Construction of Pseudomonas aeruginosa SDK-6 with synthetic lipase gene cassette and optimization of different parameters using response surface methodology for over-expression of recombinant lipase.

Lipases are industrially important enzymes having vast applications in various fields. Cloning and expression of lipase enzyme-encoding genes in suitable host lead to their widespread use in different fields. The present study represents the first attempt towards the expression of the synthetic lipase gene in Pseudomonas aeruginosa. An alkalophilic lipase gene (GenBank accession number: NP_388152) from Bacillus subtilis was synthetically designed and introduced in the pJN105 vector and subsequently cloned in Pseudomonas aeruginosa SDK-6. Agarose gel electrophoresis confirmed the transformation of SDK-6, exhibiting a band difference of ~ 700 bp between native and recombinant pJN105. Further amplification of cloned lipase gene was confirmed using PCR amplification with Lip 1 and Lip 2 primers respectively, followed by restriction analysis. Approximately 15-fold increase in lipase production was observed in recombinant Pseudomonas as compared to the native strain. One factor at a time (OFAT) analysis revealed L-arabinose, inoculum size (0.5%; v/v), and agitation (120 rpm) as significant factors affecting the over-expression of lipase enzyme. Optimization of enzyme induction conditions by central composite design (CCD) led to 1.60-fold increase in the production of lipase at 0.65% (w/v) inducer concentration, OD600-1.075 before induction and 35 °C post induction temperature with overall lipase production of 50.50 IU/mL. Statistical validation of observed value via ANOVA showed an F-value of 138.70 at p < 0.01 with R2 of 0.9921.

Enhancing parasitic organism detection in microscopy images through deep learning and fine-tuned optimizer.

Parasitic organisms pose a major global health threat, mainly in regions that lack advanced medical facilities. Early and accurate detection of parasitic organisms is vital to saving lives. Deep learning models have uplifted the medical sector by providing promising results in diagnosing, detecting, and classifying diseases. This paper explores the role of deep learning techniques in detecting and classifying various parasitic organisms. The research works on a dataset consisting of 34,298 samples of parasites such as Toxoplasma Gondii, Trypanosome, Plasmodium, Leishmania, Babesia, and Trichomonad along with host cells like red blood cells and white blood cells. These images are initially converted from RGB to grayscale followed by the computation of morphological features such as perimeter, height, area, and width. Later, Otsu thresholding and watershed techniques are applied to differentiate foreground from background and create markers on the images for the identification of regions of interest. Deep transfer learning models such as VGG19, InceptionV3, ResNet50V2, ResNet152V2, EfficientNetB3, EfficientNetB0, MobileNetV2, Xception, DenseNet169, and a hybrid model, InceptionResNetV2, are employed. The parameters of these models are fine-tuned using three optimizers: SGD, RMSprop, and Adam. Experimental results reveal that when RMSprop is applied, VGG19, InceptionV3, and EfficientNetB0 achieve the highest accuracy of 99.1% with a loss of 0.09. Similarly, using the SGD optimizer, InceptionV3 performs exceptionally well, achieving the highest accuracy of 99.91% with a loss of 0.98. Finally, applying the Adam optimizer, InceptionResNetV2 excels, achieving the highest accuracy of 99.96% with a loss of 0.13, outperforming other optimizers. The findings of this research signify that using deep learning models coupled with image processing methods generates a highly accurate and efficient way to detect and classify parasitic organisms.

On PVDF composite as partially absorbable smart implants.

