Reversible association of proteins into sub-visible amorphous aggregates using short solubility controlling peptide tags.

Careful analysis of sub-visible amorphous aggregates, where proteins associate non-covalently in either native or denatured states without forming a specific quaternary structure, may shed insight into the mechanisms of protein aggregation and solubility. Here we report a biophysical and biochemical analysis of our model protein, a bovine pancreatic trypsin inhibitor variant (BPTI-19A), whose oligomerization were controlled by attaching solubility controlling peptide tags (SCP tags) to its C terminus, which are short peptides composed of a single type of amino acid that modulate protein solubility. The dynamic light scattering and static light scattering at 25°C indicated that 11 out of 15 SCP tags merely affected the hydrodynamic radius and light scattering intensity of our reference variants BPTI-19A and BPTI-C2G. On the other hand, hydrophobic SCP tags composed of 5 Ile (C5I) or 5 Leu (C5L) were associated into sub-visible aggregates. Circular dichroism indicated that all tagged BPTI variants had the same secondary structure contents as the reference BPTI-19A at 25°C, suggesting that BPTI-C5I and C5L kept their native structure upon association. Furthermore, the thermal denaturation of all of the BPTI variants was fully reversible and typical of natively folded small globular proteins, as monitored by CD at 222 nm. However, the thermal stability of BPTI-19A tagged with hydrophobic residues decreased with increasing protein concentration and tag's hydrophobicity, and BPTI-C5I and C5L were partially denatured at 37°C. Biochemical stability assessed by limited proteolysis with pepsin correlated with the extent of the variants' aggregation, and the large sub-visible aggregates formed by BPTI-C5I and C5L significantly increased their resistance to pepsin proteolysis. Altogether, these observations indicated that hydrophobic SCP tags led to the reversible association of native-like proteins into sub-visible soluble amorphous aggregates resistant to pepsin digestion.

Antioxidant, antimicrobial, toxicity and analgesic properties of ethanol extract of Solena amplexicaulis root.

BACKGROUND: This study was subjected to investigate different pharmacological properties of ethanol extract of Solena amplexicaulis root. RESULTS: The extract contains flavonoid, alkaloid, saponin and steroid compounds. The extract exhibited excellent antioxidant activity in DPPH radical scavenging activity. The extract also showed potent activity in brine shrimp lethality bioassay. The LC50 value was found to 44.677 µg/ml. The extract showed better anti-bacterial activity against gram-negative bacteria. In antifungal assay, the maximum 79.31% of anti-mycotic activity was observed against Aspergillus ochraceus while minimum 44.2% against Rhizopus oryzae. MIC value ranged between 1500-3000 µg/ml. The extract was found moderately toxic with a 24-hr LD50 value of 81.47 mg/kg in Swiss albino mice. The degree of inhibition by the ethanolic extract of the root was found less than that of standard analgesic drug diclofenac sodium. The extract also showed moderate anti-inflammatory and antinociceptive activity and anti-diabetic property. Reducing power of the extract was comparable with standard ascorbic acid. Moderate in vitro thrombolytic activity, lipid peroxidation inhibition property, metal chelating ability and stress-protective activity was also observed. CONCLUSION: Ethanol extract of Solena amplexicaulis root can be valuable for treatment of different diseases.

Knowledge, attitudes and biosecurity practices among the small-scale dairy farmers in Sylhet district, Bangladesh.

BACKGROUND: In the context of zoonosis, Bangladesh's small-scale dairying is yet to frame satisfactory levels due to poor biosecurity practices. OBJECTIVES: This study intended to reveal the degree of knowledge, attitudes and biosecurity practices among Sylhet district, Bangladesh's small-scale dairy farmers. We also focused on the association between biosecurity practices and the incidence of non-specific enteritis in humans. METHODS: A questionnaire-based survey was conducted on the farmers' KAP via personal interviews of 15 farmers from the randomly selected fifteen small-scale dairy farms. The questionnaire was developed with 6 questions for knowledge, 6 questions for attitude and 12 questions for the practice of biosecurity measures. Alongside that, data on the number of non-specific enteritis cases experienced by the farmers or their family members were also recorded. Spearman correlation was used to find out the correlation among KAP variables and between practice scores and non-specific enteritis incidences. RESULTS: We found an insignificant (p > 0.05) influence of demographic characteristics over knowledge, attitude and biosecurity practices. Significant (p < 0.05) and strong correlations were found in knowledge-attitude (r = 0.65), knowledge-practice (r = 0.71) and attitude-practice (r = 0.64). Incidences of non-specific enteritis and biosecurity measures' practice were also strongly correlated (r = -0.9232) and statistically significant (p < 0.05). CONCLUSIONS: Our study suggests that increasing knowledge and developing a good attitude are necessary to increase the adaptation of biosecurity measures as three of these factors are correlated. Moreover, farm biosecurity measures are closely related to human health.

