The CRISPR/Cas9 System for Targeted Genome Engineering in Free-Living Fungi: Advances and Opportunities for Lichenized Fungi

Studies using whole genome sequencing, computational and gene expression, targeted genome engineering techniques for generating site-specific sequence alterations through non-homologous end joining (NHEJ) by genomic double-strand break (DSB) repair pathway with high precision, resulting in gene inactivation have elucidated the complexity of gene expression, and metabolic pathways in fungi. These tools and the data generated are crucial for precise generation of fungal products such as enzymes, secondary metabolites, antibiotics etc. Artificially engineered molecular scissors, zinc finger nucleases (ZFNs), Transcriptional activator-like effector nucleases (TALENs; that use protein motifs for DNA sequence recognition in the genome) and CRISPR associated protein 9 (Cas9;CRISPR/Cas9) system (RNA-DNA recognition) are being used in achieving targeted genome modifications for modifying traits in free-living fungal systems. Here, we discuss the recent research breakthroughs and developments which utilize CRISPR/Cas9 in the metabolic engineering of free-living fungi for the biosynthesis of secondary metabolites, enzyme production, antibiotics and to develop resistance against post-harvest browning of edible mushrooms and fungal pathogenesis. We also discuss the potential and advantages of using targeted genome engineering in lichenized fungal (mycobiont) cultures to enhance their growth and secondary metabolite production in vitro can be complemented by other molecular approaches

Pre-Clinical Evaluation of a Novel Nanoemulsion-Based Hepatitis B Mucosal Vaccine

Hepatitis B virus infection remains an important global health concern despite the availability of safe and effective prophylactic vaccines. Limitations to these vaccines include requirement for refrigeration and three immunizations thereby restricting use in the developing world. A new nasal hepatitis B vaccine composed of recombinant hepatitis B surface antigen (HBsAg) in a novel nanoemulsion (NE) adjuvant (HBsAg-NE) could be effective with fewer administrations.Physical characterization indicated that HBsAg-NE consists of uniform lipid droplets (349+/-17 nm) associated with HBsAg through electrostatic and hydrophobic interactions. Immunogenicity of HBsAg-NE vaccine was evaluated in mice, rats and guinea pigs. Animals immunized intranasally developed robust and sustained systemic IgG, mucosal IgA and strong antigen-specific cellular immune responses. Serum IgG reached > or = 10(6) titers and was comparable to intramuscular vaccination with alum-adjuvanted vaccine (HBsAg-Alu). Normalization showed that HBsAg-NE vaccination correlates with a protective immunity equivalent or greater than 1000 IU/ml. Th1 polarized immune response was indicated by IFN-gamma and TNF-alpha cytokine production and elevated levels of IgG(2) subclass of HBsAg-specific antibodies. The vaccine retains full immunogenicity for a year at 4 degrees C, 6 months at 25 degrees C and 6 weeks at 40 degrees C. Comprehensive pre-clinical toxicology evaluation demonstrated that HBsAg-NE vaccine is safe and well tolerated in multiple animal models.Our results suggest that needle-free nasal immunization with HBsAg-NE could be a safe and effective hepatitis B vaccine, or provide an alternative booster administration for the parenteral hepatitis B vaccines. This vaccine induces a Th1 associated cellular immunity and also may provide therapeutic benefit to patients with chronic hepatitis B infection who lack cellular immune responses to adequately control viral replication. Long-term stability of this vaccine formulation at elevated temperatures suggests a direct advantage in the field, since potential excursions from cold chain maintenance could be tolerated without a loss in therapeutic efficacy

A rare syndrome of central diabetes insipidus with spastic cerebellar ataxia

The syndrome of central diabetes insipidus (cDI) and spastic cerebellar ataxia is rare with only a few reports in the literature. We report the case of a 21-year-old patient who was diagnosed to have central diabetes insipidus at the age of 7 years and presented to us at the age of 21 years with progressive spastic cerebellar ataxia that evolved over four years. His MRI showed normal hyperintense signal from the posterior pituitary. The persistence of posterior pituitary signal in patients with cDI is unusual and is observed in the familial variety of cDI, the possible etiology in our patient. A brief review of the literature on the rare syndromic association of cerebellar ataxia and cDI has been discussed

Evaluation of natural products for the management of damping-off of tomato incited by Pythium aphanidermatum (Edson) fitz

The effect of various natural products from plants and animals were evaluated for the presence of antifungal activity against Pythium aphanidermatum causing damping-off of tomato. Among the plant species tested, bulbs of A. sativum (10%) and Allium cepa var. aggregatum (20%), leaves of Lawsonia inermis, Piper betle (20% each), Eucalyptus globulus and Vitex negundo (40% each) exhibited complete inhibition of the mycelial growth of P. aphanidermatum. Out of 4 animal excreta screened, Pig dung (40%) extract totally inhibited the mycelial growth of P. aphanidermatum. Garlic bulb, eucalyptus leaf and hen litter extract were found to retain the fungitoxicity when extracted at 80 °C for 10 minutes. A. sativum bulb followed by E. globulus leaf extracted in acetone was found to be significantly superior over other natural products. Among the natural products tested, the minimum mycelial dry weight of P. aphanidermatum was obtained with garlic bulb followed by eucalyptus leaf extract. The plant products combined together retained strong inhibitory effect in solid and liquid media against P. aphanidermatum. Upon mixing, the more loss of toxicity was observed when botanicals were mixed with animal excreta, particularly with pig dung. Among the natural products tested, the water extracts of A. sativum plus E. globulus and A. sativum plus L. inermis combinations recorded the maximum percentage of seed germination, growth and vigour of tomato seedlings, respectively. Also, when the various natural products tested by seed treatment, soil drenching and seed treatment plus soil drenching, garlic plus eucalyptus treatment recorded maximum seedling emergence and lesser damping off incidence in all three methods of application. Among the three methods, seed treatment plus soil drenching (St+Sd) was found to be superior than other two methods

