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Genomic surveillance detected clonal Escherichia coli sequence type-361 isolates carrying bla NDM-5 , bla KPC-3 , bla CTX-M-15 , and rmtB1 from a patient in Ukraine and four wounded foreign soldiers evacuated to Germany. Isolates were non-susceptible to carbapenems, aminoglycosides, and cefiderocol and aztreonam/avibactam due to a PBP3 YRIN insertion and the bla CMY-145 AmpC β-lactamase. Coordinated surveillance efforts across civilian, military, and veteran healthcare systems are essential to prevent further spread as international volunteers return home after medical evacuation from Ukraine., Competing Interests: The authors declare no conflict of interest.

Carriage of CTX-M-type extended-spectrum β-lactamase (ESBL) is rare in Pseudomonas aeruginosa . During routine surveillance of an endemic ST-621 P. aeruginosa at a large hospital, isolate MRSN 100690 carrying bla CTX-M-15 was cultured from a patient (P2). This was the first detection of this ESBL in the endemic ST-621 lineage. All 1 488 bacterial isolates collected from the same facility in the 12 months prior to the incidence of 100 690 were screened for the presence of bla CTX-M-15 . A set of 183 isolates was identified, in which corresponding patient metadata was evaluated for spatiotemporal overlaps with P2. The resulting three isolates, along with 100 690, were long-read sequenced using the Oxford Nanopore MinION platform to determine a potential donor of bla CTX-M-15 . The screen revealed a single Klebsiella michiganensis isolate, MRSN 895358, which carried an IncA/C2 plasmid harbouring bla CTX-M-15 . Notably, the patient harbouring 895358, P1, occupied the same hospital room as P2 9 months prior. Genomic alignment revealed that both isolates shared an identical 80.8 kb region containing the IncA/C2 plasmid replicon and bla CTX-M-15 . This region was plasmid bound in 895 358, but chromosomally bound in 100 690 due to Tn 4661 -mediated transposition. ESBL bla CTX-M-15 was acquired and subsequently integrated into the chromosome of a ST-621 P. aeruginosa , likely initiated by plasmid transfer from a K. michiganensis strain.

Background: Quantitating the contribution of phenotype-responsible elements in hypervirulent Klebsiella pneumoniae is needed., Methods: Isogenic mutants of four hypervirulent clinical isolates that produced K1 (ST23), K2 (ST86), K20 (ST1544), or K54 (ST29) capsules (mean 2.2 log 10 LD 50 (range 1.5-2.9)) were created to measure the effects on LD 50 in a murine model of the hypervirulence-associated plasmid (pVir), iucA, p rmpA, p rmpA2 (truncated), irp2, and clbBC., Findings: Curing pVir had the greatest increase in survival (mean LD 50 to 7.6 (range 7.0-9.0, p ≤ 0.0001), a dosage comparable to classical K. pneumoniae. Results also showed increased mean LD 50 s for Δ p rmpA (5.9, p ≤ 0.0001), ΔiucA (3.6, p ≤ 0.0001), Δirp2 (3.4), ΔrmpAΔiucA (6.3, p ≤ 0.0001), and ΔpVirΔirp2 (8.7, p ≤ 0.0001). Notably ΔpVir had an additional mean LD 50 increase of 1.3 compared to the pVir-encoded Δ p rmpAΔiucA (p ≤ 0.01), suggesting presence of additional pVir-virulence genes. Truncated p RmpA2 did not contribute to virulence. Odd ratios in the absence of pVir/yersiniabactin, pVir, p RmpA/aerobactin, p RmpA, aerobactin, yersiniabactin, and colibactin demonstrated a 250-fold, 67-fold, 20-fold, 16.7-fold, 9.6-fold, and 1.7-fold decrease in lethality respectively., Interpretation: These data can guide countermeasure development., Funding: This work was supported by NIH R21 AI123558-01 and 1R21AI141826-01A1 (Dr. Russo) and the Department of Veterans Affairs VA Merit Review (I01 BX004677-01) (Dr. Russo). This study was also partially funded by the U.S. Defense Health Program (DHP) Operations and Maintenance., Competing Interests: Declaration of interests The authors declare that they have no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

In 2003-2023, amid 5,436 Acinetobacter baumannii isolates collected globally through the Multidrug-Resistant Organism Repository and Surveillance Network, 97 were ST19PAS, 34 of which carbapenem-resistant. Strains (n = 32) sampled after 2019 harboured either bla OXA-23 , bla OXA-72 , and/or bla NDM-5 . Phylogenetic analysis of the 97 isolates and 11 publicly available ST19 genomes revealed three sub-lineages of carbapenemase-producing isolates from mainly Ukraine and Georgia, including an epidemic clone carrying all three carbapenemase genes. Infection control and global surveillance of carbapenem-resistant A. baumannii remain important.

Distinguishing hypervirulent (hvKp) from classical Klebsiella pneumoniae (cKp) strains is important for clinical care, surveillance, and research. Some combinations of iucA, iroB, peg-344, rmpA, and rmpA2 are most commonly used, but it is unclear what combination of genotypic or phenotypic markers (e.g., siderophore concentration, mucoviscosity) most accurately predicts the hypervirulent phenotype. Furthermore, acquisition of antimicrobial resistance may affect virulence and confound identification. Therefore, 49 K . pneumoniae strains that possessed some combinations of iucA, iroB, peg-344, rmpA, and rmpA2 and had acquired resistance were assembled and categorized as hypervirulent hvKp (hvKp) ( N = 16) or cKp ( N = 33) via a murine infection model. Biomarker number, siderophore production, mucoviscosity, virulence plasmid's Mash/Jaccard distances to the canonical pLVPK, and Kleborate virulence score were measured and evaluated to accurately differentiate these pathotypes. Both stepwise logistic regression and a CART model were used to determine which variable was most predictive of the strain cohorts. The biomarker count alone was the strongest predictor for both analyses. For logistic regression, the area under the curve for biomarker count was 0.962 ( P = 0.004). The CART model generated the classification rule that a biomarker count = 5 would classify the strain as hvKP, resulting in a sensitivity for predicting hvKP of 94% (15/16), a specificity of 94% (31/33), and an overall accuracy of 94% (46/49). Although a count of ≥4 was 100% (16/16) sensitive for predicting hvKP, the specificity and accuracy decreased to 76% (25/33) and 84% (41/49), respectively. These findings can be used to inform the identification of hvKp.IMPORTANCEHypervirulent Klebsiella pneumoniae (hvKp) is a concerning pathogen that can cause life-threatening infections in otherwise healthy individuals. Importantly, although strains of hvKp have been acquiring antimicrobial resistance, the effect on virulence is unclear. Therefore, it is of critical importance to determine whether a given antimicrobial resistant K. pneumoniae isolate is hypervirulent. This report determined which combination of genotypic and phenotypic markers could most accurately identify hvKp strains with acquired resistance. Both logistic regression and a machine-learning prediction model demonstrated that biomarker count alone was the strongest predictor. The presence of all five of the biomarkers iucA, iroB, peg-344, rmpA, and rmpA2 was most accurate (94%); the presence of ≥4 of these biomarkers was most sensitive (100%). Accurately identifying hvKp is vital for surveillance and research, and the availability of biomarker data could alert the clinician that hvKp is a consideration, which, in turn, would assist in optimizing patient care., Competing Interests: The authors declare no conflict of interest.

