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Insertion of new material into the Escherichia coli peptidoglycan (PG) sacculus between the cytoplasmic membrane and the outer membrane requires a well-organized balance between synthetic and hydrolytic activities to maintain cell shape and avoid lysis. Since most bacteria carry multiple enzymes carrying the same type of PG hydrolytic activity, we know little about the specific function of given enzymes. Here we show that the DD-carboxy/endopeptidase PBP4 localizes in a PBP1A/LpoA and FtsEX dependent fashion at midcell during septal PG synthesis. Midcell localization of PBP4 requires its non-catalytic domain 3 of unknown function, but not the activity of PBP4 or FtsE. Microscale thermophoresis with isolated proteins shows that PBP4 interacts with NlpI and the FtsEX-interacting protein EnvC, an activator of amidases AmiA and AmiB, which are needed to generate denuded glycan strands to recruit the initiator of septal PG synthesis, FtsN. The domain 3 of PBP4 is needed for the interaction with NlpI and EnvC, but not PBP1A or LpoA. In vivo crosslinking experiments confirm the interaction of PBP4 with PBP1A and LpoA. We propose that the interaction of PBP4 with EnvC, whilst not absolutely necessary for mid-cell recruitment of either protein, coordinates the activities of PBP4 and the amidases, which affects the formation of denuded glycan strands that attract FtsN. Consistent with this model, we found that the divisome assembly at midcell was premature in cells lacking PBP4, illustrating how the complexity of interactions affect the timing of cell division initiation. Author summary: Peptidoglycan biosynthesis is a major target for antibacterials. The covalently closed peptidoglycan mesh, called sacculus, protects the bacterium from lysis due to its turgor. Sacculus growth is facilitated by the balanced activities of synthases and hydrolases, and disturbing this balance leads to cell lysis and bacterial death. Because of the large number and possible redundant functions of peptidoglycan hydrolases, it has been difficult to decipher their individual functions. In this paper we show that the DD-endopeptidase PBP4 localizes at midcell during septal peptidoglycan synthesis in Escherichia coli and is involved in the timing of the assembly and activation of the division machinery. This shows that inhibition of certain hydrolases could weaken the cells and might enhance antibiotic action. [ABSTRACT FROM AUTHOR]

The peptidoglycan (PG) layer is a critical component of the bacterial cell wall and serves as an important target for antibiotics in both gram-negative and gram-positive bacteria. The hydrolysis of septal PG (sPG) is a crucial step of bacterial cell division, facilitated by FtsEX through an amidase activation system. In this study, we present the cryo-EM structures of Escherichia coli FtsEX and FtsEX-EnvC in the ATP-bound state at resolutions of 3.05 Å and 3.11 Å, respectively. Our PG degradation assays in E. coli reveal that the ATP-bound conformation of FtsEX activates sPG hydrolysis of EnvC-AmiB, whereas EnvC-AmiB alone exhibits autoinhibition. Structural analyses indicate that ATP binding induces conformational changes in FtsEX-EnvC, leading to significant differences from the apo state. Furthermore, PG degradation assays of AmiB mutants confirm that the regulation of AmiB by FtsEX-EnvC is achieved through the interaction between EnvC-AmiB. These findings not only provide structural insight into the mechanism of sPG hydrolysis and bacterial cell division, but also have implications for the development of novel therapeutics targeting drug-resistant bacteria. The septal peptidoglycan hydrolysis machinery is a crucial structure for bacterial cell division. This study analyses the cryo-EM structure and enzymatic function of E. coli FtsEX, with and without the lytic-factor protein EnvC, characterizing the molecular basis of FtsEX regulation of peptidoglycan hydrolysis, which could be used to develop novel treatments targeting drug-resistant bacteria. [ABSTRACT FROM AUTHOR]

Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia, an important reaction in the NAD+ salvage pathway. This paper reports a new nicotinamidase from the deep-sea extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831 (OiNIC). The enzyme was active towards nicotinamide and several analogues, including the prodrug pyrazinamide. The enzyme was more nicotinamidase (kcat/Km = 43.5 mM−1s−1) than pyrazinamidase (kcat/Km = 3.2 mM−1s−1). Mutational analysis was carried out on seven critical amino acids, confirming for the first time the importance of Cys133 and Phe68 residues for increasing pyrazinamidase activity 2.9- and 2.5-fold, respectively. In addition, the change in the fourth residue involved in the ion metal binding (Glu65) was detrimental to pyrazinamidase activity, decreasing it 6-fold. This residue was also involved in a new distinct structural motif DAHXXXDXXHPE described in this paper for Firmicutes nicotinamidases. Phylogenetic analysis revealed that OiNIC is the first nicotinamidase described for the order Bacillales. [ABSTRACT FROM AUTHOR]

Dihydropyrimidinase is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. These metalloenzymes possess very similar active sites and may use a similar mechanism for catalysis. However, whether the substrates and inhibitors of other cyclic amidohydrolases can inhibit dihydropyrimidinase remains unclear. This study investigated the inhibition of dihydropyrimidinase by flavonoids and substrates of other cyclic amidohydrolases. Allantoin, dihydroorotate, 5-hydantoin acetic acid, acetohydroxamate, orotic acid, and 3-amino-1,2,4-triazole could slightly inhibit dihydropyrimidinase, and the IC50 values of these compounds were within the millimolar range. The inhibition of dihydropyrimidinase by flavonoids, such as myricetin, quercetin, kaempferol, galangin, dihydromyricetin, and myricitrin, was also investigated. Some of these compounds are known as inhibitors of allantoinase and dihydroorotase. Although the inhibitory effects of these flavonoids on dihydropyrimidinase were substrate-dependent, dihydromyricetin significantly inhibited dihydropyrimidinase with IC50 values of 48 and 40 μM for the substrates dihydrouracil and 5-propyl-hydantoin, respectively. The results from the Lineweaver−Burk plot indicated that dihydromyricetin was a competitive inhibitor. Results from fluorescence quenching analysis indicated that dihydromyricetin could form a stable complex with dihydropyrimidinase with the Kd value of 22.6 μM. A structural study using PatchDock showed that dihydromyricetin was docked in the active site pocket of dihydropyrimidinase, which was consistent with the findings from kinetic and fluorescence studies. This study was the first to demonstrate that naturally occurring product dihydromyricetin inhibited dihydropyrimidinase, even more than the substrate analogs (>3 orders of magnitude). These flavonols, particularly myricetin, may serve as drug leads and dirty drugs (for multiple targets) for designing compounds that target several cyclic amidohydrolases. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-β-lactamases (MβLs), enzymes able to hydrolyze most β-lactam antibiotics. SPM-1 is an MβL produced only by P. aeruginosa, while other MβLs are found in different bacteria. Despite similar active sites, the resistance profile of MβLs towards β-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MβLs in that it contains “atypical” second sphere residues (S84, G121). Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II) requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II)-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards β-lactam antibiotics, the enzyme stability and the Zn(II) binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site. [ABSTRACT FROM AUTHOR]

The Gram-negative bacteria type VI secretion system (T6SS) has been found to play an important role in interbacterial competition, biofilm formation and many other virulence-related processes. The bacteria harboring T6SS inject the effectors into their recipient’s cytoplasm or periplasm to kill them and meanwhile, to avoid inhibiting itself, the cognate immunity proteins were produced to acts as the effector inhibitor. Tae4 (type VI amidase effector 4) and Tai4 (type VI amidase immunity 4) are newly identified T6SS effector-immunity (EI) pairs. We have recently solved the structures of StTae4-Tai4 and EcTae4-Tai4 complexes from the human pathogens Salmonella typhimurium and Enterobacter cloacae, respectively. It is very interesting and important to discover whether there is cross-neutralization between St- and EcTai4 and whether their effector inhibition mechanism is conserved. Here, we determined the crystal structure of StTae4 in complex with EcTai4. The solution conformation study revealed it is a compact heterotetramer that consists of an EcTai4 homodimer binding two StTae4 molecules in solution, different from that in crystal. A remarkable shift can be observed in both the flexible winding loop of StTae4 and protruding loop of EcTai4 and disulfide bonds are formed to stabilize their overall conformations. The in vitro and in vivo interactions studies showed EcTai4 can efficiently rescue the cells from the toxicity of its cognate effectors StTae4, but can not neutralize the toxic activities of the effectors from other families. These findings provide clear structural evidence to support the previous observation of cross-immunity within T6SS families and provide a basis for understanding their important roles in polymicrobial environments. [ABSTRACT FROM AUTHOR]

