Metabolic flux regulates growth transitions and antibiotic tolerance in uropathogenic Escherichia coli.

Reducing growth and limiting metabolism are strategies that allow bacteria to survive exposure to environmental stress and antibiotics. During infection, uropathogenic Escherichia coli (UPEC) may enter a quiescent state that enables them to reemerge after the completion of successful antibiotic treatment. Many clinical isolates, including the well-characterized UPEC strain CFT073, also enter a metabolite-dependent, quiescent state in vitro that is reversible with cues, including peptidoglycan-derived peptides and amino acids. Here, we show that quiescent UPEC is antibiotic tolerant and demonstrate that metabolic flux in the tricarboxylic acid (TCA) cycle regulates the UPEC quiescent state via succinyl-CoA. We also demonstrate that the transcriptional regulator complex integration host factor and the FtsZ-interacting protein ZapE, which is important for E. coli division during stress, are essential for UPEC to enter the quiescent state. Notably, in addition to engaging FtsZ and late-stage cell division proteins, ZapE also interacts directly with TCA cycle enzymes in bacterial two-hybrid assays. We report direct interactions between the succinate dehydrogenase complex subunit SdhC, the late-stage cell division protein FtsN, and ZapE. These interactions may enable communication between oxidative metabolism and the cell division machinery in UPEC. Moreover, these interactions are conserved in an E. coli K-12 strain. This work suggests that there is coordination among the two fundamental and essential pathways that regulate overall growth, quiescence, and antibiotic susceptibility. IMPORTANCE: Uropathogenic Escherichia coli (UPEC) are the leading cause of urinary tract infections (UTIs). Upon invasion into bladder epithelial cells, UPEC establish quiescent intracellular reservoirs that may lead to antibiotic tolerance and recurrent UTIs. Here, we demonstrate using an in vitro system that quiescent UPEC cells are tolerant to ampicillin and have decreased metabolism characterized by succinyl-CoA limitation. We identify the global regulator integration host factor complex and the cell division protein ZapE as critical modifiers of quiescence and antibiotic tolerance. Finally, we show that ZapE interacts with components of both the cell division machinery and the tricarboxylic acid cycle, and this interaction is conserved in non-pathogenic E. coli, establishing a novel link between cell division and metabolism.

A marine-derived fatty acid targets the cell membrane of Gram-positive bacteria.

IMPORTANCE: With the lack of new antibiotics in the drug discovery pipeline, coupled with accelerated evolution of antibiotic resistance, new sources of antibiotics that target pathogens of clinical importance are paramount. Here, we use bacterial cytological profiling to identify the mechanism of action of the monounsaturated fatty acid (Z)-13-methyltetra-4-decenoic acid isolated from the marine bacterium Olleya marilimosa with antibacterial effects against Gram-positive bacteria. The fatty acid antibiotic was found to rapidly destabilize the cell membrane by pore formation and membrane aggregation in Bacillus subtilis, suggesting that this fatty acid may be a promising adjuvant used in combination to enhance antibiotic sensitivity.

ELF3 mediates IL-1α induced differentiation of mesenchymal stem cells to inflammatory iCAFs.

Stromal cells in the tumor microenvironment regulate the immune landscape and tumor progression. Yet, the ontogeny and heterogeneity of reactive stromal cells within tumors is not well understood. Carcinoma-associated fibroblasts exhibiting an inflammatory phenotype (iCAFs) have been identified within multiple cancers; however, mechanisms that lead to their recruitment and differentiation also remain undefined. Targeting these mechanisms therapeutically may be important in managing cancer progression. Here, we identify the ELF3 transcription factor as the canonical mediator of IL-1α-induced differentiation of prostate mesenchymal stem cells to an iCAF phenotype, typical of the tumor microenvironment. Furthermore, IL-1α-induced iCAFs were subsequently refractive to TGF-ß1 induced trans-differentiation to a myofibroblast phenotype (myCAF), another key carcinoma-associated fibroblast subtype typical of reactive stroma in cancer. Restricted trans-differentiation was associated with phosphorylation of the YAP protein, indicating that interplay between ELF3 action and activation of the Hippo pathway are critical for restricting trans-differentiation of iCAFs. Together, these data show that the IL-1α/ELF3/YAP pathways are coordinate for regulating inflammatory carcinoma-associated fibroblast differentiation.

