Inter domain linker region affects properties of CBM6 in GH5_34 arabinoxylanases and alters oligosaccharide product profile.

Understanding the relation between enzyme domain structure and catalytic activity is crucial for optimal engineering of novel enzymes for lignocellulose bioconversion. Xylanases with varying specificities are commonly used to valorise the hemicellulose arabinoxylan (AX), yet characterization of specific arabinoxylanases remain limited. Two homologous GH5_34 arabinoxylanases, HhXyn5A and CtXyn5A, in which the two domains are connected by a 40-residue linker, exhibit distinct activity on AX, yielding different reaction product patterns, despite high sequence identity, conserved active sites and similar domain composition. In this study, the carbohydrate binding module 6 (CBM6), or the inter domain linker together with CBM6, were swapped to investigate their influence on hydrolytic activity and oligosaccharide product pattern on cereal AXs. The variants, with only CBM6 swapped, displayed reduced activity on commercial wheat and rye AX, as well as on extracted oat fibre, compared to the original enzymes. Additionally, exchange of both linker and CBM6 resulted in a reduced ratio of enzyme produced in soluble form in Escherichia coli cultivations, causing loss of activity of both HhXyn5A and CtXyn5A variants. Analysis of oligosaccharide product patterns applying HPAEC-PAD revealed a decreased number of reaction products for CtXyn5A with swapped CBM6, which resembled the product pattern of HhXyn5A. These findings emphasize the importance of the CBM6 interactions with the linker and the catalytic domain for enzyme activity and specificity, and underlines the role of the linker in enzyme structure organisation and product formation, where alterations in linker interactions with the catalytic and/or CBM6 domains, influence enzyme-substrate association and specificity.

The dyeing effect of acridine orange for multiple plasmid systems is sensitive to temperature.

The Goldview dyeing of the natural multiplasmid system of Lactobacillus plantarum PC518 was affected by temperature. The article want to identify the specific molecules that cause temperature sensitivity, then experiment on the universality of temperature sensitivity, and finally preliminarily analyze the influencing factors. At 5°C and 25°C, single pDNA, multiplasmid system, and linear DNA samples were electrophoretic on agarose gel prestained by Goldview 1, 2, 3, and acridine orange (AO), respectively. Eighteen vectors of Escherichia coli and two vectors shortened by cloning were mixed into multiplasmid systems with different member numbers, and then electrophoresis with AO staining was performed within the range of 5°C-45°C, with a linearized multiplasmid system as the control. The lane profiles (peaks) were captured with Image Lab 5.1 software. After electrophoresis, the nine-plasmid-2 system was dyed with AO solutions of different ionic strengths to detect the effect of ionic strength on temperature sensitivity. It was measured that the UV-visible absorption spectra of the nine-plasmid-2 system dissolved in AO solutions with different ionic strengths and pH. Further, a response surface model was constructed using Design-Expert.V8.0.6 software. The electrophoresis result showed that the multiplasmid system from L. plantarum PC518 stained by AO staining showed a weak band at 5°C and five bands at 25°C, which was similar to the result of staining with Goldview 1, 2, and 3. The synthetic nine-plasmid-1 system and nine-plasmid-2 system displayed different band numbers on the electrophoresis gel in the electrophoresis temperature range of 5°C-45°C, namely 3, 4, 6, 4, and 2 bands, as well as 2, 6, 7, 8, and 5 bands. Using the 1× Tris-acetate-EDTA (TAE)-AO solution, the poststaining results of the nine-plasmid-2 system in the temperature range of 5°C-45°C were 4, 6, 9, 9, and 7 bands, respectively. Further, using 5×, 10×, or 25× TAE buffer, the AO poststaining results at 5°C were 4, 2, and 1 bands, respectively. The ultraviolet spectral results from 5°C to 25°C showed that there was a significant difference (3.5 times) in the fluctuation amplitude at the absorption peak of 261.2 nm between 0× and 1-10× TAE-AO solution containing the nine-plasmid-2 system. Specifically, the fluctuation amplitudes of 0×, 1×, 5×, and 10× samples were 0.032, 0.109, 0.112, and 0.110, respectively. At the same time, using 1× and 10× TAE buffer, the AO-stained linear nine-plasmid-2 system remained stable and did not display temperature sensitivity. The response surface models of the AO-stained nine-plasmid-2 system intuitively displayed that the absorbance of the 1× TAE samples increased significantly with increasing temperature compared to the 0× TAE samples, regardless of the pH value. The findings confirmed a temperature-dependent effect in AO staining of natural or synthetic multiplasmid systems, with the optimum staining result occurring at 25°C. Ion strength was a necessary condition for the temperature sensitivity mechanism. This study layed the groundwork for further investigation into the reasons or underlying mechanisms of temperature sensitivity in AO staining of multiplasmid systems.