Rapidly growing tumor cells require a nutrient-rich environment in order to thrive, therefore, restricting access to certain key amino acids, such as arginine, often results in the death of malignant cells, which frequently display defective cell cycle check-point control. Healthy cells, by contrast, become quiescent and remain viable under arginine restriction, displaying full recovery upon return to arginine-rich conditions. The use of arginase therapy to restrict available arginine for selectively targeting malignant cells is currently under investigation in human clinical trials. However, the suitability of this approach for veterinary uses is unexplored. As a prelude to in vivo studies in canine malignancies, we examined the in vitro effects of arginine-deprivation on canine lymphoid and osteosarcoma cell lines. Two lymphoid and 2 osteosarcoma cell lines were unable to recover following 6 days of arginine deprivation, but all remaining cell lines displayed full recovery upon return to arginine-rich culture conditions. These remaining cell lines all proved susceptible to cell death following the addition of arginase to the cultures. The lymphoid lines were particularly sensitive to arginase, becoming unrecoverable after just 3 days of treatment. Two of the osteosarcoma lines were also susceptible over this time-frame; however the other 3 lines required 6-8 days of arginase treatment to prevent recovery. In contrast, adult progenitor cells from the bone marrow of a healthy dog were able to recover fully following 9 days of culture in arginase. Over 3 days in culture, arginase was more effective than asparaginase in inducing the death of lymphoid lines. These results strongly suggest that short-term arginase treatment warrants further investigation as a therapy for lymphoid malignancies and osteosarcomas in dogs. [ABSTRACT FROM AUTHOR]

We introduce PFunkel, a versatile method for extensive, researcher-defined DNA mutagenesis using a ssDNA or dsDNA template. Once the template DNA is prepared, the method can be completed in a single day in a single tube, and requires no intermediate DNA purification or sub-cloning. PFunkel can be used for site-directed mutagenesis at an efficiency approaching 100%. More importantly, PFunkel allows researchers the unparalleled ability to efficiently construct userdefined libraries. We demonstrate the creation of a library with site-saturation at four distal sites simultaneously at 70% efficiency. We also employ PFunkel to create a comprehensive codon mutagenesis library of the TEM-1 ß-lactamase gene. We designed this library to contain 18,081 members, one for each possible codon substitution in the gene (287 positions in TEM-1 x 63 possible codon substitutions). Deep sequencing revealed that ∼97% of the designed single codon substitutions are present in the library. From such a library we identified 18 previously unreported adaptive mutations that each confer resistance to the ß-lactamase inhibitor tazobactam. Three of these mutations confer resistance equal to or higher than that of the most resistant reported TEM-1 allele and have the potential to emerge clinically. [ABSTRACT FROM AUTHOR]