Androgen receptor variant-7 regulation by tenascin-c induced src activation.

BACKGROUND: Bone metastatic prostate cancer does not completely respond to androgen-targeted therapy and generally evolves into lethal castration resistant prostate cancer (CRPC). Expression of AR-V7- a constitutively active, ligand independent splice variant of AR is one of the critical resistant mechanisms regulating metastatic CRPC. TNC is an extracellular matrix glycoprotein, crucial for prostate cancer progression, and associated with prostate cancer bone metastases. In this study, we investigated the mechanisms that regulate AR-V7 expression in prostate cancer cells interacting with osteogenic microenvironment including TNC. METHODS: Prostate cancer/preosteoblast heterotypical organoids were evaluated via immunofluorescence imaging and gene expression analysis using RT-qPCR to assess cellular compartmentalization, TNC localization, and to investigate regulation of AR-V7 in prostate cancer cells by preosteoblasts and hormone or antiandrogen action. Prostate cancer cells cultured on TNC were assessed using RT-qPCR, Western blotting, cycloheximide chase assay, and immunofluorescence imaging to evaluate (1) regulation of AR-V7, and (2) signaling pathways activated by TNC. Identified signaling pathway induced by TNC was targeted using siRNA and a small molecular inhibitor to investigate the role of TNC-induced signaling activation in regulation of AR-V7. Both AR-V7- and TNC-induced signaling effectors were targeted using siRNA, and TNC expression assessed to evaluate potential feedback regulation. RESULTS: Utilizing heterotypical organoids, we show that TNC is an integral component of prostate cancer interaction with preosteoblasts. Interaction with preosteoblasts upregulated both TNC and AR-V7 expression in prostate cancer cells which was suppressed by testosterone but elevated by antiandrogen enzalutamide. Interestingly, the results demonstrate that TNC-induced Src activation regulated AR-V7 expression, post-translational stability, and nuclear localization in prostate cancer cells. Treatment with TNC neutralizing antibody, Src knockdown, and inhibition of Src kinase activity repressed AR-V7 transcript and protein. Reciprocally, both activated Src and AR-V7 were observed to upregulate autocrine TNC gene expression in prostate cancer cells. CONCLUSION: Overall, the findings reveal that prostate cancer cell interactions with the cellular and ECM components in the osteogenic microenvironment plays critical role in regulating AR-V7 associated with metastatic CRPC. Video Abstract.

Peptidoglycan Sensing Prevents Quiescence and Promotes Quorum-Independent Colony Growth of Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the leading cause of human urinary tract infections (UTIs), and many patients experience recurrent infection after successful antibiotic treatment. The source of recurrent infections may be persistent bacterial reservoirs in vivo that are in a quiescent state and thus are not susceptible to antibiotics. Here, we show that multiple UPEC strains require a quorum to proliferate in vitro with glucose as the carbon source. At low cell density, the bacteria remain viable but enter a quiescent, nonproliferative state. Of the clinical UPEC isolates tested to date, 35% (51/145) enter this quiescent state, including isolates from the recently emerged, multidrug-resistant pandemic lineage ST131 (i.e., strain JJ1886) and isolates from the classic endemic lineage ST73 (i.e., strain CFT073). Moreover, quorum-dependent UPEC quiescence is prevented and reversed by small-molecule proliferants that stimulate colony formation. These proliferation cues include d-amino acid-containing peptidoglycan (PG) tetra- and pentapeptides, as well as high local concentrations of l-lysine and l-methionine. Peptidoglycan fragments originate from the peptidoglycan layer that supports the bacterial cell wall but are released as bacteria grow. These fragments are detected by a variety of organisms, including human cells, other diverse bacteria, and, as we show here for the first time, UPEC. Together, these results show that for UPEC, (i) sensing of PG stem peptide and uptake of l-lysine modulate the quorum-regulated decision to proliferate and (ii) quiescence can be prevented by both intra- and interspecies PG peptide signaling.IMPORTANCE Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). During pathogenesis, UPEC cells adhere to and infiltrate bladder epithelial cells, where they may form intracellular bacterial communities (IBCs) or enter a nongrowing or slowly growing quiescent state. Here, we show in vitro that UPEC strains at low population density enter a reversible, quiescent state by halting division. Quiescent cells resume proliferation in response to sensing a quorum and detecting external signals, or cues, including peptidoglycan tetra- and pentapeptides.

