Unusual modifications of protein biomarkers expressed by plasmid, prophage, and bacterial host of pathogenic Escherichia coli identified using top-down proteomic analysis.

RATIONALE: Pathogenic bacteria often carry prophage (bacterial viruses) and plasmids (small circular pieces of DNA) that may harbor toxin, antibacterial, and antibiotic resistance genes. Proteomic characterization of pathogenic bacteria should include the identification of host proteins and proteins produced by prophage and plasmid genomes. METHODS: Protein biomarkers of two strains of Shiga toxin-producing Escherichia coli (STEC) were identified using antibiotic induction, matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry (MS/MS) with post-source decay (PSD), top-down proteomic (TDP) analysis, and plasmid sequencing. Alphafold2 was also used to compare predicted in silico structures of the identified proteins to prominent fragment ions generated using MS/MS-PSD. Strain samples were also analyzed with and without chemical reduction treatment to detect the attachment of pendant groups bound by thioester or disulfide bonds. RESULTS: Shiga toxin was detected and/or identified in both STEC strains. For the first time, we also identified the osmotically inducible protein (OsmY) whose sequence unexpectedly had two forms: a full and a truncated sequence. The truncated OsmY terminates in the middle of an α-helix as determined by Alphafold2. A plasmid-encoded colicin immunity protein was also identified with and without attachment of an unidentified cysteine-bound pendant group (~307 Da). Plasmid sequencing confirmed top-down analysis and the identification of a promoter upstream of the immunity gene that is activated by antibiotic induction, that is, SOS box. CONCLUSIONS: TDP analysis, coupled with other techniques (e.g., antibiotic induction, chemical reduction, plasmid sequencing, and in silico protein modeling), is a powerful tool to identify proteins (and their modifications), including prophage- and plasmid-encoded proteins, produced by pathogenic microorganisms.

Toxin and phage production from pathogenic E. coli by antibiotic induction analyzed by chemical reduction, MALDI-TOF-TOF mass spectrometry and top-down proteomic analysis.

RATIONALE: Shiga toxin-producing Escherichia coli (STEC) are an ongoing threat to public health and agriculture. Our laboratory has developed a rapid method for identification of Shiga toxin (Stx), bacteriophage, and host proteins produced from STEC. We demonstrate this technique on two genomically sequenced STEC O145:H28 strains linked to two major outbreaks of foodborne illness occurring in 2007 (Belgium) and 2010 (Arizona). METHODS: Our approach was to induce expression of stx, prophage, and host genes by antibiotic exposure, chemically reduce samples, and identify protein biomarkers from unfractionated samples using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, tandem mass spectrometry (MS/MS), and post-source decay (PSD). The protein mass and prominent fragment ions were used to identify protein sequences using top-down proteomic software developed in-house. Prominent fragment ions are the result of polypeptide backbone cleavage resulting from the aspartic acid effect fragmentation mechanism. RESULTS: The B-subunit of Stx and acid-stress proteins HdeA and HdeB were identified in both STEC strains in their intramolecular disulfide bond-intact and reduced states. In addition, two cysteine-containing phage tail proteins were detected and identified from the Arizona strain but only under reducing conditions, which suggests that bacteriophage complexes are bound by intermolecular disulfide bonds. An acyl carrier protein (ACP) and a phosphocarrier protein were also identified from the Belgium strain. ACP was post-translationally modified with attachment of a phosphopantetheine linker at residue S36. The abundance of ACP (plus linker) was significantly increased on chemical reduction, suggesting the release of fatty acids bound to the ACP + linker at a thioester bond. MS/MS-PSD revealed dissociative loss of the linker from the precursor ion as well as fragment ions with and without the attached linker consistent with its attachment at S36. CONCLUSIONS: This study demonstrates the advantages of chemical reduction in facilitating the detection and top-down identification of protein biomarkers of pathogenic bacteria.

Managing Mortality and Aging Risks with a Time-Varying Lee-Carter Model.