For various orthopedic needs, several studies have been testified on non-absorbable implants, prepared with different metals/alloys, and composites. But yet little has been stated on the partially absorbable smart implants of thermoplastic composites for online health monitoring of veterinary patients. This article highlights the in-house development of affordable, polyvinylidene fluoride (PVDF) composite-based partially absorbable smart implants (with online sensing capability) for orthopedic needs in canines. Hydroxyapatite (HAp) and chitosan (CS) nanoparticles were reinforced in the PVDF matrix by a melt processing route with various weight proportions (wt.%) to fabricate a partially absorbable smart implant for the canine. The study suggests that the 8.0 wt.% HAp and 2.0 wt.% CS in PVDF is the superlative composition/proportion of reinforcement for preparing feedstock stock filaments (for 3D printing of partially absorbable smart implants), based on rheological, mechanical, thermal, dielectric, and voltage-current-resistance (V-I-R) characteristics. For the selected composition/proportion of PVDF composite, acceptable mechanical properties (such as modulus of toughness (MoT) 2.0 MPa, Young's modulus (E) 889 MPa), and dielectric properties (dielectric constant (εr) 9.6 at room temperature (30°C) and 20 MHz) for online sensing capabilities (for health monitoring) was observed. The results are braced by attenuated total reflection Fourier transform infrared (ATR-FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS) analysis.

Bee-safe peptidomimetic acaricides achieved by comparative genomics.

The devastating Varroa mite (Varroa destructor Anderson and Trueman) is an obligatory ectoparasite of the honey bee, contributing to significant colony losses in North America and throughout the world. The limited number of conventional acaricides to reduce Varroa mites and prevent disease in honey bee colonies is challenged with wide-spread resistance and low target-site selectivity. Here, we propose a biorational approach using comparative genomics for the development of honey bee-safe and selective acaricides targeting the Varroa mite-specific neuropeptidergic system regulated by proctolin, which is lacking in the honey bee. Proctolin is a highly conserved pentapeptide RYLPT (Arg-Tyr-Leu-Pro-Thr) known to act through a G protein-coupled receptor to elicit myotropic activity in arthropod species. A total of 33 different peptidomimetic and peptide variants were tested on the Varroa mite proctolin receptor. Ligand docking model and mutagenesis studies revealed the importance of the core aromatic residue Tyr2 in the proctolin ligand. Peptidomimetics were observed to have significant oral toxicity leading to the paralysis and death of Varroa mites, while there were no negative effects observed for honey bees. We have demonstrated that a taxon-specific physiological target identified by advanced genomics information offers an opportunity to develop Varroa mite-selective acaricides, hence, expedited translational processes.

On 3D printed biomedical sensors for non-enzymatic glucose sensing applications.

Some studies have been reported in the recent past on smart sensors for non-enzymatic glucose sensing applications. Nevertheless, little has been reported on the in-house development of low-cost 3D printed smart biomedical sensors with tunable sensitivity. This study reports investigations on the in-house, low-cost fabrication of polyvinyl difluoride (PVDF) matrix-based 3D printed tunable non-enzymatic glucose sensors. For fabrication of smart sensors, Cu (4%) doped ZnO nanoparticles have been reinforced (in different weight proportions (wt%) in PVDF matrix through melt processing. The results suggest that 4% reinforcement (of 4% Cu doped-ZnO), processed at 190°C, 40 rpm screw speed on twin screw extrusion (TSE) followed by post heat treatment (HT) at 60°C are the best settings for fabrication of feedstock filaments (for bio-sensor 3D printing). Finally, a PVDF-based sensor to support bioreceptor and transducer requirements has been successfully prepared (with 4D properties (i.e. one-way programing feature), optical, morphological, bond strength, piezoelectric and mechanical characteristics). The 3D printed electro-active sensor, (of selected composition) resulted in acceptable mechanical, piezoelectric, and dielectric properties (modulus of toughness (MoT) 1.46 MPa, Young's modulus (YM) 1221.7 MPa, piezoelectric coefficient 19.3pC/N and dielectric constant 6.5). The results have been supported by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), current-voltage-resistance (I-V-R), and Fourier transformed infrared (FTIR) analysis.

Residual effect of defeated stripe rust resistance genes/QTLs in bread wheat against prevalent pathotypes of Puccinia striiformis f. sp. tritici.

