Time-course changes in fungal elicitor-induced lignan synthesis and expression of the relevant genes in cell cultures of Linum album

Linum album has been shown to accumulate anti-tumor podophyllotoxin (PTOX) and its related lignans. In the present study, we examined the effects of five fungal extracts on the production of lignans in L. album cell cultures. Fusarium graminearum extract induced the highest increase of PTOX [140μgg(-1) dry weight (DW) of the L. album cell culture] which is seven-fold greater than the untreated control, while Rhizopus stolonifer extract enhanced the accumulation of lariciresinol, instead of PTOX, up to 365μgg(-1) DW, which was 8.8-fold greater than the control. Quantitative PCR analyses showed that expression of the enzyme genes responsible for the PTOX biosynthesis cascade, such as pinoresinol-lariciresinol reductase (PLR), phenylalanine ammonia-lyase (PAL), cinnamoyl-CoA reductase (CCR) and cinnamyl-alcohol dehydrogenase (CAD) genes, were also up-regulated in a fungal extract-selective fashion. These results provide evidence that the fungal extracts used in this study differentially increase the production of PTOX or larisiresinol via the up-regulation of the genes in lignan biosynthesis in L. album cell cultures, and suggest that such selective actions of fungal elicitors on the lignan synthesis will lead to more efficient metabolic engineering-based production of PTOX and other beneficial lignans using L. album cell cultures.     

Genetic diversity of Salmonella Paratyphi A isolated from enteric fever patients in Bangladesh from 2008 to 2018

BackgroundThe proportion of enteric fever cases caused by Salmonella Paratyphi A is increasing and may increase further as we begin to introduce typhoid conjugate vaccines (TCVs). While numerous epidemiological and genomic studies have been conducted for S. Typhi, there are limited data describing the genomic epidemiology of S. Paratyphi A in especially in endemic settings, such as Bangladesh.Principal findingsWe conducted whole genome sequencing (WGS) of 67 S. Paratyphi A isolated between 2008 and 2018 from eight enteric disease surveillance sites across Bangladesh. We performed a detailed phylogenetic analysis of these sequence data incorporating sequences from 242 previously sequenced S. Paratyphi A isolates from a global collection and provided evidence of lineage migration from neighboring countries in South Asia. The data revealed that the majority of the Bangladeshi S. Paratyphi A isolates belonged to the dominant global lineage A (67.2%), while the remainder were either lineage C (19.4%) or F (13.4%). The population structure was relatively homogenous across the country as we did not find any significant lineage distributions between study sites inside or outside Dhaka. Our genomic data showed presence of single point mutations in gyrA gene either at codon 83 or 87 associated with decreased fluoroquinolone susceptibility in all Bangladeshi S. Paratyphi A isolates. Notably, we identified the pHCM2- like cryptic plasmid which was highly similar to S. Typhi plasmids circulating in Bangladesh and has not been previously identified in S. Paratyphi A organisms.SignificanceThis study demonstrates the utility of WGS to monitor the ongoing evolution of this emerging enteric pathogen. Novel insights into the genetic structure of S. Paratyphi A will aid the understanding of both regional and global circulation patterns of this emerging pathogen and provide a framework for future genomic surveillance studies.     

Computational and Pharmacological Studies on the Antioxidant,Thrombolytic, Anti-Inflammatory,and Analgesic Activity of Molineria capitulata

Molineria capitulata is an ornamental plant that has traditionally been used to treat several chronic diseases. The present study was designed to examine the antioxidant, cytotoxic, thrombolytic, anti-inflammatory, and analgesic activities of a methanolic extract of M. capitulata leaves (MEMC) using both experimental and computational models. Previously established protocols were used to perform qualitative and quantitative phytochemical screening in MEMC. A computational study, including molecular docking and ADME/T analyses, was performed. The quantitative phytochemical analysis revealed the total phenolic and flavonoid contents as 148.67 and 24 mg/g, respectively. Antioxidant activity was assessed by examining the reducing power of MEMC, resulting in absorbance of 1.87 at 400 µg/mL, demonstrating a strong reduction capacity. The extract exhibited significant protection against blood clotting and showed the highest protein denaturation inhibition at 500 µg/mL. In both the acetic acid-induced writhing and formalin-induced paw-licking models, MEMC resulted in significant potential pain inhibition in mice. In the computational analysis, 4-hydroxybenzaldehyde, orcinol glucoside, curcapital, crassifogenin C, and 2,6-dimethoxy-benzoic acid displayed a strong predictive binding affinity against the respective receptors. These findings indicated that M. capitulata possesses significant pharmacological activities to an extent supported by computational studies.     