Influential existing research has suggested that rather than being static, mortality declines decelerate at young ages and accelerate at old ages. Without accounting for this feature, the forecast mortality rates of the popular Lee-Carter (LC) model are less reliable in the long run. To provide more accurate mortality forecasting, we introduce a time-varying coefficients extension of the LC model by adopting the effective kernel methods. With two frequently used kernel functions, Epanechnikov (LC-E) and Gaussian (LC-G), we demonstrate that the proposed extension is easy to implement, incorporates the rotating patterns of mortality decline and is straightforwardly extensible to multi-population cases. Using a large sample of 15 countries over 1950-2019, we show that LC-E and LC-G, as well as their multi-population counterparts, can consistently improve the forecasting accuracy of the competing LC and Li-Lee models in both single- and multi-population scenarios.

A discussion on the robust vector autoregressive models: novel evidence from safe haven assets.

The vector autoregressive (VAR) model has been popularly employed in operational practice to study multivariate time series. Despite its usefulness in providing associated metrics such as the impulse response function (IRF) and forecast error variance decomposition (FEVD), the traditional VAR model estimated via the usual ordinary least squares is vulnerable to outliers. To handle potential outliers in multivariate time series, this paper investigates two robust estimation methods of the VAR model, the reweighted multivariate least trimmed squares and the multivariate MM-estimation. The robust information criteria are also proposed to select the appropriate number of temporal lags. Via extensive simulation studies, we show that the robust VAR models lead to much more accurate estimates than the original VAR in the presence of outliers. Our empirical results include logged daily realized volatilities of six common safe haven assets: futures of gold, silver, Brent oil and West Texas Intermediate (WTI) oil and currencies of Swiss Francs and Japanese Yen. Our sample covers July 2017-June 2020, which includes the history-writing price drop of WTI on April 20, 2020. Our baseline results suggest that the traditional VAR model may significantly overestimate some parameters, as well as IRF and FEVD metrics. In contrast, robust VAR models provide more reliable results, the validity of which is verified via various approaches. Empirical implications based on robust estimates are further illustrated.

Definition of customer requirements in big data using word vectors and affinity propagation clustering.

Customer requirements (CRs) have a significant impact on product design. The existing methods of defining CRs, such as customer surveys and expert evaluations, are time-consuming, inaccurate and subjective. This paper proposes an automatic CRs definition method based on online customer product reviews using the big data analysis. Word vectors are defined using a continuous bag of words (CBOW) model. Online customer reviews are searched by a crawling method and filtered by the parts of speech and frequency of words. Filtered words are then clustered into groups by an affinity propagation (AP) clustering method based on trained word vectors. Exemplars in each clustering group are finally used to define CRs. The proposed method is verified by case studies of defining CRs for product design. Results show that the proposed method has better performance to determine CRs compared to existing CRs definition methods.

Multiple investigations of aqueous Eu(III)-oxalate complexes: the reduction in coordination number and validation of spectral linear correlation.

Detailed information on the An(iii)/Ln(iii) complexation properties in solution is essential for separation chemistry and the prediction of their potential for radionuclide migration from nuclear waste repositories into natural aquifers. In the present study, to better reveal and confirm the structural information of [Eu(Ox)x (H2O)h-2x]3-2x (h = 8, 9; x = 0-3) aqueous species, especially the variable coordination number (CN), and explore the validity of the spectral linear correlation between the luminescence lifetime and the residual hydration number in the first coordination sphere of Eu(iii) compounds in solution, a comparison between the spectral results and the theoretical calculations in a wide parametric space in terms of the pH value and oxalate concentration was carried out by combining time-resolved luminescence spectroscopy (TRLS) with speciation modelling and density functional theory (DFT) calculations. We have found direct and clear evidence for the 9-fold to 8-fold coordination number reduction of Eu(iii) atoms upon coordination with more than one oxalate in an aqueous medium, and as well systematically validated the applicability of the spectral linear correlation in an aqueous system (otherwise solid state) involving multiple species with the support of relatively reliable and clear speciation modelling.

A retrospective analysis of the dynamic transmission routes of the COVID-19 in mainland China.