Metabolic Pathway Construction and Optimization of Escherichia coli for High-Level Ectoine Production.

Ectoine is widely produced by various bacteria as a natural cell protectant against environment stress, e.g., osmotic and temperature stress. Its protective properties therefore exhibit high commercial value, especially in agriculture, medicine, cosmetics, and biotechnology. Here, we successfully constructed an engineered Escherichia coli for the heterologous production of ectoine. Firstly, the ectABC genes from Halomonas elongata were introduced into E. coli MG1655 to produce ectoine without high osmolarity. Subsequently, lysA gene was deleted to weaken the competitive L-lysine biosynthesis pathway and ectoine bioconversion was further optimized, leading to an increase of ectoine titer by 16.85-fold. Finally, at the low cell density of 5 OD600/mL in Erlenmeyer flask, the concentration of extracellular ectoine was increased to 3.05 mg/mL. At the high cell density of 15 OD600/mL, 12.7 g/L of ectoine was achieved in 24 h and the overall yield is 1.27 g/g glycerol and sodium aspartate. Our study herein provides a feasible and valuable biosynthesis pathway of ectoine with a potential for large-scale industrial production using simple and cheap feedstocks.

The Probiotic Bacterium Phaeobacter inhibens Downregulates Virulence Factor Transcription in the Shellfish Pathogen Vibrio coralliilyticus by N-Acyl Homoserine Lactone Production.

Phaeobacter inhibens S4Sm acts as a probiotic bacterium against the oyster pathogen Vibrio coralliilyticus Here, we report that P. inhibens S4Sm secretes three molecules that downregulate the transcription of major virulence factors, metalloprotease genes, in V. coralliilyticus cultures. The effects of the S4Sm culture supernatant on the transcription of three genes involved in protease activity, namely, vcpA, vcpB, and vcpR (encoding metalloproteases A and B and their transcriptional regulator, respectively), were examined by reverse transcriptase quantitative PCR (qRT-PCR). The expression of vcpB and vcpR were reduced to 36% and 6.6%, respectively, compared to that in an untreated control. We constructed a V. coralliilyticus green fluorescent protein (GFP) reporter strain to detect the activity of inhibitory compounds. Using a bioassay-guided approach, the molecules responsible for V. coralliilyticus protease inhibition activity were isolated from S4Sm supernatant and identified as three N-acyl homoserine lactones (AHLs). The three AHLs are N-(3-hydroxydecanoyl)-l-homoserine lactone, N-(dodecanoyl-2,5-diene)-l-homoserine lactone, and N-(3-hydroxytetradecanoyl-7-ene)-l-homoserine lactone, and their half maximal inhibitory concentrations (IC50s) against V. coralliilyticus protease activity were 0.26 µM, 3.7 µM, and 2.9 µM, respectively. Our qRT-PCR data demonstrated that exposures to the individual AHLs reduced the transcription of vcpR and vcpB Combinations of the three AHLs (any two or all three AHLs) on V. coralliilyticus produced additive effects on protease inhibition activity. These AHL compounds may contribute to the host protective effects of S4Sm by disrupting the quorum sensing pathway that activates protease transcription of V. coralliilyticusIMPORTANCE Probiotics represent a promising alternative strategy to control infection and disease caused by marine pathogens of aquaculturally important species. Generally, the beneficial effects of probiotics include improved water quality, control of pathogenic bacteria and their virulence, stimulation of the immune system, and improved animal growth. Previously, we isolated a probiotic bacterium, Phaeobacter inhibens S4Sm, which protects oyster larvae from Vibrio coralliilyticus RE22Sm infection. We also demonstrated that both antibiotic secretion and biofilm formation play important roles in S4Sm probiotic activity. Here, we report that P. inhibens S4Sm, an alphaproteobacterium and member of the Roseobacter clade, also secretes secondary metabolites that hijack the quorum sensing ability of V. coralliilyticus RE22Sm, suppressing virulence gene expression. This finding demonstrates that probiotic bacteria can exert their host protection by using a multipronged array of behaviors that limit the ability of pathogens to become established and cause infection.

