Production and progeny testing of androgenetic rosy barb Puntius conchonius

Protocol for androgenetic cloning of the rosy barb, Puntius conchonius, with contrasting gray and golden strains is described. At the intensity of 4.2 W/m2, UV irradiation for 3.0 min inactivates the maternal genome in eggs of the gray barb. Following activation by the golden barb sperm, 24-min old eggs are shocked at 41 degrees C for 2 min to restore diploidy. Maternal genomic inactivation is confirmed by the (i) golden body color, (ii) karyotyping, and (iii) progeny testing of F1-F3 progenies. Estimates of stage-specific mortality of haploid and diploid androgenotes indicate no change in the time scale or developmental sequence, when sperm of related strain is used for activation, and when haploid genome regulates the development. Survival of androgenetic clones remains constant for the F1, F2, and F3 progenies and is about 15% and 7% at hatching and sexual maturity, respectively. Homozygosity of the androgenotes is shown to inflict greater mortality. Between F1 and F3 generations, the heterozygosity of the androgenetic clone is decreased, as evidenced by reduction in size hierarchy. Though the reproductive performance of the F1, F2, and F3 supermales is superior to the normal ones, the realized fecundity remains equal around 80 progenies per brood. The 92 crosses involving 16 supermales and 10 normal dams yield 75-100% male progenies, confirming the possible operation of XXfemale symbol:XYmale symbol sex determination system. The frequency of unexpected occurrence of female progenies is about 8%, the causes for which are discussed.     

Identification of genes encoding exported Mycobacterium tuberculosis proteins using a Tn552'phoA in vitro transposition system

Secreted and cell envelope-associated proteins are important to both Mycobacterium tuberculosis pathogenesis and the generation of protective immunity to M. tuberculosis. We used an in vitro Tn552'phoA transposition system to identify exported proteins of M. tuberculosis. The system is simple and efficient, and the transposon inserts randomly into target DNA. M. tuberculosis genomic libraries were targeted with Tn552'phoA transposons, and these libraries were screened in M. smegmatis for active PhoA translational fusions. Thirty-two different M. tuberculosis open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the M. tuberculosis databases. This collection of exported proteins includes factors that are known to participate in the immune response of M. tuberculosis and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms.     

Marker-aided selection and validation of various $${ }$$ gene combinations for rice blast resistance in elite rice variety ADT 43

Rice blast caused by fungal pathogen Pyricularia oryzae has a major impact on reducing yield potential of rice. In this study, homozygous plants were selected using microsatellite markers from the \(\hbox {BC}_{3}\hbox {F}_{2}\) population pyramided with four major genes in elite rice variety ADT 43. Background and selected lines with various blast resistance gene combinations were screened under natural conditions to study the effects of various gene combinations. Upon inspection of lines with different gene combinations, the three-gene pyramided line Pi54+Pi33+Pi1 was found to be highly resistant with the score of 3.3 followed by other three-gene pyramided lines Pi54+Pi2+Pi1 and Pi33+Pi2+Pi1, with the scores of 3.9 and 3.8, respectively. Two-gene pyramided lines Pi54+Pi1, Pi33+Pi1 and Pi2+Pi1 were found to be moderately resistant with a mean score of 4.0 each. In the case of monogenic lines, positive plants for Pi54 performed almost equal to three-gene pyramided lines with a mean score of 3.6. Lines with Pi2 and Pi1 were found to be moderately resistant and moderately susceptible with the mean scores of 4.1 and 4.5, respectively.     

Fabrication of Sub‐Micrometer‐Thick Solid Electrolyte Membranes of β‐Li3PS4 via Tiled Assembly of Nanoscale,Plate‐Like Building Blocks

Solid electrolytes represent a critical component in future batteries that provide higher energy and power densities than the current lithium‐ion batteries. The potential of using ultrathin films is among the best merits of solid electrolytes for considerably reducing the weight and volume of each battery unit, thereby significantly enhancing the energy density. However, it is challenging to fabricate ultrathin membranes of solid electrolytes using the conventional techniques. Here, a new strategy is reported for fabricating sub‐micrometer‐thick membranes of β‐Li₃PS₄ solid electrolytes via tiled assembly of shape‐controlled, nanoscale building blocks. This strategy relies on facile, low‐cost, solution‐based chemistry to create membranes with tunable thicknesses. The ultrathin membranes of β‐Li₃PS₄ show desirable ionic conductivity and necessary compatibility with metallic lithium anodes. The results of this study also highlight a viable strategy for creating ultrathin, dense solid electrolytes with high ionic conductivities for the next‐generation energy storage and conversion systems.     

