Combatting persister cells: The daunting task in post-antibiotics era.

Over the years, much attention has been drawn to antibiotic resistance bacteria, but drug inefficacy caused by a subgroup of special phenotypic variants - persisters - has been largely neglected in both scientific and clinical field. Interestingly, this subgroup of phenotypic variants displayed their power of withstanding sufficient antibiotics exposure in a mechanism different from antibiotic resistance. In this review, we summarized the clinical importance of bacterial persisters, the evolutionary link between resistance, tolerance, and persistence, redundant mechanisms of persister formation as well as methods of studying persister cells. In the light of our recent findings of membrane-less organelle aggresome and its important roles in regulating bacterial dormancy depth, we propose an alternative approach for anti-persister therapy. That is, to force a persister into a deeper dormancy state to become a VBNC (viable but non-culturable) cell that is incapable of regrowth. We hope to provide the latest insights on persister studies and call upon more research interest into this field.

Photothermal therapy may be a double-edged sword by inducing the formation of bacterial antibiotic tolerance.

Photothermal nanoparticles are thought to be the most suitable candidates against infectious disease by disrupting the cell membrane or inhibiting cellular metabolism. However, cells with low-metabolic activity states may be endowed with greater ability against harsh environments including antibiotic treatment. For now, it remains unexplored whether and how photothermal therapy (PTT) gives rise to bacterial antibiotic tolerance. In this study, we showed that although it exhibits excellent bactericidal ability, PTT with typical photothermal nanoparticle gold nanocages (AuNCs) can give rise to a subpopulation of cells with great ability of antibiotic tolerance. The subpopulation exhibits delayed growth and decreased cellular ATP levels, indicating a low metabolic state. Specifically, after AuNCs attach to the surface of a bacterial cell, photothermal manipulation can induce cell membrane shrinkage and block the bacterial respiratory chain. Besides, heat shock induces protein aggregation and leads to the dysfunction of a number of important proteins. The heat shock protein DnaK is closely associated with protein aggregation and plays a vital role in modulating antibiotic tolerance, providing a potential therapeutic target.

AtPPRT1, an E3 Ubiquitin Ligase, Enhances the Thermotolerance in Arabidopsis.

E3 ubiquitin ligase plays a vital role in the ubiquitin-mediated heat-related protein degradation pathway. Herein, we report that the expression of AtPPRT1, a C3HC4 zinc-finger ubiquitin E3 ligase gene, was induced by heat stress, and the ß-glucuronidase (GUS) gene driven by the AtPPRT1 promoter has shown increased activity after basal and acquired thermotolerance. To further explore the function of AtPPRT1 in heat stress response (HSR), we used the atpprt1 mutant and AtPPRT1-overexpressing lines (OE2 and OE10) to expose in heat shock. In this study, the atpprt1 mutant had a lower germination and survival rate than those of Col-0 when suffered from the heat stress, whereas OEs enhanced basal and acquired thermotolerance in Arabidopsis seedlings. When compared to Col-0 and OEs, loss-of-function in AtPPRT1 resulted in lower chlorophyll retention and higher content of reactive oxygen species (ROS) after heat treatment. Moreover, the transcript levels of AtPPRT1 and several heat-related genes (AtZAT12, AtHSP21 and AtHSFA7a) were upregulated to greater extents in OEs and lower extents in atpprt1 compared to Col-0 after heat treated. Hence, we suggest that AtPPRT1 may act as a positive role in regulating the high temperature by mediating the degradation of unknown target proteins.

AtPPRT1 negatively regulates salt stress response in Arabidopsis seedlings.

Salt stress is one of the environmental factors that negatively affect plant growth and development. We have previously reported a putative C3HC4 zinc-finger ubiquitin E3 ligase (AtPPRT1) negatively regulates Abscisic acid (ABA) and drought stress response. According to previous studies, the accumulation of ABA in plants can further regulate the salt stress response. Therefore, in this study, we further analyzed whether AtPPRT1 negatively regulates the salt stress response. The results showed that AtPPRT1 expression was induced by salt stress. Furthermore, under salt stress, the ß-glucuronidase (GUS) gene driven by the AtPPRT1 promoter has shown increased activity in the hypocotyl and petioles of Arabidopsis seedlings. Additionally, seedlings of the T-DNA insertion mutant atpprt1 showed significant growth advantage under salt stress, whereas overexpressing AtPPRT1 (OE lines) in Arabidopsis seedlings displayed hypersensitive under salt stress. Etiolated atpprt1 seedlings also demonstrated significantly elongated hypocotyl lengths in salt stress. The elevated or reduced salt tolerance of atpprt1 and AtPPRT1 overexpressing lines was confirmed by the changes in chlorophyll content and 3,3'-Diaminobenzidine (DAB) staining. The above data suggest that AtPPRT1 has a negative effect on salt tolerance in Arabidopsis seedlings.