Antagonism of Quorum Sensing Phenotypes by Analogs of the Marine Bacterial Secondary Metabolite 3-Methyl-N-(2'-Phenylethyl)-Butyramide.

Quorum sensing (QS) antagonists have been proposed as novel therapeutic agents to combat bacterial infections. We previously reported that the secondary metabolite 3-methyl-N-(2'-phenylethyl)-butyramide, produced by a marine bacterium identified as Halobacillus salinus, inhibits QS controlled phenotypes in multiple Gram-negative reporter strains. Here we report that N-phenethyl hexanamide, a structurally-related compound produced by the marine bacterium Vibrio neptunius, similarly demonstrates QS inhibitory properties. To more fully explore structure-activity relationships within this new class of QS inhibitors, a panel of twenty analogs was synthesized and biologically evaluated. Several compounds were identified with increased attenuation of QS-regulated phenotypes, most notably N-(4-fluorophenyl)-3-phenylpropanamide against the marine pathogen Vibrio harveyi (IC50 = 1.1 µM). These findings support the opportunity to further develop substituted phenethylamides as QS inhibitors.

Replication of Zika Virus in Human Prostate Cells: A Potential Source of Sexually Transmitted Virus.

Background: While Zika virus (ZIKV) is mainly transmitted by mosquitoes, numerous cases of sexual transmission have been reported during recent outbreaks. Little is known about which host cell types or entry factors aid in mediating this sexual transmission. Methods: In this study, we investigated ZIKV cell tropism by infecting 2 types of human prostate cells with 3 contemporary ZIKV isolates from persons infected in the Americas. We used real-time quantitative polymerase chain reaction and immunofluorescence analyses to measure infection and flow cytometry to detect entry factor expression. Results: Here we show that ZIKV infects, replicates, and produces infectious virus in prostate stromal mesenchymal stem cells, epithelial cells, and organoids made with a combination of these cells. We also show that prostate cells express several well-characterized flavivirus attachment factors. In contrast, dengue virus does not infect or does not replicate in these prostate cells, although it is known to use similar receptors. Conclusions: Our results indicate that ZIKV favors infection of stromal cells more so than epithelial cells in organoids, possibly indicating a preference for stem cells in general. Overall, these results suggest that ZIKV replication occurs in the human prostate and can account for ZIKV secretion in semen, thus leading to sexual transmission.

Influence of the neural microenvironment on prostate cancer.

BACKGROUND: Nerves are key factors in prostate cancer (PCa), but the functional role of innervation in prostate cancer is poorly understood. PCa induced neurogenesis and perineural invasion (PNI), are associated with aggressive disease. METHOD: We denervated rodent prostates chemically and physically, before orthotopically implanting cancer cells. We also performed a human neoadjuvant clinical trial using botulinum toxin type A (Botox) and saline in the same patient, before prostatectomy. RESULT: Bilateral denervation resulted in reduced tumor incidence and size in mice. Botox treatment in humans resulted in increased apoptosis of cancer cells in the Botox treated side. A similar denervation gene array profile was identified in tumors arising in denervated rodent prostates, in spinal cord injury patients and in the Botox treated side of patients. Denervation induced exhibited a signature gene profile, indicating translation and bioenergetic shutdown. Nerves also regulate basic cellular functions of non-neoplastic epithelial cells. CONCLUSION: Nerves play a role in the homeostasis of normal epithelial tissues and are involved in prostate cancer tumor survival. This study confirms that interactions between human cancer and nerves are essential to disease progression. This work may make a major impact in general cancer treatment strategies, as nerve/cancer interactions are likely important in other cancers as well. Targeting the neural microenvironment may represent a therapeutic approach for the treatment of human prostate cancer.