A bio-sustainable approach for reducing Eucalyptus tree-caused agricultural ecosystem hazards employing Trichoderma bio-sustained spores and mycorrhizal networks.

The presence of the exotic Eucalyptus tree in crop-growing soil and the accumulation of its undecomposed leaves is a significant ecological hazard. The waxy coating on the leaves and the phenolic compounds takes a long time to break down under normal conditions. It is necessary to explore various fungi that can degrade these leaves for an eco-friendly solution to this problem. In this study, spores of nine native Trichoderma strains were produced on wheat agar using a lactic acid-induced sporulation strategy (LAISS). Trichoderma biosustained spores and Serendipita indica (SI) spores were applied to a rice field with accumulated Eucalyptus leaves under continuous ponding (CP) and alternate flooding and wetting conditions (AFW). Among the strains, TI04 (Trichoderma viride) and TI15 (Trichoderma citrinoviride) showed faster (5 days) and massive sporulation (1.06-1.38 × 1011 CFU/g) in LAISS. In vitro, TI04 and TI15 biosustained on Eucalyptus leaves and improved rice seedling growth and SI infection under greenhouse conditions. In the rice-field experiment, Trichoderma-treatment had a threefold yield (percentage) increase from control, with TI04 (CP) increasing the yield by 30.79, TI04 (AFW) by 29.45, TI15 (CP) by 32.72, and TI15 (AFW) rising by 31.91. Remarkably, unfilled grain yield significantly decreased in all the Trichoderma treatments. Under AFW conditions, TI04 and TI15 showed a higher pH increase. Furthermore, TI04 and TI15 under AFW had higher water productivity (t ha-1 cm-1) of 0.0763 and 0.0791, respectively, and the highest rates (percentage) of SI colonization of 86.36 and 83.16, respectively. According to the findings, LAISS-produced Trichoderma spores can be applied to break down persistent wastes and restore agricultural ecosystems through increased mycorrhizae networking.

Optimization of industrial (3000 L) production of Bacillus subtilis CW-S and its novel application for minituber and industrial-grade potato cultivation.

A commercial plant probiotic product was developed employing Bacillus subtilis CW-S in submerged fermentation. The effects of molasses and urea on cell growth were investigated with the goal of low-cost manufacturing. Plackett-Burman and Central-Composite Design (CCD) were utilized to optimize production parameters to maximize productivity. The stability of the formulated product and its efficacy in cultivating minituber in aeroponics and industrial-grade potatoes in the field were assessed. The results showed that the medium BS10 (molasses and urea) produced satisfactory cell density (7.19 × 108 CFU/mL) as compared to the control (1.51 × 107 CFU/mL) and BS1-BS9 (expensive) media (1.84 × 107-1.37 × 109 CFU/mL). According to validated CCD results, optimized parameters fitted well in pilot (300 L; 2.05 × 109 CFU/mL) and industrial (3000 L; 2.01 × 109 CFU/mL) bioreactors, resulting in a two-fold increase in cell concentration over laboratory (9.84 × 108 CFU/mL) bioreactors. In aeroponics, CW-S produced excellent results, with a significant increase in the quantity and weight of minitubers and the survival rate of transplanted plantlets. In a field test, the yield of industrial-grade (> 55 mm) potatoes was increased with a reduction in fertilizer dose. Overall, the findings suggest that CW-S can be produced commercially utilizing the newly developed media and optimized conditions, making plant probiotics more cost-effective and accessible to farmers for crop cultivation, particularly in aeroponic minituber and industrial-grade potato production.

Epitope design of L1 protein for vaccine production against Human Papilloma Virus types 16 and 18.