A tryptophan-based assay method to search regulatory compounds for transcriptionally controlled tumor protein.

Transcriptionally controlled tumor protein (TCTP) is a highly conserved protein performing a large number of cellular functions by binding with various partner proteins. The importance of its roles in many diseases requires an assay method to find regulatory compounds. However, the molecular characteristics of TCTP made it difficult to search for chemicals interacting with it. In this study, a tryptophan-based assay method was designed and Y151W mutant TCTP was constructed to search binding chemicals. Since there is no tryptophan in the native sequence of TCTP, the incorporation of tryptophan in the Y151W mutant was very effective to establish the method. A flavonoid library was employed to the assay with the method. With the native and Y151W mutant TCTPs, three flavonoids such as morin, myricetin and isobavachalcone have been found to interact with TCTP. Combined with native gel electrophoresis, the binding region of isobavachalcone was suggested to be the flexible loop of TCTP. This approach can be easily applicable to find binding compounds of proteins with similar molecular characteristics of TCTP.

A rapid, inexpensive and effective method for the efficient isolation of genomic DNA from Gram-negative bacteria.

Currently, there are several protocols to extract bacterial DNA based on different principles. However, the quantity and the quality of the DNA obtained by each method are highly variable and microorganism dependent. In most of these classical crude methods, highly toxic and hazardous organic solvents such as phenol and chloroform are used for deproteinization, whereas in certain protocols, expensive enzymes including RNases and Proteinases are used. This study was designed to introduce a simple, rapid, inexpensive and effective genomic DNA isolation procedure for Gram-negative bacteria, without the usage of toxic chemicals and costly enzymes. This novel method was compared with another classical method known as the salting-out method, which uses proteinase-K. Concentration and yield of the extracted DNA were determined by gel electrophoresis by comparing the gel band intensity of the sample DNA to that of a DNA quantitation standard and by the Quantus™ fluorometer. According to the results, the yield of extracted DNA was higher in the novel method compared to the salting-out method. Moreover, the entire process was accomplished in less than 2 h with the novel method. Purity and integrity of extracted genomic DNA by both methods were similar. In addition, the quality of DNA was determined using Multicopy Associated Filamentation (MAF) gene amplification by polymerase chain reaction (PCR). Thus, the described technique is non-toxic, less time and fund consuming, efficient and a well-suited method for routine DNA isolation from Gram negative bacteria.

Comprehensive assessment of 12 commercial DNA-binding dyes as alternatives to ethidium bromide for agarose gel electrophoresis.

Staining and visualization of the nucleic acid bands on agarose gels using ethidium bromide (EB) has been a widely used technique in molecular biology. Although it is an efficient dye for this purpose, EB is known to be mutagenic and genotoxic in humans. This led to the emergence of various alternative dyes, which were claimed to be safer and more efficient than EB. However, these dyes portray varied sensitivity and interference with the electrophoretic mobility of nucleic acids. This work aimed at assessing ten nucleic acid-binding dyes and two prestained dyes for these properties by three staining techniques, such as precasting, preloading, and poststaining. Of these, preloading was not suitable for any of the dye while poststaining worked optimal for most of them. Precasting was suitable for only four dyes viz. DNA Stain G, SYBR™ safe, EZ-Vision® in-gel, and LabSafe™. Poststaining was, in general, a costlier method than precasting. The work gives a comprehensive understanding of the performance of nucleic acid-binding dyes for routine molecular biology experiments.

Sensitive and rapid analysis of plasmid DNA topology isoforms by capillary gel electrophoresis with laser induced fluorescence in uncoated capillary.