An outbreak involving an extensively antibiotic-resistant Acinetobacter baumannii strain in three military treatment facilities was identified. Fifty-nine isolates recovered from 30 patients over a 4-year period were found among a large collection of isolates using core genome multilocus sequence typing (MLST). They differed by only 0 to 18 single nucleotide polymorphisms (SNPs) and carried the same resistance determinants except that the aphA6 gene was missing in 25 isolates. They represent a novel sublineage of GC1 lineage 1 that likely originated in Afghanistan. IMPORTANCE A. baumannii is recognized as one of the most important nosocomial pathogens, and carbapenem-resistant strains pose a particularly difficult treatment challenge. Outbreaks linked to this pathogen are reported worldwide, particularly during periods of societal upheaval, such as natural disasters and conflicts. Understanding how this organism enters and establishes itself within the hospital environment is key to interrupting transmission, but few genomic studies have examined these transmissions over a prolonged period. Though historical, this report provides an in-depth analysis of nosocomial transmission of this organism across continents and within and between different hospitals., Competing Interests: The authors declare no conflict of interest.

Calcium-permeable channels in the plasma membrane play vital roles in plant growth, development, and response to environmental stimuli. Arabidopsis possesses 20 glutamate receptor-like proteins that share similarities with animal ionotropic glutamate receptors and mediate Ca 2+ influx in plants. Calcium-dependent protein kinases (CDPKs) phosphorylate serine (Ser)-860 of glutamate receptor-like (GLR)3.7 protein, which interacts with 14-3-3ω and plays an essential role in salt and abscisic acid response in Arabidopsis by modulating Ca 2+ signaling. However, the significance of CDPK- mediated phosphorylation status of Ser residues of GLR3.6 with regard to the functioning of GLR3.6 remains to be elucidated. In this study, we performed an in vitro kinase assay using CDPK16 and peptides containing the 14-3-3ω interacting domain of GLR3.6. We showed that Ser861/862 of GLR3.6 are required for the interaction with 14-3-3ω and that Ser856 of GLR3.6 is specifically phosphorylated by CDPK16 but not by CDPK3 and CDPK34. In addition, the expression of GLR3.6 was quickly downregulated by salt stress, and plants of glr3.6 mutants and GLR3.6 -overexpression lines presented shorter and longer root lengths, respectively, under normal growth conditions than Col. Overexpression of the GLR3.6-Ser856 to Ala mutation resulted in a less sensitive phenotype in response to salt stress similar to glr3.6 . Our results indicated that the Ser861/862 residues of GLR3.6 are required for interaction with 14-3-3ω. Additionally, the phosphorylation status of Ser856 residue of GLR3.6, which is mediated specifically by CDPK16, regulates root growth in normal and salt stress and conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Silamparasan, Chang and Jinn.)

Background: Extra-intestinal pathogenic Escherichia coli (ExPEC) are a leading cause of bloodstream and urinary tract infections worldwide. Over the last two decades, increased rates of antibiotic resistance in E. coli have been reported, further complicating treatment. Worryingly, specific lineages expressing extended-spectrum β-lactamases (ESBLs) and fluoroquinolone resistance have proliferated and are now considered a serious threat. Obtaining contemporary information on the epidemiology and prevalence of these circulating lineages is critical for containing their spread globally and within the clinic., Methods: Whole-genome sequencing (WGS), phylogenetic analysis, and antibiotic susceptibility testing were performed for a complete set of 2075 E. coli clinical isolates collected from 1776 patients at a large tertiary healthcare network in the USA between October 2019 and September 2020., Results: The isolates represented two main phylogenetic groups, B2 and D, with six lineages accounting for 53% of strains: ST-69, ST-73, ST-95, ST-131, ST-127, and ST-1193. Twenty-seven percent of the primary isolates were multidrug resistant (MDR) and 5% carried an ESBL gene. Importantly, 74% of the ESBL-E.coli were co-resistant to fluoroquinolones and mostly belonged to pandemic ST-131 and emerging ST-1193. SNP-based detection of possible outbreaks identified 95 potential transmission clusters totaling 258 isolates (12% of the whole population) from ≥ 2 patients. While the proportion of MDR isolates was enriched in the set of putative transmission isolates compared to sporadic infections (35 vs 27%, p = 0.007), a large fraction (61%) of the predicted outbreaks (including the largest cluster grouping isolates from 12 patients) were caused by the transmission of non-MDR clones., Conclusion: By coupling in-depth genomic characterization with a complete sampling of clinical isolates for a full year, this study provides a rare and contemporary survey on the epidemiology and spread of E. coli in a large US healthcare network. While surveillance and infection control efforts often focus on ESBL and MDR lineages, our findings reveal that non-MDR isolates represent a large burden of infections, including those of predicted nosocomial origins. This increased awareness is key for implementing effective WGS-based surveillance as a routine technology for infection control., (© 2022. The Author(s).)

Carbapenem-resistant Enterobacterales pose an urgent threat to human health worldwide. Klebsiella pneumoniae sequence type (ST) 14, initially identified in the Middle East and South-Asia and co-harbouring the carbapenemase genes bla OXA-232 and bla NDM-1, is now emerging globally. One such strain was detected in the USA in 2013 from a patient initially treated in India that also carried armA , a 16S rRNA methyltransferase that confers resistance to all clinically relevant aminoglycosides. Genetic and phenotypic changes were observed in 14 serial isolates collected from this chronically infected patient. The index isolate carried five plasmids, including an IncFIB-IncHI1B (harbouring armA and bla NDM-1 ), an IncFIA ( bla CTX-M-15 ) and a ColE-like ( bla OXA-232 ), and was extensively resistant to antibiotics. Four years later, a subsequent isolate had accumulated 34 variants, including a loss-of-function mutation in romA , resulting in tigecycline non-susceptibility. Importantly, this isolate now only carried two plasmids, including a large mosaic molecule made of fragments, all harbouring distinct toxin-antitoxin systems, from three of the canonical plasmids. Of the original acquired antibiotic resistance genes, this isolate only retained bla CTX-M-15 , and as a result susceptibility to the carbapenems and amikacin was restored. Long-read sequencing of a subset of five representative isolates, collected between 2013 and 2017, allowed for the elucidation of the complex plasmid patterns and revealed the role of IS 26 -mediated plasmid reshuffling in the evolution of this clone. Such investigations of the mechanisms underlying plasmid stability, together with global and local surveillance programmes, are key to a better understanding of plasmid host range and dissemination.

Objective: It is unclear whether tea infusions with or without sucrose supplementation alter oral biofilm development, so we evaluated the effect of unsweetened and sucrose-sweetened black and green tea infusions on in vitro saliva-derived biofilms., Design: Biofilms were developed from human saliva for 20 h in cell-free 25% human saliva within static glass-bottom microplates. During biofilm development, biofilms were treated with either (i) unsweetened black tea, (ii) unsweetened green tea, (iii) 10% sucrose-sweetened black tea, (iv) 10% sucrose-sweetened green tea (v) deionized water (negative control), or (vi) 10% sucrose (positive control). Biofilms were incubated at 37 °C in 5% CO 2 . After 20 h of development, biofilms were imaged using a CLSM, and biofilm architecture and viability were evaluated., Results: All the tea infusions reduced biofilm biomass and altered some other biofilm architectural outcomes (e.g., biofilm surface area) compared to the control groups. Statistically significant differences in biofilm biomass, number of objects, surface area, and convex-hull porosity were observed between biofilms treated with green and black tea. The addition of sugar to tea did not significantly modify the ability of tea to alter biofilm architecture. Only the treatment of biofilms with unsweetened black tea significantly reduced bacterial viability., Conclusions: While both teas reduced biofilm biomass and altered biofilm architecture, black tea had an enhanced effect that may relate to this tea's observed antimicrobial activity. The addition of sucrose to tea infusions did not appear to reduce the impact of either tea in modifying oral biofilm architecture., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