Albisporachelin, a New Hydroxamate Type Siderophore from the Deep Ocean Sediment-Derived Actinomycete Amycolatopsisalbispora WP1T.

Marine actinobacteria continue to be a rich source for the discovery of structurally diverse secondary metabolites. Here we present a new hydroxymate siderophore produced by Amycolatopsis albispora, a recently described species of this less explored actinomycete genus. Strain WP1T was isolated from sediments collected at -2945 m in the Indian Ocean. The new siderophore, designated albisporachelin, was isolated from iron depleted culture broths and the structure was established by 1D and 2D NMR and MS/MS experiments, and application of a modified Marfey's method. Albisporachelin is composed of one N-methylated-formylated/hydroxylated l-ornithine (N-Me-fh-l-Orn), one l-serine (l-Ser), one formylated/hydroxylated l-ornithine (fh-l-Orn) and a cyclo-N-methylated-hydroxylated l-ornithine (cyclo-N-Me-h-l-Orn).

A Human Gut Commensal Ferments Cranberry Carbohydrates To Produce Formate.

Commensal bifidobacteria colonize the human gastrointestinal tract and catabolize glycans that are impervious to host digestion. Accordingly, Bifidobacterium longum typically secretes acetate and lactate as fermentative end products. This study tested the hypothesis that B. longum utilizes cranberry-derived xyloglucans in a strain-dependent manner. Interestingly, the B. longum strain that efficiently utilizes cranberry xyloglucans secretes 2.0 to 2.5 mol of acetate-lactate. The 1.5 acetate:lactate ratio theoretical yield obtained in hexose fermentations shifts during xyloglucan metabolism. Accordingly, this metabolic shift is characterized by increased acetate and formate production at the expense of lactate. α-l-Arabinofuranosidase, an arabinan endo-1,5-α-l-arabinosidase, and a ß-xylosidase with a carbohydrate substrate-binding protein and carbohydrate ABC transporter membrane proteins are upregulated (>2-fold change), which suggests carbon flux through this catabolic pathway. Finally, syntrophic interactions occurred with strains that utilize carbohydrate products derived from initial degradation from heterologous bacteria.IMPORTANCE This was a study of bacterial metabolism of complex cranberry carbohydrates termed xyloglucans that are likely not digested prior to reaching the colon. This is significant, as bifidobacteria interact with this dietary compound to potentially impact human host health through energy and metabolite production by utilizing these substrates. Specific bacterial strains utilize cranberry xyloglucans as a nutritive source, indicating unknown mechanisms that are not universal in bifidobacteria. In addition, xyloglucan metabolism proceeds by using an alternative pathway that could lead to further research to investigate mechanisms underlying this interaction. Finally, we observed cross-feeding between bacteria in which one strain degrades the cranberry xyloglucan to make it available to a second strain. Similar nutritive strategies are known to occur within the gut. In aggregate, this study may lead to novel foods or supplements used to impact human health through rational manipulation of the human microbiome.

Kinetics of the BrO + HO2 reaction over the temperature range T = 246-314 K.