Background: Staphylococcus aureus is a frequent cause of serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, and sepsis. Its adherence to various host structures is crucial for the establishment of diseases. Adherence may be mediated by a variety of adhesins, among them the autolysin/adhesins Atl and Aaa. Aaa is composed of three N-terminal repeated sequences homologous to a lysin motif (LysM) that can confer cell wall attachment and a Cterminally located cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain having bacteriolytic activity in many proteins. Methodology/Principal Findings: Here, we show by surface plasmon resonance that the LysM domain binds to fibrinogen, fibronectin, and vitronectin respresenting a novel adhesive function for this domain. Moreover, we demonstrated that the CHAP domain not only mediates the bacteriolytic activity, but also adherence to fibrinogen, fibronectin, and vitronectin, thus demonstrating for the first time an adhesive function for this domain. Adherence of an S. aureus aaa mutant and the complemented aaa mutant is slightly decreased and increased, respectively, to vitronectin, but not to fibrinogen and fibronectin, which might at least in part result from an increased expression of atl in the aaa mutant. Furthermore, an S. aureus atl mutant that showed enhanced adherence to fibrinogen, fibronectin, and endothelial cells also demonstrated increased aaa expression and production of Aaa. Thus, the redundant functions of Aaa and Atl might at least in part be interchangeable. Lastly, RT-PCR and zymographic analysis revealed that aaa is negatively regulated by the global virulence gene regulators agr and SarA. Conclusions/Significance: We identified novel functions for two widely distributed protein domains, LysM and CHAP, i.e. the adherence to the extracellular matrix proteins fibrinogen, fibronectin, and vitronectin. The adhesive properties of Aaa might promote S. aureus colonization of host extracellular matrix and tissue, suggesting a role for Aaa in the pathogenesis of S. aureus infections. [ABSTRACT FROM AUTHOR]

Accumulating evidence suggests that breast cancer metastatic progression is modified by germline polymorphism, although specific modifier genes have remained largely undefined. In the current study, we employ the MMTV-PyMT transgenic mouse model and the AKXD panel of recombinant inbred mice to identify AT--rich interactive domain 4B (Arid4b; NM_194262) as a breast cancer progression modifier gene. Ectopic expression of Arid4b promoted primary tumor growth in vivo as well as increased migration and invasion in vitro, and the phenotype was associated with polymorphisms identified between the AKR/J and DBA/2J alleles as predicted by our genetic analyses. Stable shRNA--mediated knockdown of Arid4b caused a significant reduction in pulmonary metastases, validating a role for Arid4b as a metastasis modifier gene. ARID4B physically interacts with the breast cancer metastasis suppressor BRMS1, and we detected differential binding of the Arid4b alleles to histone deacetylase complex members mSIN3A and mSDS3, suggesting that the mechanism of Arid4b action likely involves interactions with chromatin modifying complexes. Downregulation of the conserved Tpx2 gene network, which is comprised of many factors regulating cell cycle and mitotic spindle biology, was observed concomitant with loss of metastatic efficiency in Arid4b knockdown cells. Consistent with our genetic analysis and in vivo experiments in our mouse model system, ARID4B expression was also an independent predictor of distant metastasis-free survival in breast cancer patients with ER+ tumors. These studies support a causative role of ARID4B in metastatic progression of breast cancer. [ABSTRACT FROM AUTHOR]

There is increasing evidence that regulation of local chromatin structure is a critical mechanism underlying the consolidation of long-term memory (LTM), however considerably less is understood about the specific mechanisms by which these epigenetic effects are mediated. Furthermore, the importance of histone acetylation in Drosophila memory has not been reported. The histone deacetylase (HDAC) Rpd3 is abundant in the adult fly brain, suggesting a post-mitotic function. Here, we investigated the role of Rpd3 in long-term courtship memory in Drosophila. We found that while modulation of Rpd3 levels predominantly in the adult mushroom body had no observed impact on immediate recall or onehour memory, 24-hour LTM was severely impaired. Surprisingly, both overexpression as well as RNAi-mediated knockdown of Rpd3 resulted in impairment of long-term courtship memory, suggesting that the dose of Rpd3 is critical for normal LTM. [ABSTRACT FROM AUTHOR]

Mycobacteriophages are dsDNA viruses that infect mycobacterial hosts. The mycobacteriophage Ms6 accomplishes lysis by producing two cell wall hydrolytic enzymes, Lysin A (LysA) that possesses a central peptidoglycan recognition protein (PGRP) super-family conserved domain with the amidase catalytic site, that cleaves the amide bond between the Nacetylmuramic acid and L-alanine residues in the oligopeptide crosslinking chains of the peptidoglycan and Lysin B (LysB) a mycolylarabinogalactan esterase that hydrolyzes the mycolic acids from the mycolyl-arabinogalactan-peptidoglycan complex. Examination of the endolysin (lysA) DNA sequence revealed the existence of an embedded gene (lysA241) encoded in the same reading frame and preceded by a consensus ribosome-binding site. In the present work we show that, even though lysA is essential for Ms6 viability, phage mutants that express only the longer (Lysin384) or the shorter (Lysin241) endolysin are viable, but defective in the normal timing, progression and completion of host cell lysis. In addition, both endolysins have peptidoglycan hydrolase activity and demonstrated broad growth inhibition activity against various Grampositive bacteria and mycobacteria [ABSTRACT FROM AUTHOR]