Influence of Ni and Sn Perovskite NiSn(OH)₆ Nanoparticles on Energy Storage Applications

New NiSn(OH)6 hexahydroxide nanoparticles were synthesised through a co-precipitation method using various concentrations of Ni2+ and Sn4+ ions (e.g., 1:0, 0:1, 1:2, 1:1, and 2:1; namely, N, S, NS-3, NS-2, and NS-1) with an ammonia solution. The perovskite NiSn(OH)6 was confirmed from powder X-ray diffraction and molecule interactions due to different binding environments of Ni, Sn, O, and water molecules observed from an FT-IR analysis. An electronic transition was detected from tin (Sn 3d) and nickel (Ni 2p) to oxygen (O 2p) from UV-Vis/IR spectroscopy. Photo luminescence spectroscopy (PL) identified that the emission observed at 400–800 nm in the visible region was caused by oxygen vacancies due to various oxidation states of Ni and Sn metals. A spherical nanoparticle morphology was observed from FE-SEM; this was due to the combination of Ni2+ and Sn4+ increasing the size and porosity of the nanoparticle. The elemental (Ni and Sn) distribution and binding energy of the nanoparticle were confirmed by EDAX and XPS analyses. Among the prepared various nanoparticles, NS-2 showed a maximum specific capacitance of 607 Fg−1 at 1 Ag−1 and 56% capacitance retention (338 Fg−1 and 5 Ag−1), even when increasing the current density five times, and excellent cycle stability due to combining Ni2+ with Sn4+, which improved the ionic and electrical conductivity. EIS provided evidence for NS-2’s low charge transfer resistance compared with other prepared samples. Moreover, the NS-2//AC (activated carbon) asymmetric supercapacitor exhibited the highest energy density and high-power density along with excellent cycle stability, making it the ideal material for real-time applications

Effect of Yttrium doping on antibacterial and antioxidant property of LaTiO3

Abstract The advancement of multidrug-resistant bacterial strains and their adverse effects is one of the most significant global health issues. The perovskite nanomaterial with combined antioxidant and antibacterial activities in one molecule has the potential for improved therapeutic solutions. In this work, Yttrium-doped Lanthanum Titanate (LaTi1 −x Y x O3, where x = 0, 0.05, and 0.1) was synthesized using auto combustion technique. Excellent crystalline structure with a tetragonal system is revealed by X-ray diffraction analysis (XRD). UV–Visible diffuse reflectance spectroscopy (UV–Vis DRS), Fourier transform infrared (FTIR), and photoluminescence (PL) were used to study its optical characteristics. The field emission scanning electron microscope (FE-SEM) shows rod-like pellet-shaped Yttrium-doped nanostructures, and the elements present were confirmed with the Energy Dispersive X-Ray Analysis (EDAX). Various concentrations of the synthesized materials were tested for antibacterial activity against Gram-positive (Staphylococcus aureus 902) and Gram-negative (E. coli 443) strains using the agar-well diffusion method with gentamicin antibiotic as a positive control. High antibacterial activity of 87.1% and 83.3% was shown by 10% Yttrium-doped LaTiO3 (LY(0.1)TO) at 500 mg/mL against both positive and negative stains, respectively. Moreover, the antioxidant properties of synthesized materials were assessed with IC50 values of 352.33 µg/mL, 458.055 µg/mL, and 440.163 µg/mL for samples LaTi1 − x Y x O3, where x = 0, 0.05, and 0.1 respectively. The antibacterial and antioxidant capabilities of the proposed samples illustrate their applicability in various biomedical applications

Epoxy composite reinforced with jute/basalt hybrid – Characterisation and performance evaluation using machine learning techniques

Epoxy resins, prized for their versatile properties, are derived from bio-based materials, contributing to sustainability and eco-friendliness in both production and application. This study focuses on the application of gradient boosting machine learning techniques in the field of machining to predict the surface roughness and also the contour based experimental validation of the numerical results. The turning experiments, conducted via Taguchi's L27 array, aimed to explore the effects of depth of cut, feed rate, and spindle speed. Higher spindle speeds, lower feed rates, and shallower cuts led to smoother surfaces in turned jute/basalt epoxy composites. Machine learning models (Gradient Boosting Machine, AdaBoost, and XGBoost) were then used to predict surface roughness. Amongst these, XGBoost outperformed GBM and AdaBoost, exhibiting maximum and average prediction errors of 3.78 % and 2.24 %, respectively. XGBoost accurately predicted 2D surface roughness contours that closely matched experimental contours for training and test cases. Taguchi's Orthogonal Matrix identified minimum surface roughness values as 0.773 μm (experimental), 0.800 μm (GBM), 0.880 μm (AdaBoost), and 0.774 μm (XGBoost). All were achieved at 1500 rpm spindle speed, 0.05 mm/rev feed rate, and 0.3 mm depth of cut