Simultaneous quantitation of 17 female sex hormones in illegal products by validated liquid chromatography-high resolution mass spectrometry and liquid chromatography-tandem mass spectrometry methods.

The consumption of food and drugs adulterated with female sex hormones can have an extremely adverse effect on human health. Therefore, developing appropriate monitoring methods for the identification of various exogenous female sex hormones is crucial for minimizing and eliminating the related health risks. Herein, 17 female hormones categorized into two groups: estrogen and progestin, were analyzed using reversed-phase liquid chromatography coupled to Orbitrap or triple quadrupole mass spectrometry. The fragmentation patterns for all compounds were discovered, and fragmented structures were also derived from them through liquid chromatography-high-resolution mass spectrometry followed by qualitative sample analysis. In addition, a quantitative analysis of 67 samples of illicit drugs and dietary supplements was performed using the validated liquid chromatography-tandem mass spectrometry method. Female hormone components were detected in two samples of an unauthorized injectable solution and a tablet-type drug. Medroxyprogesterone was detected in the samples in the range of 96.4-206 ng/g. Notably, eight components similar in structure to steroids were simultaneously detected as male sex hormones by confirming their fragmentation ion patterns using liquid chromatography-high-resolution mass spectrometry. The developed methods thus offer a dependable and practically applicable approach for the screening and detection of exogenous female sex hormones in real food and drug samples to ensure public health.

Co-introduction of plasmids harbouring the carbapenemase genes, blaNDM-1 and blaOXA-232, increases fitness and virulence of bacterial host.

BACKGROUND: Bacterial isolates with multiple plasmids harbouring different carbapenemase genes have emerged and been identified repeatedly, despite a general notion that plasmids confer fitness cost in bacterial host. In this study, we investigated the effects of plasmids with carbapenemase genes on the fitness and virulence of bacteria. METHODS: Different plasmids harbouring the carbapenemase genes, blaNDM-1 and blaOXA-232, were isolated from a carbapenem-resistant K. pneumoniae strain. Each plasmid was conjugated into the Escherichia coli strain DH5α, and a transconjugant with both plasmids was also obtained by transformation. Their in vitro competitive ability, biofilm formation, serum resistance, survival ability within macrophage and fruit fly, and fly killing ability were evaluated. RESULTS: The transconjugants with a single plasmid showed identical phenotypes to the plasmid-free strain, except that they decreased fly survival after infection. However, significantly increased fitness, virulence and biofilm production were observed consistently for the transconjugant with both plasmids, harbouring blaNDM-1 and blaOXA-232. CONCLUSIONS: Our data indicate that bacteria carrying multiple plasmids encoding different carbapenemases may have increased fitness and virulence, emphasizing the need for diverse strategies to combat antimicrobial resistance.

Intrinsic negative feedback governs activation surge in two-component regulatory systems.

PhoP and PhoQ comprise a two-component system in the bacterium Salmonella enterica. PhoQ is the sensor kinase/phosphatase that modifies the phosphorylation state of the regulator PhoP in response to stimuli. The amount of phosphorylated PhoP surges after activation, then declines to reach a steady-state level. We now recapitulate this surge in vitro by incubating PhoP and PhoQ with ATP and ADP. Mathematical modeling identified PhoQ's affinity for ADP as the key parameter dictating phosphorylated PhoP levels, as ADP promotes PhoQ's phosphatase activity toward phosphorylated PhoP. The lid covering the nucleotide-binding pocket of PhoQ governs the kinase to phosphatase switch because a lid mutation that decreased ADP binding compromised PhoQ's phosphatase activity in vitro and resulted in sustained expression of PhoP-dependent mRNAs in vivo. This feedback mechanism may curtail futile ATP consumption because ADP not only stimulates PhoQ's phosphatase activity but also inhibits ATP binding necessary for the kinase reaction.

Huge Anterior Skull Base Defect Reconstruction on Communicating Between Cranium and Nasal Cavity: Combination Flap of Galeal Flap and Reverse Temporalis Flap.