A Velocity Map Imaging Study of the Reactions of O+ (4S) With CH4.

We present a velocity map imaging study of the key ion-molecule reactions occurring in the O+(4S3/2) + CH4 (X 1A1) system at collision energies of 1.84 and 2.14 eV. In addition to charge transfer to form CH 4 + (X 2B2), we also present data on formation of CH 3 + (X 1A1'), for which the experimentally determined images provide clear confirmation that the products arise from dissociative charge transfer rather than hydride transfer. Experimental data are also presented on the formation of HCO+ through a transient [OCH4]+ complex living many rotational periods. Plausible reaction pathways and intermediate structures are presented to give insight into the routes for formation of these reaction products.

Expression Pattern and Function Analysis of AtPPRT1, a Novel Negative Regulator in ABA and Drought Stress Responses in Arabidopsis.

Abscisic acid (ABA) plays a fundamental role in plant growth and development, as well as in the responses to abiotic stresses. Previous studies have revealed that many components in ABA and drought stress signaling pathways are ubiquitinated by E3 ligases. In this study, AtPPRT1, a putative C3HC4 zinc-finger ubiquitin E3 ligase, was explored for its role in abiotic stress response in Arabidopsis thaliana. The expression of AtPPRT1 was induced by ABA. In addition, the ß-glucuronidase (GUS) gene driven by the AtPPRT1 promoter was more active in the root hair zone and root tips of primary and major lateral roots of young seedlings in the presence of ABA. The assays for seed germination, stomatal aperture, root length, and water deficit demonstrated that the AtPPRT1-overexpressing Arabidopsis was insensitive to ABA and sensitive to drought stress compared with wild-type (WT) plants. The analysis by quantitative real-time PCR (qRT-PCR) revealed that the expression of three stress-inducible genes (AtRAB18, AtERD10, and AtKIN1) were upregulated in the atpprt1 mutant and downregulated in AtPPRT1-overexpressing plants, while two ABA hydrolysis genes (AtCYP707A1 and AtCYP707A3) were downregulated in the atpprt1 mutant and upregulated in AtPPRT1-overexpressing plants in the presence of ABA. AtPPRT1 was localized in the mitochondria. Our findings indicate that AtPPRT1 plays a negative role in ABA and drought stress responses.

AtPUB48 E3 ligase plays a crucial role in the thermotolerance of Arabidopsis.

As the global temperature gradually increases, thermotolerance is vital to the growth and survival for plants. Ubiquitin-mediated protein degradation is a central regulator of many key cellular and physiological processes, including responses to biotic and abiotic stresses. E3 Ubiquitin-ligases, as the major components in the ubiquitination pathway, confer specificity of substrate recognition. Herein, we report that AtPUB48 expression was induced by heat stress, including basal and acquired thermotolerance. AtPUB48-overexpressing lines (OEs) of plants were generated to detect the functions of AtPUB48 in the heat response signaling pathway in Arabidopsis. Seeds of Atpub48-2 mutant had a lower germination rate than those of wild-type (WT) and OE plants when suffered from high temperatures. On the contrary, overexpression of AtPUB48 in Arabidopsis enhanced basal and acquired thermotolerance in seed germination and seedling growth. Moreover, the transcript expression levels of several heat-related downstream genes were highly improved in the OE lines under heat stress, although there were lower levels in the Atpub48-2 mutant compared with that of WT. An in vitro ubiquitination assay confirmed that AtPUB48 with U-box and ARM-repeats functioned as an E3 ubiquitin ligase. The subcellular localization showed that AtPUB48 localized to the nucleus. Collectively, these data imply that AtPUB48 acts as a novel regulator in the heat response signaling pathway. AtPUB48 may target the unknown substrate receptor to 26S proteasome proteolysis.

Velocity Map Imaging Study of Ion-Radical Chemistry: Charge Transfer and Carbon-Carbon Bond Formation in the Reactions of Allyl Radicals with C(.).