Effects of halogen substitution on Watson–Crick base pairing: A possible mechanism for radiosensitivity

The halogen substituent effect on geometries and charge distributions of the A–T base pair derivatives was evaluated using density functional theory at B3LYP/6-31G1 level. The results indicate that all of the substitutions affect geometries and charge distributions of the atoms contributing hydrogen bonds. These changes would be the reason of the radiosensitization of these compounds incorporating DNA.     

Timing of enteral feeding in cerebral malaria in resource-poor settings: a randomized trial

Background

Early start of enteral feeding is an established treatment strategy in intubated patients in intensive care since it reduces invasive bacterial infections and length of hospital stay. There is equipoise whether early enteral feeding is also beneficial in non-intubated patients with cerebral malaria in resource poor settings. We hypothesized that the risk of aspiration pneumonia might outweigh the potential benefits of earlier recovery and prevention of hypoglycaemia.

Method and Findings

A randomized trial of early (day of admission) versus late (after 60 hours in adults or 36 hours in children) start of enteral feeding was undertaken in patients with cerebral malaria in Chittagong, Bangladesh from May 2008 to August 2009. The primary outcome measures were incidence of aspiration pneumonia, hypoglycaemia and coma recovery time. The trial was terminated after inclusion of 56 patients because of a high incidence of aspiration pneumonia in the early feeding group (9/27 (33%)), compared to the late feeding group (0/29 (0%)), p = 0.001). One patient in the late feeding group, and none in the early group, had hypoglycaemia during admission. There was no significant difference in overall mortality (9/27 (33%) vs 6/29 (21%), p = 0.370), but mortality was 5/9 (56%) in patients with aspiration pneumonia.

Conclusions

In conclusion, early start of enteral feeding is detrimental in non-intubated patients with cerebral malaria in many resource-poor settings. Evidence gathered in resource rich settings is not necessarily transferable to resource-poor settings.

Trial Registration

Controlled-Trials.com ISRCTN57488577     

PET translates neurophysiology into images: A review to stimulate a network between neuroimaging and basic research

In the last decades there has been a progressive advance in the development of techniques able to explore in humans neurophysiologic and neurochemical processes. Positron emission tomography (PET) is a very powerful technique allowing to study a quite variable range of physiological and biochemical processes in the healthy subjects and in diseases. Apart from its capacity to provide pathophysiological information, PET is also important for the objective assessment of therapeutic efficacy. Initial studies were performed measuring cerebral metabolic rate for glucose (CMRglc) and cerebral blood flow (CBF), representing an indirect index of synaptic activity. The advent of receptor tracers allowed measuring other important physiological parameters, such as receptor occupancy, and endogenous release. In neuropsychiatric disorders, as Alzheimer disease, schizophrenia, epilepsy and Huntington disease, PET has been useful to elaborate hypothesis of the pathogenesis, to relate symptoms to biological variables and to study individuals at increased risk. The new concepts of neurovascular unit and default network, preferentially active at rest, can significantly change the approach of PET, with images reflecting a complex scenario, not merely limited to neural activity, but involving the activity of the entire neurovascular unit and the multifunctional role of astrocytes. To detect dysfunction of the dialog between glutamatergic neurons and astrocytes could lead to a better understanding of altered functional brain images. In this direction a professional network between PET researchers and basic scientists, could give a determinant improvement in the capability to understand the complex physiological and pathophysiological cerebral world.     

Mechanisms of β-adrenergic receptors agonists in mediating pro and anti-apoptotic pathways in hyperglycemic Müller cells

Molecular Biology Reports - The current study aimed to investigate the stimulatory effect of beta-adrenergic receptors (β-ARs) on brain derived neurotropic factor (BDNF) and cAMP response...     