INTRODUCTION: Traditionally, galeal flap or cranialization was often used to reconstruct the skull base defect caused by trauma or tumor removal. However, in the case of huge skull base defect, galeal flap is not enough to block the communication between nasal cavity and intracranial space. In this study, authors suggest combination flap of galea and reverse temporalis muscle as a method for reconstruction of huge skull base defect. MATERIALS AND METHODS: From 2016 to 2019, retrospective review was conducted, assessing 7 patients with bone defect which is not just opening of frontal sinus but extends to frontal sinus and cribriform plate. Reconstructions were done by combination of galeal flap and reverse temporalis muscle flap transposition. RESULTS: Defects were caused by nasal cavity tumor with intracranial extension or brain tumor with nasal cavity extension. There was no major complication in every case. During the follow up period, no patient had signs of complication such as ascending infection, herniation and CSF rhinorrhea. Postoperative radiologic images of all patients that were taken at least 6 months after the surgery showed that flaps maintained the lining and the volume well. DISCUSSION: Conventional reconstruction of skull base defect with galeal flap is not effective enough to cover the large sized defect. In conclusion, galeal flap in combination with reverse temporalis muscle flap can effectively block the communication of nasal cavity and intracranium.

ATP reduction by MgtC and Mg2+ homeostasis by MgtA and MgtB enables Salmonella to accumulate RpoS upon low cytoplasmic Mg2+ stress.

RpoS is one of several alternative sigma factors known to alter gene expression profiles by RpoS-associated RNA polymerase in response to a variety of stresses. The enteric bacteria Salmonella enterica and Escherichia coli accumulate RpoS under low Mg2+ concentrations via a common mechanism in which the PhoP regulator activates expression of antiadaptor proteins that, by sequestering the adaptor RssB, prevent RpoS degradation by the protease ClpXP. Here, we demonstrate that this genetic program alone does not fully support RpoS accumulation when cytoplasmic Mg2+ concentration drops to levels that impair protein synthesis. Under these circumstances, only S. enterica continues RpoS accumulation in a manner dependent on other PhoP-activated programs (i.e. ATP reduction by the MgtC protein and Mg2+ import by the MgtA and MgtB transporters) that maintain translation homeostasis. Moreover, we provide evidence that the mgtC gene, which is present in S. enterica but not in E. coli, is responsible for the differences in RpoS accumulation between these two bacterial species. Our results suggest that bacteria possess a mechanism to control RpoS accumulation responding to cytoplasmic Mg2+ levels, the difference of which causes distinct RpoS accumulation in closely related bacterial species.

Expanding the use of internal mammary artery perforators as a recipient vessel in free tissue transfer: An anatomical analysis by computed tomography angiography in breast cancer patients.

BACKGROUND: The internal mammary artery (IMA) perforator has assumed recent prominence as recipient vessels in free autologous breast reconstruction. However, anatomical understanding is unclear, due to limited cadaver and clinical studies. We evaluated the usability of these vessels by evaluating perforator size, dominance, laterality, interspace location, and relationship with breast volume. METHODS: A retrospective review assessed 197 female patients with breast cancer who had undergone computed tomography angiography (CTA) of the chest wall. The average age and body mass index (BMI) of patients was 49.0 ± 6.5 years and 24.2 ± 5 .8 kg/m2 , respectively. The average volume of breasts was 437 ± 190 mL. Our analysis focused on the anatomy of IMA perforator and its relationship to volume and BMI. RESULTS: A total of 377 hemi-chest evaluations were performed. Most patients (95.5%) had sizeable perforating artery identified on CTA. Among all sizeable perforators identified, the mean diameter of the most dominant arterial perforator was 1.8 ± 0 .8 mm. The right hemi-chest had significantly larger perforators than the left (1.9 ± 0 .9 mm vs. 1.7 ± 0 .7 mm, p = 0.002). The first intercostal space (ICS) had a slightly greater of perforators than second ICS (34.6% vs. 29.8%, p = 0.172). However, second ICS had a greater number of most dominant perforators compared to first ICS (38.9% vs. 34.7%, p = 0.357). Perforators from first ICS emerge medial to the sternal edge and breast footprint. When dividing groups with 0-1 versus 2-3 reliable perforators, breast volume was significantly higher in the later (422.0 mL vs. 461.2 mL, p = 0.019). CONCLUSION: These results are expected to encourage microsurgeons to use the IMA perforator by providing a clear anatomical roadmap.

Violation of Ohm's law in a Weyl metal.

