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Abstract Many bacterial species that cannot sporulate, such as the model bacterium Escherichia coli, can nevertheless survive for years, following exhaustion of external resources, in a state termed long-term stationary phase (LTSP). Here we describe the dynamics of E. coli adaptation during the first three years spent under LTSP. We show that during this time, E. coli continuously adapts genetically through the accumulation of mutations. For nonmutator clones, the majority of mutations accumulated appear to be adaptive under LTSP, reflected in an extremely convergent pattern of mutation accumulation. Despite the rapid and convergent manner in which populations adapt under LTSP, they continue to harbor extensive genetic variation. The dynamics of evolution of mutation rates under LTSP are particularly interesting. The emergence of mutators affects overall mutation accumulation rates as well as the mutational spectra and the ultimate spectrum of adaptive alleles acquired under LTSP. With time, mutators can evolve even higher mutation rates through the acquisition of additional mutation rate–enhancing mutations. Different mutator and nonmutator clones within a single population and time point can display extreme variation in their mutation rates, resulting in differences in both the dynamics of adaptation and their associated deleterious burdens. Despite these differences, clones that vary greatly in their mutation rates tend to coexist within their populations for many years, under LTSP.

Conditions that sustain constant bacterial growth are seldom found in nature. Oligotrophic environments and competition among microorganisms force bacteria to be able to adapt quickly to rough and changing situations. A particular lifestyle composed of continuous cycles of growth and starvation is commonly referred to as feast and famine. Bacteria have developed many different mechanisms to survive in nutrient-depleted and harsh environments, varying from producing a more resistant vegetative cell to complex developmental programmes. As a consequence of prolonged starvation, certain bacterial species enter a dynamic nonproliferative state in which continuous cycles of growth and death occur until 'better times' come (restoration of favourable growth conditions). In the laboratory, microbiologists approach famine situations using batch culture conditions. The entrance to the stationary phase is a very regulated process governed by the alternative sigma factor RpoS. Induction of RpoS changes the gene expression pattern, aiming to produce a more resistant cell. The study of stationary phase revealed very interesting phenomena such as the growth advantage in stationary phase phenotype. This review focuses on some of the interesting responses of gram-negative bacteria when they enter the fascinating world of stationary phase.

Chitosan is a chiral polyglucosamine polysaccharide that selectively binds to S -enantiomer of ketorolac rather than R -enantiomer. In this paper, a novel chiral stationary phase was prepared by molecular imprinting of chitosan using racemic ketorolac as a template. This imprinting process resulted in a high enantioselectivity (59.66% ee and enantioselectivity coefficient of 2.6) as evaluated by the batch rebinding study. The prepared stationary phase enabled chiral resolution of racemic ketorolac when packed into a solid-phase extraction cartridge. Moreover, promising results were obtained when an HPLC column packed with the proposed stationary phase was tested for chiral separation. The selectivity factor ( α ) was calculated to be 5.3 indicating the enantioselectivity of the prepared stationary phase. The chromatographic resolution trials revealed a mixed hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) separation modes in addition to the imprinted cavities. These results showed that racemic compounds could be cheaper and more available templates for imprinting of enantioselective polymers for chiral stationary phases.

The use of stationary-phase optimized selectivity in liquid chromatography (SOS-LC) was shown to be successful for HPLC to analyze complex mixtures using a Phase OPtimized Liquid Chromatography (POPLC) kit. This commercial kit contains five stationary-phase types of varying lengths, which can be coupled to offer an improved separation of compounds. Recently, stationary-phase optimized selectivity supercritical fluid chromatography (SOS-SFC) has been introduced, transferring the methodology to SFC. In this study, the applicability of a customized POPLC expert kit for isocratic SFC runs was explored. Five stationary-phase chemistries were selected as potentially most suitable for achiral separations of polar compounds: aminopropyl (amino), cyanopropyl (CN), diol, ethylpyridine (EP), and silica. The retention factors (k) on the individual stationary phases were used for the prediction of the best stationary-phase combination, based on the POPLC algorithm (via the included software). As an alternative, the best column combination was predicted using multiple linear regression (MLR) models on the results obtained from a simplex centroid mixture design with only three stationary-phase types (amino, silica, and EP). A third approach applied the isocratic POPLC algorithm on the same three stationary-phase data. The proposed combinations were assembled and tested. The predicted and experimental retention factors were compared. The predictions based on the POPLC algorithm provided a stationary phase showing a complete separation of the mixture. The stationary phase suggested by the MLR models, on the other hand, showed co-elution of two compounds, due to an unexpected experimental retention shift. Overall, the customized POPLC kit showed good potential to be applied in SFC.