Broad-range Glycosidase Activity Profiling

Plants produce hundreds of glycosidases. Despite their importance in cell wall (re)modeling, protein and lipid modification, and metabolite conversion, very little is known of this large class of glycolytic enzymes, partly because of their post-translational regulation and their elusive substrates. Here, we applied activity-based glycosidase profiling using cell-permeable small molecular probes that react covalently with the active site nucleophile of retaining glycosidases in an activity-dependent manner. Using mass spectrometry we detected the active state of dozens of myrosinases, glucosidases, xylosidases, and galactosidases representing seven different retaining glycosidase families. The method is simple and applicable for different organs and different plant species, in living cells and in subproteomes. We display the active state of previously uncharacterized glycosidases, one of which was encoded by a previously declared pseudogene. Interestingly, glycosidase activity profiling also revealed the active state of a diverse range of putative xylosidases, galactosidases, glucanases, and heparanase in the cell wall of Nicotiana benthamiana. Our data illustrate that this powerful approach displays a new and important layer of functional proteomic information on the active state of glycosidases.Carbohydrates are present in all kingdoms of life and are particularly prominent in plants (1). Plants produce carbohydrates as one of their major constituents through their photosynthetic activity. The simplest synthesized forms of carbohydrates are monosaccharide sugars such as glucose, which provides energy for various cellular activities. Carbohydrates also exist in very complex forms. Monosaccharide sugars are attached to one another through covalent glycosidic linkage, which generates di-, oligo-, and polysaccharides. Carbohydrates also attach to non-carbohydrate species (lipids, proteins, hormones) through a glycosidic linkage to form glycoconjugates (2).Glycosidic bonds are hydrolyzed by a group of enzymes termed glycosyl hydrolases (GHs)¹ or glycosidases (3). Because of the tremendous carbohydrate diversity, there are a vast variety of glycosidases, including glucosidases, xylosidases, and galactosidases, that preferentially hydrolyze their respective glycoside substrates. In general, the number of glycosidase-related genes in plants (for instance, Arabidopsis) is relatively high when compared with that in other sequenced organisms (for instance, human) (4). This signifies the unique importance of glycosidases in plants as opposed to other organisms. Based on protein sequence similarities, glycosidases are classified into different GH families. Members of the same GH family share a common mechanism of glycosidic bond cleavage (5).Mechanistically, glycosidases are classified as retaining or inverting enzymes (6). To hydrolyze the glycosidic bond, both retaining and inverting enzymes carry two catalytic glutamate or aspartate residues (or both) (7). Of these two catalytic residues, one acts as a proton donor and the other as a nucleophile/base. The distance between these catalytic residues in the active site of the glycosidases determines the mechanism of hydrolysis. Retaining enzymes have two catalytic residues separated by a distance of ∼5.5 Å, and their hydrolysis mechanism retains the net anomeric configuration of the C1 atom in the sugar molecule. In contrast, inverting enzymes have catalytic residues that are ∼10 Å apart, and these enzymes invert the overall anomeric configuration of the C1 carbon atom in the released sugar (8).Both retaining and inverting glycosidases are present abundantly in plants. The genome of Arabidopsis thaliana encodes for 400 glycosidases, of which 260 are retaining enzymes and 140 are inverting enzymes. Genetic, molecular, and biochemical approaches revealed that glycosidases are localized in different cellular compartments and are important for various biological processes. The majority of plant glycosidases reside in the cell wall, and these enzymes can play major roles in cell wall restructuring (9). Other characterized glycosidases reside in other compartments to regulate glycosylation of proteins and hormones. Despite the importance of GH enzymes, physiological and biochemical functions are assigned to only a few glycosidases (9).Activity-based protein profiling (ABPP) is a powerful tool for monitoring the active state of multiple enzymes without knowledge of their natural substrates (10, 11). ABPP involves chemical probes that react with active site residues in an activity-dependent manner. Thus ABPP displays the availability and reactivity of active site residues in proteins, which are hallmarks for enzyme activity (12). ABPP is particularly attractive because the profiling can be done without purifying the enzymes and can be performed in cell extracts or in living cells. Another key advantage of ABPP is that the activities of large multigene enzyme families can be monitored using broad-range probes. ABPP has had a significant impact on plant science. After the introduction of probes for papain-like cysteine proteases (13, 14), these probes revealed increased protease activities in the tomato and maize apoplasts during immune responses (15, 16) and that these immune proteases are targeted by unrelated inhibitors secreted by fungi, oomycetes, and nematodes (17–24). Likewise, probes for the proteasome displayed unexpected increased proteasome activity during immune responses (25) and revealed that the bacterial effector molecule syringolin A targets the nuclear proteasome (26). We anticipate that more regulatory mechanisms will be discovered through the use of probes introduced for serine hydrolases, metalloproteases, vacuolar processing enzymes, ATP binding proteins, and glutathione transferases (27–32).Cyclophellitol-aziridine-based probes were previously used in animal proteomes to target retaining glucosidases (33). Here we established and applied glycosidase profiling in plants. We discovered that cyclophellitol-aziridine-based probes targeted an unexpectedly broad range of glycosidases representing members of at least seven different GH families. We used these probes to study the active state of glycosidases present in living cells, in different organs and plant species, and in the apoplast of Nicotiana benthamiana.     