Ohm's law is a fundamental paradigm in the electrical transport of metals. Any transport signatures violating Ohm's law would give an indisputable fingerprint for a novel metallic state. Here, we uncover the breakdown of Ohm's law owing to a topological structure of the chiral anomaly in the Weyl metal phase. We observe nonlinear I-V characteristics in Bi0.96Sb0.04 single crystals in the diffusive limit, which occurs only for a magnetic-field-aligned electric field (E∥B). The Boltzmann transport theory with the charge pumping effect reveals the topological-in-origin nonlinear conductivity, and it leads to a universal scaling function of the longitudinal magnetoconductivity, which completely describes our experimental results. As a hallmark of Weyl metals, the nonlinear conductivity provides a venue for nonlinear electronics, optical applications, and the development of a topological Fermi-liquid theory beyond the Landau Fermi-liquid theory.

Polymorphic Elastocapillarity: Kinetically Reconfigurable Self-Assembly of Hair Bundles by Varying the Drain Rate.

We report various patterns formed by draining liquid from hair bundles. Hair-like fibers arranged in triangular bundles self-assemble into various cross sections when immersed in liquid then removed. The combinations of their length and the kinetics, represented by the drain rate, lead to various polymorphic self-assemblies: concave hexagonal, triangular, circular, or inverted triangular patterns. The equilibrium of these shapes is predicted by elastocapillarity, the balance between the bending strain energy of the hairs and the surface energy of the liquid. Shapes with a larger strain energy, such as the inverted triangular bundles, are obtained at the higher liquid drain rates. This polymorphic self-assembly is fully reversible by rewetting and draining and can have applications in multifunctional dynamic textures.

Remote Perturbations in Tertiary Contacts Trigger Ligation of Lysine to the Heme Iron in Cytochrome c.

Perturbations in protein structure define the mechanism of allosteric regulation and biological information transfer. In cytochrome c (cyt c), ligation of Met80 to the heme iron is critical for the protein's electron-transfer (ET) function in oxidative phosphorylation and for suppressing its peroxidase activity in apoptosis. The hard base Lys is a better match for the hard ferric iron than the soft base Met is, suggesting the key role of the protein scaffold in favoring Met ligation. To probe the role of the protein structure in the maintenance of Met ligation, mutations T49V and Y67R/M80A were designed to disrupt hydrogen bonding and packing of the heme coordination loop, respectively. Electronic absorption, nuclear magnetic resonance, and electron paramagnetic resonance spectra reveal that ferric forms of both variants are Lys-ligated at neutral pH. A minor change in the tertiary contacts in T49V, away from the heme coordination loop, appears to be sufficient to execute a change in ligation, suggesting a cross-talk between the different regions of the protein structure and a possibility of built-in conformational switches in cyt c. Analyses of thermodynamic stability, kinetics of Lys binding and dissociation, and the pH-dependent changes in ligation provide a detailed characterization of the Lys coordination in these variants and relate these properties to the extent of structural perturbations. The findings emphasize the importance of the hydrogen-bonding network in controlling ligation of the native Met80 to the heme iron.

tRNAPro -mediated downregulation of elongation factor P is required for mgtCBR expression during Salmonella infection.

Bacterial ribosome requires elongation factor P to translate fragments harbouring consecutive proline codons. Given the abundance of ORFs with potential EF-P regulated sites, EF-P was assumed to be constitutively expressed. Here, we report that the intracellular pathogen Salmonella enterica serovar Typhimurium decreases efp mRNA levels during course of infection. We determined that the decrease in efp mRNA is triggered by low levels of charged tRNAPro , a condition that Salmonella experiences when inside a macrophage phagosome. Surprisingly, downregulation of EF-P selectively promotes expression of the virulence mgtC gene and contributes to Salmonella's ability to survive inside macrophages. The decrease in EF-P levels induces ribosome stalling at the consecutive proline codons of the mgtP open reading frame in the mgtCBR leader RNA, and thus allows formation of a stem-loop structure promoting transcription of the mgtC gene. The substitution of proline codons in the mgtP gene eliminates EF-P-mediated mgtC expression and thus Salmonella's survival inside macrophages. Our findings indicate that Salmonella benefits virulence genes by decreasing EF-P levels and inducing the stringent response inside host.

Directed C-H Activation and Tandem Cross-Coupling Reactions Using Palladium Nanocatalysts with Controlled Oxidation.