The fourth outbreak of the Coronaviruses, known as the COVID-19, has occurred in Wuhan city of Hubei province in China in December 2019. We propose a time-varying sparse vector autoregressive (VAR) model to retrospectively analyze and visualize the dynamic transmission routes of this outbreak in mainland China over January 31-February 19, 2020. Our results demonstrate that the influential inter-location routes from Hubei have become unidentifiable since February 4, 2020, whereas the self-transmission in each provincial-level administrative region (location, hereafter) was accelerating over February 4-15, 2020. From February 16, 2020, all routes became less detectable, and no influential transmissions could be identified on February 18 and 19, 2020. Such evidence supports the effectiveness of government interventions, including the travel restrictions in Hubei. Implications of our results suggest that in addition to the origin of the outbreak, virus preventions are of crucial importance in locations with the largest migrant workers percentages (e.g., Jiangxi, Henan and Anhui) to controlling the spread of COVID-19.

Macroscopic and spectroscopic characterization of U(VI) sorption on biotite.

Knowledge of the geochemical behavior of uranium is critical for the safe disposal of radioactive wastes. Biotite, a Fe(II)-rich phyllosilicate, is a common rock-forming mineral and a major component of granite or granodiorite. This work comprehensively studied the sorption of U(VI) on biotite surface with batch experiments and analyzed the uranium speciation with various spectroscopic techniques, including X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and time-resolved fluorescence spectra (TRFS). Our results indicated that uranyl ions could penetrate into the interlayer of biotite, this ion-exchange process was pH-dependent and only favorable under acidic condition. Instead of precipitation or reduction to uraninite, the TRFS results strongly suggests U(VI) forms surface complexes under the neutral and alkaline condition, though the number and structure of surface species could not be identified accurately. Besides, the oxidation of biotite with peroxide hydrogen showed that structural Fe(II) would have a very low redox reactivity. With leaching experiments, zeta potential analysis and thermodynamics calculation, we discussed the possible reasons for inhibition of U(VI) reduction at the biotite-water interface. Our results may provide insight on interaction mechanism of uranium at mineral-water interface and help us understand the migration behavior of uranium in natural environments.

Sorption of U(VI) onto natural soils and different mineral compositions: The batch method and spectroscopy analysis.

This research studied the sorption behavior of uranium(VI) onto two different kinds of soils: surface soil and undersurface soil that taken from the depth of 30 m undersurface. The soil samples were collected from a low and medium-level radioactive waste disposal site in the southwest of China. The effects of pH, solid-liquid ratio and contact time on the adsorption behavior were studied by batch adsorption method. The experiment results show that the mineral composition of soil and the speciation of U in natural groundwater are two main influencing factors. Muscovite and clinochlore, two of the main minerals of soil samples, dominate the sorption behavior of uranium onto natural soils at weak acidic and near neutral pH range. Under neutral and weak alkaline conditions, the thermodynamic calculation results show that Ca2+ and CO32- have significant influence on the species of uranium in aqueous solution. The U sorption reduced sharply due to the formation of the CaUO2(CO3)32- (aq) complex. This work provides a better insight of the sorption behavior of uranium onto natural soils, and gives an in-depth understanding about the influence of aqueous and surface speciation.

The Sources and Diversity of Immigrant Population Change in Australia, 1981-2011.

Australia has one of the largest percentages of immigrant populations in the developed world with a highly regulated system of immigration control and regular censuses to track their changes over time. However, the ability to explain the population change through the demographic components of immigration, emigration, and death by age and sex is complicated because of differences in measurement and sources of information. In this article, we explore three methods for reconciling the demographic accounts from 1981 to 2011 for the Australia-born and 18 foreign-born population groups. We then describe how the immigrant populations have changed and what has contributed most to that change. We find that the sources of immigrant population change have varied considerably by age, sex, country of birth, and period of immigration. Immigrants from Europe are currently the oldest and slowest-growing populations, whereas those from elsewhere are growing rapidly and exhibit relatively young population age structures. Studying these patterns over time helps us to understand the nature of international migration and its long-term contributions to population change and composition.

A Versatile and Robust Approach to Stimuli-Responsive Protein Multilayers with Biologically Enabled Unique Functions.