The work describes the use of SYBR Gold to improve the detection sensitivity of plasmid DNA topoisomers by capillary gel electrophoresis with laser induced fluorescence in an uncoated capillary. The impact of different dyes, including ethidium bromide, SYBR Green and SYBR Gold, was compared based on detection and separation of DNA plasmid topoisomers. Use of SYBR Gold enabled improvement of detection sensitivity by 15-fold while maintaining good separation resolution of the different topoisomers. The baseline dropped with the use SYBR Gold but was overcome by the employment of a capillary with longer ineffective length (40 vs. 20 cm). Separation resolution and reproducibility were impacted by the concentration of SYBR Gold and hydroxypropyl methylcellulose. With the use of a short capillary (10 cm effective length and 50 cm total length), fast separations of supercoiled, linear, open circular, and other isoforms were accomplished within 8 min. Appropriate capillary cleaning with 0.1 M sodium hydroxide/0.1 M hydrochloric acid and capillary storage with 0.1 M hydrochloric acid ensured good separation reproducibility of 217 runs during an extended period of use.

Residual protein analysis by SDS-PAGE in clinically manufactured BM-MSC products.

Residual substances that are considered hazardous to the recipient must be removed from final cellular therapeutic products manufactured for clinical purposes. In doing so, quality rules determined by competent authorities (CAs) for the clinical use of tissue- and cell-based products can be met. In our study, we carried out residual substance analyses, and purity determination studies of trypsin and trypsin inhibitor in clinically manufactured bone marrow-derived mesenchymal stromal/stem cell products, using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. Despite being a semiquantitative method, SDS-PAGE has several benefits over other methods for protein analysis, such as simplicity, convenience of use, and affordability. Due to its convenience and adaptability, SDS-PAGE is still a commonly used method in many laboratories, despite its limits in dynamic range and quantitative precision. Our goal in this work was to show that SDS-PAGE may be used effectively for protein measurement, especially where practicality and affordability are the major factors. The results of our study suggest a validated method to guide tissue and cell manufacturing sites for making use of an agreeable, accessible, and cost-effective method for residual substance analyses in clinically manufactured cellular therapies.

Customizable BAC-based DNA markers for pulsed-field gel electrophoresis.

DNA markers are used as a size reference and sample loading control during gel electrophoresis. Most markers are designed for conventional gel electrophoresis to separate DNA smaller than 20 kb. For larger molecules, pulsed-field gel electrophoresis (PFGE) marker is required. Limited PFGE markers are available because large DNA are prone to nicking and degradation, causing smeary bands. Here, we developed a robust marker based on bacterial artificial chromosomes (BACs) with bands up to 184 kb. This marker could consistently confer intense and distinct bands for accurate gel analysis in molecular biology studies, laboratory validations or clinical diagnosis.

SURE gel electrophoresis: A method for improved detection and purification of dilute nucleic acid samples.

Agarose gel electrophoresis is performed routinely by molecular biologists as both an analytical and a preparative method for characterization of nucleic acids. Gel analysis of highly dilute DNA solutions is challenging because of the limited sensitivity of detection available with conventional methods. In this study a new approach is described for concentrating samples directly within gels called SURE (successive reloading) electrophoresis. The approach involves loading of dilute samples multiple times into a single well, with each loading followed by a brief pulse of electrical current before the next sample is loaded. The procedure generates single bands created by molecular stacking that exhibit strongly enhanced signal intensities and minimal band broadening. Using optimized voltages and time intervals as many as 20 successive loadings could be performed and up to 800 µL could be loaded into a single well. Gel extraction and fluorescent quantitation demonstrated that approximately 97 % of the DNA from each loading was incorporated into the resultant band. Highly dilute DNA samples (<0.0007 ng per microliter) could be readily detected after six loadings. The method produced good results with either TAE or TBE as electrophoresis buffers, using loading dyes with or without SDS, and in both minigels and large gels.

Detection of SARS-CoV-2 spike protein D614G mutation using µTGGE.

