Phytase activity in tobacco (Nicotiana tabacum) root exudates is exhibited by a purple acid phosphatase

Phytases are enzymes that catalyze liberation of inorganic phosphates from phytate, the major organic phosphorus in soil. Tobacco (Nicotiana tabacum) responds to phosphorus starvation with an increase in extracellular phytase activity. By a three-step purification scheme, a phosphatase with phytase activity was purified 486-fold from tobacco root exudates to a specific activity of 6,028 nkat mg(-1) and an overall yield of 3%. SDS-PAGE revealed a single polypeptide of 64 kDa, thus indicating apparent homogeneity of the final enzyme preparation. Gel filtration chromatography suggested that the enzyme was a ca. 56 kDa monomeric protein. De novo sequencing by tandem mass spectrometry resulted in a tryptic peptide sequence that shares high homology with several plant purple acid phosphatases. The identity of the enzyme was further confirmed by molybdate-inhibition assay and cDNA cloning. The purified enzyme exhibited pH and temperature optima at 5.0-5.5 and 45 degrees C, respectively, and were found to have high affinities for both p-nitrophenyl phosphate (pNPP; K(m)=13.9 microM) and phytate (K(m)=14.7 microM), but a higher kcat for pNPP (2,056 s(-1)) than phytate (908 s(-1)). Although a broad specificity of the enzyme was observed for a range of physiological substrates in soil, maximum activity was achieved using mononucleotides as substrates. We conclude that the phytase activity in tobacco root exudates is exhibited by a purple acid phosphatase and its catalytic properties are pertinent to its role in mobilizing organic P in soil.     

Evaluation on masks with exhaust valves and with exhaust holes from physiological and subjective responses

The purpose of this study was to compare the effects of wearing different kinds of masks on the ear canal temperature, heart rate, clothing microclimate, and subjective perception of discomfort. Ten subjects performed intermittent exercise on a treadmill while wearing the protective masks in a climatic chamber controlled at an air temperature of 25 degrees C and a relative humidity of 70%. Two types of mask-mask A, with exhaust valves and mask B, with exhaust holes-were used in the study. The results of this study indicated: (1) The subjects had a tendency toward lower maximum heart rate when wearing mask A than when wearing mask B. (2) Temperatures and absolute humidities (the outer surface of mask, the microclimate inside the mask, the chest wall skin and microclimate) of mask A were significantly lower than those of mask B. (3) The ear canal temperature increased significantly in mask B as compared to that in mask A. (4) The ear canal temperature showed significant augmentation along with increased temperature and humidity inside the mask microclimate. The mask microclimate temperature also affected significantly the chest microclimate temperature. (5) Mask A was rated significantly lower for perception of humidity, heat, breath resistance, tightness, unfitness, odor, fatigue, and offered less overall discomfort than mask B. (6) Subjective preference for mask A was higher. (7) The ratings of subjective overall discomfort showed significant augmentation along with increased wetness and fatigue. We discuss how the ventilation properties of masks A and B induce significantly different temperature and humidity in the microclimates of the masks and the heat loss of the body, which have profound influences on heart rate, thermal stress, and subjective perception of discomfort.     

Influenza virus in human exhaled breath: an observational study

Background

Recent studies suggest that humans exhale fine particles during tidal breathing but little is known of their composition, particularly during infection.

Methodology/Principal Findings

We conducted a study of influenza infected patients to characterize influenza virus and particle concentrations in their exhaled breath. Patients presenting with influenza-like-illness, confirmed influenza A or B virus by rapid test, and onset within 3 days were recruited at three clinics in Hong Kong, China. We collected exhaled breath from each subject onto Teflon filters and measured exhaled particle concentrations using an optical particle counter. Filters were analyzed for influenza A and B viruses by quantitative polymerase chain reaction (qPCR). Twelve out of thirteen rapid test positive patients provided exhaled breath filter samples (7 subjects infected with influenza B virus and 5 subjects infected with influenza A virus). We detected influenza virus RNA in the exhaled breath of 4 (33%) subjects–three (60%) of the five patients infected with influenza A virus and one (14%) of the seven infected with influenza B virus. Exhaled influenza virus RNA generation rates ranged from <3.2 to 20 influenza virus RNA particles per minute. Over 87% of particles exhaled were under 1 µm in diameter.

