Engineering co-culture system for production of apigetrin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Microbial cells have extensively been utilized to produce value-added bioactive compounds. Based on advancement in protein engineering, DNA recombinant technology, genome engineering, and metabolic remodeling, the microbes can be re-engineered to produce industrially and medicinally important platform chemicals. The emergence of co-culture system which reduces the metabolic burden and allows parallel optimization of the engineered pathway in a modular fashion restricting the formation of undesired byproducts has become an alternative way to synthesize and produce bioactive compounds. In this study, we present genetically engineered E. coli-based co-culture system to the de novo synthesis of apigetrin (APG), an apigenin-7-O-β-d-glucopyranoside of apigenin. The culture system consists of an upstream module including 4-coumarate: CoA ligase (4CL), chalcone synthase, chalcone flavanone isomerase (CHS, CHI), and flavone synthase I (FNSI) to synthesize apigenin (API) from p-coumaric acid (PCA). Whereas, the downstream system contains a metabolizing module to enhance the production of UDP-glucose and expression of glycosyltransferase (PaGT3) to convert API into APG. To accomplish this improvement in titer, the initial inoculum ratio of strains for making the co-culture system, temperature, and media component was optimized. Following large-scale production, a yield of 38.5 µM (16.6 mg/L) of APG was achieved. In overall, this study provided an efficient tool to synthesize bioactive compounds in microbial cells.     

Modern pollen assemblages from Reasi (Jammu and Kashmir), India: a tool for interpreting fossil pollen records

Twenty five surface samples/moss cushions were collected for palynological analysis from open areas of Reasi District, Jammu and Kashmir (India). These samples were used to investigate the relationships between extant vegetation and modern pollen spectra, which serve as modern analogue for the reliable ecological interpretation of fossil pollen records. The present vegetation in the region comprises tropical dry deciduous forests and subtropical pine forests with scattered stands of oak. The pollen analysis reveals that Pinus sp. (average 69% in the pollen assemblages), amongst the conifers, dominates the pollen rain, which can be attributed to its high pollen productivity and exceptional pollen dispersal efficiency. Cedrus sp. and Podocarpus sp. pollen contribute with an average of 16 and 5% to the total pollen rain. Other conifers such as Picea sp., Abies sp., Juniperus sp. and Tsuga sp., as well as broad-leaved taxa such as Quercus sp., Alnus sp., Betula sp., Carpinus sp., Corylus sp., Juglans sp., Ulmus sp., Salix sp., Elaeocarpus sp., Mallotus sp. and Aesculus sp., have lower averages of 1 to 4.5% in the total pollen rain which could be either due to their poor pollen dispersal efficiency or to the poor preservation in the samples. Tubuliflorae (average 25%), Poaceae (average 6.26%), Cerealia and other crop plants (average 7.68%) are other prominent taxa in the pollen rain. The nearly complete absence of members of tropical dry deciduous forests in the pollen spectra likely is due to the fact that most species in this vegetation type are not wind pollinated.     

A Wireless Triboelectric Nanogenerator

A new “wireless” paradigm for harvesting mechanical energy via a 3D‐printed wireless triboelectric nanogenerator (W‐TENG) comprised of an ecofriendly graphene polylactic acid (gPLA) nanocomposite and Teflon is demonstrated. The W‐TENG generates very high output voltages >2 kV with a strong electric field that enables the wireless transmission of harvested energy over a distance of 3 m. The W‐TENG exhibited an instantaneous peak power up to 70 mW that could be wirelessly transmitted for storage into a capacitor obviating the need for hard‐wiring or additional circuitry. Furthermore, the use of W‐TENG for wireless and secure actuation of smart‐home applications such as smart tint windows, temperature sensors, liquid crystal displays, and security alarms either with a single or a specific user‐defined passcode of mechanical pulses (e.g., Fibonacci sequence) is demonstrated. The scalable additive manufacturing approach for gPLA‐based W‐TENGs, along with their high electrical output and unprecedented wireless applications, is poised for revolutionizing the present mechanical energy harvesting technologies.     

Elucidation of the anticancer potential and tubulin isotype-specific interactions of β-sitosterol

Beta-sitosterol (β-SITO), a phytosterol present in many edible vegetables, has been reported to possess antineoplastic properties and cancer treatment potential. We have shown previously that it binds at a unique site (the ‘SITO-site’) compared to the colchicine binding site at the interface of α- and β-tubulin. In this study, we investigated the anticancer efficacy of β-SITO against invasive breast carcinoma using MCF-7 cells. Since ‘isotypes’ of β-tubulin show tissue-specific expression and many are associated with cancer drug resistance, using computer-assisted docking and atomistic molecular dynamic simulations, we also examined its binding interactions to all known isotypes of β-tubulin in αβ-tubulin dimer. β-SITO inhibited MCF-7 cell viability by up to 50%, compared to vehicle-treated control cells. Indicating its antimetastatic potential, the phytosterol strongly inhibited cell migration. Immunofluorescence imaging of β-SITO-treated MCF-7 cells exhibited disruption of the microtubules and chromosome organization. Far-UV circular dichroism spectra indicated loss of helical stability in tubulin when bound to β-SITO. Docking and MD simulation studies, combined with MM-PBSA and MM-GBSA calculations revealed that β-SITO preferentially binds with specific β-tubulin isotypes (β_II and β_III) in the αβ-tubulin dimer. Both these β-tubulin isotypes have been implicated in drug resistance against tubulin-targeted chemotherapeutics. Our data show the tubulin-targeted anticancer potential of β-SITO, and its potential clinical utility against β_II and β_III isotype-overexpressing neoplasms.     