It has been reported that the mitochondrial carrier family proteins of AtMTM1 and AtMTM2 are necessary for manganese superoxide dismutase (MnSOD) activation in Arabidopsis , and are responsive to methyl viologen (MV)-induced oxidative stress. In this study, we showed that MnSOD activity was enhanced specifically by Mn treatments. By using AtMnSOD -overexpressing and AtMnSOD -knockdown mutant plants treated with the widely used oxidative stressors including MV, NaCl, H 2 O 2 , and tert-butyl hydroperoxide (t-BH), we revealed that Arabidopsis MnSOD was crucial for root-growth control and superoxide scavenging ability. In addition, it has been reported that E . coli MnSOD activity is inhibited by Fe and that MTM1 -mutated yeast cells exhibit elevated Fe content and decreased MnSOD activity, which can be restored by the Fe 2+ -specific chelator, bathophenanthroline disulfonate (BPS). However, we showed that BPS inhibited MnSOD activity in AtMTM1 and AtMTM2 single- and double-mutant protoplasts, implying that altered Fe homeostasis affected MnSOD activation through AtMTM1 and AtMTM2. Notably, we used inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis to reveal an abnormal Fe/Mn ratio in the roots and shoots of AtMTM1 and AtMTM2 mutants under MV stress, indicating the importance of AtMTM1 in roots and AtMTM2 in shoots for maintaining Fe/Mn balance.

Pectin is a major component of the plant cell wall, forming a network that contributes to cell wall integrity and flexibility. Pectin methylesterase (PME) catalyzes the removal of methylester groups from the homogalacturonan backbone, the most abundant pectic polymer, and contributes to intercellular adhesion during plant development and different environmental stimuli stress. In this study, we identified and characterized an Arabidopsis type-II PME , PME53 , which encodes a cell wall deposited protein and may be involved in the stomatal lineage pathway and stomatal functions. We demonstrated that PME53 is expressed explicitly in guard cells as an abscisic acid (ABA)-regulated gene required for stomatal movement and thermotolerance. The expression of PME53 is significantly affected by the stomatal differentiation factors SCRM and MUTE. The null mutation in PME53 results in a significant increase in stomatal number and susceptibility to ABA-induced stomatal closure. During heat stress, the pme53 mutant highly altered the activity of PME and significantly lowered the expression level of the calmodulin AtCaM3 , indicating that PME53 may be involved in Ca 2+ -pectate reconstitution to render plant thermotolerance. Here, we present evidence that the PME53-mediated de-methylesterification status of pectin is directed toward stomatal development, movement, and regulation of the flexibility of the guard cell wall required for the heat response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wu, Yu, Wang and Jinn.)

Numerous in vitro biofilm model systems are available to study oral biofilms. Over the past several decades, increased understanding of oral biology and advances in technology have facilitated more accurate simulation of intraoral conditions and have allowed for the increased generalizability of in vitro oral biofilm studies. The integration of contemporary systems with confocal microscopy and 16S rRNA community profiling has enhanced the capabilities of in vitro biofilm model systems to quantify biofilm architecture and analyse microbial community composition. In this review, we describe several model systems relevant to modern in vitro oral biofilm studies: the constant depth film fermenter, Sorbarod perfusion system, drip-flow reactor, modified Robbins device, flowcells and microfluidic systems. We highlight how combining these systems with confocal microscopy and community composition analysis tools aids exploration of oral biofilm development under different conditions and in response to antimicrobial/anti-biofilm agents. The review closes with a discussion of future directions for the field of in vitro oral biofilm imaging and analysis., (© 2021 The Society for Applied Microbiology.)

The manganese (Mn) tracking factor for mitochondrial Mn superoxide dismutase (MnSOD) has been annotated as yMTM1 in yeast, which belongs to the mitochondrial carrier family. We confirmed that Arabidopsis AtMTM1 and AtMTM2 are functional homologs of yMTM1 as they can revive yeast MnSOD activity in yMTM1 -mutant cells. Transient expression of AtMnSOD-3xFLAG in the AtMTM1 and AtMTM2 -double mutant protoplasts confirmed that AtMTM1 and AtMTM2 are required for AtMnSOD activation. Our study revealed that AtMnSOD interacts with AtMTM1 and AtMTM2 in the mitochondria. The expression levels of AtMTM1 , AtMTM2 , and AtMnSOD respond positively to methyl viologen (MV) and metal stress. AtMTM1 and AtMTM2 are involved in Mn and Fe homeostasis, root length, and flowering time. Transient expression of chloroplast-destined AtMnSOD revealed that an evolutionarily conserved activation mechanism, like the chloroplastic-localized MnSOD in some algae, still exists in Arabidopsis chloroplasts. This study strengthens the proposition that AtMTM1 and AtMTM2 participate in the AtMnSOD activation and ion homeostasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hu, Lin, Huang, Huang, Kuo and Jinn.)

The abscisic acid (ABA) and chaperone signaling pathways are the central regulators of plant stress defense. Despite their significance and potential overlap, these systems have been described separately. In this review, we summarize information about mechanisms by which the ABA and chaperone signaling pathways might be coregulated. The central factors that join the ABA and chaperone signaling systems are the SWI/SNF chromatin-remodeling proteins, which are involved in stress memory. A benefit from coordination is that the signals sensed through both the ABA and chaperone signaling systems are perceived and stored via chromatin-remodeling factors. For improving plant stress resistance, we propose new bioengineering strategies, which we term 'bioengineering memory'., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Biofilm model systems are used to study biofilm growth and predict the effects of anti-biofilm interventions within the human oral cavity. Many in vitro biofilm model systems use a confocal laser scanning microscope (CLSM) in conjunction with image analysis tools to study biofilms. The aim of this study was to evaluate an in-house developed image analysis software program that we call BAIT (Biofilm Architecture Inference Tool) to quantify the architecture of oral multi-species biofilms following anti-biofilm interventions using a microfluidic biofilm system. Differences in architecture were compared between untreated biofilms and those treated with water (negative control), sodium gluconate ('placebo') or stannous fluoride (SnF2). The microfluidic system was inoculated with pooled human saliva and biofilms were developed over 22 h in filter-sterilized 25 % pooled human saliva. During this period, biofilms were treated with water, sodium gluconate, or SnF2 (1000, 3439 or 10 000 p.p.m. Sn 2+ ) 8 and 18 h post-inoculation. After 22 h of growth, biofilms were stained with LIVE/DEAD stain, and imaged by CLSM. BAIT was used to calculate biofilm biovolume, total number of objects, surface area, fluffiness, connectivity, convex hull porosity and viability. Image analysis showed oral biofilm architecture was significantly altered by 3439 and 10 000 p.p.m. Sn 2+ treatment regimens, resulting in decreased biovolume, surface area, number of objects and connectivity, while fluffiness increased (P<0.01). In conclusion, BAIT was shown to be able to measure the changes in biofilm architecture and detects possible antimicrobial and anti-biofilm effects of candidate agents.