Conclusions

These findings regarding influenza virus RNA suggest that influenza virus may be contained in fine particles generated during tidal breathing, and add to the body of literature suggesting that fine particle aerosols may play a role in influenza transmission.     

SARS-CoV-2 S2P spike ages through distinct states with altered immunogenicity

The SARS-CoV-2 spike is the primary target of virus-neutralizing antibodies and critical to the development of effective vaccines against COVID-19. Here, we demonstrate that the prefusion-stabilized two-proline “S2P” spike—widely employed for laboratory work and clinical studies—unfolds when stored at 4 °C, physiological pH, as observed by electron microscopy (EM) and differential scanning calorimetry, but that its trimeric, native-like conformation can be reacquired by low pH treatment. When stored for approximately 1 week, this unfolding does not significantly alter antigenic characteristics; however, longer storage diminishes antibody binding, and month-old spike elicits virtually no neutralization in mice despite inducing high ELISA-binding titers. Cryo-EM structures reveal the folded fraction of spike to decrease with aging; however, its structure remains largely similar, although with varying mobility of the receptor-binding domain. Thus, the SARS-CoV-2 spike is susceptible to unfolding, which affects immunogenicity, highlighting the need to monitor its integrity.     

Three newOphioceras species (Ascomycetes) from the tropics

Ophioceras guttulatum sp. nov.,O. hongkongense sp. nov. andO. palmae sp. nov. are described and illustrated from decaying terrestrial palms and woody substrates in freshwater habitats. They all have black perithecia with long necks, cylindrical asci with refractive apical rings and filiform ascospores.     

Economic effects and the efficacy of intervention: exploring unintended effects of management and policy on the spread of non-indigenous species

Models have been well developed describing human movements as vectors of the spread of non-indigenous species (NIS). However, to be maximally useful, predictions need to be integrated with management models of how different policies change human behaviour and lead to concurrent changes in invasion risk. Using the dispersal of freshwater organisms by recreational boaters as our study system and mandatory boat washing as our management strategy, we conducted a survey of recreational boaters (n = 580 respondents, t = 2354 boating trips) in Ontario, Canada, and performed counterfactual analysis of boater behavior across different management options. We developed a model to quantify three responses to mandatory boat washing policies: (1) the continued use of a policy lake; (2) switching to a non-policy lake (“trip redistribution”); or, (3) a reduction in boating trips (“trip loss”). We found that boater and locational traits did not have a significant effect, but even modest user fees at washing stations greatly influenced trip redistribution and loss, explaining 87% of the variation in boater choices. These results indicate that user fees can strongly reduce the effectiveness of boat washing programs to mitigate invasion risk and could have unintended local economic effects, supporting the need to minimize boater expense as a program goal. In contrast, only minor redistribution and loss occurred if users washed but did not pay, and when taken together with the lack of effect for boater and locational traits, suggest that simple human-mediated dispersal models would be sufficient to prioritize management actions under “zero fee” scenarios. Simulating management scenarios using an existing spread model for 10 aquatic NIS in Ontario further emphasized the benefit of zero fees. Although averted invasions increased monotonically with effort (number of lakes with washing stations), the relative effectiveness (number of invasions averted per unit effort) was high even with management of a single lake, given zero fees, but required washing stations at far more lakes to maximize relative effectiveness when user fees were imposed.     