We present an experimental and computational study of the dynamics of collisions of ground state carbon cations with allyl radicals, C3H5, at a collision energy of 2.2 eV. Charge transfer to produce the allyl cation, C3H5(+), is exoergic by 3.08 eV and proceeds via energy resonance such that the electron transfer occurs without a significant change in nuclear velocities. The products have sufficient energy to undergo the dissociation process C3H5(+) → C3H4(+) + H. Approximately 80% of the reaction products are ascribed to charge transfer, with ∼40% of those products decaying via loss of a hydrogen atom. We also observe products arising from the formation of new carbon-carbon bonds. The experimental velocity space flux distributions for the four-carbon products are symmetric about the centroid of the reactants, providing direct evidence that the products are mediated by formation of a C4H5(+) complex living at least a few rotational periods. The primary four-carbon reaction products are formed by elimination of molecular hydrogen from the C4H5(+) complex. More than 75% of the nascent C4H3(+) products decay by C-H bond cleavage to yield a C4H2(+) species. Quantum chemical calculations at the MP2/6-311+g(d,p) level of theory support the formation of a nonplanar cyclic C4H5(+) adduct that is produced when the p-orbital containing the unpaired electron on C(+) overlaps with the unpaired spin density on the terminal carbon atoms in allyl. Product formation then occurs by 1,2-elimination of molecular hydrogen from the cyclic intermediate to form a planar cyclic C4H3(+) product. The large rearrangement in geometry as the C4H3(+) products are formed is consistent with high vibrational excitation in that product and supports the observation that the majority of those products decay to form the C4H2(+) species.

Ion-molecule reaction dynamics: Velocity map imaging studies of N(+) and O(+) with CD3OD.

We present a study of the charge transfer reactions of the atomic ions N(+)and O(+) with methanol in the collision energy range from ∼2 to 4 eV. Charge transfer is driven primarily by energy resonance, although the widths of the product kinetic energy distributions suggest that significant interchange between relative translation and product vibration occurs. Charge transfer with CD3OD is more exoergic for N(+), and the nascent parent ion products appear to be formed in excited B̃ and C̃ electronic states, and fragment to CD2OD(+) by internal conversion and vibrational relaxation to the ground electronic state. The internal excitation imparted to the parent ion is sufficient to result in loss of one or two D atoms from the carbon atom. The less exoergic charge transfer reaction of O(+) forms nascent parent ions in the excited à state, and internal conversion to the ground state only results in ejection of single D atom. Selected isotopomers of methanol were employed to identify reaction products, demonstrating that deuterium atom loss from nascent parent ions occurs by C-D bond cleavage. Comparison of the kinetic energy distributions for charge transfer to form CD3OD(+) and CD2OD(+) by D atom loss with the known dynamics for hydride abstraction from a carbon atom provides strong evidence that the D loss products are formed by dissociative charge transfer rather than hydride (deuteride) transfer. Isotopic labeling also demonstrates that chemical reaction in the N(+) + CD3OD system to form NO(+) + CD4 does not occur in the energy range of these experiments, contrary to earlier speculation in the literature.

Velocity Map Imaging Study of Charge-Transfer and Proton-Transfer Reactions of CH3 Radicals with H3(.).

The velocity map imaging method has been applied to crossed beam studies of charge transfer and proton transfer between methyl (CH3) radicals formed by pyrolysis and H3(+) cations over the collision energy range from 1.2 to 3.4 eV. Vibrational excitation in the H3(+) reactants plays an important role both in promoting endoergic charge transfer and in supplying energy to the products of the proton-transfer reaction. Excited H3(+) reactants with vibrational energy in excess of the barrier lead to energy-resonant charge transfer via long-range collisions. A small fraction of collisions that take place at low impact parameters appear to form charge-transfer products with higher levels of internal excitation. The proton-transfer reaction exhibits direct, stripping-like dynamics. Consistent with the kinematics of proton transfer, incremental kinetic energy supplied to the reactants is strongly directed into product relative kinetic energy, as predicted by the concept of "induced repulsive energy release".

Ion imaging study of dissociative charge transfer in the N2(+) + CH4 system.

The velocity map ion imaging method is applied to the dissociative charge transfer reactions of N2(+) with CH4 studied in crossed beams. The velocity space images are collected at four collision energies between 0.5 and 1.5 eV, providing both product kinetic energy and angular distributions for the reaction products CH3(+) and CH2(+). The general shapes of the images are consistent with long range electron transfer from CH4 to N2(+) preceding dissociation, and product kinetic energy distributions are consistent with energy resonance in the initial electron transfer step. The branching ratio for CH3(+):CH2(+) is 85:15 over the full collision energy range, consistent with literature reports.