The periodic breakdowns of stripe rust resistance due to emergence of new virulent and more aggressive pathotypes of Puccinia striiformis f. sp. tritici have resulted in severe epidemics in India. This necessitates the search for new and more durable resistance sources against stripe rust. The three bread wheat cultivars PBW 343 (carries Yr9 and Yr27), PBW 621 (carries Yr17) and HD 2967 (gene not known) were highly popular among the farmers after their release in 2011. But presently all three cultivars are highly susceptible to stripe rust at seedling as well as at adult plant stages as their resistance has been broken down due to emergence of new pathotypes of the pathogen (110S119, 238S119). In previous study, the crosses of PBW 621 with PBW 343 and HD 2967 and evaluation of further generations (up to F4) against pathotype 78S84 resulted in resistant segregants. In the present study, the F5 and F6 RIL populations have been evaluated against new pathotypes of Pst. The RILs categorized based on the disease severity on the P (Penultimate leaf) and F (flag) leaf into three categories i.e., high, moderate and low level of APR (adult plant resistance) having 1-200, 201-400 and >400 values of AUDPC, respectively, upon infection with stripe rust. The various APR components (latent period, lesion growth rate, spore production and uredial density) were studied on each category, i.e., resistant, moderately resistant and susceptible. The values of APR parameters decreased as the level of resistance increased. Based on molecular analysis, the lines (representing different categories of cross PBW 621 X PBW 343) containing the genes Yr9 and Yr17 due to their interactive effect provide resistance. Based on BSA using 35k SNPs and KASP markers association with phenotypic data of the RIL population (PBW 621 X HD 2967) showed the presence of two QTLs (Q.Pst.pau-6B, Q.Pst.pau-5B) responsible for the residual resistance and two SNPs AX-94891670 and AX-94454107 were found to be associated with the trait of interest on chromosome 6B and 5B respectively. The present study concludes that in the population of both the crosses (PBW 621 X PBW 343 and PBW 621 X HD 2967) major defeated gene contributed towards residual resistance by interacting with minor gene/QTLs.

Screening and Characterization of Next-Generation Biofuels Producing Bacterial Strains.

Production of fuels from renewable resources is of utmost importance due to fast depletion of fossil resources and related environmental issues. The present study explored the intrinsic capability of microbial strains to produce alka(e)nes, the next-generation biofuel, thus to reduce the dependence upon current petroleum fuels. Eight bacterial strains, namely, SDK-1, SDK-2, SDK-6, SDK-7, SDK-8, SDK-9, SDK-10, and SDK-11 were isolated from sludge and soil samples collected from different sources using lauric acid as a substrate with a potential to produce alka(e)nes. Production of different medium- and long-chain alka(e)nes by these isolates was confirmed via gas chromatography-mass spectrometer (GC-MS) analysis. SDK-1 (7.2%), SDK-2 (3.72%), and SDK-6 (3.52%) produced significant proportion of medium-chain hydrocarbons as compared to SDK-10 and control with no production. These isolates may be further investigated for production of these alternative sources of energy. In contrary, maximum fraction of long-chain hydrocarbons is produced in SDK-8 (75.28%) followed by SDK-9 (61.51%). Similarly more than 50% of the total hydrocarbons produced in SDK-8 constitute fossil mimic hydrocarbons while only 10.78% fractions were found in SDK-10. Since these fractions resemble different hydrocarbons obtained from crude oil, hence may be explored for their wide applications in different fields. Biochemical characterization and sequencing of the 16S rRNA gene revealed the homology of SDK-1, SDK-2 and SDK-6 with Pseudomonas aeruginosa, SDK-7 and SDK-9 with Enterobacter cloacae, SDK-8 with Klebsiella pnuemoniae, SDK-10 with Enterobacter hormaechei and SDK-11 with Pseudomonas nitroreducens, respectively.

Ecdysis triggering hormone peptide in the African malaria mosquito Anopheles gambiae: The peptide structure for receptor activation.