Heat stress (HS) is expected to be of increasing worldwide concern in the near future, especially with regard to crop yield and quality as a consequence of rising or varying temperatures as a result of global climate change. HS response (HSR) is a highly conserved mechanism among different organisms but shows remarkable complexity and unique features in plants. The transcriptional regulation of HSR is controlled by HS transcription factors (HSFs) which allow the activation of HS-responsive genes, among which HS proteins (HSPs) are best characterized. Cell wall remodeling constitutes an important component of plant responses to HS to maintain overall function and growth; however, little is known about the connection between cell wall remodeling and HSR. Pectin controls cell wall porosity and has been shown to exhibit structural variation during plant growth and in response to HS. Pectin methylesterases (PMEs) are present in multigene families and encode isoforms with different action patterns by removal of methyl esters to influencing the properties of cell wall. We aimed to elucidate how plant cell walls respond to certain environmental cues through cell wall-modifying proteins in connection with modifications in cell wall machinery. An overview of recent findings shed light on PMEs contribute to a change in cell-wall composition/structure. The fine-scale modulation of apoplastic calcium ions (Ca 2+ ) content could be mediated by PMEs in response to abiotic stress for both the assembly and disassembly of the pectic network. In particular, this modulation is prevalent in guard cell walls for regulating cell wall plasticity as well as stromal aperture size, which comprise critical determinants of plant adaptation to HS. These insights provide a foundation for further research to reveal details of the cell wall machinery and stress-responsive factors to provide targets and strategies to facilitate plant adaptation.

Biofilms are surface-attached microbial communities whose architecture can be captured with confocal microscopy. Manual or automatic thresholding of acquired images is often needed to help distinguish biofilm biomass from background noise. However, manual thresholding is subjective and current automatic thresholding methods can lead to loss of meaningful data. Here, we describe an automatic thresholding method designed for confocal fluorescent signal, termed the biovolume elasticity method (BEM). We evaluated BEM using confocal image stacks of oral biofilms grown in pooled human saliva. Image stacks were thresholded manually and automatically with three different methods; Otsu, iterative selection (IS), and BEM. Effects on biovolume, surface area, and number of objects detected indicated that the BEM was the least aggressive at removing signal, and provided the greatest visual and quantitative acuity of single cells. Thus, thresholding with BEM offers a sensitive, automatic, and tunable method to maintain biofilm architectural properties for subsequent analysis.

Dormancy-associated antigens encoded by the dormancy survival regulon (DosR) genes are required for survival of Mycobacterium tuberculosis (Mtb) in macrophages. However, mechanisms underlying survival of Mtb in macrophages remains to be elucidated. A recombinant Mycobacterium smegmatis strain (rMs) expressing a fusion protein of two dormancy‑associated antigens Rv2031c and Rv2626c from Mtb was constructed in the present study. In an in vitro culture, growth rate of rMs was lower compared with Ms. A total of 24 h following infection of murine macrophages with rMs or Ms, percentage of viable cells decreased and the number of bacteria in viable cells increased compared with Ms, demonstrating that virulence and intracellular survival of rMs were enhanced. Compared with macrophages infected with Ms, necrosis of macrophages infected with rMs was increased, while apoptosis was inhibited. Macrophages infected with rMs secreted more interferon‑γ and interleukin‑6, but fewer nitric oxide and tumor necrosis factor‑α, compared with macrophages infected with Ms. The present study demonstrated that the fusion protein composed of dormancy‑associated antigens Rv2031c and Rv2626c in Ms serves a physiological function of a dormancy‑associated antigen and modulates innate immunity of host macrophages, therefore favoring intracellular bacillary survival.

The number of stomata on leaves can be affected by intrinsic development programming and various environmental factors, in addition the control of stomatal apertures is extremely important for the plant stress response. In response to elevated temperatures, transpiration occurs through the stomatal apertures, allowing the leaf to cool through water evaporation. As such, monitoring of stomata behavior to elevated temperatures remains as an important area of research. The protocol allows analysis of stomatal aperture, morphology, and density through a non-destructive imprint of Arabidopsis thaliana leaf surface. Stomatal counts were performed and observed under a scanning electron microscope., (Copyright © 2017 The Authors; exclusive licensee Bio-protocol LLC.)

Pectin is an important cell wall polysaccharide required for cellular adhesion, extension, and plant growth. The pectic methylesterification status of guard cell walls influences the movement of stomata in response to different stimuli. Pectin methylesterase (PME) has a profound effect on cell wall modification, especially on the degree of pectic methylesterification during heat response. The Arabidopsis thaliana PME34 gene is highly expressed in guard cells and in response to the phytohormone abscisic acid. The genetic data highlighted the significant role of PME34 in heat tolerance through the regulation of stomatal movement. Thus, the opening and closure of stomata is mediated by changes in response to a given stimulus, could require a specific cell wall modifying enzyme to function properly.

Pectin, a major component of the primary cell wall, is synthesized in the Golgi apparatus and exported to the cell wall in a highly methylesterified form, then is partially demethylesterified by pectin methylesterases (PMEs; EC 3.1.1.11). PME activity on the status of pectin methylesterification profoundly affects the properties of pectin and, thereby, is critical for plant development and the plant defense response, although the roles of PMEs under heat stress (HS) are poorly understood. Functional genome annotation predicts that at least 66 potential PME genes are contained in Arabidopsis ( Arabidopsis thaliana ). Thermotolerance assays of PME gene T-DNA insertion lines revealed two null mutant alleles of PME34 (At3g49220) that both consistently showed reduced thermotolerance. Nevertheless, their impairment was independently associated with the expression of HS-responsive genes. It was also observed that PME34 transcription was induced by abscisic acid and highly expressed in guard cells. We showed that the PME34 mutation has a defect in the control of stomatal movement and greatly altered PME and polygalacturonase (EC 3.2.1.15) activity, resulting in a heat-sensitive phenotype. PME34 has a role in the regulation of transpiration through the control of the stomatal aperture due to its cell wall-modifying enzyme activity during the HS response. Hence, PME34 is required for regulating guard cell wall flexibility to mediate the heat response in Arabidopsis., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Extracellular DNA (eDNA) has been identified in the matrix of many different monospecies biofilms in vitro, including some of those produced by oral bacteria. In many cases, eDNA stabilizes the structure of monospecies biofilms. Here, the authors aimed to determine whether eDNA is an important component of natural, mixed-species oral biofilms, such as plaque on natural teeth or dental implants. To visualize eDNA in oral biofilms, approaches for fluorescently stained eDNA with either anti-DNA antibodies or an ultrasensitive cell-impermeant dye, YOYO-1, were first developed using Enterococcus faecalis, an organism that has previously been shown to produce extensive eDNA structures within biofilms. Oral biofilms were modelled as in vitro "microcosms" on glass coverslips inoculated with the natural microbial population of human saliva and cultured statically in artificial saliva medium. Using antibodies and YOYO-1, eDNA was found to be distributed throughout microcosm biofilms, and was particularly abundant in the immediate vicinity of cells. Similar arrangements of eDNA were detected in biofilms on crowns and overdenture abutments of dental implants that had been recovered from patients during the restorative phase of treatment, and in subgingival dental plaque of periodontitis patients, indicating that eDNA is a common component of natural oral biofilms. In model oral biofilms, treatment with a DNA-degrading enzyme, NucB from Bacillus licheniformis, strongly inhibited the accumulation of biofilms. The bacterial species diversity was significantly reduced by treatment with NucB and particularly strong reductions were observed in the abundance of anaerobic, proteolytic bacteria such as Peptostreptococcus, Porphyromonas and Prevotella. Preformed biofilms were not significantly reduced by NucB treatment, indicating that eDNA is more important or more exposed during the early stages of biofilm formation. Overall, these data demonstrate that dental plaque eDNA is potentially an important target for oral biofilm control.