Cervical cancer accounts for about two-thirds of all cancer cases linked etiologically to Human Papilloma Virus (HPV). 15 oncogenic HPV types can cause cervical cancer, of which HPV16 and HPV18 combinedly account for about 70% of it. So, effective epitope design for the clinically relevant HPV types 16 and 18 would be of major medical benefit. Here, a comprehensive analysis is carried out to predict the epitopes against HPV types 16 and 18 through "reverse vaccinology" approach. We attempted to identify the evolutionarily conserved regions of major capsid protein (L1) as well as minor capsid protein (L2) of HPV and designed epitopes within these regions. In this study, we analyzed about 49 and 27 sequences of HPV L2 and L1 proteins respectively. Since we found that the intertype variability of L2 is higher than for L1 proteins, our analysis was emphasized on epitopes of L1 of HPV types 16 and 18. We had selected HLA-A*0201, DRB1*1501, DQB1*0602, DRB1*0401 and DQB1*0301 alleles for the prediction of T cell epitopes of L1 of HPV 16 and 18. Finally, we reported that predicted epitope sequences EEYDLQFIFQLCKITLTA, and RHGEEYDLQFIFQLCKITLTA of L1 protein of HPV 16, and LPDPNKF, PETQRLVWAC, PVPGQYDA, YNPETQRLVWAC, DTGYGAMD, PVPGQYDATK, KQDIPKVSAYQYRVFRV, RDNVSVDYKQTQLCI and YSRHVEEYDLQFIF of L1 protein of HPV 18 could be therapeutic tools for vaccine design against HPV.

Amorphous protein aggregation monitored using fluorescence self-quenching.

Biophysical understanding of amorphous protein aggregation can significantly impact diverse area of biotechnology. Here, we report the time dependent salt-induced formation of amorphous aggregation as monitored by fluorescence self-quenching and compare the results with conventional methods for detecting protein aggregation [static light scattering (LS) and dynamic light scattering (DLS)]. As a model protein, we used a bovine pancreatic trypsin inhibitor (BPTI) variant extended by two glycines (C2G) at its C terminus, and three variants where three types of Solubility Controlling Peptide tags (SCP tags) made of five serines (C5S), alanines (C5A) or aspartic acids (C5D) were added to the C terminus of C2G. All variants have a native-like BPTI structure and trypsin inhibitory activity, but different solubilities controlled by the SCP tags. The BPTIs were labeled using NHS-Fluorescein (FAM) conjugated to BPTI's lysines, and we measured the changes in fluorescence intensity occurring upon the addition of NaCl. The fluorescence of all FAM-BPTIs decreased almost immediately, albeit to a different extent, upon addition of salt and became constant after 10 min for 24 h or more. On the other hand, LS and DLS signal changes were dependent on the type of tags. Namely, C2G's LS and DLS signals changed immediately, the signals of C5S and C5A tagged FAM-BPTIs increased slowly from 10 min to 24 h, and those of C5D remained constant. These observations indicated the presence of at least one intermediate step, with increased protein-protein interaction yielding a 'molecular condensation' phase. According to this model, C2G would rapidly turn from 'condensates' to aggregates, whereas C5S and C5A tagged FAM-BPTIs would do so slowly, and the soluble C5D tagged variant would remain in the molecular condensation state.

Antioxidant, antimicrobial, toxicity and analgesic properties of ethanol extract of Solena amplexicaulis root

BACKGROUND: This study was subjected to investigate different pharmacological properties of ethanol extract ofSolena amplexicaulis root. RESULTS: The extract contains flavonoid, alkaloid, saponin and steroid compounds. The extract exhibited excellent antioxidant activity in DPPH radical scavenging activity. The extract also showed potent activity in brine shrimp lethality bioassay. The LC50 value was found to 44.677 µg/ml. The extract showed better anti-bacterial activity against gram-negative bacteria. In antifungal assay, the maximum 79.31% of anti-mycotic activity was observed against Aspergillus ochraceus while minimum 44.2% against Rhizopus oryzae. MIC value ranged between 1500 - 3000 µg/ml. The extract was found moderately toxic with a 24-hr LD50 value of 81.47 mg/kg in Swiss albino mice. The degree of inhibition by the ethanolic extract of the root was found less than that of standard analgesic drug diclofenac sodium. The extract also showed moderate anti-inflammatory and antinociceptive activity and anti-diabetic property. Reducing power of the extract was comparable with standard ascorbic acid. Moderate in vitro thrombolytic activity, lipid peroxidation inhibition property, metal chelating ability and stress-protective activity was also observed. CONCLUSION: Ethanol extract of Solena amplexicaulis root can be valuable for treatment of different diseases.