Infections by mosquito-borne diseases represent one of the leading causes of death in third world countries. The rapid progression of resistance to conventional insecticide causes a significant threat to the highly efficient preventive methods currently in place. Insect neuropeptidergic system offers potential targets to control the insect vectors. The essential roles of the neuropeptide ecdysis triggering hormone (ETH) in insect development and reproduction led us to attempt understanding of the fundamentals of the biochemical interaction between ETH and its receptor in the African malaria mosquito Anopheles gambiae. One of two ETH peptides of the African malaria mosquito (AgETH1), a small peptide hormone with 17 amino acid residues (SESPGFFIKLSKSVPRI-NH2 ), was studied to elucidate its molecular structure. N-termini deletions and mutations of conserved amino acids in the ligand revealed the critical residues for the receptor activation. The solution structure of AgETH1 using 2D 1 H-1 H nuclear magnetic resonance (NMR) spectroscopy and nuclear overhauser effect (NOE) derived constraints revealed a short alpha helix between residues 3S and 11S. The NMR solution structure of AgETH1 will be of significant assistance for designing a new class of insecticidal compounds that acts on the AgETH receptor aiming for in silico docking studies.

Predictive modeling of surface and dimensional features of vapour-smoothened FDM parts using self-adaptive cuckoo search algorithm.

Despite numerous advantages of fused deposition modeling (FDM), the inherent layer-by-layer deposition behavior leads to considerable surface roughness and dimensional variability, limiting its usability for critical applications. This study has been conducted to select optimum parameters of FDM and vapour smoothing (chemical finishing) process to maximize surface finish, hardness, and dimensional accuracy. A self-adaptive cuckoo search algorithm for predictive modelling of surface and dimensional features of vapour-smoothened FDM-printed functional prototypes has been demonstrated. The chemical finishing has been performed on hip prosthesis (benchmark) using hot vapours of acetone (using dedicated experimental set-up). Based upon the selected design of experiment technique, 18 sets of experiments (with three repetitions) were performed by varying six parameters. Afterwards, a self-adaptive cuckoo search algorithm was implemented by formulating five objective functions using regression analysis to select optimum parameters. An excellent functional relationship between output and input parameters has been developed using a self-adaptive cuckoo search algorithm which has successfully found the solution to optimization issues related to different responses. The confirmatory experiments indicated a strong correlation between predicted and actual surface finish measurements, along with hardness and dimensional accuracy.

Thermomechanical investigations of PEKK-HAp-CS composites.

In this experimental study, a composite of poly-ether-ketone-ketone by reinforcement of hydroxyapatite and chitosan has been prepared for possible applications as orthopaedic scaffolds. Initially, different weight percentages of hydroxyapatite and chitosan were reinforced in the poly-ether-ketone-ketone matrix and tested for melt flow index in order to check the flowability of different compositions/proportions. Suitable compositions revealed by the melt flow index test were then taken forward for the extrusion of filament required for fused deposition modelling. For thermomechanical investigations, Taguchi-based design of experiments has been used with input variables in the extrusion process as follows: temperature, load applied and different composition/proportions. The specimens in the form of feedstock filament produced by the extrusion process were made to undergo tensile testing. The specimens were also inspected by differential scanning calorimetry and photomicrographs. Finally, the specimen showing the best performance from the thermomechanical viewpoint has been selected to extrude the filament for the fused deposition modelling process.

A Luminescent Probe for Ratiometric Optical Detection of HgII and Turn-On Fluorescent Sensing of CuII.

Luminescent detection of heavy metals continues to be of growing importance considering their increasing inseparable roles in modern day lifestyle. To this end, we report a simple yet interesting thiourea derivative that results in detection of CuII in a turn-on fashion via a chemodosimetric redox reaction leading to oxidative cyclization of the chemosensor molecule. Elaborate studies with regard to the sensing process have been performed along with the proposal of a plausible cyclization mechanism of the molecule. Furthermore, the probe optically detects HgII , yet another heavy metal of prime importance, discernable even to the naked eye. The occurrence of a coordination complex has been proved by UV/Visible spectroscopic experiments as well as ESI-mass spectrometry.