Molecular docking and analgesic studies of Erythrina variegata?s derived phytochemicals with COX enzymes

Secondary metabolites from plants are a good source for the NSAID drug development. We studied the analgesic activity of ethanolic extract of Erythrina variegata L. (Fabaceae) followed by molecular docking analysis. The analgesic activity of Erythrina variegata L. is evaluated by various methods viz., acetic acid-induced writhing test, hot plate and tail immersion test. Subsequently, molecular docking analysis has been performed to identify compounds having activity against COX-1 and COX-2 enzymes by using GOLD docking fitness. The result of preliminary phytochemical screening revealed that the extract contains alkaloids and flavonoids. In analgesic activity tests, the extract at the doses of 50, 100 and 200 mg/kg body weight (b.w.) produced a increase in pain threshold in a dose dependent manner. In acetic acid induced writhing test, the inhibitory effect was similar to the reference drug diclofenac sodium. The extract showed 18.89% writhing inhibitory effect at the dose 200 mg/kg b.w., whereas diclofenac sodium showed 79.42% inhibition of writhing at a dose of 10 mg/kg b.w. The results of tail immersion and hot plate test also showed potential analgesic activity of the extract which is also comparable to the standard drug morphine (5 mg/kg b.w.). Docking studies shows that phaseollin of Erythrina variegata L. has the best fitness score against the COX-1 which is 56.64 and 59.63 for COX- 2 enzyme. Phaseollin of Erythrina variegata L. detected with significant fitness score and hydrogen bonding against COX-1 and COX-2 is reported for further validation.     

Streptococcus pneumoniae serotype-2 childhood meningitis in Bangladesh: a newly recognized pneumococcal infection threat

Background

Streptococcus pneumoniae is a leading cause of meningitis in countries where pneumococcal conjugate vaccines (PCV) targeting commonly occurring serotypes are not routinely used. However, effectiveness of PCV would be jeopardized by emergence of invasive pneumococcal diseases (IPD) caused by serotypes which are not included in PCV. Systematic hospital based surveillance in Bangladesh was established and progressively improved to determine the pathogens causing childhood sepsis and meningitis. This also provided the foundation for determining the spectrum of serotypes causing IPD. This article reports an unprecedented upsurge of serotype 2, an uncommon pneumococcal serotype, without any known intervention.

Methods and Findings

Cases with suspected IPD had blood or cerebrospinal fluid (CSF) collected from the beginning of 2001 till 2009. Pneumococcal serotypes were determined by capsular swelling of isolates or PCR of culture-negative CSF specimens. Multicenter national surveillance, expanded from 2004, identified 45,437 patients with suspected bacteremia who were blood cultured and 10,618 suspected meningitis cases who had a lumber puncture. Pneumococcus accounted for 230 culture positive cases of meningitis in children <5 years. Serotype-2 was the leading cause of pneumococcal meningitis, accounting for 20.4% (45/221; 95% CI 15%–26%) of cases. Ninety eight percent (45/46) of these serotype-2 strains were isolated from meningitis cases, yielding the highest serotype-specific odds ratio for meningitis (29.6; 95% CI 3.4–256.3). The serotype-2 strains had three closely related pulsed field gel electrophoresis types.

Conclusions

S. pneumoniae serotype-2 was found to possess an unusually high potential for causing meningitis and was the leading serotype-specific cause of childhood meningitis in Bangladesh over the past decade. Persisting disease occurrence or progressive spread would represent a major potential infection threat since serotype-2 is not included in PCVs currently licensed or under development.