Inference of cell cycle-dependent proteolysis by laser scanning cytometry

Mechanisms that couple protein turnover to cell cycle progression are critical for coordinating the events of cell duplication and division. Despite the importance of cell cycle-regulated proteolysis, however, technologies to measure this phenomenon are limited, and typically involve monitoring cells that are released back into the cell cycle after synchronization. We describe here the use of laser scanning cytometry (LSC), a technical merger between fluorescence microscopy and flow cytometry, to determine cell cycle-dependent changes in protein stability in unperturbed, asynchronous, cultures of mammalian cells. In this method, the ability of the LSC to accurately measure whole cell fluorescence is employed, together with RNA fluorescence in situ hybridization and immunofluorescence, to relate abundance of a particular RNA and protein in a cell to its point at the cell cycle. Parallel monitoring of RNA and protein levels is used, together with protein synthesis inhibitors, to reveal cell cycle-specific changes in protein turnover. We demonstrate the viability of this method by analyzing the proteolysis of two prominent human oncoproteins, Myc and Cyclin E, and argue that this LSC-based approach offers several practical advantages over traditional cell synchronization methods.     

A multi-satellite framework to rapidly evaluate extreme biosphere cascades: The Western US 2021 drought and heatwave

The increasing frequency and intensity of climate extremes and complex ecosystem responses motivate the need for integrated observational studies at low latency to determine biosphere responses and carbon-climate feedbacks. Here, we develop a satellite-based rapid attribution workflow and demonstrate its use at a 1–2-month latency to attribute drivers of the carbon cycle feedbacks during the 2020–2021 Western US drought and heatwave. In the first half of 2021, concurrent negative photosynthesis anomalies and large positive column CO₂ anomalies were detected with satellites. Using a simple atmospheric mass balance approach, we estimate a surface carbon efflux anomaly of 132 TgC in June 2021, a magnitude corroborated independently with a dynamic global vegetation model. Integrated satellite observations of hydrologic processes, representing the soil–plant–atmosphere continuum (SPAC), show that these surface carbon flux anomalies are largely due to substantial reductions in photosynthesis because of a spatially widespread moisture-deficit propagation through the SPAC between 2020 and 2021. A causal model indicates deep soil moisture stores partially drove photosynthesis, maintaining its values in 2020 and driving its declines throughout 2021. The causal model also suggests legacy effects may have amplified photosynthesis deficits in 2021 beyond the direct effects of environmental forcing. The integrated, observation framework presented here provides a valuable first assessment of a biosphere extreme response and an independent testbed for improving drought propagation and mechanisms in models. The rapid identification of extreme carbon anomalies and hotspots can also aid mitigation and adaptation decisions.     

Synthesis and characterization of zinc oxide nanoparticles by using polyol chemistry for their antimicrobial and antibiofilm activity

The present investigation deals with facile polyol mediated synthesis and characterization of ZnO nanoparticles and their antimicrobial activities against pathogenic microorganisms. The synthesis process was carried out by refluxing zinc acetate precursor in diethylene glycol(DEG) and triethylene glycol(TEG) in the presence and in the absence of sodium acetate for 2 h and 3 h. All synthesized ZnO nanoparticles were characterized by X-ray diffraction (XRD), UV visible spectroscopy (UV), thermogravimetric analysis (TGA), fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy(FESEM), transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDX) technique. All nanoparticles showed different degree of antibacterial and antibiofilm activity against Gram-positive Staphylococcus aureus (NCIM 2654)and Gram-negative Proteus vulgaris (NCIM 2613). The antibacterial and antibiofilm activity was inversely proportional to the size of the synthesized ZnO nanoparticles. Among all prepared particles, ZnO nanoparticles with least size (~ 15 nm) prepared by refluxing zinc acetate dihydrate in diethylene glycol for 3 h exhibited remarkable antibacterial and antibiofilm activity which may serve as potential alternatives in biomedical application.     

Metastable states of HYPK-UBA domain's seeds drive the dynamics of its own aggregation

Protein aggregation is a multi-step process that requires sequential structural transitions of monomers during their incorporation into oligomers. Such process involves the formation of various intermediate stages in protein structures. Seed-nucleation mediated oligomerization is observed in many aggregation-prone proteins. Understanding of the protein seed's structural features and mechanisms of its transition-state formation are important for knowing the details of post-nucleation aggregation process. We have identified the metastable states in the seeds of the Ubiquitin associated (UBA) domain of Huntingtin Interacting Protein K (HYPK). This is studied by monitoring the events of dynamic transitions of metastable seeds to aggregates or monomers through microscopy, biophysical and computational techniques. HYPK-UBA seeds can exist in specific metastable state(s) that show transition from closed to open conformations, thereby reorienting the helix associated hydrophobic patches to cause its self-aggregation. Metastable seeds show inter-seed exchange of monomers through simultaneous dissociation-association phenomenon. Monomer release from metastable seeds can cause the dissolution of the aggregates. Like metastable monomers, metastable seeds also show reduction in their secondary structure by altering the molecular contacts and solvent accessible hydrophobic surfaces. Induction of metastable seeds from the ground-state is a slow thermodynamic process and it results from excitable perturbations. Conclusively, we propose the concept that the thermodynamic induction of metastable states in HYPK-UBA seed potentiates the molecule to switch its conformations that increases the protein's self-aggregation by the mechanism of hydrophobic patch collapse, while also releasing the monomers from oligomeric seeds due to structural instability.     

Coexistent Brugada Syndrome and Wolff-Parkinson-White Syndrome: What is the Optimal Management?