The kinetics of the reaction between gas phase BrO and HO2 radicals, BrO + HO2 → HOBr + O2 (1), have been studied over the atmospherically relevant temperature range T = 246-314 K and at ambient pressure, p = 760 ± 20 Torr, using laser flash photolysis coupled with ultraviolet absorption spectroscopy. The reaction was initiated by the generation of bromine monoxide radicals following laser photolytic generation of Br atoms from Br2/Cl2 containing mixtures and their reaction with ozone. Subsequently, the addition of methanol vapour to the reaction mixture, in the presence of excess oxygen, afforded the efficient simultaneous post-photolysis formation of HO2 radicals using well-defined chemistry. The decay of BrO radicals, in the presence and absence of HO2, was interrogated to determine the rate coefficients for the BrO + BrO and the BrO + HO2 reactions. A detailed sensitivity analysis was performed to ensure that the BrO + HO2 reaction was unequivocally monitored. The rate coefficient for reaction (1) is described by the Arrhenius expression: where statistical errors are 1σ. The negative temperature dependence of this reaction is in general accord with those reported by previous studies of this reaction. However, the present work reports greater absolute values for k1 than those of several previous studies. An assessment of previous laboratory studies of k1 is presented. This work confirms that reaction (1) plays a significant role in HOBr formation throughout the atmosphere following both anthropogenic, biogenic and volcanic emissions of brominated species. Reaction (1) therefore contributes to an efficient ozone depleting process in the atmosphere, and further confirms the significance of interactions between two different families of reactive atmospheric trace species.

RUNX1 is essential for mesenchymal stem cell proliferation and myofibroblast differentiation.

Myofibroblasts are a key cell type in wound repair, cardiovascular disease, and fibrosis and in the tumor-promoting microenvironment. The high accumulation of myofibroblasts in reactive stroma is predictive of the rate of cancer progression in many different tumors, yet the cell types of origin and the mechanisms that regulate proliferation and differentiation are unknown. We report here, for the first time to our knowledge, the characterization of normal human prostate-derived mesenchymal stem cells (MSCs) and the TGF-ß1-regulated pathways that modulate MSC proliferation and myofibroblast differentiation. Human prostate MSCs combined with prostate cancer cells expressing TGF-ß1 resulted in commitment to myofibroblasts. TGF-ß1-regulated runt-related transcription factor 1 (RUNX1) was required for cell cycle progression and proliferation of progenitors. RUNX1 also inhibited, yet did not block, differentiation. Knockdown of RUNX1 in prostate or bone marrow-derived MSCs resulted in cell cycle arrest, attenuated proliferation, and constitutive differentiation to myofibroblasts. These data show that RUNX1 is a key transcription factor for MSC proliferation and cell fate commitment in myofibroblast differentiation. This work also shows that the normal human prostate gland contains tissue-derived MSCs that exhibit multilineage differentiation similar to bone marrow-derived MSCs. Targeting RUNX1 pathways may represent a therapeutic approach to affect myofibroblast proliferation and biology in multiple disease states.

Generalized Hypermobility, Knee Hyperextension, and Outcomes After Anterior Cruciate Ligament Reconstruction: Prospective, Case-Control Study With Mean 6 Years Follow-up.

PURPOSE: To determine whether generalized hypermobility and contralateral knee hyperextension affect failure rates and patient-related outcomes after anterior cruciate ligament reconstruction (ACLR). METHODS: A total of 226 consecutive patients presenting with acute ACL tears were prospectively evaluated for generalized hypermobility by a modified Beighton criteria. Minimum 2-year follow-up was achieved for 183 knees (81%). Patients underwent ACLR with either bone-patellar-tendon (BPTB) autograft (n = 46), quadrupled hamstring (HT) autograft (n = 85), or allograft tissue (n = 52). KT-1000 measurements, International Knee Documentation Committee (IKDC), Cincinnati, and Lysholm scores were obtained. RESULTS: Forty-one of 183 consecutive patients were categorized as hypermobile. At mean 6 years' follow-up (range 2-12.5 years), IKDC (P = .003), Cincinnati (P = .001), and Lysholm scores (P < .001) were significantly better in the Non-Hypermobile group for patients with an intact graft. The failure rate was higher in the Hypermobile group (10 knees, 24.4% failure rate) compared with the Nonhypermobile group (11 knees, 7.7% failure rate) (P = .006). The overall ACL injury rate (ACL graft injury, excessive graft laxity, plus contralateral ACL tear) was higher in the Hypermobile group (34.1%) compared with the Nonhypermobile group (12.0%) (P = .002). Heel height >5 cm (P = .009) and fifth metacarpophalangeal (MCP) extension >90° (P = .006) were independently predictive of failure for the entire study population. CONCLUSIONS: Graft failure rates were higher and inferior subjective outcomes were observed after ACLR in patients with generalized hypermobility. Heel height and fifth MCP hyperextension were most predictive of ACL injury/reinjury and poorer outcome scores. Nearly one-third of hypermobile patients sustained a contralateral ACL tear, ipsilateral graft failure, or had excessive graft laxity. LEVEL OF EVIDENCE: Level III, case control study.