BACKGROUND: The accurate and expeditious detection of SARS-CoV-2 mutations is critical for monitoring viral evolution, assessing its impact on transmission, virulence, and vaccine efficacy, and formulating public health interventions. In this study, a detection system utilizing micro temperature gradient gel electrophoresis (µTGGE) was developed for the identification of the D614 and G614 variants of the SARS-CoV-2 spike protein. METHODS: The in vitro synthesized D614 and G614 gene fragments of the SARS-CoV-2 spike protein were amplified via polymerase chain reaction and subjected to µTGGE analysis. RESULTS: The migration patterns exhibited by the D614 and G614 variants on the polyacrylamide gel were distinctly dissimilar and readily discernible by µTGGE. In particular, the mid-melting pattern of D614 was shorter than that of G614. CONCLUSIONS: Our results demonstrate the capability of µTGGE for the rapid, precise, and cost-effective detection of SARS-CoV-2 spike protein D614 and G614 variants without the need for sequencing. Therefore, this approach holds considerable potential for use in point-of-care mutation assays for SARS-CoV-2 and other pathogens.

Investigation of the seasonal prevalence, phenotypic, and genotypic characteristics of Listeria monocytogenes in slaughterhouses in Burdur.

AIM: This study examined Listeria monocytogenes isolates from two slaughterhouses in Burdur province, southern Turkey, over four seasons for antibiotic resistance, serogroups, virulence genes, in vitro biofilm forming capacity, and genetic relatedness. METHODS AND RESULTS: Carcass (540) and environment-equipment surface (180) samples were collected from two slaughterhouses (S1, S2) for 1 year (4 samplings). Of the 89 (12.4%) positive isolates, 48 (53.9%) were from animal carcasses, and 41 (46.1%) from the environment-equipment surfaces. Autumn was the peak season for Listeria monocytogenes compared to summer and spring (P < 0.05). In addition, the most common serotype between seasons was 1/2c. Except for plcA and luxS genes, all isolates (100%) harbored inlA, inlC, inlJ, hlyA, actA, iap, flaA genes. Listeria monocytogenes isolates were identified as belonging to IIc (1/2c-3c; 68.5%), IVb (4b-4d-4e; 29.2%), and IIa (1/2a-3a; 2.2%) in the screening using multiplex polymerase chain reaction-based serogrouping test. A total of 65 pulsotypes and 13 clusters with at least 80% homology were determined by using pulsed field gel electrophoresis on samples that had been digested with ApaI. Thirty-four (38.2%) of the isolates were not resistant to any of the 14 antibiotics tested. The antibiotic to which the isolates showed the most resistance was rifampicin (44.9%). Serotype 1/2c was the most resistant serotype to antibiotics. Despite having biofilm-associated genes (inlA, inlB, actA, flaA, and luxS), a minority (11%) of isolates formed weak biofilm. CONCLUSION: This study revealed seasonal changes prevalence of Listeria monocytogenes, particularly higher in autumn, posing a greater risk of meat contamination. Notably, Serotype 1/2c showed significant prevalence and antibiotic resistance. Indistinguishable isolates indicated cross-contamination, underscoring the importance of prioritized training for slaughterhouse personnel in sanitation and hygiene protocols.

Use of therapeutic plasma exchange to remove lipoprotein X in a patient with vanishing bile duct syndrome presenting with cholestasis, pseudohyponatremia, and hypercholesterolemia: A case report and review of literature.

INTRODUCTION: Lipoprotein X (Lp-X) is an abnormal lipoprotein found in multiple disease conditions, including liver dysfunction and cholestasis. High Lp-X concentrations can interfere with some laboratory testing that may result in spurious results. The detection of Lp-X can be challenging, and there is currently a lack of consensus regarding the management of Lp-X other than treating the underlying disease. CASE PRESENTATION: A 42-year-old female with Hodgkin's lymphoma treated with dexamethasone, high dose cytarabine and cisplatin and vanishing bile duct syndrome confirmed by liver biopsy presented with cholestasis, pseudohyponatremia (sodium, 113 mmol/L; reference range 136-146 mmL/L; serum osmolality, 303 mOsm/kg), and hypercholesterolemia (> 2800 mg/dL, reference range < 200 mg/dL). Lp-X was confirmed by lipoprotein electrophoresis (EP). Although she did not manifest any specific signs or symptoms, therapeutic plasma exchange (TPE) was initiated based on laboratory findings of extreme hypercholesterolemia, spuriously abnormal serum sodium, and HDL values, and the potential for short- and long-term sequelae such as hyperviscosity syndrome, xanthoma, and neuropathy. During the hospitalization, she was treated with four 1.0 plasma volume TPE over 6 days using 5% albumin for replacement fluid. After the first TPE, total cholesterol (TC) decreased to 383 mg/dL and sodium was measured at 131 mmol/L. The patient was transitioned into outpatient maintenance TPE to eliminate the potential of Lp-X reappearance while the underlying disease was treated. Serial follow-up laboratory testing with lipoprotein EP showed the disappearance of Lp-X after nine TPEs over a 10-week period. LITERATURE REVIEW: There are seven and four case reports of Lp-X treated with TPE and lipoprotein apheresis (LA), respectively. While all previous case reports showed a reduction in TC levels, none had monitored the disappearance of Lp-X after completing a course of therapeutic apheresis. CONCLUSION: Clinicians should have a heightened suspicion for the presence of abnormal Lp-X in patients with cholestasis, hypercholesterolemia, and pseudohyponatremia. Once Lp-X is confirmed by lipoprotein EP, TPE should be initiated to reduce TC level and remove abnormal Lp-X. Most LA techniques are not expected to be beneficial since Lp-X lacks apolipoprotein B. Therefore, we suggest that inpatient course of TPE be performed every other day until serum sodium, TC and HDL levels become normalized. Outpatient maintenance TPE may also be considered to keep Lp-X levels low while the underlying disease is treated. Serum sodium, TC, and HDL levels should be monitored while on maintenance TPE.

