were found between the two transportation plants. These results suggest an the influence of working hours on specific sleep-wake patterns. The polyphasic sleep pattern shown by irregular shift group could be a strategy to cope with sleep deprivation, which may account for their difficulty to resist falling asleep behind the wheel.     

GEDAE-LaB: A Free Software to Calculate the Energy System Contributions during Exercise

Purpose

The aim of the current study is to describe the functionality of free software developed for energy system contributions and energy expenditure calculation during exercise, namely GEDAE-LaB.

Methods

Eleven participants performed the following tests: 1) a maximal cycling incremental test to measure the ventilatory threshold and maximal oxygen uptake (V˙O₂max); 2) a cycling workload constant test at moderate domain (90% ventilatory threshold); 3) a cycling workload constant test at severe domain (110% V˙O₂max). Oxygen uptake and plasma lactate were measured during the tests. The contributions of the aerobic (A_MET), anaerobic lactic (LA_MET), and anaerobic alactic (AL_MET) systems were calculated based on the oxygen uptake during exercise, the oxygen energy equivalents provided by lactate accumulation, and the fast component of excess post-exercise oxygen consumption, respectively. In order to assess the intra-investigator variation, four different investigators performed the analyses independently using GEDAE-LaB. A direct comparison with commercial software was also provided.

Results

All subjects completed 10 min of exercise at moderate domain, while the time to exhaustion at severe domain was 144 ± 65 s. The A_MET, LA_MET, and AL_MET contributions during moderate domain were about 93, 2, and 5%, respectively. The A_MET, LA_MET, and AL_MET contributions during severe domain were about 66, 21, and 13%, respectively. No statistical differences were found between the energy system contributions and energy expenditure obtained by GEDAE-LaB and commercial software for both moderate and severe domains (P > 0.05). The ICC revealed that these estimates were highly reliable among the four investigators for both moderate and severe domains (all ICC ≥ 0.94).

Conclusion

These findings suggest that GEDAE-LaB is a free software easily comprehended by users minimally familiarized with adopted procedures for calculations of energetic profile using oxygen uptake and lactate accumulation during exercise. By providing availability of the software and its source code we hope to facilitate future related research.     

Kunitz-type protease inhibitors group B from Solanum palustre

Five Kunitz protease inhibitor group B genes were isolated from the genome of the diploid non-tuber-forming potato species Solanum palustre. Three of five new genes share 99% identity to the published KPI-B genes from various cultivated potato accessions, while others exhibit 96% identity. Spls-KPI-B2 and Spls-KPI-B4 proteins contain unique substitutions of the most conserved residues usually involved to trypsin and chymotrypsin-specific binding sites of Kunitz-type protease inhibitor (KPI)-B, respectively. To test the inhibition of trypsin and chymotrypsin by Spls-KPI proteins, five of them were produced in E. coli purified using a Ni-sepharose resin and ion-exchange chromatography. All recombinant Spls-KPI-B inhibited trypsin; K(i) values ranged from 84.8 (Spls-KPI-B4), 345.5 (Spls-KPI-B1), and 1310.6 nM (Spls-KPI-B2) to 3883.5 (Spls-KPI-B5) and 8370 nM (Spls-KPI-B3). In addition, Spls-KPI-B1 and Spls-KPI-B4 inhibited chymotrypsin. These data suggest that regardless of substitutions of key active-center residues both Spls-KPI-B4 and Spls-KPI-B1 are functional trypsin-chymotrypsin inhibitors.     

The binding of heparin to spike glycoprotein inhibits SARS-CoV-2 infection by three mechanisms

