Surfacing activity and food utilisation in the obligatory air-breathing fish ophiocephalus striatus as a function of body weight

Reared in cylindrical aquaria containing different depths of water (2.5 to 70 cm) the obligatory air-breathing fish Ophiocephalus striatus, belonging to different weight classes (0.1, 0.75, 10, 20 and 41 g), was forced to swim vertically a longer or shorter distance per surfacing. Surfacing frequency was a depth-dependent, activity in individuals weighing less than 20 g in all weight classes, the frequency was nearly 2 times more in the series fed ad libitum on fish muscle, than in the one where the fish were starved. Owing to the sustained surfacing activity and the consequent fatigue, the test individuals hung to the surface for a definite period. Neither frequency nor duration of hanging was depth-dependent. Mean hanging durations for the feeding series were 1.1, 3.8, 5.9, 7.9 and 8.8 hr/day in the 0.1, 0.75, 10, 20 and 41 g weight classes, respectively; the corresponding values for the starving series were 1.3, 15.0, 13.0, 12.7 and 12.8 hr/day. The distance swum by the feeding fish increased from 57 to 681 m/day and from 61 to 507 m/day in the 0.1 and 41 g individuals exposed to the minimum and maximum aquarium depths. Feeding rate, which was a depth-dependent activity, decreased from 280 to 113 g cal/g live fish/day with increasing weight. Rate and efficiency of conversion also decreased with increasing body weight; in larger fish conversion was dependent on volume rather than on depth of the aquarium. O₂ uptake of feeding fish was about 6 times higher than the starving ones of the tested weight classes at different aquarium depths.This paper is part of a thesis submitted by the author (from A.P.A. College, PALNI) to Madurai University in partial fulfillment of the requirement of the Ph. D. degree. Contribution No. 12 under a research scheme granted to Dr. T. J. Pandian by the UGC (New Delhi). The author is grateful to Dr. T. J. Pandian for valuable guidance, financial support and encouragements.     

Effects of feeding and starvation on growth and swimming activity in an obligatory air-breathing fish

Reared in (tubular) aquaria containing different depths of water, Ophiocephalus striatus (0.7 g, 4.5 cm body length), an obligatory air-breathing tropical fish, swam long or short distances to enable themselves to exchange atmospheric air. In each tested depth (2.5, 5.0, 15.5, 31.0 and 40.0 cm) series, one group was starved, while the other was fed ad libitum twice a day on fish muscle. In the shallowest water (2.5 cm depth), the feeding group surfaced 1,294 times, travelling 64.7 m at an energy cost of 20.4 mg dry fish substance/g live fish/day, against those exposed to the deepest water (40 cm depth), which expended 35.8 mg/g/day, swimming 1,503.4 m on 1,879 visits to the surface. The starving group surfaced only 482 times, travelling 24.1 m at an expense of 5.8 mg/g/day in the shallowest water, while those at 40 cm depth surfaced 504 times, swimming 403.2 m at an energy cost of 7.4 mg/g/day. Owing to the sustained swimming activity and the consequent fatigue, the test individuals belonging to both groups in all the tested series hang to the surface for a definite interval, repaying the O₂ debt. Observations were also made to assess the duration of hanging to precisely estimate the distance travelled. Irrespective of changes in depths of water, the duration of hanging to surface was only 3.0 hr/day for the feeding groups, while it was as much as 15.5 hr/day for the starving groups. The maximum sustained metabolic level of O.striatus reared in 40 cm depth was equivalent to 1.23 ml O₂/g/hr, which is about 2 times higher than the value reported for the active metabolism of swimming Oncorhynchus nerka at 15°C in Brett's (1964) respirometer. O.striatus reared in 2.5 cm depth fed 32.0 mg and converted 6.7 mg dry food/g live fish/day, while those exposed to the deepest water fed 49.1 mg, but converted only 5.5 mg/g/day. Culturing obligatory air-breathing fishes in shallow waters will be advantageous.     

Treatment planning and dosimetric comparison study on two different volumetric modulated arc therapy delivery techniques

Aim

To compare and evaluate the performance of two different volumetric modulated arc therapy delivery techniques.

Background

Volumetric modulated arc therapy is a novel technique that has recently been made available for clinical use. Planning and dosimetric comparison study was done for Elekta VMAT and Varian RapidArc for different treatment sites.