Heat stress response (HSR) is a conserved mechanism developed to increase the expression of heat shock proteins (HSPs) via a heat shock factor (HSF)-dependent mechanism. Signaling by the stress phytohormone abscisic acid (ABA) is involved in acquired thermotolerance as well. Analysis of Arabidopsis (Arabidopsis thaliana) microarray databases revealed that the expression of HSFA6b, a class A HSF, extensively increased with salinity, osmotic, and cold stresses, but not heat. Here, we show that HSFA6b plays a pivotal role in the response to ABA and in thermotolerance. Salt-inducible HSFA6b expression was down-regulated in ABA-insensitive and -deficient mutants; however, exogenous ABA application restored expression in ABA-deficient, but not -insensitive plants. Thus, ABA signaling is required for proper HSFA6b expression. A transcriptional activation assay of protoplasts revealed that ABA treatment and coexpression of an ABA signaling master effector, ABA-RESPONSIVE ELEMENT-BINDING PROTEIN1, could activate the HSFA6b promoter. In addition, HSFA6b directly bound to the promoter of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN2A and enhanced its expression. Analysis of ABA responses in seed germination, cotyledon greening, and root growth as well as salt and drought tolerance in HSFA6b-null, overexpression, and dominant negative mutants revealed that HSFA6b is a positive regulator participating in ABA-mediated salt and drought resistance. Thermoprotection tests showed that HSFA6b was required for thermotolerance acquisition. Our study reveals a network in which HSFA6b operates as a downstream regulator of the ABA-mediated stress response and is required for heat stress resistance. This new ABA-signaling pathway is integrated into the complex HSR network in planta., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Aim: Urea transporters (UT) are a family of transmembrane proteins that specifically transport urea. UT inhibitors exert diuretic activity without affecting electrolyte balance. The purpose of this study was to discover novel UT inhibitors and determine the inhibition mechanism., Methods: The primary screening urea transporter B (UT-B) inhibitory activity was conducted in a collection of 10 000 diverse small molecules using mouse erythrocyte lysis assay. After discovering a hit with a core structure of 1-phenylamino-4-phenylphthalazin, the UT-B inhibitory activity of 160 analogs were examined with a stopped-flow light scattering assay and their structure-activity relationship (SAR) was analyzed. The inhibition mechanism was further investigated using in silico assays., Results: A phenylphthalazine compound PU1424, chemically named 5-(4-((4-methoxyphenyl) amino) phthalazin-1-yl)-2-methylbenzene sulfonamide, showed potent UT-B inhibition activity, inhibited human and mouse UT-B-mediated urea transport with IC50 value of 0.02 and 0.69 μmol/L, respectively, and exerted 100% UT-B inhibition at higher concentrations. The compound PU1424 did not affect membrane urea transport in mouse erythrocytes lacking UT-B. Structure-activity analysis revealed that the analogs with methoxyl group at R4 and sulfonic amide at R2 position exhibited the highest potency inhibition activity on UT-B. Furthermore, in silico assays validated that the R4 and R2 positions of the analogs bound to the UT-B binding pocket and exerted inhibition activity on UT-B., Conclusion: The compound PU1424 is a novel inhibitor of both human and mouse UT-B with IC50 at submicromolar ranges. Its binding site is located at the So site of the UT-B structure.

Coaggregation, the specific recognition and adherence of different microbial species, is thought to enhance biofilm formation. To date, no studies have focused on the ability of microorganisms isolated from a broad range of environments to coaggregate with each other and it is unclear whether coaggregation promotes the transmission of microorganisms between environmental niches. We aimed to evaluate the coaggregation ability of 29 bacteria and one fungus, isolated from a range of different environments, and to characterize the cell-surface polymers that mediate coaggregation between selected pairs. Strains were categorized as belonging to one of the four microbial archetypes: aquatic, broad environment, human opportunistic pathogen or human oral. A total of 23 of the 30 strains (77%) coaggregated with at least one other and 21/30 (70%) coaggregated with strains belonging to other archetypes. Nasopharyngeal bacteria belonging to the human opportunistic pathogen archetype showed the least number of coaggregations, and five Haemophilus influenzae strains did not coaggregate. Protease and sugar treatments indicated that coaggregation between strains of different archetypes was often mediated by lectin-saccharide interactions (9 of 15 evaluated pairs). In conclusion, coaggregation can occur between taxonomically disparate species isolated from discrete environments. We propose that these organisms be labeled as 'cross-environment coaggregating organisms'. The ability to coaggregate may aid species to colonize non-indigenous biofilms., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Acinetobacter baumannii is an emerging opportunistic nosocomial pathogen. Two factors that may enhance persistence in healthcare settings are antimicrobial resistance and biofilm-forming ability. The aim of this work was to determine whether A. baumannii isolates that persist in healthcare settings (endemic), can be differentiated from sporadic isolates based upon their ability to resist antibiotics and their biofilm-forming capability., Methods: Two hundred and ninety A. baumannii isolates were isolated over 17 months in the Detroit Medical Center (DMC). The isolates were genotyped using repetitive extragenic palindromic-PCR (REP-PCR). REP-types appearing greater than 10 times during active surveillance were considered endemic. The in vitro biofilm-forming ability and antibiotic resistance profile of each isolate were evaluated. Isolates were tested for the presence of two genetic markers-one implicated in biofilm formation (bap) and the other in antibiotic resistance (blaOXA-23)., Results: Of the 290 isolates evaluated, 84% carried bap and 36% carried blaOXA-23 . Five unique REP-PCR banding-types were detected >10 times (endemic) and constituted 58% of the 290 isolates. These five endemic REP-PCR types were 5.1 times more likely than sporadic isolates to carry both bap and blaOXA-23 . Furthermore, endemic isolates were resistant to 3 more antibiotic classes, on average, than sporadic isolates and four of the five endemic REP-PCR types formed denser biofilms in vitro than sporadic isolates., Conclusions: Endemic A. baumannii isolates are more likely than sporadic isolates to possess factors that increase virulence and enhance survival within a large healthcare system.

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

The rate of desorption of chemisorbed chlorobenzene molecules from the Si(111)-7 × 7 surface, induced by nonlocal charge injection from an STM tip, depends on the surface temperature. Between 260 and 313 K, we find an Arrhenius thermal activation energy of 450 ± 170 meV, consistent with the binding energy of physisorbed chlorobenzene on the same surface. Injected electrons excite the chlorobenzene molecule from the chemisorption state to an intermediate physisorption state, followed by thermal desorption. We find a second thermal activation energy of 21 ± 4 meV in the lower temperature region between 77 and 260 K, assigned to surface phonon excitation.

Plant nucleotide-binding leucine-rich repeat (NB-LRR) proteins serve as intracellular sensors to detect pathogen effectors and trigger immune responses. Transcription of the NB-LRR-encoding Resistance (R) genes needs to be tightly controlled to avoid inappropriate defense activation. How the expression of the NB-LRR R genes is regulated is poorly understood. The Arabidopsis (Arabidopsis thaliana) suppressor of npr1-1, constitutive 1 (snc1) mutant carries a gain-of-function mutation in a Toll/Interleukin1 receptor-like (TIR)-NB-LRR-encoding gene, resulting in the constitutive activation of plant defense responses. A snc1 suppressor screen identified modifier of snc1,9 (mos9), which partially suppresses the autoimmune phenotypes of snc1. Positional cloning revealed that MOS9 encodes a plant-specific protein of unknown function. Expression analysis showed that MOS9 is required for the full expression of TIR-NB-LRR protein-encoding RECOGNITION OF PERONOSPORA PARASITICA 4 (RPP4) and SNC1, both of which reside in the RPP4 cluster. Coimmunoprecipitation and mass spectrometry analyses revealed that MOS9 associates with the Set1 class lysine 4 of histone 3 (H3K4) methyltransferase Arabidopsis Trithorax-Related7 (ATXR7). Like MOS9, ATXR7 is also required for the full expression of SNC1 and the autoimmune phenotypes in the snc1 mutant. In atxr7 mutant plants, the expression of RPP4 is similarly reduced, and resistance against Hyaloperonospora arabidopsidis Emwa1 is compromised. Consistent with the attenuated expression of SNC1 and RPP4, trimethylated H3K4 marks are reduced around the promoters of SNC1 and RPP4 in mos9 plants. Our data suggest that MOS9 functions together with ATXR7 to regulate the expression of SNC1 and RPP4 through H3K4 methylation, which plays an important role in fine-tuning their transcription levels and functions in plant defense.