The transition from growth to stationary phase is a natural response of bacteria to starvation and stress. When stress is alleviated and more favorable growth conditions return, bacteria resume proliferation without a significant loss in fitness. Although specific adaptations that enhance the persistence and survival of bacteria in stationary phase have been identified, mechanisms that help maintain the competitive fitness potential of nondividing bacterial populations have remained obscure. Here, we demonstrate that staphylococci that enter stationary phase following growth in media supplemented with excess glucose, undergo regulated cell death to maintain the competitive fitness potential of the population. Upon a decrease in extracellular pH, the acetate generated as a byproduct of glucose metabolism induces cytoplasmic acidification and extensive protein damage in nondividing cells. Although cell death ensues, it does not occur as a passive consequence of protein damage. Instead, we demonstrate that the expression and activity of the ClpXP protease is induced, resulting in the degeneration of cellular antioxidant capacity and, ultimately, cell death. Under these conditions, inactivation of either clpX or clpP resulted in the extended survival of unfit cells in stationary phase, but at the cost of maintaining population fitness. Finally, we show that cell death from antibiotics that interfere with bacterial protein synthesis can also be partly ascribed to the corresponding increase in clpP expression and activity. The functional conservation of ClpP in eukaryotes and bacteria suggests that ClpP-dependent cell death and fitness maintenance may be a widespread phenomenon in these domains of life.

A spherical chiral porous organic polymer (POPs) COP-1 is synthesized by the Friedel–Crafts alkylation reaction of Boc-3-(4-biphenyl)-L-alanine (BBLA) and 4,4′-bis(chloromethyl)-1,1′-biphenyl (BCMBP), which was used as a novel chiral stationary phase (CSPs) for mixed-mode high-performance liquid chromatography (HPLC) enantioseparation. The racemic compounds were resolved in normal-phase liquid chromatography (NPLC) using n-hexane/isopropanol as mobile phase and reversed-phase liquid chromatography (RPLC) using methanol/water as mobile phase. The COP-1-packed column exhibited excellent separation performance toward various racemic compounds including alcohols, amines, ketones, esters, epoxy compounds, organic acids, and amino acids in NPLC and RPLC modes. The effects of analyte mass and column temperature on the separation efficiency of racemic compounds were investigated. In addition, the chiral resolution ability of the COP-1-packed column not only can be complementary in RPLC/NPLC modes but also exhibit a good chiral recognition complementarity with Chiralpak AD-H column and chiral porous organic cage (POC) NC1-R column. The relative standard deviations (RSD) ( n  = 5) of the retention time, resolution value, and peak area by repeated separation of 1-(4-chiorophenyl)ethanol are all below 3.0%. The COP-1 column shows high column efficiency (e.g., 17,320 plates m −1 for 1-(4-chlorophenyl)ethanol on COP-1 column in NPLC), high enantioselectivity, and good reproducibility toward various racemates. This work demonstrates that chiral POPs microspheres are promising chiral materials for HPLC enantioseparation. Graphical abstract

Homochiral metal-organic frameworks (MOFs) have attracted considerable attention in many fields of research, such as chiral catalysis and chiral chromatography. However, only few homochiral MOFs can be effectively used in capillary electrochromatography (CEC) and their performances are far from adequate. In this study, we successfully synthesized achiral nanocrystalline MIL-53. A facile post-synthetic modification strategy was then implemented to functionalize the product, yielding a homochiral MOF: l-His-NH-MIL-53. This MOF was then employed as a chiral coating in open-tubular CEC mode (OT-CEC), and, as such, it exhibited high enantioselectivities for several racemic drugs. The homochiral MOF and the fabricated capillary coating were systematically characterized using transmission electron microscopy, scanning electron microscopy (with energy-dispersive X-ray spectrometry), Fourier-transform infrared spectroscopy, X-ray diffractometry, thermogravimetric analysis, circular dichroism spectroscopy, Brunauer-Emmett-Teller surface area measurements, and X-ray photoelectron spectroscopy. This study is expected to provide a new strategy for the design and establishment of MOF-based chiral OT-CEC systems. [Display omitted] •l-His-NH-MIL-53 was prepared as a novel chiral stationary phase for OT-CEC.•Multiple methods were employed to characterize the MOF and MOF coating.•MOF-modified capillary exhibited high enantioselectivity for several racemic drugs.