Controlled oxidation of palladium nanoparticles provided high-valent PdIV oxo-clusters which efficiently promote directed C-H halogenation reactions. In addition, palladium nanoparticles can undergo changes in oxidation states to provide both high-valent PdIV and low-valent Pd0 species within one system, and thus a tandem reaction of C-H halogenation and cross-coupling (C-N, C-C, and C-S bond formation) was successfully established.

Fumarate-Mediated Persistence of Escherichia coli against Antibiotics.

Bacterial persisters are a small fraction of quiescent cells that survive in the presence of lethal concentrations of antibiotics. They can regrow to give rise to a new population that has the same vulnerability to the antibiotics as did the parental population. Although formation of bacterial persisters in the presence of various antibiotics has been documented, the molecular mechanisms by which these persisters tolerate the antibiotics are still controversial. We found that amplification of the fumarate reductase operon (FRD) inEscherichia coliled to a higher frequency of persister formation. The persister frequency ofE. coliwas increased when the cells contained elevated levels of intracellular fumarate. Genetic perturbations of the electron transport chain (ETC), a metabolite supplementation assay, and even the toxin-antitoxin-relatedhipA7mutation indicated that surplus fumarate markedly elevated theE. colipersister frequency. AnE. colistrain lacking succinate dehydrogenase (SDH), thereby showing a lower intracellular fumarate concentration, was killed ∼1,000-fold more effectively than the wild-type strain in the stationary phase. It appears thatSDHandFRDrepresent a paired system that gives rise to and maintainsE. colipersisters by producing and utilizing fumarate, respectively.

Lon-mediated proteolysis of the FeoC protein prevents Salmonella enterica from accumulating the Fe(II) transporter FeoB under high-oxygen conditions.

The Salmonella Feo system consists of the FeoA, FeoB, and FeoC proteins and mediates ferrous iron [Fe(II)] import. FeoB is an inner membrane protein that, along with contributions from two small hydrophilic proteins, FeoA and FeoC, transports Fe(II). We previously reported that FeoC binds to and protects the FeoB transporter from FtsH-mediated proteolysis. In the present study, we report proteolytic regulation of FeoC that occurs in an oxygen-dependent fashion. While relatively stable under low-oxygen conditions, FeoC was rapidly degraded by the Lon protease under high-oxygen conditions. The putative Fe-S cluster of FeoC seemed to function as an oxygen sensor to control FeoC stability, as evidenced by the finding that mutation of the putative Fe-S cluster-binding site greatly increased FeoC stability under high-oxygen conditions. Salmonella ectopically expressing the feoB and feoC genes was able to accumulate FeoB and FeoC only under low-oxygen conditions, suggesting that FeoC proteolysis prevents Salmonella from accumulating the FeoB transporter under high-oxygen conditions. Finally, we propose that Lon-mediated FeoC proteolysis followed by FtsH-mediated FeoB proteolysis helps Salmonella to avoid uncontrolled Fe(II) uptake during the radical environmental changes encountered when shifting from low-iron anaerobic conditions to high-iron aerobic conditions.

25-hydroxyvitamin D status is associated with chronic cerebral small vessel disease.

BACKGROUND AND PURPOSE: The aim of this study was to determine the association between 25-hydroxyvitamin D (25(OH)D) and neuroimaging correlates of cerebral small vessel disease. METHODS: We identified 759 consecutive patients with acute ischemic stroke or transient ischemic attack. Lacunes, white matter hyperintensity, and cerebral microbleed (CMB) were assessed using MR images. Deep CMB was defined as the presence of CMB in basal ganglia, thalamus, or brain stem. The association between 25(OH)D and small vessel disease was tested using linear and logistic regression analyses. RESULTS: Mean age was 68 (±13) years. Mean level of 25(OH)D was 34.1±17.8 nmol/L. On bivariate analysis, a 25-nmol/L decrease in 25(OH)D was associated with lacunes (regression coefficient, 0.23; 95% confidence interval [CI], 0.02-0.45), severe white matter hyperintensity (odds ratio, 2.05; 95% CI, 1.41-3.08), and deep CMB (odds ratio, 1.28; 95% CI, 1.01-1.63). Also, 25(OH)D deficiency (≤25 nmol/L) was associated with lacunes (regression coefficient, 0.5; 95% CI, 0.04-0.95), severe white matter hyperintensity (odds ratio, 2.74; 95% CI, 1.31-6.45), and deep CMB (odds ratio, 1.68; 95% CI, 1.03-2.78). The association remained significant even after multivariable adjustment and in the subgroup of previously healthy patients. CONCLUSIONS: 25(OH)D is inversely associated with lacunes, white matter hyperintensity, and deep CMB. Our findings suggest that 25(OH)D is linked to small vessel disease, and in future trials it should be tested whether 25(OH)D supplementation can prevent small vessel disease.