Activation of Cu/Zn superoxide dismutases (CuZnSODs) is aided by Cu incorporation and disulfide isomerization by Cu chaperone of SOD (CCS). As well, an Fe-S cluster scaffold protein, ISU, might alter the incorporation of Fe or Mn into yeast MnSOD (ySOD2), thus leading to active or inactive ySOD2. However, metallochaperones involved in the activation of FeSODs are unknown. Recently, we found that a chloroplastic chaperonin cofactor, CPN20, could mediate FeSOD activity. To investigate whether Fe incorporation in FeSOD is affected by CPN20, we used inductively coupled plasma mass spectrometry to analyze the ability of CPN20 to bind Fe. CPN20 could bind Fe, and the Fe binding to FeSOD was increased with CPN20 incubation. Thus, CPN20 might be an Fe chaperone for FeSOD activation, a role independent of its well-known co-chaperonin activity.

The Ca ( 2+) /calmodulin (CaM) signaling pathway mediates the heat stress (HS) response and acquisition of thermotolerance in plants. We showed that the rice CaM1-1 isoform can interpret a Ca ( 2+) signature difference in amplitude, frequency, and temporal-spatial properties in regulating transcription of nucleoplasmic small heat-shock protein gene (sHSPC/N) during HS. Ca ( 2+) and A23187 treatments under HS generated an intense and sustained increase in [Ca ( 2+) ]cyt and accelerated the expression of CaM1-1 and sHSPC/N genes, which suggests that HS-induced apoplastic Ca ( 2+) influx was responsible for the [Ca ( 2+) ]cyt transient and downstream HS signaling. Here, we discuss an emerging paradigm in the oscillation regulation of CaM1-1 expression during HS and highlight the areas that need further investigation.

We investigated heat shock (HS)-triggered Ca(2+) signalling transduced by a Ca(2+) sensor, calmodulin (CaM), linked to early transcriptome changes of HS-responsive genes in rice. We observed a biphasic [Ca(2+) ](cyt) signature in root cells that was distinct from that in epicotyl and leaf cells, which showed a monophasic response after HS. Treatment with Ca(2+) and A23187 generated an intense and sustained increase in [Ca(2+) ](cyt) in response to HS. Conversely, treatment with Ca(2+) chelator, L-type Ca(2+) channel blocker and CaM antagonist, but not intracellular Ca(2+) release inhibitor, strongly inhibited the increased [Ca(2+) ](cyt) . HS combined with Ca(2+) and A23187 accelerated the expression of OsCaM1-1 and sHSPC/N genes, which suggests that the HS-induced apoplastic Ca(2+) influx is responsible for the [Ca(2+) ](cyt) response and downstream HS signalling. In addition, the biphasic response of OsCaM1-1 in the nucleus followed the Ca(2+) signature, which may provide the information necessary to direct HS-related gene expression. Overexpression of OsCaM1-1 induced the expression of Ca(2+) /HS-related AtCBK3, AtPP7, AtHSF and AtHSP at a non-inducing temperature and enhanced intrinsic thermotolerance in transgenic Arabidopsis. Therefore, HS-triggered rapid increases in [Ca(2+) ](cyt) , together with OsCaM1-1 expression and its nuclear localization, are important in mediating downstream HS-related gene expression for the acquisition of thermotolerance in rice., (© 2012 Blackwell Publishing Ltd.)

The critical role that the major histocompatibility complex plays in the immune recognition of parasites and pathogens makes its evolutionary dynamics exceptionally relevant to ecology, population biology, and conservation studies. The black muntjac is a rare deer endemic to a small mountainous region in eastern China. We found that this species has two DQA loci through cDNA expression and sequence variation analysis. The level of variation at both DQA loci was found to be extremely low (three alleles for DQA1 and four alleles for DQA2), possibly because of past bottlenecks and the species' relatively solitary behavior pattern. The ratio of d(N)/d(S) in the putative peptide binding region of the DQA2 locus (13.36, P = 0.012) was significantly larger than one but not that of DQA1 (0.94, P = 0.95), suggesting strong positive selection at the DQA2 but not at the DQA1 locus. This difference might reflect different sets of evolutionary selection pressures acting on the two loci. The phylogenetic tree showed that DQA1 alleles from two species of Cervidae and two of Bovidae grouped together, as did the DQA2 alleles. However, different genes from the four species were located in separate branches. These results lead us to suggest that these DQA alleles are derived from primordial DQA genes from a common ancestor and are maintained in Cervidae and Bovidae since their divergence around 25.5-27.8 million years ago.

This study investigated the wear phenomena of chamfered acetabular cup liners. The liners have three parameters at the lengthened rim: the length of the elongation, the depth of the cup and the chamfer angle. Using published wear volume equations for cylindrically elongated liners, this study analysed the volume of the chamfer to obtain the exact theoretical wear volume of the chamfered liner. The criteria described in our previous paper were used to verify the accuracy of the proposed methodology. The results showed that a shallow cup depth (<2 mm) and a chamfer angle of about 30° could significantly reduce the wear volume arising from the elongation. When the chamfer angle was increased further, this effect became progressively less significant. The results suggest that a chamfer angle up to about 30° is appropriate, but the angle should not be larger than required to obtain the minimum femoral range of motion (ROM). The results of this study can also be integrated with the analysis of the kinetics and kinematics of liners. A compromise design incorporating a lower wear volume, sufficient ROM and a lower contact stress should be possible.

Copper-zinc superoxide dismutase (CuZnSOD; CSD) is an important antioxidant enzyme for oxidative stress protection. To date, two activation pathways have been identified in many species. One requiring the CCS, Cu chaperone for SOD, to insert Cu and activate CSD (referred to as CCS-dependent pathway), and the other works independently of CCS (referred to as CCS-independent pathway). In our previous study, we suggest an unidentified factor will work with glutathione (GSH) for CSD activation in the absence of the CCS. Here, two models of the CCS-independent mechanism are proposed. The role of the unidentified factor may work as a scaffold protein, which provides a platform for the CSD protein and Cu-GSH to interact, or as a Cu carrier, which itself can bind Cu and interact with CSD proteins. We also suggest that the CSD protein conformation at C-terminal is important in providing a docking site for unidentified factor to access.

Superoxide dismutases (SODs) are important antioxidant enzymes that catalyze the disproportionation of superoxide anion to oxygen and hydrogen peroxide to guard cells against superoxide toxicity. The major pathway for activation of copper/zinc SOD (CSD) involves a copper chaperone for SOD (CCS) and an additional minor CCS-independent pathway reported in mammals. We characterized the CCS-dependent and -independent activation pathways for three CSDs localized in different cellular compartments in Arabidopsis (Arabidopsis thaliana). The main activation pathway for CSD1 in the cytoplasm involved a CCS-dependent and -independent pathway, which was similar to that for human CSD. Activation of CSD2 in chloroplasts depended totally on CCS, similar to yeast (Saccharomyces cerevisiae) CSD. Peroxisome-localized CSD3 via a CCS-independent pathway was similar to nematode (Caenorhabditis elegans) CSD in retaining activity in the absence of CCS. In Arabidopsis, glutathione played a role in CCS-independent activation, as was reported in humans, but an additional factor was required. These findings reveal a highly specific and sophisticated regulation of CSD activation pathways in planta relative to other known CCS-independent activation.