Coexistent Brugada syndrome and Wolff-Parkinson-White (WPW) syndrome is rare, and as such poses management challenges. The overlap of symptoms attributable to each condition, the timing of ventricular stimulation after accessory pathway ablation and the predictive value of programmed stimulation in Brugada syndrome are controversial. We describe a case of coexistent Brugada syndrome and WPW syndrome in a symptomatic young adult. We discuss our treatment approach and the existing literature along with the challenges in management of such cases.     

The role of hydroxyproline in collagen folding: conformational energy calculations on oligopeptides containing proline and hydroxyproline

We have observed that the rate of folding of the enzymatically hydroxylated form of poly(Gly-Pro-Pro) into the triple-helical conformation is considerably higher than that of the unhydroxylated polypeptide [R. K. Chopra and V. S. Ananthanarayanan (1982) Proc. Natl. Acad. Sci. USA 79 , 7180–7184]. In this study, we examine a plausible kinetic pathway for triple-helix formation by selecting peptide models for the unhydroxylated collagen molecule, and computing their conformational energies before and after proline hydroxylation. Starting with the available data on the preferred conformations of proline- and hydroxyproline-containing peptide sequences, energy minimization was carried out on the following pairs of peptides: Gly-Ala-Pro-Gly-Ala and Gly-Ala-Hyp-Gly-Ala; Gly-Pro-Pro-Gly-Ala and Gly-Pro-Hyp-Gly-Ala; Gly-Ala-Pro-Gly-Ala-Pro and Gly-Ala-Hyp-Gly-Ala-Hyp. It was found that, with each pair of peptides, the energetically most favorable conformation (I) has an extended structure at the Gly-Ala or Gly-Pro segment and a β-bend at the Pro-Gly or Hyp-Gly segment. In the Hyp-containing peptides, this conformation is further stabilized by a (Hyp_{i + 2})OH…OC(Gly_i) hydrogen bond. Conformation I is lower in energy by about 6–13 kcal/mol of the peptide than the fully extended conformations that resemble the single collagen polypeptide chain and contain no intramolecular hydrogen bond. In contrast to the proline counterpart, the hydroxyproline-containing peptides are found capable of adopting a partially extended conformation that does not contain the β-bend but retains the (Hyp)OH…OC(Gly) hydrogen bond. The energy of this conformation is intermediate between conformation I and the fully extended conformation. The continuation of the β-bend along the chain is restricted by stereochemical constraints that are more severe in the latter two pairs of peptides than in the first pair. Such a restriction may be considered to trigger the “unbending” of the minimum energy conformation leading to its straightening into the fully extended conformation; the latter, in turn, would lead to triple-helix formation through favorable interchain interactions. We propose that the partially extended conformation in the Hyp-containing peptides could serve as a kinetic intermediate on the way to forming the fully extended conformation. Because of the (Hyp_{i + 2})OH…OC(Gly_i) hydrogen bond, this conformation would also serve to lock the trans geometry at the Gly-Ala(Pro) and Ala(Pro)-Hyp peptide bonds, thereby enhancing the rate of their helix formation. A scheme for collagen folding in proposed on the basis of these results.     

Consequences of adaptation to larval crowding on sexual and fecundity selection in Drosophila melanogaster

Sexual selection is a major force influencing the evolution of sexually reproducing species. Environmental factors such as larval density can manipulate adult condition and influence the direction and strength of sexual selection. While most studies on the influence of larval crowding on sexual selection are either correlational or single-generation manipulations, it is unclear how evolution under chronic larval crowding affects sexual selection. To answer this, we measured the strength of sexual selection on male and female Drosophila melanogaster that had evolved under chronic larval crowding for over 250 generations in the laboratory, along with their controls which had never experienced crowding, in a common garden high-density environment. We measured selection coefficients on male mating success and sex-specific reproductive success, as separate estimates allowed dissection of sex-specific effects. We show that experimental evolution under chronic larval crowding decreases the strength of sexual and fecundity selection in males but not in females, relative to populations experiencing crowding for the first time. The effect of larval crowding in reducing reproductive success is almost twice in females than in males. Our study highlights the importance of studying how evolution in a novel, stressful environment can shape adult fitness in organisms.     

CD, 1H NMR and molecular modeling studies of the interaction of Ca2+ with substance P and Ala7-substance P in a non-polar solvent.

The biologically relevant conformation of substance P is likely to be dictated by the lipid milieu wherein the hormone would interact with its receptor. Assuming that specific constraints to the hormone structure may be imparted by its interaction with Ca2+ ions in the low dielectric lipid medium, the interaction of substance P and its inactive analog, Ala7-substance P, has been characterized in a lipid-mimetic solvent. Circular dichroism (CD) and NMR spectral methods were employed to study the conformation of the free and Ca2+-bound forms of the peptides and the conformational changes that occur on Ca2+ binding. The results show that both peptides assume a helical structure in the non-polar solvent used, a mixture of acetonitrile and trifluoroethanol. The N-terminal region is, however, less ordered in the analog peptide compared with the native hormone. Ca2+ addition causes significant conformational changes in both the peptides. However, while substance P binds two Ca2+ ions in a cooperative manner, Ala7-substance P binds only one Ca2+ ion with a relatively weaker affinity. Computations of the minimum-energy conformations of the free and Ca2+-bound peptides were performed using interproton distances derived from nuclear Overhauser enhancement spectra of the two peptides, as well as the information provided by changes in proton chemical shifts caused by Ca2+ addition. Taken together, the results of this study suggest that differences in the interaction of substance P and Ala7-substance P with Ca2+ in the non-polar milieu, which in turn leads to differences in their Ca2+-bound conformations, may be the basis for the differences in their biological potencies.