The iron-sensing fur regulator controls expression timing and levels of salmonella pathogenicity island 2 genes in the course of environmental acidification.

In order to survive inside macrophages, Salmonella produces a series of proteins encoded by genes within Salmonella pathogenicity island 2 (SPI-2). In the present study, we report that Fur, a central regulator of iron utilization, negatively controls the expression of SPI-2 genes. Time course analysis of SPI-2 expression after the entry of Salmonella into macrophages revealed that SPI-2 genes are induced earlier and at higher levels in the absence of the Fur regulator. It was hypothesized that Fur repressed the SPI-2 expression that was activated during acidification of the phagosome. Indeed, as pH was lowered from pH 7.0 to pH 5.5, the lack of Fur enabled SPI-2 gene expression to be induced at higher pH and to be expressed at higher levels. Fur controlled SPI-2 genes via repression of the SsrB response regulator, a primary activator of SPI-2 expression. Fur repressed ssrB expression both inside macrophages and under acidic conditions, which we ascribe to the direct binding of Fur to the ssrB promoter. Our study suggests that Salmonella could employ iron inside the phagosome to precisely control the timing and levels of SPI-2 expression inside macrophages.

Structural insights into the molecular mechanism of Escherichia coli SdiA, a quorum-sensing receptor.

Escherichia coli SdiA is a quorum-sensing (QS) receptor that responds to autoinducers produced by other bacterial species to control cell division and virulence. Crystal structures reveal that E. coli SdiA, which is composed of an N-terminal ligand-binding domain and a C-terminal DNA-binding domain (DBD), forms a symmetrical dimer. Although each domain shows structural similarity to other QS receptors, SdiA differs from them in the relative orientation of the two domains, suggesting that its ligand-binding and DNA-binding functions are independent. Consistently, in DNA gel-shift assays the binding affinity of SdiA for the ftsQP2 promoter appeared to be insensitive to the presence of autoinducers. These results suggest that autoinducers increase the functionality of SdiA by enhancing the protein stability rather than by directly affecting the DNA-binding affinity. Structural analyses of the ligand-binding pocket showed that SdiA cannot accommodate ligands with long acyl chains, which was corroborated by isothermal titration calorimetry and thermal stability analyses. The formation of an intersubunit disulfide bond that might be relevant to modulation of the DNA-binding activity was predicted from the proximal position of two Cys residues in the DBDs of dimeric SdiA. It was confirmed that the binding affinity of SdiA for the uvrY promoter was reduced under oxidizing conditions, which suggested the possibility of regulation of SdiA by multiple independent signals such as quorum-sensing inducers and the oxidation state of the cell.

Development of colistin resistance in pmrA-, phoP-, parR- and cprR-inactivated mutants of Pseudomonas aeruginosa.

OBJECTIVES: Colistin susceptibility in Pseudomonas aeruginosa is associated with a lipopolysaccharide (LPS) structure that is controlled by the modulation of several two-component regulatory systems. In this study, we attempted to elucidate the role of these two-component systems in the development of colistin resistance in P. aeruginosa. METHODS: pmrA-, phoP-, parR- or cprR-inactivated mutants were constructed from a colistin-susceptible P5 strain. Colistin-resistant mutants (P5R, P5ΔpmrA-R, P5ΔphoP-R, P5ΔparR-R and P5ΔcprR-R) were developed in vitro from a wild-type strain (P5) and pmrA-, phoP-, parR- or cprR-inactivated mutants by serial passage in colistin-containing media. Expression levels of the pmrA, phoP, parR, cprR and arnB genes were determined and amino acid alterations of two-component regulatory systems during development of colistin resistance were also investigated. RESULTS: While P5ΔpmrA-R, P5ΔparR-R and P5ΔcprR-R showed elevated expression of the phoP gene, the expression levels of the pmrA, parR and cprR genes were not different between gene-inactivated mutants and the adapted colistin-resistant mutants. P5ΔphoP-R showed no significant elevation in expression of any of the pmrA, parR or cprR genes. The arnB gene was overexpressed in all in vitro-selected colistin-resistant mutants compared with colistin-susceptible wild-type and gene-inactivated mutants. Three amino acid alterations in PhoQ and three in ParS were identified in induced colistin-resistant mutants. CONCLUSIONS: Our data suggest that individual two-component systems may not be essential for the acquisition of colistin resistance in P. aeruginosa. The PhoPQ two-component system may play a major role in the development of colistin resistance in our strains, but alternative or compensatory pathways may exist.