Protein-based materials call for innovative processing techniques to integrate their unique biologically enabled functions with other materials of complementary features. Herein, we report the covalent protein layer-by-layer assembly via orthogonal "Tag-Catcher" reactions as a facile and robust approach to make entirely protein-based multilayers on a variety of substrates. Programmed assembly of native telechelic proteins not only endows the materials valuable stimuli-sensitive behaviors, but also unique properties unparalleled by any synthetic counterparts. As proof of concept, super uranyl-binding protein (SUP) is immobilized on silica gel by this method with tunable capacity and enhanced capability for uranyl sequestration. Not only is the capturing performance enhanced in the multilayer setup, it also confers resilience to recycling, allowing efficient harvest of uranyl with an average of ∼90% and ∼60% recovery rate in over 10 cycles from water and synthetic seawater, respectively. The approach is the first entirely protein-based multilayers covalently assembled by the layer-by-layer method. It provides a platform for immobilizing proteins with synergistic enhancement of function and resilience and expands the scope and capability of genetically encoded protein-based materials.

Influence of Fe(II) on the Se(IV) sorption under oxic/anoxic conditions using bentonite.

79Se, one of the key radionuclides for nuclear waste disposal, threatens the quality of the environment, as well as human health. Therefore, it needs to be permanently isolated from the biosphere. The aim of the study was to investigate the effects of Fe(II)/Fe(III) on the removal of 79Se using bentonite in the pH range of 2.0-10.0 under oxic/anoxic conditions. Under oxic conditions, Se(IV) prefers to form inner-sphere complexes with Fe(III)-oxyhydroxide, derived from the oxidization of Fe(II) using oxygen. Interestingly, Se(IV) will interact with Fe(III) and form a poorly soluble ferric selenite at pH ∼4 under oxic conditions. Under anoxic conditions, however, the concentration of Fe(II) is closely related to the sorption process of Se(IV) on bentonite. When the concentration of Fe(II) was less than 1%, Fe(II) combined with the hydroxyl, forming Fe(OH)2, which generated a disproportionation at pH ∼8 and formed a new sorbent, Fe3O4. However, when the concentration of Fe(II) was increased to 5%, reduction precipitation was the primary way to remove Se(IV) in aqueous solution. XANES (X-ray Absorption Near Edge Structure) spectra showed that higher pH values are beneficial for the formation of the final thermodynamic reduction product, Fe selenide. These results suggested that Fe(II) significantly affect the Se(IV) sorption. Overall, this study confirmed the significant role of Fe(II) on the retardation of 79Se and on remediation for Se(IV) contamination in the hydrosphere.

BODIPY-based fluorescent thermometer as a lysosome-targetable probe: how the oligo(ethylene glycols) compete photoinduced electron transfer.

A novel BODIPY-based fluorescent thermometer, which shows a lysosome-targeting property, was successfully prepared. Due to the electron-donating ability of the oligo(ethylene glycols), the photoinduced electron-transfer pathway from morpholine to BODIPY dye is blocked. The fluorescence of the thermometer quenched by intramolecular rotation at room temperature was progressively enhanced during heating due to the increased microviscosity around the fluorophore.

BosR functions as a repressor of the ospAB operon in Borrelia burgdorferi.

The Lyme disease spirochete, Borrelia burgdorferi, must abundantly produce outer surface lipoprotein A (OspA) in the tick vector but downregulate OspA in mammals in order to evade the immune system and maintain its natural enzootic cycle. Here, we show that BosR binds two regulatory elements of the ospAB operon and that increasing BosR expression leads to downregulation of OspA. Both regulatory sequences, cisI and cisII, showed strong BosR-binding and cisII bound much tighter than cisI. A promoterless bosR gene fused with an inducible promoter was introduced into an rpoS mutant and a wild-type strain to assess RpoS-independent and -dependent downregulation of OspA by BosR. With the induction of BosR expression, OspA expression was reduced more significantly in the RpoS-deficient than wild-type background, but not completely repressed. In the presence of constitutive expression of OspC, DbpA and DbpB, increasing BosR production resulted in complete repression of OspA in the RpoS mutant. Taken together, the study clearly demonstrated BosR serves as a repressor that binds both regulatory elements of the ospAB operon and shuts off expression.