Ion imaging study of reaction dynamics in the N+ + CH4 system.

The velocity map ion imaging method is applied to the ion-molecule reactions of N(+) with CH(4). The velocity space images are collected at collision energies of 0.5 and 1.8 eV, providing both product kinetic energy and angular distributions for the reaction products CH(4)(+), CH(3)(+), and HCNH(+). The charge transfer process is energy resonant and occurs by long-range electron transfer that results in minimal deflection of the products. The formation of the most abundant product, CH(3)(+), proceeds by dissociative charge transfer rather than hydride transfer, as reported in earlier publications. The formation of HCNH(+) by C-N bond formation appears to proceed by two different routes. The triplet state intermediates CH(3)NH(+) and CH(2)NH(2)(+) that are formed as N(+)((3)P) approaches CH(4) may undergo sequential loss of two hydrogen atoms to form ground state HCNH(+) products on a spin-allowed pathway. However, the kinetic energy distributions for formation of HCNH(+) extend past the thermochemical limit to form HCNH(+) + 2H, implying that HCNH(+) may also be formed in concert with molecular hydrogen, and requiring that intersystem crossing to the singlet manifold must occur in a significant (~25%) fraction of reactive collisions. We also report GAUSSIAN G2 calculations of the energies and structures of important singlet and triplet [CNH(4)(+)] complexes that serve as precursors to product formation.

Imaging ion-molecule reactions: charge transfer and C-N bond formation in the C(+) + NH3 system.

The velocity mapping ion imaging method is applied to the ion-molecule reactions occurring between C(+) and NH(3). The velocity space images are collected over the relative collision energy range from 1.5 to 3.3 eV, allowing both product kinetic energy distributions and angular distributions to be obtained from the data. The charge transfer process appears to be direct, dominated by long-range electron transfer that results in minimal deflection of the products. The product kinetic energy distributions are consistent with a process dominated by energy resonance. The kinetic energy distributions for C-N bond formation appear to scale with the total available energy, providing strong evidence that energy in the [CNH(3)](+) precursor to products is distributed statistically. The angular distributions for C-N bond formation show pronounced forward-backward symmetry, as expected for a complex that resembles a prolate symmetric top decaying along its symmetry axis.

Zero kinetic energy photoelectron spectroscopy of tetracene using laser desorption for vaporization.

Far infrared (FIR) spectroscopy of polycyclic aromatic hydrocarbons is of particular interest to astrophysics since vibrational modes in this range are representative of the molecular size and shape. This information is hence important for identification of chemical compositions and for modeling of the IR spectrum observed in the outer space. In this work, we report neutral and cation FIR spectroscopy of tetracene vaporized from a laser desorption source. Results from two-color resonantly enhanced multiphoton ionization and two-color zero kinetic energy photoelectron spectroscopy will be presented. Several skeletal vibrational modes of the first electronically excited state of the neutral species and those of the cation are assigned, with the aid of ab initio and density functional calculations. The adiabatic ionization potential is determined to be 55 918 +/- 7 cm(-1). Interestingly, all observed vibrational modes can be rationalized based on a simple Huckle calculation, i.e., by observing the addition or elimination of nodal planes due to electronic excitation and/or ionization. Limited by the Franck-Condon principle and the rigidity of the molecular frame of tetracene, only IR forbidden modes are observed in this work.

Electronic polarization spectroscopy of metal phthalocyanine chloride compounds in superfluid helium droplets.

We report the electronic polarization spectroscopy of two metal phthalocyanine chloride compounds (MPcCl, M=Al,Ga) embedded in superfluid helium droplets and oriented in a dc electric field. For both compounds, the laser induced fluorescence spectra show preference for perpendicular excitation relative to the orientation field. This result indicates that the permanent dipoles of both compounds are predominantly perpendicular to the transition dipole. Since the permanent dipole derives from the metal chloride, while the transition dipole derives from the phthalocyanine chromophore, in the plane of phthalocyanine, this qualitative result is not surprising. However, quantitative modeling reveals that this intuitive model is inadequate and that the transition dipole might have tilted away from the molecular plane of phthalocyanine. The out of plane component of the transition dipole amounts to approximately 10% if the permanent dipole is assumed to be approximately 4 debye. The origin for this tilt is puzzling, and we tentatively attribute it to the transition of nonbonding orbitals, either from the chlorine atom or from the bridge nitrogen atom, to the pi* orbitals of the phthalocyanine chromophore. On the other hand, although unlikely, we cannot completely exclude the possibility that both our high level density functional theory calculation and ab initio results severely deviate from reality. The droplet matrix induces redshifts in the origin of the electronic transition and produces discrete phonon wings. Nevertheless, in dc electric fields, all phonon wings and the zero phonon line demonstrate the same dependence on the polarization direction of the excitation laser. Although electronic excitation does couple to the superfluid helium matrix and the resulting phonon wings add complications to the electronic spectrum, this coupling does not affect the direction of the electronic transition dipole. Electronic polarization spectroscopy in superfluid helium droplets is thus still informative in revealing the permanent dipole and its relation relative to the transition dipole.