Histone deacetylase inhibitors (HDI) dampen cellular innate immune response by decreasing interferon production and have been shown to increase the growth of vesicular stomatitis virus and HSV. As attenuated tumour-selective oncolytic vaccinia viruses (VV) are already undergoing clinical evaluation, the goal of this study is to determine whether HDI can also enhance the potency of these poxviruses in infection-resistant cancer cell lines. Multiple HDIs were tested and Trichostatin A (TSA) was found to potently enhance the spread and replication of a tumour selective vaccinia virus in several infection-resistant cancer cell lines. TSA significantly decreased the number of lung metastases in a syngeneic B16F10LacZ lung metastasis model yet did not increase the replication of vaccinia in normal tissues. The combination of TSA and VV increased survival of mice harbouring human HCT116 colon tumour xenografts as compared to mice treated with either agent alone. We conclude that TSA can selectively and effectively enhance the replication and spread of oncolytic vaccinia virus in cancer cells. [ABSTRACT FROM AUTHOR]

An understanding of the integrated relationships among the principal cellular functions that govern the bioenergetic reactions of an organism is necessary to determine how cells remain viable and optimise their fitness in the environment. Urease is a complex enzyme that catalyzes the hydrolysis of urea to ammonia and carbonic acid. While the induction of urease activity by several microorganisms has been predominantly considered a stress-response that is initiated to generate a nitrogen source in response to a low environmental pH, here we demonstrate a new role of urease in the optimisation of cellular bioenergetics. We show that urea hydrolysis increases the catabolic efficiency of Streptococcus thermophilus, a lactic acid bacterium that is widely used in the industrial manufacture of dairy products. By modulating the intracellular pH and thereby increasing the activity of b-galactosidase, glycolytic enzymes and lactate dehydrogenase, urease increases the overall change in enthalpy generated by the bioenergetic reactions. A cooperative altruistic behaviour of urease-positive microorganisms on the urease-negative microorganisms within the same environment was also observed. The physiological role of a single enzymatic activity demonstrates a novel and unexpected view of the non-transcriptional regulatory mechanisms that govern the bioenergetics of a bacterial cell, highlighting a new role for cytosol-alkalizing biochemical pathways in acidogenic microorganisms. [ABSTRACT FROM AUTHOR]

Background: Children with ependymoma may experience a relapse in up to 50% of cases depending on the extent of resection. Key biological events associated with recurrence are unknown. Methodology/Principal Findings: To discover the biology behind the recurrence of ependymomas, we performed CGHarray and a dual-color gene expression microarray analysis of 17 tumors at diagnosis co-hybridized with the corresponding 27 first or subsequent relapses from the same patient. As treatment and location had only limited influence on specific gene expression changes at relapse, we established a common signature for relapse. Eighty-seven genes showed an absolute fold change ≥2 in at least 50% of relapses and were defined as the gene expression signature of ependymoma recurrence. The most frequently upregulated genes are involved in the kinetochore (ASPM, KIF11) or in neural development (CD133, Wnt and Notch pathways). Metallothionein (MT) genes were downregulated in up to 80% of the recurrences. Quantitative PCR for ASPM, KIF11 and MT3 plus immunohistochemistry for ASPM and MT3 confirmed the microarray results. Immunohistochemistry on an independent series of 24 tumor pairs at diagnosis and at relapse confirmed the decrease of MT3 expression at recurrence in 17/24 tumor pairs (p = 0.002). Conversely, ASPM expression was more frequently positive at relapse (87.5% vs 37.5%, p = 0.03). Loss or deletion of the MT genes cluster was never observed at relapse. Promoter sequencing after bisulfite treatment of DNA from primary tumors and recurrences as well as treatment of short-term ependymoma cells cultures with a demethylating agent showed that methylation was not involved in MT3 downregulation. However, in vitro treatment with a histone deacetylase inhibitor or zinc restored MT3 expression. Conclusions/Significance: The most frequent molecular events associated with ependymoma recurrence were overexpression of kinetochore proteins and down-regulation of metallothioneins. Metallothionein-3 expression is epigenetically controlled and can be restored in vitro by histone deacetylase inhibitors. [ABSTRACT FROM AUTHOR]