Antibiotic-Resistance Profiles and Genetic Diversity of Shigella Isolates in China: Implications for Control Strategies.

The urgent need for comprehensive and systematic analyses of Shigella as the key pathogen led us to meticulously explore the epidemiology and molecular attributes of Shigella isolates. Accordingly, we procured 24 isolates (10 from Xinjiang and 14 from Wuhan, China) and performed serotype identification and antimicrobial susceptibility testing. Resistance gene detection and homology analysis by polymerase chain reaction and pulsed-field gel electrophoresis (PFGE), respectively, were performed for genetic diversity analysis. All isolates were identified as Shigella flexneri, with 70% (35.4-91.9%) and 30% (8.1-64.6%) of the Xinjiang isolates and 85.7% (56.2-97.5%) and 14.3% (2/14, 2.5-43.9%) of the Wuhan isolates belonging to serotype 2a and serotype 2b, respectively. All isolates displayed resistance to at least two antibiotics and complete resistance to ampicillin. Multidrug resistance (MDR) was recorded in 70.8% (48.8-86.6%) of isolates, with Xinjiang isolates exhibiting relatively higher resistance to ampicillin-sulbactam, piperacillin, ceftriaxone, and aztreonam. Conversely, Wuhan isolates displayed higher MDR and resistance to tetracycline, ciprofloxacin, levofloxacin, and cefepime relative to Xinjiang isolates. Molecular scrutiny of antibiotic-resistance determinants revealed that blaTEM was the main mechanism of ampicillin resistance, blaCTX-M was the main gene for resistance to third- and fourth-generation cephalosporins, and tetB was the predominant gene associated with tetracycline resistance. Four Xinjiang and seven Wuhan isolates shared T1-clone types (>85%), and two Xinjiang and one Wuhan isolates were derived from the T6 clone with a high similarity of 87%. Six PFGE patterns (T1, T2, T5, T6-3, T8, and T10) of S. flexneri were associated with MDR. Thus, there is a critical need for robust surveillance and control strategies in managing Shigella infections, along with the development of targeted interventions and antimicrobial stewardship programs tailored to the distinct characteristics of Shigella isolates in different regions of China.

Alzheimer's Disease: A Molecular Model and Implied Path to Improved Therapy.

Amyloid-associated neurodegenerative diseases, including Alzheimer's disease (AD), are characterized by the in-brain accumulation of ß-sheet structured protein aggregates called amyloids. However, neither a disease model nor therapy is established. We review past data and present new, preliminary data and opinions to help solve this problem. The following is the data-derived model/hypothesis. (1) Amyloid-forming proteins have innate immunity functions implemented by conversion to another sheet conformation, α-sheet. (2) In health, α-sheet structured, amyloid-forming proteins inactivate microbes by co-assembly with microbe α-sheets. Amyloid-forming proteins then undergo α-to-ß-sheet conversion. (3) In disease, α-sheet-structured, amyloid-forming proteins over-accumulate and are neuron-toxic. This hypothesis includes formation by virus capsid subunits of α-sheets. In support, we find that 5-10 mM methylene blue (MB) at 54 °C has a hyper-expanding, thinning effect on the phage T4 capsid, as seen by negative stain- and cryo-electron microscopy after initial detection by native gel electrophoresis (AGE). Given the reported mild anti-AD effect of MB, we propose the following corollary hypothesis. (1) Anti-AD MB activity is, at least in part, caused by MB-binding to amyloid α-sheet and (2) MB induces the transition to α-sheet of T4 capsid subunits. We propose using AGE of drug incubated T4 to test for improved anti-AD activity.