Anti-pathogenic Potential of Coral Associated Bacteria Isolated from Gulf of Mannar Against Pseudomonas aeruginosa

Infections of Pseudomonas aeruginosa are of great concern because of its increasing resistance towards conventional antibiotics. Quorum sensing system of P. aeruginosa acts as a global regulator of almost all the virulence factors and majorly its biofilm formation. In the present study, quenching of QS system of P. aeruginosa has been explained with bioactives from bacteria associated with the coral Acropora digitifera. Isolated bioactives inhibited the expression of various virulence traits of P. aeruginosa like biofilm formation, and the production of extracellular enzymes like protease and elastase. This study also emphasises the potential of coral associated bacteria in producing bioactive agents with anti-pathogenic properties.     

Design of a new fluorescent probe: Pyrrole/imidazole hairpin polyamides with pyrene conjugation at their γ-turn

Fluorophores that are conjugated with N-methylpyrrole-N-methylimidazole (Py–Im) polyamides postulates versatile applications in biological and physicochemical studies. Here, we show the design and synthesis of new types of pyrene-conjugated hairpin Py–Im polyamides (1–5). We evaluated the steady state fluorescence of the synthesized conjugates (1–5) in the presence and absence of oligodeoxynucleotides 5′-CGTATGGACTCGG-3′ (ODN 1) and 5′-CCGAGTCCATACG-3′ (ODN 2) and observed a distinct increase in emission at 386 nm with conjugates 4 and 5. Notably, conjugate 5 that contains a β-alanine linker had a stronger binding affinity (K_D = 1.73 × 10^?8 M) than that of conjugate 4 (K_D = 1.74 × 10^?6 M). Our data suggests that Py–Im polyamides containing pyrene fluorophore with a β-alanine linker at the γ-turn NH₂ position can be developed as the competent fluorescent DNA-binding probes.     

Flavonoid from Carica papaya inhibits NS2B-NS3 protease and prevents Dengue 2 viral assembly

Dengue virus belongs to the virus family Flaviviridae. Dengue hemorrhagic disease caused by dengue virus is a public health problem worldwide. The viral non structural 2B and 3 (NS2B-NS3) protease complex is crucial for virus replication and hence, it is considered to be a good anti-viral target. Leaf extracts from Carica papaya is generally prescribed for patients with dengue fever, but there are no scientific evidences for its anti-dengue activity; hence we intended to investigate the anti-viral activity of compounds present in the leaves of Carica papaya against dengue 2 virus (DENV-2). We analysed the anti-dengue activities of the extracts from Carica papaya by using bioinformatics tools. Interestingly, we find the flavonoid quercetin with highest binding energy against NS2B-NS3 protease which is evident by the formation of six hydrogen bonds with the amino acid residues at the binding site of the receptor. Our results suggest that the flavonoids from Carica papaya have significant anti-dengue activities.

Abbreviations

ADME - Absorption, distribution, metabolism and excretion, BBB - Blood brain barrier, CYP - Cytochrome P450, DENV - – Dengue virus, DHF - Dengue hemorrhagic fever, DSS - Dengue shock syndrome, GCMS - – Gas chromatography- Mass spectrometry, MOLCAD - Molecular Computer Aided Design, NS - Non structural, PDB - Protein data bank, PMF - Potential Mean Force.     

Production of stable bispecific IgG1 by controlled Fab-arm exchange

The manufacturing of bispecific antibodies can be challenging for a variety of reasons. For example, protein expression problems, stability issues, or the use of non-standard approaches for manufacturing can result in poor yield or poor facility fit. In this paper, we demonstrate the use of standard antibody platforms for large-scale manufacturing of bispecific IgG1 by controlled Fab-arm exchange. Two parental antibodies that each contain a single matched point mutation in the CH3 region were separately expressed in Chinese hamster ovary cells and manufactured at 1000 L scale using a platform fed-batch and purification process that was designed for standard antibody production. The bispecific antibody was generated by mixing the two parental molecules under controlled reducing conditions, resulting in efficient Fab-arm exchange of >95% at kg scale. The reductant was removed via diafiltration, resulting in spontaneous reoxidation of interchain disulfide bonds. Aside from the bispecific nature of the molecule, extensive characterization demonstrated that the IgG1 structural integrity was maintained, including function and stability. These results demonstrate the suitability of this bispecific IgG1 format for commercial-scale manufacturing using standard antibody manufacturing techniques.