The Set2-Rpd3S pathway is important for the control of transcription memory. Mutation of components of this pathway results in cryptic transcription initiation within the coding region of approximately 30% of yeast genes. Specifically, deletion of the Set2 histone methyltransferase or Rco1, a component of the Rpd3S histone deacetylase complex leads to hyperacetylation of certain open reading frames (ORFs). We used this mutant as a system to study the role of histone modifications and co-activator recruitment in preinitiation complex (PIC) formation. Specifically, we looked at the dependence of promoters on the bromodomain-containing RSC complex and the Bdf1 protein. We found that the dependence of cryptic promoters for these proteins varied. Overall, our data indicate that cryptic promoters are independently regulated, and their activation is dependent on factors that govern gene activation at canonical promoters. [ABSTRACT FROM AUTHOR]

Background: Human immunodeficiency virus (HIV) infection that persists despite antiretroviral therapy (ART) is a daunting problem. Given the limited evidence that resting CD4+ T cell infection (RCI) is affected by the histone deacetylase (HDAC) inhibitor valproic acid (VPA), we measured the stability of RCI and residual viremia in patients who added VPA with or without raltegravir (RAL), or enfuvirtide (ENF) with or without VPA, to standard ART. Methods: Patients with plasma HIV RNA<50 c/mL added sustained-release VPA (Depakote ERH) twice daily, RAL 400 mg twice daily, or ENF 90 mcg twice daily. Change in RCI was measured by outgrowth assays. Low-level viremia was quantitated by single-copy plasma HIV RNA assay (SCA). Results: In three patients on standard ART a depletion of RCI was observed after 16 weeks of VPA, but this effect waned over up to 96 weeks of further VPA. In two patients ENF added to stable ART had no effect on RCI. Simultaneous intensification with ENF and addition of VPA had no effect on RCI frequency in one patient, and resulted in a 46% decline in a second. No significant depletion of RCI (>50%) was seen in six volunteers after the addition of RAL and VPA. In 4 of the 6 patients this lack of effect might be attributed to intermittent viremia, low VPA levels, or intermittent study therapy adherence. Overall, there was no effect of the addition of RAL or ENF on low-level viremia measured by SCA. Conclusions: The prospective addition of VPA and RAL, VPA and ENF, or ENF failed to progressively reduce the frequency of RCI, or ablate intermittent and low-level viremia. New approaches such as more potent HDAC inhibition, alone or in combination with intensified ART or other agents that may disrupt proviral latency must be pursued. [ABSTRACT FROM AUTHOR]

Peptidoglycan-recognition proteins (PGRPs) are evolutionarily conserved molecules that are structurally related to bacterial amidases. Several Drosophila PGRPs have lost this enzymatic activity and serve as microbe sensors through peptidoglycan recognition. Other PGRP family members, such as Drosophila PGRP-SC1 or mammalian PGRP-L, have conserved the amidase function and are able to cleave peptidoglycan in vitro. However, the contribution of these amidase PGRPs to host defense in vivo has remained elusive so far. Using an RNA-interference approach, we addressed the function of two PGRPs with amidase activity in the Drosophila immune response. We observed that PGRP-SC1/2- depleted flies present a specific over-activation of the IMD (immune deficiency) signaling pathway after bacterial challenge. Our data suggest that these proteins act in the larval gut to prevent activation of this pathway following bacterial ingestion. We further show that a strict control of IMD-pathway activation is essential to prevent bacteria-induced developmental defects and larval death. [ABSTRACT FROM AUTHOR]