Influence of nutrient, toxic metal and herbicide contents on the soil bacterial communities in tropical vegetable growing areas.

The relationship between bacterial diversity and the bioavailability of nutrients, toxic metals and the herbicide oxyfluorfen in a tropical vegetable growing area was evaluated. The study was conducted in a vegetable growing area located in the mountainous region of Rio de Janeiro (Brazil), and samples were collected in areas of vegetable cultivation and areas of environmental reserve. Fertility analyses and determination of the pseudototal levels of toxic metals in the soil samples were performed. The profile of the soil bacterial community was determined by amplification of the 16S rRNA gene and separation by DGGE. The results showed that the levels of toxic metals and elements associated with soil fertility were higher in vegetable production areas. These differences in the physical and chemical characteristics of the soil favored the presence of a greater number of OTUs in the cultivation areas (17.3-27 OTUs) than in the areas of environmental reserve (13-22 OTUs). Therefore, this study demonstrates that the presence of toxic metals and the herbicide oxyfluorfen and the increase in fertility in soils in areas with intensive vegetable cultivation resulting from the intensive management adopted in these areas promotes a differentiation of the bacterial profiles in soils in tropical vegetable growing areas.

Proteomic profiling of Arabidopsis G-protein ß subunit AGB1 mutant under salt stress.

Salt stress is a limiting environmental factor that inhibits plant growth in most ecological environments. The functioning of G-proteins and activated downstream signaling during salt stress is well established and different G-protein subunits and a few downstream effectors have been identified. Arabidopsis G-protein ß-subunit (AGB1) regulates the movement of Na+ from roots to shoots along with a significant role in controlling Na+ fluxes in roots, however, the molecular mechanism of AGB1 mediated salt stress regulation is not well understood. Here, we report the comparative proteome profiles of Arabidopsis AGB1 null mutant agb1-2 to investigate how the absence of AGB1 modulates the protein repertoire in response to salt stress. High-resolution two-dimensional gel electrophoresis (2-DE) showed 27 protein spots that were differentially modulated between the control and NaCl treated agb1-2 seedlings of which seven were identified by mass spectrometry. Functional annotation and interactome analysis indicated that the salt-responsive proteins were majorly associated with cellulose synthesis, structural maintenance of chromosomes, DNA replication/repair, organellar RNA editing and indole glucosinolate biosynthesis. Further exploration of the functioning of these proteins could serve as a potential stepping stone for dissection of molecular mechanism of AGB1 functions during salt stress and in long run could be extrapolated to crop plants for salinity stress management.

Accelerated Screening of Alternative DNA Base-Organic Molecule-Base Architectures via Integrated Theory and Experiment.

Organic molecule-mediated noncanonical DNA self-assembly expands the standard DNA base-pairing alphabets. However, only a very limited number of small molecules have been recognized as mediators because of the tedious and complicated experiments like crystallization and microscopy imaging. Here we present an integrative screening protocol incorporating molecular dynamics (MD) for fast theoretical simulation and native polyacrylamide gel electrophoresis for convenient experimental validation. Melamine, the molecule that was confirmed mediating noncanonical DNA base-pairing, and 38 other candidate molecules were applied to demonstrate the feasibility of this protocol. We successfully identified seven stable noncanonical DNA duplex structures, and another eight novel structures with sub-stability. In addition, we discovered that hairpins at both ends can significantly stabilize the noncanonical DNA structures, providing a guideline to design small organic molecule-incorporated DNA structures. Such an efficient screening protocol will accelerate the design of alternative DNA-molecule architectures beyond Watson-Crick pairs. Considering the wide range of potential mediators, it will also facilitate applications such as noncovalent, highly dense loading of drug molecules in DNA-based delivery system and probe design for sensitive detection of certain molecules.