Materials and methods

Ten patients were selected for the planning comparison study. This includes 2 head and neck, 2 oesophagus, 1 bladder, 3 cervix and 2 rectum cases. Total dose of 50 Gy was given for all the plans. All plans were done for RapidArc using Eclipse and for Elekta VMAT with Monaco treatment planning system. All plans were generated with 6 MV X-rays for both RapidArc and Elekta VMAT. Plans were evaluated based on the ability to meet the dose volume histogram, dose homogeneity index, radiation conformity index, estimated radiation delivery time, integral dose and monitor units needed to deliver the prescribed dose.

Results

RapidArc plans achieved the best conformity (CI_95% = 1.08 ± 0.07) while Elekta VMAT plans were slightly inferior (CI_95% = 1.10 ± 0.05). The in-homogeneity in the PTV was highest with Elekta VMAT with HI equal to 0.12 ± 0.02 Gy when compared to RapidArc with 0.08 ± 0.03. Significant changes were observed between the RapidArc and Elekta VMAT plans in terms of the healthy tissue mean dose and integral dose. Elekta VMAT plans show a reduction in the healthy tissue mean dose (6.92 ± 2.90) Gy when compared to RapidArc (7.83 ± 3.31) Gy. The integral dose is found to be inferior with Elekta VMAT (11.50 ± 6.49) × 10⁴ Gy cm³ when compared to RapidArc (13.11 ± 7.52) × 10⁴ Gy cm³. Both Varian RapidArc and Elekta VMAT respected the planning objective for all organs at risk. Gamma analysis result for the pre-treatment quality assurance shows good agreement between the planned and delivered fluence for 3 mm DTA, 3% DD for all the evaluated points inside the PTV, for both VMAT and RapidArc techniques.

Conclusion

The study concludes that a variable gantry speed with variable dose rate is important for efficient arc therapy delivery. RapidArc presents a slight improvement in the OAR sparing with better target coverage when compared to Elekta VMAT. Trivial differences were noted in all the plans for organ at risk but the two techniques provided satisfactory conformal avoidance and conformation.     

A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data

Microsomal cytochrome b₅ (cytb₅) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb₅ (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb₅ NMR resonances and site-directed mutagenesis data were used to characterize the cytb₅ interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb₅ using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb₅ and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb₅. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb₅. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb₅-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes.     

Biophysical characterization of anticoagulant hemextin AB complex from the venom of snake Hemachatus haemachatus

Hemextin AB complex from the venom of Hemachatus haemachatus is the first known natural anticoagulant that specifically inhibits the enzymatic activity of blood coagulation factor VIIa in the absence of factor Xa. It is also the only known heterotetrameric complex of two three-finger toxins. Individually only hemextin A has mild anticoagulant activity, whereas hemextin B is inactive. However, hemextin B synergistically enhances the anticoagulant activity of hemextin A and their complex exhibits potent anticoagulant activity. In this study we characterized the nature of molecular interactions leading to the complex formation. Circular dichroism studies indicate the stabilization of β-sheet in the complex. Hemextin AB complex has an increased apparent molecular diameter in both gas and liquid phase techniques. The complex formation is enthalpically favorable and entropically unfavorable with a negative change in the heat capacity. Thus, the anticoagulant complex shows less structural flexibility than individual subunits. Both electrostatic and hydrophobic interactions are important for the complexation; the former driving the process and the latter helping in the stabilization of the tetramer. The tetramer dissociates into dimers and monomers with the increase in the ionic strength of the solution and also with increase in the glycerol concentration in the buffer. The two dimers formed under each of these conditions display distinct differences in their apparent molecular diameters and anticoagulant properties. Based on these results, we have proposed a model for this unique anticoagulant complex.     

The Slowing Rate of CpG Depletion in SARS-CoV-2 Genomes Is Consistent with Adaptations to the Human Host