Dimensionless Analysis for Investigating the Quality Characteristics of Aluminium Matrix Composites Prepared through Fused Deposition Modelling Assisted Investment Casting.

The aluminium matrix composites (AMCs) have become a tough competitor for various categories of metallic alloys, especially ferrous materials, owing to their tremendous servicing in the diversified application. In this work, additional efforts have been made to formulate a mathematical model, by using dimensionless analysis, able to predict the mechanical characteristics of the AMCs that have already been optimized and characterized by the authors. Here, the experimental and statistical data obtained from the Taguchi L18 orthogonal array and analysis of variance (ANOVA) have been used. They permit collection of the output responses and allow the identification of significant process parameters, respectively, which thereafter were used to design the mathematical model. Second order polynomial equations have been obtained from the specific output response and the relevant input parameter were incorporated with the highest level of contribution. The obtained quadratic equations indicate the regression values (R2) equal to unity, hence, proving the performances of the fit. The results demonstrate that the developed mathematical models present very high accuracy for predicting the output responses.

Investigations on joining of orthopaedic scaffold with rapid tooling.

Poly lactic acid reinforced with hydroxyapatite and chitosan is one of the commonly used thermoplastic composite materials in three-dimensional printing of orthopaedic scaffolds. This article is an extension of work reported on hydroxyapatite- and chitosan-reinforced, poly lactic acid-based biocompatible feedstock filament for three-dimensional printing of functional prototypes on low cost commercial fused deposition modelling setup. In this work, joining of orthopaedic scaffolds has been performed with rapid tooling by using friction stir welding process. Finally, the mechanical properties (such as tensile strength and hardness) of the orthopaedic scaffolds joints were studied with support of the photomicrographs and thermal images. The results of study suggest that best settings for joining of micro cracks in orthopaedic scaffolds of poly lactic acid-hydroxyapatite-chitosan are rotational speed of 1000 r/min with length of consumable tool as 4 mm and staring time as 40 s.

Multi-Material Additive Manufacturing of Sustainable Innovative Materials and Structures.

This paper highlights the multi-material additive manufacturing (AM) route for manufacturing of innovative materials and structures. Three different recycled thermoplastics, namely acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), and high impact polystyrene (HIPS) (with different Young's modulus, glass transition temperature, rheological properties), have been selected (as a case study) for multi-material AM. The functional prototypes have been printed on fused deposition modelling (FDM) setup as tensile specimens (as per ASTM D638 type-IV standard) with different combinations of top, middle, and bottom layers (of ABS/PLA/HIPS), at different printing speed and infill percentage density. The specimens were subjected to thermal (glass transition temperature and heat capacity) and mechanical testing (peak load, peak strength, peak elongation, percentage elongation at peak, and Young's modulus) to ascertain their suitability in load-bearing structures, and the fabrication of functional prototypes of mechanical meta-materials. The results have been supported by photomicrographs to observe the microstructure of the analyzed multi-materials.

"Turn-on" Pb2+ sensing and rapid detection of biothiols in aqueous medium and real samples.

Detection of lead has continued to be of immense interest in the present industrial as well as environmental diaspora. To this end, we report a prudent Schiff base which enables the sensitive detection of Pb2+ ions in mixed aqueous medium. The probe is afforded by simple synthetic and purification processes. Further, the probe employs simple mechanistic detection of Pb2+ and also successively detects a host of biothiols. The sensor is also used to detect Pb2+ ions in real water samples and consecutive detection of a variety of biothiols, including functionalized thiouracils, in onion and garlic extract. The fact that the Pb2+-L ensemble is 'non-selective' towards any particular thiol substituted analyte in real samples could as well prove to be an interesting contribution for chemical and biological detoxification processes.