Sometimes faster can be better: Microneedling IPG strips enables higher throughput for integrative top-down proteomics.

Passive rehydration of immobilized pH gradient (IPG) strips for two-dimensional gel electrophoresis (2DE) has, to our knowledge, never been quantitatively evaluated to determine an ideal rehydration time. Seeking to increase throughput without sacrificing analytical rigor, we report that a substantially shorter rehydration time is accomplished when surface area of IPG strips is increased via microneedling. Rehydration for 4 h, post microneedling, provides comparable results to overnight rehydration in final analyses by 2DE, while also shortening the overall protocol by 1 day.

Controlled Experimental Infection in Pigs with a Strain of Yersinia enterocolitica Harboring Genetic Markers for Human Pathogenicity: Colonization and Stability.

Yersinia enterocolitica (Ye) is one of the major causes of foodborne zoonosis. The BT4/O:3 bioserotype is most commonly isolated in human infections. Pigs are considered the main reservoir of Ye, and hence, understanding the dynamics of infection by this pathogen at the individual and group levels is crucial. In the present study, an experimental model was validated in Large White pigs infected with a BT4/O:3 strain. This study showed that Ye contamination in pigs may occur via the introduction of the bacteria not only by mouth but also by snout, with a colonization process consisting of three periods corresponding to three contamination statuses of pigs: P1, corresponding to the 24 h following ingestion or inhalation of Ye with the appearance of bacteria in tonsils or in feces; P2, from 2 days postinoculation (dpi), corresponding to expansion of Ye and colonization of the digestive system and extraintestinal organs associated with an IgG serological response; and P3, after 21 dpi, corresponding to regression of colonization with intermittent Ye detection in tonsils and feces. Although the inoculated strain persisted up to 56 dpi in all pigs, genetic variations with the loss of the gene yadA (a gene involved in human infection) and the emergence of two new multilocus variable-number tandem-repeat analysis (MLVA) profiles were observed in 33% of the 30 isolates studied. This experimental infection model of pigs by Ye provides new insights into the colonization steps in pigs in terms of bacterial distribution over time and bacterial genetic stability.

Characterization of host and escherichia coli strains causing recurrent urinary tract infections based on molecular typing.

BACKGROUND: Escherichia coli is the leading pathogen responsible for urinary tract infection (UTI) and recurrent UTI (RUTI). Few studies have dealt with the characterization of host and bacteria in RUTI caused by E. coli with genetically identical or different strains. This study aimed to investigate the host and bacterial characteristics of E. coli RUTI based on molecular typing. RESULTS: Patients aged 20 years or above who presented with symptoms of UTI in emergency department or outpatient clinics between August 2009 and December 2010 were enrolled. RUTI was defined as patients had 2 or more infections in 6 months or 3 or more in 12 months during the study period. Host factors (including age, gender, anatomical/functional defect, and immune dysfunction) and bacterial factors (including phylogenicity, virulence genes, and antimicrobial resistance) were included for analysis. There were 41 patients (41%) with 91 episodes of E. coli RUTI with highly related PFGE (HRPFGE) pattern (pattern similarity > 85%) and 58 (59%) patients with 137 episodes of E. coli RUTI with different molecular typing (DMT) pattern, respectively. There was a higher prevalence of phylogenetic group B2 and neuA and usp genes in HRPFGE group if the first episode of RUTI caused by HRPFGE E. coli strains and all episodes of RUTI caused by DMT E. coli strains were included for comparison. The uropathogenic E. coli (UPEC) strains in RUTI were more virulent in female gender, age < 20 years, neither anatomical/ functional defect nor immune dysfunction, and phylogenetic group B2. There were correlations among prior antibiotic therapy within 3 months and subsequent antimicrobial resistance in HRPFGE E. coli RUTI. The use of fluoroquinolones was more likely associated with subsequent antimicrobial resistance in most types of antibiotics. CONCLUSIONS: This study demonstrated that the uropathogens in RUTI were more virulent in genetically highly-related E. coli strains. Higher bacterial virulence in young age group (< 20 years) and patients with neither anatomical/functional defect nor immune dysfunction suggests that virulent UPEC strains are needed for the development of RUTI in healthy populations. Prior antibiotic therapy, especially the fluoroquinolones, within 3 months could induce subsequent antimicrobial resistance in genetically highly-related E. coli RUTI.