Heparin, a naturally occurring glycosaminoglycan, has been found to have antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. To elucidate the mechanistic basis for the antiviral activity of heparin, we investigated the binding of heparin to the SARS-CoV-2 spike glycoprotein by means of sliding window docking, molecular dynamics simulations, and biochemical assays. Our simulations show that heparin binds at long, positively charged patches on the spike glycoprotein, thereby masking basic residues of both the receptor-binding domain (RBD) and the multifunctional S1/S2 site. Biochemical experiments corroborated the simulation results, showing that heparin inhibits the furin-mediated cleavage of spike by binding to the S1/S2 site. Our simulations showed that heparin can act on the hinge region responsible for motion of the RBD between the inactive closed and active open conformations of the spike glycoprotein. In simulations of the closed spike homotrimer, heparin binds the RBD and the N-terminal domain of two adjacent spike subunits and hinders opening. In simulations of open spike conformations, heparin induces stabilization of the hinge region and a change in RBD motion. Our results indicate that heparin can inhibit SARS-CoV-2 infection by three mechanisms: by allosterically hindering binding to the host cell receptor, by directly competing with binding to host heparan sulfate proteoglycan coreceptors, and by preventing spike cleavage by furin. Furthermore, these simulations provide insights into how host heparan sulfate proteoglycans can facilitate viral infection. Our results will aid the rational optimization of heparin derivatives for SARS-CoV-2 antiviral therapy.     

Changes in the proteome of Salmonella enterica serovar Thompson as stress adaptation to sublethal concentrations of thymol

Thymol is a natural biocide and component of some essential oils from herbs. Its inhibitory effect on the growth of different microorganisms is well documented. The precise targets of the antibacterial action of thymol is not yet been fully established, the action seems to take place in different ways. The strain Salmonella enterica serovar Thompson MCV1 was grown in the presence of a sublethal concentration (0.01%) of thymol. The proteins extracted from treated and untreated cells were subjected to 2‐D PAGE, followed by in‐gel spot digestion and subsequent MALDI‐TOF analysis. The analysis of gels showed many proteins that were either upregulated or downregulated by the presence of thymol, with significant changes in proteins belonging to different functional classes. In particular, the thioredoxin‐1 was not expressed in the treated cells, indicating that its absence could be a consequence of the stress caused by the presence of thymol. On the other hand, different chaperon proteins were upregulated or de novo synthesis such as GroEL and DnaK, key proteins in the protection mechanism toward thermal stress. Outer membrane proteins were upregulated in treated cells; indeed the bacterial envelope stress response is trigged by the accumulation of misfolded outer membrane proteins. Moreover, the thymol seems to impair the citrate metabolic pathway, as well as many enzymes involved in the synthesis of ATP. Definitely, thymol plays a role in altering very different pathways of cell metabolism.     

Caspase-dependent apoptosis of the HCT-8 epithelial cell line induced by the parasite Giardia intestinalis

Giardia intestinalis is a flagellated protozoan which causes enteric disease worldwide. Giardia trophozoites infect epithelial cells of the proximal small intestine and can cause acute or chronic diarrhea. The mechanism of epithelial injury in giardiasis remains unknown. A number of enteric pathogens, including protozoan parasites, are able to induce enterocyte apoptosis. The aim of this work was to assess whether G. intestinalis strain WB clone C6 is able to induce apoptosis in the human intestinal epithelial cell line HCT-8, and to investigate the role of caspases in this process. Results demonstrated that the parasite induces cell apoptosis, as confirmed by DNA fragmentation analysis, detection of active caspase-3 and degradation of the caspase-3 substrate PARP [poly(ADP-ribose) polymerase]. Furthermore, G. intestinalis infection induces activation of both the intrinsic and the extrinsic apoptotic pathways, down-regulation of the antiapoptotic protein Bcl-2 and up-regulation of the proapoptotic Bax, suggesting a possible role for caspase-dependent apoptosis in the pathogenesis of giardiasis.     

Single muscle fiber proteomics reveals unexpected mitochondrial specialization

Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high‐sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype‐specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type‐resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity.     

Molecular cloning and characterization of an almond 9-hydroperoxide lyase, a new CYP74 targeted to lipid bodies

Oxylipin metabolism represents one of many defence mechanisms employed by plants. It begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases to form fatty acid hydroperoxides that are substrates for several enzymes, including specialized cytochrome P450s known as CYP74s. The targeting of a new CYP74, a 9-hydroperoxide lyase (HPL) from almonds, to the endomembrane system and lipid bodies, both as enzyme activity in almond seeds and as GFP fusions transiently expressed in tobacco protoplasts, is described. Such association of a CYP74 with lipid bodies has not been reported previously. Also described are the properties of a 9-HPL gene, the developmental regulation of its expression, the production and characterization of recombinant 9-HPL in Escherichia coli, and the developmental correlation between gene expression, enzyme activity, and the appearance of volatile C9 aldehydes from HPL action.