Prevalence of chronic kidney disease and prediabetes and associated risk factors: a community-based screening in Zhuhai, Southern China.

BACKGROUND: The prevalence of chronic kidney disease (CKD) and prediabetes has increased in China, and at different rates in different locations. Therefore a community-based screening research was conducted in order to determine the prevalence of CKD and prediabetes, and to analyze associated risk factors of CKD and prediabetes in a city of Southern China. METHODS: A total of 7801 community residents aged 18 year and older from 6 communities were screened by a stratified random cluster sampling method. An estimated glomerular filtration rate (eGFR), albuminuria, fasting plasma glucose (FPG), and homeostatic model assessment of insulin resistance (HOMA-IR) were measured. Age-standardized prevalence was calculated by the direct method with the use of data on the population distribution in China in 2006. Multivariate logistic analysis was used to analyze the risk factors of CKD and prediabetes, and association of insulin resistance (IR) with CKD and prediabetes was analyzed. RESULTS: The age-standardized prevalence of CKD was 12.5%, eGFR < 60 ml×min(-1)×1.73 m(-2) was 2.7% and ACR (albumin to creatinine ratio) > 30 mg/g was 10.3%. The age-standardized prevalence of prediabetes was 12.1%. Logistic regression suggests that IR was a common independent risk factor of CKD and prediabetes. Further analysis show that HOMA-IR was increased with the aggravation of kidney injury and FPG. CONCLUSION: CKD and prediabetes have become a major public health problem in Zhuhai, Southern China; insulin resistance may be an important risk factor.

RpoS regulates essential virulence factors remaining to be identified in Borrelia burgdorferi.

BACKGROUND: Since the RpoN-RpoS regulatory network was revealed in the Lyme disease spirochete Borrelia burgdorferi a decade ago, both upstream and downstream of the pathway have been intensively investigated. While significant progress has been made into understanding of how the network is regulated, most notably, discovering a relationship of the network with Rrp2 and BosR, only three crucial virulence factors, including outer surface protein C (OspC) and decorin-binding proteins (Dbps) A and B, are associated with the pathway. Moreover, for more than 10 years no single RpoS-controlled gene has been found to be critical for infection, raising a question about whether additional RpoS-dependent virulence factors remain to be identified. METHODOLOGY/PRINCIPAL FINDINGS: The rpoS gene was deleted in B. burgdorferi; resulting mutants were modified to constitutively express all the known virulence factors, OspC, DbpA and DbpB. This genetic modification was unable to restore the rpoS mutant with infectivity. CONCLUSIONS/SIGNIFICANCE: The inability to restore the rpoS mutant with infectivity by simultaneously over-expressing all the three virulence factors allows us to conclude RpoS also regulates essential genes that remain to be identified in B. burgdorferi.

Whole-body imaging of infection using bioluminescence.

Bioluminescence imaging is a powerful technique to visualize and monitor biological processes in numerous systems. This unit describes two strategies for bioluminescence imaging that can be used to study bacterial infection in mice. One method is to express a luciferase gene in the bacteria; the second method is to use bacteria that express both a luciferase and ß-lactamase along with a substrate containing caged luciferin, which is released by ß-lactamase hydrolysis and reacts with luciferase to generate light. For both strategies, bioluminescent signals are imaged using an IVIS live animal imaging system (Caliper Life Sciences). The bioluminescence images are analyzed to localize bioluminescent bacteria, quantify signal, and determine the wavelengths of the signals produced. The correlation of bacterial numbers with signal intensity in vivo can be determined, allowing a quantitative measure of bacterial numbers in mice in real time. Methods are described in detail to facilitate successful application of these emerging technologies in nearly any experimental system.

[Primary peripheral T-cell lymphoma of the penis: a case report and review of the literature].