Bamboo is distinguished by its rapid growth, for growth more than 100 cm per day. Because of the rapid growth, tissues have significant ATP requirements, which results in intense reduction of oxygen and thus oxidative stress. For this reason, bamboo may have a special and efficient scavenger system to release the stress during fast cell division and elongation. Here, we investigated superoxide dismutase (SOD, E.C.1.15.1.1), the first line of antioxidant enzymes, in green bamboo (Bambusa oldhamii). The SOD activity profile in this species was complex, with 5 genes and 7 isozymes of CuZnSOD and 4 genes and 1 isozyme of MnSOD. We isolated one of each of the green bamboo CuZnSOD and MnSOD genes, and their activities were stable under a broad range of pH and temperature treatments, even at room temperature for more than 3 days. Bamboo SODs showed developmental and tissue-specific regulation, and both transcript and protein levels were responsive to abscisic acid, UV-B and high-light treatments. The complexity of the cis-elements in promoter regions implied that the regulation mechanisms of SOD might help accomplish the unique fast-growth phenotype of green bamboo., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Apoplastic Ca(2+) concentration controls membrane permeability, cell wall stabilization, and cell integrity; however, little is known about its role in thermotolerance in plants. Here, we report that the acquired thermotolerance of etiolated rice seedlings (Oryza sativa) was abolished by an exogenously supplied Ca(2+) chelator, EGTA, related to increased cellular content leakage during heat shock (HS) treatment. Thermotolerance was restored by the addition of Ca(2+) during EGTA incubation. Pectin methylesterase (EC 3.1.1.11), a cell-wall remodeling enzyme, was activated in response to HS, and its elevated activity was related to the recovery of the HS-released Ca(2+) concentration. EGTA interfered with the capability of HS to increase oscillation of [Ca(2+)]cyt content. We assume that heat-activated PME activity is involved in cell-wall-localized Ca(2+). The removal of apoplastic Ca(2+) might participate in HS signaling to induce HS protein expression and cell-wall remodeling to retain plasma membrane integrity, prevent cellular content leakage and confer thermoprotection., (© 2010 Landes Bioscience)

In Arabidopsis thaliana, heat shock factor binding protein (AtHSBP) is a negative regulator of the heat shock response (HSR), and defective AtHSBP leads to seed abortion. We found that the wild-type and AtHSBP-knockout plants did not differ in ovule phenotypes at flower position 3, which indicates that the seed abortion occurs after fertilization and during embryogenesis. The conserved residues of the hydrophobic heptad repeat (HR) domains in AtHSBP were mutated and examined for their subcellular localization and interacting ability with heat shock factors (AtHSFs). The HR domains at the C terminus of AtHSBP are important for retaining AtHSBP in the cytoplasm under normal growth conditions and for interacting with AtHSFs, which negatively affects the DNA-binding capacity and transactivation activity of AtHSFs during the HSR.

Synthesis of heat shock proteins (HSPs) in response to heat shock (HS) is essential for thermotolerance. The effect of a Ca(2+) chelator, EGTA, was investigated before a lethal HS treatment in soybean (Glycine max) seedlings with acquired thermotolerance induced by preheating. Such seedlings became non-thermotolerant with EGTA treatment. The addition of Ca(2+), Sr(2+) or Ba(2+) to the EGTA-treated samples rescued the seedlings from death by preventing the increased cellular leakage of electrolytes, amino acids, and sugars caused by EGTA. It was confirmed that EGTA did not affect HSP accumulation and physiological functions but interfered with the recovery of HS-released Ca(2+) concentration which was required for thermotolerance. Pectin methylesterase (PME, EC 3.1.1.11), a cell wall remodelling enzyme, was activated in response to HS, and its elevated activity caused an increased level of demethylesterified pectin which was related to the recovery of the HS-released Ca(2+) concentration. Thus, the recovery of HS-released Ca(2+) in Ca(2+)-pectate reconstitution through PME activity is required for cell wall remodelling during HS in soybean which, in turn, retains plasma membrane integrity and co-ordinates with HSPs to confer thermotolerance.

Heat shock response (HSR) is a universal mechanism in all organisms. It is under tight regulation by heat shock factors (HSFs) and heat shock proteins (HSPs) after heat shock (HS) to prevent stress damage. On the attenuation of HSR, HSP70 and HSF Binding Protein1 (HSBP1) interact with HSF1 and thus dissociate trimeric HSF1 into an inert monomeric form in humans. However, little is known about the effect of HSBP with thermal stress in plants. This report describes our investigation of the role of AtHSBP in Arabidopsis (Arabidopsis thaliana) by genetic and molecular approaches. AtHSBP was heat inducible and ubiquitously expressed in all tissues; AtHSBP was also crucial for seed development, as demonstrated by AtHSBP-knockout lines showing seed abortion. Thermotolerance results showed that AtHSBP participates in acquired thermotolerance but not basal thermotolerance and is a negative regulator of HSR. Subcellular localization revealed that the cytosol-localized AtHSBP translocated to the nucleus in response to HS. Protoplast two-hybrid assay results confirmed that AtHSBP interacts with itself and with the HSFs, AtHSFA1a, AtHSFA1b, and AtHSFA2. AtHSBP also negatively affected AtHSFA1b DNA-binding capacity in vitro. Quantitative polymerase chain reaction and western-blot analysis demonstrated that altered levels of AtHSBP lead to differential HSP expression, mainly during the recovery from HS. These studies provide a new insight into HSBP in plants and reveal that AtHSBP is a negative regulator of HSR and required for seed development.

Mainland serow is an endanged artiodactyl of southern Anhui province, China, that is often subject to poaching. To provide an easy, rapid and reliable marker for identification of bushmeat, skin and other tissues of the species, we developed a sequence characterized amplified region (SCAR) based on a species-specific random amplified polymorphic DNA (RAPD) marker. Initially, a 1012-bp species-specific DNA fragment of mainland serow was detected by a RAPD primer S1193. Then, a serow-specific primer pair (SCF/SCR) was designed according to the specific RAPD fragment, resulting in a 438-bp SCAR for the species. Finally, the reliability of the SCAR primers was tested by a common multiplex polymerase chain reaction using the combination of the SCAR and cyt b universal primers. The results that all mainland serow samples presented two target bands but the others failed to produce the SCAR indicated that the designed primers were highly diagnostic. Therefore, the SCAR probe developed in this study will be useful for quick authentication of mainland serow tissue samples for conservation biology and bushmeat regulation., (© 2009 Blackwell Publishing Ltd.)