Carbon dots (CDs) derived crosslinked covalent organic nanomaterials (CONs) possessing high specific surface area and abundant surface functional groups are considered to be potential candidates for multimodal chromatographic separations. Typically, the synthesis of CDs and CONs requires harsh reaction conditions and toxic organic solvents, hence, the pursuit of facile and mild preparation strategies is the goal of researchers. In this work, 3-aminopropyltriethoxysilane and D-glucose were used as nitrogen and carbon sources, respectively, to prepare amino-CDs (AmCDs) by rapid low-temperature polymerization rather than the common high-temperature and high-pressure reaction. Then, surface functionalization of the aminated silica gel was carried out in a deep eutectic solvent by using hydrophilic AmCDs and 1,3,5-triformylbenzene (TFB) as the functional monomers. Consequently, a novel N-rich CDs derived CON surface-functionalized silica gel (AmCDs-CON@SiO 2 ) was obtained under mild reaction conditions. The combination of AmCDs and TFB created an ideal CON based chromatographic stationary phase. The incorporation of TFB not only contributed to the successful construction of a crosslinked CON, but also enhanced the interaction forces. The developed AmCDs-CON@SiO 2 has a great potential for versatile applications in liquid chromatography. This study proposes a simple stationary phase preparation strategy by the surface modification of silica gel with CDs-based CON. Moreover, this study verified the application potential of CDs derived CON in chromatographic separation. This not only promotes the development of CDs in the field of liquid chromatographic stationary phase, but also provides some reference value for the wide application of cross-linked CON. Graphical Abstract

Chiral metal-organic frameworks (CMOFs) with enantiomeric subunits have been employed in chiral chemistry. In this study, a CMOF formed from 6-methoxyl-(8S,9R)-cinchonan-9-ol-3-carboxylic acid (HQA) and ZnCl2, {(HQA)(ZnCl2)(2.5H2O)}n, was constructed as a chiral stationary phase (CSP) via an in situ fabrication approach and used for chiral amino acid and drug analyses for the first time. The {(HQA)(ZnCl2)(2.5H2O)}n nanocrystal and the corresponding chiral stationary phase were systematically characterised using a series of analytical techniques including scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, circular dichroism, X-ray photoelectron spectroscopy, thermogravimetric analysis, and Brunauer-Emmett-Teller surface area measurements. In open-tubular capillary electrochromatography (CEC), the novel chiral column exhibited strong and broad enantioselectivity toward a variety of chiral analytes, including 19 racemic dansyl amino acids and several model chiral drugs (both acidic and basic). The chiral CEC conditions were optimised, and the enantioseparation mechanisms are discussed. This study not only introduces a new high-efficiency member of the MOF-type CSP family but also demonstrates the potential of improving the enantioselectivities of traditional chiral recognition reagents by fully using the inherent characteristics of porous organic frameworks. [Display omitted] •{(HQA)(ZnCl2)(2.5H2O)}n was prepared as a novel chiral stationary phase for OT-CEC for enantioseparation.•Numerous characterization methods were applied to characterize the MOF and MOF-coated capillary.•MOF-coated capillary exhibited high enantioselecivity for several racemic amino acids and drugs.

In radar target recognition, the canonical scatterer model (CSM) serves as an effective alternative to the scatterer center model (SCM) for efficiently characterizing electromagnetic (EM) scattering properties of complex targets. Based on physical optics (PO) and the stationary phase method (SPM), this paper analytically derives the novel extension of the CSM for six canonical scatterers: plate, dihedral, trihedral, cylinder, cone, and sphere. The proposed polarization-dependent framework isolates the polarimetric response from CSMs’ intrinsic geometries, reducing the full-polarimetric matrix to an explicit function exclusively governed by bistatic radar spatial configurations. Experimental validation demonstrates mean relative percentage errors (MRPEs) in radar cross section (RCS) of 0.3%, 2%, 2.6%, 3%, 6%, and 7%. This model constitutes a foundational prototype for scattering dictionaries addressing both forward and inverse EM scattering problems, possessing significant practical utility in radar target recognition and image interpretation.