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Uracil is not always a mistakenly occurring base in DNA. Uracils in DNA genomes are known to be important in the life cycles of Bacillus subtilis phages (PBS1/2) and the malarial parasite, Plasmodium falciparum; and have been implicated in the development of fruit fly and antibody maturation in B-lymphocytes. Availability of a sensitive, specific and robust technique for the detection uracils in genes/genomes is essential to understand its varied biological roles. Mycobacterium smegmatis UdgX (MsmUdgX), identified and characterised in our laboratory, forms covalent complexes with the uracil sites in DNA in a specific manner. MsmUdgX cleaves the glycosidic bond between uracil and the deoxyribose sugar in DNA to produce uracilate and oxocarbenium ions. The oxocarbenium ion is then captured into a covalent complex by the nucleophilic attack of a histidine side chain of MsmUdgX. Here, we describe the use of a fusion protein, mCherry tagged MsmUdgX (mChUdgX), which combines the property of MsmUdgX to covalently and specifically bind the uracil sites in the genome, with the sensitivity of fluorescent detection of mCherry as a reporter. We show that both the purified mChUdgX and the Escherichia coli cell-extracts overexpressing mChUdgX provide high sensitivity and specificity of detecting uracils in DNA.

The ESCRT pathway functions at different subcellular membranes to induce their negative curvature, and it has been largely characterized in model eukaryotes belonging to Opisthokonta. But searches of the genomes of many nonopisthokonts belonging to various supergroups indicate that some of them may harbour fewer ESCRT components. Of the genomes explored thus far, one of the most minimal set of ESCRT components was identified in the human pathogen Giardia lamblia, which belongs to Excavata. Here we report that an ESCRT-mediated pathway most likely operates at the peripheral vesicles, which are located at the cell periphery and the bare zone of this protist. Functional comparison of all the identified putative giardial ESCRT components, with the corresponding well-characterized orthologues from Saccharomyces cerevisiae, indicated that only some of the ESCRT components could functionally substitute for the corresponding yeast proteins. While GlVps25, GlVps2, and all three paralogues of GlVps4, tested positive in functional complementation assays, GlVps22, GlVps20, and GlVps24 did not. Binary interactions of either GlVps22 or GlVps25, with other ESCRT-II components from Giardia or yeast indicate that the giardial Vps36 orthologue is either completely missing or highly diverged. Interactions within the giardial ESCRT-III components also differ from those in yeast; while GlVps46a interacts preferentially with Vps24 compared to Vps2, GlVps46b, like the yeast orthologue, interacts with both.

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic-acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the receptor-binding domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan-engineered RBD protein fragment that is expressed at a purified yield of 214 mg/l in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over 4 weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ∼415 against replicative virus, comparing favorably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance, and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

Despite the crucial impact of leptin signaling on metabolism and body weight, little is known about the structure of the liganded leptin receptor (LEP-R) complex. Here, we applied single-particle electron microscopy (EM) to characterize the architecture of the extracellular region of LEP-R alone and in complex with leptin. We show that unliganded LEP-R displays significant flexibility in a hinge region within the cytokine homology region 2 (CHR2) that is connected to rigid membrane-proximal FnIII domains. Leptin binds to CHR2 in order to restrict the flexible hinge and the disposition of the FnIII "legs." Through a separate interaction, leptin engages the Ig-like domain of a second liganded LEP-R, resulting in the formation of a quaternary signaling complex. We propose that the membrane proximal domain rigidification in the context of a liganded cytokine receptor dimer is a key mechanism for the transactivation of Janus kinases (Jaks) bound at the intracellular receptor region.