Abscission is a developmental program that results in the active shedding of infected or nonfunctional organs from a plant body. Here, we establish a signaling pathway that controls abscission in Arabidopsis thaliana from ligand, to receptors, to downstream effectors. Loss of function mutations in Inflorescence Deficient in Abscission (IDA), which encodes a predicted secreted small protein, the receptor-like protein kinases HAESA (HAE) and HAESA-like 2 (HSL2), the Mitogen-Activated Protein Kinase Kinase 4 (MKK4) and MKK5, and a dominant-negative form of Mitogen-Activated Protein Kinase 6 (MPK6) in a mpk3 mutant background all have abscission-defective phenotypes. Conversely, expression of constitutively active MKKs rescues the abscission-defective phenotype of hae hsl2 and ida plants. Additionally, in hae hsl2 and ida plants, MAP kinase activity is reduced in the receptacle, the part of the stem that holds the floral organs. Plants overexpressing IDA in a hae hsl2 background have abscission defects, indicating HAE and HSL2 are epistatic to IDA. Taken together, these results suggest that the sequential action of IDA, HAE and HSL2, and a MAP kinase cascade regulates the programmed separation of cells in the abscission zone.

The copper chaperone for superoxide dismutase (CCS) has been identified as a key factor integrating copper into copper/zinc superoxide dismutase (CuZnSOD) in yeast (Saccharomyces cerevisiae) and mammals. In Arabidopsis (Arabidopsis thaliana), only one putative CCS gene (AtCCS, At1g12520) has been identified. The predicted AtCCS polypeptide contains three distinct domains: a central domain, flanked by an ATX1-like domain, and a C-terminal domain. The ATX1-like and C-terminal domains contain putative copper-binding motifs. We have investigated the function of this putative AtCCS gene and shown that a cDNA encoding the open reading frame predicted by The Arabidopsis Information Resource complemented only the cytosolic and peroxisomal CuZnSOD activities in the Atccs knockout mutant, which has lost all CuZnSOD activities. However, a longer AtCCS cDNA, as predicted by the Munich Information Centre for Protein Sequences and encoding an extra 66 amino acids at the N terminus, could restore all three, including the chloroplastic CuZnSOD activities in the Atccs mutant. The extra 66 amino acids were shown to direct the import of AtCCS into chloroplasts. Our results indicated that one AtCCS gene was responsible for the activation of all three types of CuZnSOD activity. In addition, a truncated AtCCS, containing only the central and C-terminal domains without the ATX1-like domain failed to restore any CuZnSOD activity in the Atccs mutant. This result indicates that the ATX1-like domain is essential for the copper chaperone function of AtCCS in planta.

Accumulation of class I small heat shock proteins (sHSPs) is induced by the proline analog, azetidine-2-carboxylic acid (Aze) in soybean seedlings to a level similar to that induced by exposure to 40 degrees C. However, only the treatment with 10 mM Aze for 6 h and subsequently with 10 mM proline for 24 h protected the seedlings from damage during subsequent exposure to 45 degrees C as assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. A chaperone activity assay showed that the purified class I sHSPs induced by Aze were functional in vitro and protected proteins from thermal denaturation. Amino acid composition analysis indicated that Aze was not incorporated into de novo synthesized class I sHSPs. Accumulation of class I sHSPs in the soluble post-ribosomal supernatant fraction was found to be important for acquisition of thermotolerance. We suggest that both the accumulation of class I sHSPs and their presence in the soluble fraction are important for establishment of thermotolerance.

The cytosolic class I small heat shock proteins (sHSP-CI) represent the most abundant sHSP in plants. Here, we report the characterization and the expression profile of nine members of the sHSP-CI gene family in rice (Oryza sativa Tainung No.67), of which Oshsp16.9A, Oshsp16.9B, Oshsp16.9C, Oshsp16.9D and Oshsp17.9B are clustered on chromosome 1, and Oshsp17.3, Oshsp17.7, Oshsp17.9A and Oshsp18.0 are clustered on chromosome 3. Oshsp17.3 and Oshsp18.0 are linked by a 356-bp putative bi-directional promoter. Individual gene products were identified from the protein subunits of a heat shock complex (HSC) and from in vitro transcription/ translation products by two-dimensional gel electrophoreses (2-DE). All sHSP-CI genes except Oshsp17.9B were induced strongly after a 2-h heat shock treatment. The genes on chromosome 3 were induced rapidly at 32 and 41 degrees C, whereas those on chromosome 1 were induced slowly by similar conditions. Seven of these genes, except Oshsp16.9D and Oshsp17.9B, were induced by arsenite (As), but only genes on chromosome 3 were strongly induced by azetidine-2-carboxylic acid (Aze, a proline analog) and cadmium (Cd). A similar expression profile of all sHSP-CI genes at a lower level was evoked by ethanol, H2O2 and CuCl2 treatments. Transient expression assays of the promoter activity by linking to GUS reporter gene also supported the in vivo selective expression of the sHSP-CI genes by Aze treatment indicating the differential induction of rice sHSP-CI genes is most likely regulated at the transcriptional level. Only Oshsp16.9A abundantly accumulated in mature dry seed also suggested additionally prominent roles played by this HSP in development.

Background: Many researchers suggest that adult mammalian central nervous system (CNS) is incapable of completing self-repair or regeneration. And there are accumulating lines of evidence which suggest that endogenous neural stem cells (NSCs) are activated in many pathological conditions, including stroke in the past decades, which might partly account for rehabilitation afterwards. In this study, we investigated whether there was endogenous neural stem cell activation in intracerebral hemorrhagic (ICH) rat brains., Methods: After ICH induction by stereotactical injection of collagenase type VII into globus pallidus, 5-Bromo-2 Deoxyuridine (BrdU) was administered intraperitoneally to label newborn cells. Immunohistochemical method was used to detect Nestin, a marker for neural stem cells, and BrdU., Results: Nestin-positive or BrdU-Labeled cells were predominantly located at 2 sites: basal ganglion around hemotoma, ependyma and nearby subventricular zone (SVZ). No positive cells for the 2 markers were found in the 2 sites of normal control group and sham group, as well as in non-leisioned parenchyma, both hippocampi and olfactory bulbs in the 4 groups. Nestin+ cells presented 4 types of morphology, and BrdU+ nucleus were polymorphologic. Positive cell counting around hemotoma showed that at day 2, Nestin+ cells were seen around hemotoma in model group, the number of which increased at day 4, day 7 (P <0.01), peaked at day 14 (P <0.05), and reduced significantly by day 28 (P <0.01)., Conclusion: Endogenous neural stem cells were activated in experimental intracerebral hemorrhagic rat brains.

Prostate epithelial cells accumulate the highest zinc levels of any cells in the body. Evidence indicates that zinc plays critical roles in the normal function and pathology of the prostate gland. We have identified two important effects of zinc in the prostate epithelial cells: the inhibition of m-aconitase and the induction of mitochondrial apoptogenesis. However, at the present time, the effects of zinc on prostatic cells in in vivo conditions have not yet been reported. The objectives of this in vivo study were to investigate the effect of zinc on: tumorogenicity in nude mice, zinc accumulation in tumor tissues, and the levels of mitochondrial membrane permeability related proteins, Bax/Bcl-2. A tumorigenicity animal model was established using male nude mice (4-6 weeks old) with inoculation of PC-3 cells (5-10x10(6)/mL) prepared in 10% Matrigel. The mice were treated with zinc by ALZET osmotic pumps (Durect Corporation), with a releasing rate of 0.25 micro l/h for 28 days. Zinc concentrations of the tumor tissues were determined by Atomic Absorption Spectrophotometer method. Frozen sections of tumor tissues were prepared for TUNEL assay. The levels of Bax and Bcl-2 in the tumor tissues were determined by Western blot analyses. Our study demonstrated that in vivo treatment of zinc increased zinc accumulation and citrate production in PC-3 cell induced tumor tissues and inhibited tumor growth. The inhibitory effect of zinc appears to result from zinc-induced apoptosis by regulation of mitochondrial membrane permeability-related Bax/Bcl-2 proteins.