Virtual screening and biophysical studies lead to HSP90 inhibitors

Heat shock protein 90 (HSP90) is a molecular chaperone that plays important functional roles in cells. The chaperone activity of HSP90 is regulated by the hydrolysis of ATP at the protein’s N-terminal domain. HSP90, in particular the N-terminal domain, is a current inhibition target for therapeutic treatments of cancers. This paper describes an application of virtual screening, thermal shift assaying and protein NMR spectroscopy leading to the discovery of HSP90 inhibitors that contain the resorcinol structure. The resorcinol scaffold can be found in a class of HSP90 inhibitors that are currently undergoing clinical trials. The proved success of the resorcinol moiety in HSP90 inhibitors validates this combined virtual screen and biophysical technique approach, which may be applied for future inhibitor discovery work for HSP90 as well as other targets.     

Lipopolysaccharide pretreatment increases protease-activated receptor-2 expression and monocyte chemoattractant protein-1 secretion in vascular endothelial cells

Background

This study investigated whether lipopolysaccharide (LPS) increase protease-activated receptor-2 (PAR-2) expression and enhance the association between PAR-2 expression and chemokine production in human vascular endothelial cells (ECs).

Methods

The morphology of ECs was observed through microphotography in cultured human umbilical vein ECs (EA. hy926 cells) treated with various LPS concentrations (0, 0.25, 0.5, 1, and 2 μg/mL) for 24 h, and cell viability was assessed using the MTT assay. Intracellular calcium imaging was performed to assess agonist (trypsin)-induced PAR-2 activity. Western blotting was used to explore the LPS-mediated signal transduction pathway and the expression of PAR-2 and adhesion molecule monocyte chemoattractant protein-1 (MCP-1) in ECs.

Results

Trypsin stimulation increased intracellular calcium release in ECs. The calcium influx was augmented in cells pretreated with a high LPS concentration (1 μg/mL). After 24 h treatment of LPS, no changes in ECs viability or morphology were observed. Western blotting revealed that LPS increased PAR-2 expression and enhanced trypsin-induced extracellular signal-regulated kinase (ERK)/p38 phosphorylation and MCP-1 secretion. However, pretreatment with selective ERK (PD98059), p38 mitogen-activated protein kinase (MAPK) (SB203580) inhibitors, and the selective PAR-2 antagonist (FSLLRY-NH2) blocked the effects of LPS-activated PAR-2 on MCP-1 secretion.

Conclusions

Our findings provide the first evidence that the bacterial endotoxin LPS potentiates calcium mobilization and ERK/p38 MAPK pathway activation and leads to the secretion of the pro-inflammatory chemokine MCP-1 by inducing PAR-2 expression and its associated activity in vascular ECs. Therefore, PAR-2 exerts vascular inflammatory effects and plays an important role in bacterial infection-induced pathological responses.

     

Biomechanical consequences of subtalar joint arthroereisis in treating posterior tibial tendon dysfunction: a theoretical analysis using finite element analysis

Subtalar joint arthroereisis (SJA) has been introduced to control the hyperpronation in cases of flatfoot. The objective of this study is to evaluate the biomechanical consequence of SJA to restore the internal stress and load transfer to the intact state from the attenuated biomechanical condition induced by posterior tibial tendon dysfunction (PTTD). A three-dimensional finite element model of the foot and ankle complex was constructed based on clinical images of a healthy female (age 28 years, height 165 cm, body mass 54 kg). The boundary and loading condition during walking was acquired from the gait experiment of the model subject. Five sets of simulations (conditions) were completed: intact condition, mild PTTD, severe PTTD, mild PTTD with SJA, severe PTTD with SJA. The maximum von Mises stress of the metatarsal shafts and the load transfer along the midfoot during stance were analyzed. Generally, SJA deteriorated the joint force of the medial cuneonavicular and calcaneocuboid joints during late stance, while that of the metatarsocuneiform joints during early stance were over-corrected. Only the calcaneocuboid joint force at 45% stance demonstrated a trend of improvement. Besides, SJA exaggerated the increased stress of the metatarsals compared to the PTTD conditions, except that of the first metatarsal. Our study did not support the hypothesis that SJA can restore the internal load transfer and midfoot stress. SJA cannot compensate the salvage of midfoot stability attributed by PTTD and could be biomechanically insufficient to restore the biomechanical environment. Additional procedures such as orthotic intervention may be necessary.