A critical review of past earthquakes reveals damage/failure of important lifeline facilities like elevated water tanks in recurrence, leading to serious hazards even after the event. In the context of such hazards indicating deficiencies in the existing seismic design strategy of such structures, dynamic characteristics of elevated tanks supported by cylindrical shaft (shaft staging) are comprehensively studied in the first part of present investigation. The same is made through analytical formulations developed and validated by rigorous finite element analysis and small-scale experimentation. Subsequent examination on seismic response of representative tanks indicates that the columns of the frame-supported tanks (tanks with frame staging) and the wall of the shaft-staging of such reinforced concrete ( R / C ) tanks are susceptible to tension, particularly at tank empty condition. Such possibility seems to be further aggravated if the effect of soil–structure interaction (SSI) is ignored in design. Identifying the deficiencies in current design, simple design procedure of such elevated tanks utilizing the formulations developed is proposed. Torsional vulnerability of shaft-supported elevated water tanks is also identified to be marginal as opposed to the same for frame supported ones.

The frame stagings with a single row of columns placed along the periphery of a circle, are generally adopted for elevated water tanks to support the tank container. Apart from this usual staging configuration, some alternate configurations are also used in practice. These alternate configurations are made by adding few structural members to the usual configuration. These staging configurations are advantageous for adoption from a few different viewpoints. The present paper aims to observe the effect of soil–structure interaction on two dynamic characteristics namely, the impulsive lateral period which regulates lateral seismic behaviour and the impulsive torsional-to-lateral period ratio which regulates torsional vulnerability of the structure. The analytical expressions for these two dynamic characteristics have been derived considering the effect of soil-flexibility for elevated water tanks with these alternate configurations. These formulations have been validated against the results of finite element analysis for a few example tanks. A parametric study with limited example tanks based on these formulations shows that the frame staging with all kinds of alternate configurations having less panel heights, more number of columns, larger column diameter and stiffer circumferential beams compared to columns encounters the strongest influence of soil–structure interaction effect. The study on the example tanks with different alternate configurations shows that the design of elevated tanks based on a fixed base assumption may lead to wrong assessment of seismic base shear. The underestimation of base shear may lead to unsafe design whereas overestimation may cause uneconomic design. Neglecting soil-flexibility may also cause overlooking the possibility of occurring axial tension in columns and wrong assessment of torsional vulnerability of the staging structures.

The complexes [VIVO(L)(bpy)] and [VVO(L)(OMe)(OHMe)] (H2L  Schiff base condensate of salicylaldehyde and an α-amino acid and bpy = 2,2′-bipyridine) have been synthesized. The [VIVO(L)(OMe)(OHMe)]− species have been electrogenerated in solutions. The VO 3+ − VO 2+ E 1 2 values of [VO(L)(OHMe)] are lower than those of [VO(L)(bpy)] by ≈750 mV. Upon exposure to air [VO(L)(OMe)(OHMe)]− is spontaneously oxidized to [VO(L)(OMe)(OHMe)]. The [VO(L)(bpy)] and [VO(L)(OMe) (OHMe)]− complexes display dxy1 type axial EPR spectra. The 51V hyperfine constants follow the order [VO(L)(OMe)(OHMe)]− > [VO(L)(bpy)]. The dxy → dxz, dyz transition in these complexes appear in the region 700–720 nm.

Water soluble manganese(III) complexes of sulphonated azophenol and salicylaldimine ligands 3–6, have been isolated: Na3[Mn(LS)2]·nH2O and Na5[Mn(LSS)2]·nH2O (n = 4 or 5) where H3LS and H4LSS represent mono- and di-sulphonated ligands, respectively. The coordination sphere of the complexes are of the type Mn(ONO)2 where the O and N atoms are of phenolic and azo/azomethine type. The complexes are uniformly high-spin (t23e1) in character (∼5 μB). The manganese(IV)-manganese(III) couple in water has cyclic voltammetric E 1 2 values in the range 0.30–0.50 V vs SCE and it is estimated that the potentials increase by 70 mV for each sulphonate substitution. The manganese(IV) complexes [Mn(LS)2]2− and [Mn(LSS)2]4− have been quantitatively generated in solution both chemically and electrochemically. Their X-band EPR spectra consist of a strong resonance near g = 2. In frozen water-ethylene glycol glass (77 K) the g = 2 resonance shows 55Mn hyperfine structure (A, 94–97 G). Forbidden lines are also resolved and with their help the axial zero-field splitting parameter D is estimated to be 0.012 cm−1. It is small compared to the X-band quantum, 0.31 cm−1.