Depletion of CpG dinucleotides in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genomes has been linked to virus evolution, host-switching, virus replication, and innate immune responses. Temporal variations, if any, in the rate of CpG depletion during virus evolution in the host remain poorly understood. Here, we analyzed the CpG content of over 1.4 million full-length SARS-CoV-2 genomes representing over 170 million documented infections during the first 17 months of the pandemic. Our findings suggest that the extent of CpG depletion in SARS-CoV-2 genomes is modest. Interestingly, the rate of CpG depletion is highest during early evolution in humans and it gradually tapers off, almost reaching an equilibrium; this is consistent with adaptations to the human host. Furthermore, within the coding regions, CpG depletion occurs predominantly at codon positions 2-3 and 3-1. Loss of ZAP (Zinc-finger antiviral protein)-binding motifs in SARS-CoV-2 genomes is primarily driven by the loss of the terminal CpG within the motifs. Nonetheless, majority of the CpG depletion in SARS-CoV-2 genomes occurs outside ZAP-binding motifs. SARS-CoV-2 genomes selectively lose CpGs-motifs from a U-rich context; this may help avoid immune recognition by TLR7. SARS-CoV-2 alpha-, beta-, and delta-variants of concern have reduced CpG content compared to sequences from the beginning of the pandemic. In sum, we provide evidence that the rate of CpG depletion in virus genomes is not uniform and it greatly varies over time and during adaptations to the host. This work highlights how temporal variations in selection pressures during virus adaption may impact the rate and the extent of CpG depletion in virus genomes.     

Dose-dependent proteomic analysis of glioblastoma cancer stem cells upon treatment with γ-secretase inhibitor

Notch signaling has been demonstrated to have a central role in glioblastoma (GBM) cancer stem cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation, a dose-dependent quantitative mass spectrometry (MS)-based proteomic study has been performed based on the global proteome profiling and a target verification phase where both Immunoassay and a multiple reaction monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation, as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data, possibly due to their crosstalk with Notch Signaling. Overall, this quantitative proteomic-based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs.     

Novel calcium-binding GTPase (AtCBG) involved in ABA-mediated salt stress signaling in Arabidopsis

We have identified a novel Ca²⁺-signal sensing GTPase (643 amino acid residues with an estimated molecular mass of 79 kDa) from the Arabidopsis genome database. This protein contains a RHO-like GTPase domain at the N-terminus (15–184 amino acids) and two calcium-binding EF-hand motifs (199–227 and 319–347 amino acids, respectively). It has the capability to bind calcium and hydrolyze GTP; in addition, its GTPase activity is regulated by changes in Ca²⁺ concentration. The expression of this gene was induced by ABA and salt stresses, and specific knock-out mutants were highly sensitive to ABA and salt treatments. These findings suggest that this protein is a novel ABA- and salt stress-related Ca²⁺ signal transducer.     

Involvement of hypoxia-inducing factor-1alpha-dependent plasminogen activator inhibitor-1 up-regulation in Cyr61/CCN1-induced gastric cancer cell invasion

Cysteine-rich 61 (Cyr61/CCN1), one of the members of CCN family, has been implicated in the progression of human malignancies. Previously, our studies have demonstrated that Cyr61/CCN1 has a role in promoting gastric cancer cell invasion, but the mechanism is not clear yet. Here, we found that hypoxia-inducing factor-1alpha (HIF-1alpha) protein, but not mRNA, expression was significantly elevated in gastric cancer cells overexpressing Cyr61. Supportively, a profound reduction of endogenous HIF-1alpha protein was noted in one highly invasive cell line, TSGH, when transfected with antisense Cyr61. By comparison, the induction kinetics of HIF-1alpha protein by recombinant Cyr61 (rCyr61) was distinct from that of insulin-like growth factor-1 and CoCl(2) treatment, both well known for induction of HIF-1alpha. Using cycloheximide and MG132, we demonstrated that the Cyr61-mediated HIF-1alpha up-regulation was through de novo protein synthesis, rather than increased protein stability. rCyr61 could also activate the PI3K/AKT/mTOR and ERK1/2 signaling pathways, both of which were essential for HIF-1alpha protein accumulation. Blockage of HIF-1alpha activity in Cyr61-expressing cells by transfecting with a dominant negative (DN)-HIF-1alpha strongly inhibited their invasion ability, suggesting that elevation in HIF-1alpha protein is vital for Cyr61-mediated gastric cancer cell invasion. In addition, several HIF-1alpha-regulated invasiveness genes were examined, and we found that only plasminogen activator inhibitor-1 (PAI-1) showed a significant increase in mRNA and protein levels in cells overexpressing Cyr61. Treatment with PAI-1-specific antisense oligonucleotides or function-neutralizing antibodies abolished the invasion ability of the Cyr61-overexpressing cells. Transfection with dominant negative-HIF-1alpha to block HIF-1alpha activity also effectively reduced the elevated PAI-1 level. In conclusion, our data provide a detailed mechanism by which Cyr61 promoted gastric cancer cell invasive ability via an HIF-1alpha-dependent up-regulation of PAI-1.