Symptomatic hyponatremia following lateral medullary infarction: a case report.

BACKGROUND: Hyponatremia has been reported from patients with severe neurological disease, and the syndrome of inappropriate secretion of antidiuretic hormone and cerebral salt wasting syndrome are the two main etiologies of hyponatremia after brain injury. Here we describe a patient with a lateral medullary infarction who experienced symptomatic hyponatremia with finding suggestive of syndrome of inappropriate secretion of antidiuretic hormone followed by cerebral salt wasting syndrome. CASE PRESENTATION: A 70-year-old Korean man visited emergency room complaining of sudden onset vertigo and gait disturbance. Neurological exam showed left side ataxia, Horner syndrome, and right side hypesthesia. Brain magnetic resonance imaging disclosed acute infarction involving left lateral medulla. His neurological status was stabilized, but he began to complain of non-vertiginous dizziness and general weakness five days after admission. Serum sodium level dropped from 131 mEq/mL to 122 mEq/mL with reduced serum osmolarity of 265 mOsm/L. The diagnosis of syndrome of inappropriate secretion of antidiuretic hormone was made and we restricted fluid intake, but his symptoms worsened and his mental status became drowsy. Follow up serum sodium level was 108 mEq/L with volume loss, suggesting cerebral salt wasting syndrome. We treated him with hypertonic saline and his consciousness was recovered. CONCLUSION: This case shows symptomatic hyponatremia after lateral medullary infarction, providing insight about distinct pathogenesis of syndrome of inappropriate secretion of antidiuretic hormone and cerebral salt wasting syndrome.

Carotid artery calcification is associated with deep cerebral microbleeds.

BACKGROUND/AIMS: The aim of the present study was to determine the association between arterial calcification and cerebral microbleeds (CMB) relative to their distribution. METHODS: We identified 834 consecutive patients with acute ischemic stroke or transient ischemic attack who underwent CT angiography and susceptibility-weighted imaging. RESULTS: Internal carotid artery (ICA) calcification and CMB were found in 660 patients (79.1%) and 335 patients (40.2%), respectively. ICA calcification was independently associated with CMB of any location (odds ratio, OR, 2.86, 95% CI 2.01-4.08, p < 0.0001). The association between calcification and deep CMB was stronger (OR 3.51, 95% CI 2.39-5.14, p < 0.0001). However, ICA calcification was not associated with CMB in a strictly lobar area. CONCLUSION: ICA calcification is an independent risk factor for deep CMB but not for a strictly lobar CMB. Our findings might contribute to the elucidation of the pathogenesis of cerebral small vessel disease.

Colistin heteroresistance in Citrobacter freundii clinical isolates from Republic of Korea.

We investigated colistin heteroresistance in Citrobacter freundii isolates from Korean hospitals. Using population analysis profiling (PAP), we detected colistin heteroresistance in 31.3% of isolates. Among these, ST217 was the most prevalent clone (58.5%), particularly within colistin-heteroresistant isolates (80.0%). Interestingly, the second most common clone, ST248, was not found in heteroresistant isolates. We identified amino acid changes in PhoQ, PmrA, and PmrB, along with mRNA overexpression in pmrB and arnD. Colistin monotherapy showed no efficacy, but a combination of colistin and ciprofloxacin successfully eradicated all five isolates, even at 0.5 × minimum inhibitory concentrations. This study underscores the high prevalence of colistin heteroresistance in C. freundii isolates, limiting the effectiveness of colistin monotherapy. Combining colistin with ciprofloxacin may offer a viable treatment option for C. freundii infections.