OBJECTIVE: To report a case of primary peripheral T-cell lymphoma of the penis. METHODS: We analyzed the clinicopathological characteristics of the case of primary peripheral T-cell lymphoma using histological, cytochemical and immunohistochemical methods and by review of the literature. RESULTS: The patient was a 65 years old man and presented with a diffuse enlargement of the penis as the initial sign, followed by erosive ulcer in the caput penis and inguinal lymphadenectasis. The tumor was pathohistologically manifested as an epidermal ulcer, with tumorous necrosis around the capillary, infiltrative growth and atypical changes of the neoplastic cells and proliferation of capillaries. Immunohistochemically, the tumor cells were positive for CD43 and CD3, but negative for CD20, CD79a, CD34, CD30, CD56 and CD34. Clinically it responded to the chemotherapy designed for peripheral T-cell lymphoma. CONCLUSION: Primary peripheral T-cell lymphoma of the penis is an extremely rare malignant tumor, the diagnosis of which relies on histopathological examination, immunohistochemical staining and differentiation between squamous cell carcinoma and other types of lymphoma.

Common and unique contributions of decorin-binding proteins A and B to the overall virulence of Borrelia burgdorferi.

As an extracellular bacterium, the Lyme disease spirochete Borrelia burgdorferi resides primarily in the extracellular matrix and connective tissues and between host cells during mammalian infection, where decorin and glycosaminoglycans are abundantly found, so its interactions with these host ligands potentially affect various aspects of infection. Decorin-binding proteins (Dbps) A and B, encoded by a 2-gene operon, are outer surface lipoproteins with similar molecular weights and share approximately 40% identity, and both bind decorin and glycosaminoglycans. To investigate how DbpA and DbpB contribute differently to the overall virulence of B. burgdorferi, a dbpAB mutant was modified to overproduce the adhesins. Overproduction of either DbpA or DbpB resulted in restoration of the infectivity of the mutant to the control level, measured by 50% infectious dose (ID(50)), indicating that the two virulence factors are interchangeable in this regard. Overproduction of DbpA also allowed the mutant to disseminate to some but not all distal tissues slightly slower than the control, but the mutant with DbpB overproduction showed severely impaired dissemination to all tissues that were analyzed. The mutant with DbpA overproduction colonized all tissues, albeit generating bacterial loads significantly lower than the control in heart and joint, while the mutant overproducing DbpB remained severely defective in heart colonization and registered bacterial loads substantially lower than the control in joint. Taken together, the study indicated that DbpA and DbpB play a similar role in contribution to infectivity as measured by ID(50) value but contribute differently to dissemination and tissue colonization.

Both decorin-binding proteins A and B are critical for the overall virulence of Borrelia burgdorferi.

Both decorin-binding proteins (DbpA and DbpB) of the Lyme disease spirochete Borrelia burgdorferi bind decorin and glycosaminoglycans, two important building blocks of proteoglycans that are abundantly found in the extracellular matrix (ECM) and connective tissues as well as on cell surfaces of mammals. As an extracellular pathogen, B. burgdorferi resides primarily in the ECM and connective tissues and between host cells during mammalian infection. The interactions of B. burgdorferi with these host ligands mediated by DbpA and DbpB potentially influence various aspects of infection. Here, we show that both DbpA and DbpB are critical for the overall virulence of B. burgdorferi in the murine host. Disruption of the dbpBA locus led to nearly a 10(4)-fold increase in the 50% infectious dose (ID50). Complementation of the mutant with either dbpA or dbpB reduced the ID50 from over 10(4) to roughly 10(3) organisms. Deletion of the dbpBA locus affected colonization in all tissues of infected mice. The lack of dbpA alone precluded the pathogen from colonizing the heart tissue, and B. burgdorferi deficient for DbpB was recovered only from 42% of the heart specimens of infected mice. Although B. burgdorferi lacking either dbpA or dbpB was consistently grown from joint specimens of almost all infected mice, it generated bacterial loads significantly lower than the control. The deficiency in either DbpA or DbpB did not reduce the bacterial load in skin, but lack of both significantly did. Taken together, the study results indicate that neither DbpA nor DbpB is essential for mammalian infection but that both are critical for the overall virulence of B. burgdorferi.