Al3+ sensing through different turn-on emission signals vis-à-vis two different excitations: Applications in biological and environmental realms.

A rationally designed Schiff base chemosensor (L) could render specific detection of Al3+ ions with two distinct turn-on emission signals, separated by over 100 nm upon excitation at two different wavelengths. The utility of the probe lies in facilitating sensing in 80% aqueous medium with an emission close to 600 nm via an intramolecular charge transfer (ICT) mechanism. The biocompatible and cell permeable probe could readily sense Al3+ in live HeLa cells as well. The affinity of the probe for Al3+could be leveraged to specifically study DNA- Al3+ interaction in solution.

Role of time-resolved-CTA in intracranial arteriovenous malformation evaluation at 128-slice CT in comparison with digital subtraction angiography.

Introduction The present study aimed to evaluate the accuracy of time-resolved-computed tomographic angiography (TR-CTA) on a 128-slice CT scanner vis-à-vis cerebral digital subtraction angiography (DSA) in defining the morphological and haemodynamic characteristics of cerebral arteriovenous malformation (AVM). Methods Twenty-one patients (age range 10-46, mean 24.8 years) with clinical suspicion of AVM and three patients (age range 23-35, mean 24.3 years) with diagnosed AVM who were on follow-up underwent DSA and TR-CTA, on average 1.5 days apart. Three independent neuroradiologists analysed both studies in a blinded fashion based on the following parameters: AVM location, arterial feeder territories, venous drainage pattern, nidus flow characteristics, venous outflow obstruction, arterial feeder enlargement, external carotid artery feeder, location of aneurysm if any, leptomeningeal and transdural recruitment, neoangiogenesis, and pseudophlebitic pattern. Results The TR-CTA correctly demonstrated AVM in all 21 positive cases. It concordantly detected location (21/21), venous drainage pattern (21/21), nidus flow characteristics (21/21), and the venous outflow obstruction (9/9). However, discordance was seen in the demonstration of the arterial feeder (2/45) ( p = 0.49), arterial enlargement (13/17) ( p = 0.103), external carotid artery feeder (0/1), aneurysmal location (3/5) ( p = 0.40), leptomeningeal recruitment (1/3) ( p = 0.40), neoangiogenesis (0/4) ( p = 0.028) and in the pseudophlebitic pattern (2/5) ( p = 0.167) demonstration. Conclusions The results suggest that TR-CTA can provide the important features of cerebral AVM which are required in patient management.

Time resolved computed tomography angiography in the evaluation of brain arteriovenous malformation: a feasibility study.

Background and purpose Digital subtraction angiography is the current gold standard for diagnosing as well as the follow-up of cerebral arteriovenous malformations. However, as it is invasive, relatively expensive and time-consuming, a non-invasive alternative is of interest. We aimed to evaluate the feasibility of time resolved computed tomography angiography (TR-CTA) in a series of five diagnosed cranial arteriovenous malformation patients, demonstrated by conventional digital subtraction angiography with respect to acquisition, depiction of angiographic phases and radiation exposure. Materials and methods Five patients demonstrating a cranial arteriovenous malformation on digital subtraction angiography were studied with TR-CTA. The TR-CTA imaging was done by using a 128-detector computed tomography scanner. Digital subtraction angiography and TR-CTA studies were independently read by two blinded observers, by using a standardised scoring sheet. TR-CTA results were analysed with digital subtraction angiography as the criterion standard. Results TR-CTA generated comparable angiographic phases. In all five cases, there was complete agreement between digital subtraction angiography and TR-CTA regarding the size, arterial feeders, nidal morphology and venous drainage of the arteriovenous malformation. Conclusions TR-CTA imaging as a technique is feasible, providing images with good temporal and spatial resolution at an acceptable radiation dose. It appears to be a promising non-invasive adjunct to digital subtraction angiography.