Numbness Around the Total Knee Arthroplasty Surgical Scar: Prevalence and Effect on Functional Outcome.

BACKGROUND: Numbness around the surgical scar can be a source of discomfort or dissatisfaction in a proportion of patients undergoing total knee arthroplasty (TKA). Literature reports wide variation in its prevalence and the consequence of numbness on the outcome of TKA is not clear. We investigated the prevalence of numbness, along with contributing factors, and assessed its effect on the functional outcome of TKA. METHODS: In total, 258 knees were included in this prospective patient-reported outcome measure case-control study. Demographic details, type and length of incision, pre-operative and 1-year post-operative Knee Society Scores were recorded and compared. RESULTS: The prevalence of numbness at 1 year was 53%, with a female preponderance. Patients older than 70 years were less affected. Discomfort due to numbness was recorded in 8.7% of the patients, 75% of which were female. The length of the incision correlated positively with the presence of numbness. The Knee Society Scores did not correlate with the presence or area of numbness. CONCLUSION: Our findings indicate a high prevalence of numbness after TKA. Nevertheless, numbness does not affect the functional outcome.

Neuronal Trans-Differentiation in Prostate Cancer Cells.

BACKGROUND: Neuroendocrine (NE) differentiation in prostate cancer (PCa) is an aggressive phenotype associated with therapy resistance. The complete phenotype of these cells is poorly understood. Clinical classification is based predominantly on the expression of standard NE markers. METHODS: We analyzed the phenotype of NE carcinoma of the prostate utilizing in vitro methods, in silico, and immunohistochemical analyses of human disease. RESULTS: LNCaP cells, subjected to a variety of stressors (0.1% [v/v] fetal bovine serum, cyclic AMP) induced a reproducible phenotype consistent with neuronal trans-differentiation. Cells developed long cytoplasmic processes resembling neurons. As expected, serum deprived cells had decreased expression in androgen receptor and prostate specific antigen. A significant increase in neuronal markers also was observed. Gene array analysis demonstrated that LNCaP cells subjected to low serum or cAMP showed statistically significant manifestation of a human brain gene expression signature. In an in silico experiment using human data, we identified that only hormone resistant metastatic prostate cancer showed enrichment of the "brain profile." Gene ontology analysis demonstrated categories involved in neuronal differentiation. Three neuronal markers were validated in a large human tissue cohort. CONCLUSION: This study proposes that the later stages of PCa evolution involves neuronal trans-differentiation, which would enable PCa cells to acquire independence from the neural axis, critical in primary tumors. Prostate 76:1312-1325, 2016. © 2016 Wiley Periodicals, Inc.

Contributions of tropodithietic acid and biofilm formation to the probiotic activity of Phaeobacter inhibens.