Risk assessment of submicron PM-bound hexavalent chromium during wintertime

This study reports health risk assessment of PM₁-bound carcinogenic hexavalent chromium [Cr(VI)] from central part of Indo-Gangetic plain (IGP) (PM₁: particulate matter with aerodynamic diameter ≤1µm). Cr(VI) concentration has been estimated utilizing spectrophotometer with a modified novel method. Average ratio of Cr(VI)/Cr_T was 0.39 ± 0.07 (Cr_T: Total chromium) in the central IGP (Kanpur). Our study reports that mass fraction of Cr(VI) averaging at 0.39 is ～3 times higher than that assumed conventionally [Cr(VI)/Cr_T: 1/7]. Cancer risk assessment has been performed by assessing excess cancer risk (ECR) for the Cr(VI). ECR determined due to Cr(VI) was 57 and 14.3 (in one million) for adults and children, respectively. Our study suggests that risk due to Cr(VI) reported in previous studies were being underestimated by a factor of three. The Cr(VI)/Cr_T average ratio of 0.39 determined in this study was utilized to calculate risk assessment due to Cr(VI) from other locations in the IGP. Owing to large population of India (～125 million), the cancer risk due to Cr(VI) inhalation itself would become very significant. Thus, future research should focus on metal speciation of PM-bound samples from different locations to better constraint the toxicological risk assessment on a regional-to-global scale.     

Designing B‐ and T‐cell multi‐epitope based subunit vaccine using immunoinformatics approach to control Zika virus infection

The Zika virus is a rapidly spreading Aedes mosquito‐borne sickness, which creates an unanticipated linkage birth deformity and neurological turmoil. This study represents the use of the combinatorial immunoinformatics approach to develop a multiepitope subunit vaccine using the structural and nonstructural proteins of the Zika virus. The designed subunit vaccine consists of cytotoxic T‐lymphocyte and helper T‐lymphocyte epitopes accompanied by suitable adjuvant and linkers. The presence of humoral immune response specific B‐cell epitopes was also confirmed by B‐cell epitope mapping among vaccine protein. Further, the vaccine protein was characterized for its allergenicity, antigenicity, and physiochemical parameters and found to be safe and immunogenic. Molecular docking and molecular dynamics studies of the vaccine protein with the toll‐like receptor‐3 were performed to ensure the binding affinity and stability of their complex. Finally, in silico cloning was performed for the effective expression of vaccine construct in the microbial system (Escherichia coli K12 strain). Aforementioned approaches result in the multiepitope subunit vaccine which may have the ability to induce cellular as well as humoral immune response. Moreover, this study needs the experimental validation to prove the immunogenic and protective behavior of the developed subunit vaccine.     

A hypercellulolytic mutant of Fusarium oxysporum

Multiple mutagenesis of Fusarium oxysporum DSM 841 resulted in enhanced yields of cellulases. The hypercellulolytic mutant (NTG-19) secretes high levels of extracellular cellulases on different cellulosic substrates. Addition of surfactant, Tween-80, further increased enzyme secretion by about 30%. The results on hydrolysis of wheat straw by parent strain, DSM 841 and mutant NTG-19 cellulases also revealed a significant improvement in the hydrolytic potential of the cellulolytic enzymes from the mutant NTG-19.     

Alkaline phosphatase secretion-negative mutant of Bacillus licheniformis 749/C.

An alkaline phosphatase secretion-blocked mutant of Bacillus licheniformis 749/C was isolated. This mutant had defects in the phoP and phoR regions of the chromosome. The selection procedure was based on the rationale that N-methyl-N'-nitro-N-nitrosoguanidine can induce mutations of closely linked multiple genes. The malate gene and the phoP and phoR genes are located at the 260-min position in the Bacillus subtilis chromosome; hence, the malate gene could be used as a marker for the mutation of the phoP and phoR regions of the chromosome. In a two-step selection procedure, strains defective in malate utilization were first selected with the cephalosporin C procedure. Second, these malate-defective strains were further screened in a dye medium to select strains with defects in alkaline phosphatase secretion. One stable mutant (B. licheniformis 749/cNM 105) had a total secretion block for alkaline phosphatase and had the following additional characteristics: (i) the amount of alkaline phosphatase synthesized was comparable to that in the wild type; (ii) the alkaline phosphatase was membrane bound; (iii) the mutant strain alkaline phosphatase, in contrast to that of the wild type, could not be extracted with MgCl2, although the amounts of protein extracted from each strain were comparable; (iv) the sodium dodecyl sulfate-polyacrylamide gel pattern of MgCl2-extracted proteins from the mutant strain was different from that of the wild-type proteins; (v) the mutant, unlike the wild type, could not use malate as a sole source of carbon; and (vi) the outside surface of the wall of the mutant cells contained an additional electron-dense layer that was not present on the wild-type cell wall surface.