Conformational identification of tryptamine embedded in superfluid helium droplets using electronic polarization spectroscopy.

We report electronic polarization spectroscopy of tryptamine embedded in superfluid helium droplets. In a dc electric field, dependence of laser induced fluorescence from tryptamine on the polarization direction of the excitation laser is measured. Among the three observed major conformers A, D, and E, conformers D and E display preference for perpendicular excitation relative to the orientation field, while conformer A is insensitive to the polarization direction of the excitation laser. We attribute the behavior of conformer A to the fact that the angle between the permanent dipole and the transition dipole is close to the magic angle. Using a linear variation method, we can reproduce the polarization preference of the three conformers and determine the angle between the transition dipole and the permanent dipole. Since the side chain exerts small effect on the direction of the transition dipole in the frame of the indole chromophore, all three conformers have a common transition dipole more or less in the indole plane at an angle of approximately 60 degrees relative to the long axis of the chromophore. The orientation of the side chain, on the other hand, determines the size and direction of the permanent dipole, thereby affecting the angle between the permanent dipole and the transition dipole. For conformer D in the droplet, our results agree with the Anti(ph) structure, rather than the Anti(py) structure. Our work demonstrates that polarization spectroscopy is effective in conformational identification for molecules that contain a known chromophore. Although coupling of the electronic transition with the helium matrix is not negligible, it does not affect the direction of the transition dipole.

The kinetics of the reactions of C2 (a 3pi(u)) with alcohols.

The reactions of C2 (a 3pi(u)) radicals with a series of alcohols have been studied at about 6.5 Torr total pressure and room temperature using the pulsed laser photolysis/laser-induced fluorescence technique. The relative concentration of C2 (a 3pi(u)) radicals, which are generated via the photolysis of C2Cl4 with the focused output from the fourth harmonic of a Nd:YAG laser (266 nm), was monitored by laser-induced fluorescence (LIF) in the (0, 0) band of the C2 (d 3pi(g)<--a 3pi(u)) transition at 516.5 nm. Under pseudo-first-order conditions, we measured the time evolution of C2 (a 3pi(u)) and determined the rate constants for reactions of C2 (a 3pi(u)) with alcohols. The rate constants increase linearly with the number of C atoms in the alcohols. All of them are larger than those for reactions of C2 (a 3pi(u)) with alkanes (C1-C5). Based on the bond dissociation energy and linear free energy correlations, we believe the reactions of C2 (a 3pi(u)) with alcohols proceed via the mechanism of hydrogen abstraction. The experimental results show that the H-atom on the C-H bonds is activated at the presence of the OH substituent group in the alcohol molecule. The theoretical calculations for the reaction of C2 (a 3pi(u)) with methanol also support these hypotheses.

Kinetics of C2(a3Piu) radical reactions with alkanes by LIF.

The reactions of C2(a3Piu) radicals with a series of alkanes have been studied at room temperature and 6.5 torr total pressure using the pulsed laser photolysis/laser-induced fluorescence technique. C2(a3Piu) radicals were generated by photolysis of C2Cl4 with the focused output from the fourth harmonic of a Nd: YAG laser at 266 nm. The relative concentration of C2(a3Piu) radicals was monitored on the (0,0) band of the C2(d3Pig <-- a3Piu) transition at 516.5 nm by laser-induced fluorescence. From the analysis of the relative concentration-time behavior of C2(a3Piu) under pseudofirst-order conditions, the rate constants for the reactions of C2(a3Piu) with alkanes (C1-C8) were determined. The rate constant increases linearly with the increasing of the number of CH2 groups in the alkanes. The experimental results indicate that the reaction of C2(a3Piu) with small alkanes (C1-C8) follows the typical hydrogen abstraction process. Based on the correlation of the experimental results with the bond dissociation energy of the alkanes, the reactions of C2(a3Piu) with small alkanes likely proceed via the mechanism of hydrogen abstraction.