BACKGROUND: The probiotic bacterium Phaeobacter inhibens strain S4Sm, isolated from the inner shell surface of a healthy oyster, secretes the antibiotic tropodithietic acid (TDA), is an excellent biofilm former, and increases oyster larvae survival when challenged with bacterial pathogens. In this study, we investigated the specific roles of TDA secretion and biofilm formation in the probiotic activity of S4Sm. RESULTS: Mutations in clpX (ATP-dependent ATPase) and exoP (an exopolysaccharide biosynthesis gene) were created by insertional mutagenesis using homologous recombination. Mutation of clpX resulted in the loss of TDA production, no decline in biofilm formation, and loss of the ability to inhibit the growth of Vibrio tubiashii and Vibrio anguillarum in co-colonization experiments. Mutation of exoP resulted in a ~60% decline in biofilm formation, no decline in TDA production, and delayed inhibitory activity towards Vibrio pathogens in co-colonization experiments. Both clpX and exoP mutants exhibited reduced ability to protect oyster larvae from death when challenged by Vibrio tubiashii. Complementation of the clpX and exoP mutations restored the wild type phenotype. We also found that pre-colonization of surfaces by S4Sm was critical for this bacterium to inhibit pathogen colonization and growth. CONCLUSIONS: Our observations demonstrate that probiotic activity by P. inhibens S4Sm involves contributions from both biofilm formation and the production of the antibiotic TDA. Further, probiotic activity also requires colonization of surfaces by S4Sm prior to the introduction of the pathogen.

Kinetics of the ClO + HO2 reaction over the temperature range T = 210-298 K.

The rate coefficient for the atmospherically important radical reaction: which leads to ozone depletion, has been studied over the temperature range T = 210-298 K and at ambient pressure p = 760 ± 20 Torr. The reaction was studied using laser flash photolysis radical generation coupled with broadband charge coupled device absorption spectroscopy employing a two-dimensional charge-coupled-device (CCD) detection system. ClO radicals were generated following the photolysis of Cl2 and Cl2O gas mixtures diluted in nitrogen and oxygen. ClO radicals were monitored using broadband fingerprinting of their characteristic vibronic (A(2)Π â† X(2)Π) spectral structure, representing a definitive monitoring of this radical. Addition of hydroperoxy radical precursors to the gas mixture (methanol and oxygen) subsequently led to a competition for photolytically generated Cl atoms and a simultaneous prompt formation of both ClO and HO2 radicals. Detailed analysis and modelling of the radical production routes provided a degree of constraint into numerical integration simulations which were then used to interrogate and fit to ClO temporal profiles to extract the rate coefficient k1. The ambient temperature (T = 298 K) rate coefficient reported is k1 = (8.5 ± 1.5) × 10(-12) cm(3) molecule(-1) s(-1). The rate coefficient, k1, is described by the Arrhenius expression:where errors are 1σ statistical only. This significant rate coefficient is greater than previously reported, with a stronger negative temperature dependence than previously observed. Consequently this suggests that the contribution of to ozone loss, in particular at mid-latitudes might be currently underestimated in models. This work reports atmospheric pressure kinetic parameters for this reaction which are greater than those reported from low pressure studies, perhaps supporting ClO and HO2 association as predicted by previous theoretical studies of this process and highlighting the need for further pressure dependent experimental studies of the title reaction, which has been demonstrated here to be effective as an ozone loss process over a wide temperature range.

Kinetics of the ClO + CH3O2 reaction over the temperature range T = 250-298 K.

The kinetics of the potentially atmospherically important ClO + CH3O2 reaction (1) have been studied over the range T = 250-298 K at p = 760 Torr using laser flash photolysis radical generation, coupled with time resolved ultraviolet absorption spectroscopy, employing broad spectral monitoring using a charge coupled device detector array. ClO radicals were monitored unequivocally using this technique, and introduction of CH3O2 precursors ensured known initial methylperoxy radical concentrations. ClO temporal profiles were thereafter analysed to extract kinetic parameters for reaction (1). A detailed sensitivity analysis was also performed to examine any potential systematic variability in k1 as a function of kinetic or physical uncertainties. The kinetic data recorded in this work show good agreement with the most recent previous study of this reaction, reported by Leather et al. The current work reports an Arrhenius parameterisation for k1, given by: . This work therefore concurs with that of Leather et al. implying that the title reaction is potentially less significant in the atmosphere than inferred from preceding studies. However, reaction (1) is evidently a non-terminating radical reaction, whose effects upon atmospheric composition therefore need to be ascertained through atmospheric model studies.