Modulation Domain Research Articles

Roles of Conserved Active Site Residues in the Ketosynthase Domain of an Assembly Line Polyketide Synthase.

Ketosynthase (KS) domains of assembly line polyketide synthases (PKSs) catalyze intermodular translocation of the growing polyketide chain as well as chain elongation via decarboxylative Claisen condensation. The mechanistic roles of ten conserved residues in the KS domain of Module 1 of the 6-deoxyerythronolide B synthase were interrogated via site-directed mutagenesis and extensive biochemical analysis. Although the C211A mutant at the KS active site exhibited no turnover activity, it was still a competent methylmalonyl-ACP decarboxylase. The H346A mutant exhibited reduced rates of both chain translocation and chain elongation, with a greater effect on the latter half-reaction. H384 contributed to methylmalonyl-ACP decarboxylation, whereas K379 promoted C-C bond formation. S315 played a role in coupling decarboxylation to C-C bond formation. These findings support a mechanism for the translocation and elongation half-reactions that provides a well-defined starting point for further analysis of the key chain-building domain in assembly line PKSs.

Performance Analysis of Spatial Modulation in Overlay Cognitive Radio Communications

We study the application of spatial modulation (SM) in overlay cognitive radio (CR) networks, in which the primary and secondary networks work concurrently over the same spectrum band. We assume that the direct link between the primary transmitter and receiver is not available, i.e. broken, and that the CR transmitter assists the primary network as a relay to amplify-and-forward the transmitted symbols of the primary. SM is used in the secondary to split the transmission space into two amplitude-phase modulation (APM) and spatial domains. In the proposed scheme, the secondary transmitter retransmits the primary symbols in APM domain, while its own information is transmitted by the index of transmitting antenna, similar to space shift keying, without causing any interference to the primary receiver. We analyze the performance of the optimal detectors at both the primary and secondary receivers for the case of phase shift keying modulation in terms of the average symbol error rate (ASER). We also study the asymptotic behavior of the ASER at both the primary and secondary at high signal-to-noise ratios. Simulation results show that the SM can be effectively used in overlay CR systems.

Integrated acoustic echo and noise suppression in modulation domain

The quality of speech transmission in mobile communication systems deteriorates due to the presence of background noise and acoustic echo. The background noises are the disturbances from the surroundings and acoustic echo is induced due to the reverberation of loudspeaker signal in the near end environment. In conventional acoustic echo suppression setup, the echo path effect is modelled either in time or in frequency domain, and to cancel the echo, a replica of the echo is created by estimating the echo path response adaptively in the corresponding domain. Recently, the modulation domain analysis, which captures the human perceptual properties, is widely being used in speech processing. Modulation domain conveys the temporal variation of the acoustic magnitude spectra which acts as an information bearing signal. In this work, a novel integrated system for acoustic echo and noise suppression in the modulation domain is developed. So far, no work in this context in modulation domain has been found as reported. An efficient method for modelling the echo path and estimating the echo in the modulation domain is introduced and implemented. The effects of echo and noise are suppressed using the modulation spectral manipulation and the performance of the proposed system is found to be better than other conventional integrated systems.

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases

The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzyme-substrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs.

Reversible Glucan Phosphorylation Requires Unique Carbohydrate Binding Platforms

Research into starch metabolism and the fatal neurodegenerative epilepsy called Lafora disease are surprisingly linked by a family of enzymes that we recently discovered called glucan phosphatases. Plants release the energy in transitory starch via a recently identified three‐step process: starch phosphorylation, degradation, and dephosphorylation. Dikinases phosphorylate the outer starch glucose units to make them water‐soluble and enzyme accessible so that amylases can release maltose and glucose. Following amylase activity, the phosphate must be removed by glucan phosphatases. In the absence of glucan phosphates, plants cannot access the energy stored in starch and the starch granules grow in size while plant growth is stunted.We recently determined the structure of the Arabidopsis glucan phosphatases Starch Excess 4 (SEX4) and Like Sex Four2 (LSF2) with phospho‐glucan product bound at 1.62Å and 2.3Å, respectively. The glucose moiety closest to the SEX4 active site is positioned with the oxygen of the C6 carbon directed into the catalytic cleft and we find that SEX4 preferentially dephosphorylates C6 hydroxyls. Alternatively, the glucose moiety closest to the LSF2 active site is positioned with the C3 carbon towards the catalytic cleft and we find that LSF2 exclusively dephosphorylates C3 hydroxyls. Using structural and biochemical insights, we completely reversed SEX4 specificity, providing a method for engineering glucan phosphatase activity.The human EPM2A gene encodes laforin and recessive mutations in EPM2A result in Lafora disease (LD). In the absence of laforin activity, glycogen transforms into a hyper‐phosphorylated, water‐insoluble, starch‐like Lafora body (LB) that drives neuronal apoptosis, neurodegeneration, and eventual death of LD patients. The physiological function of laforin is to dephosphorylate glycogen, yet the mechanism of glycogen dephosphorylation by laforin was unknown. Additionally, LD missense mutations are dispersed throughout laforin, bringing to question the structural mechanism(s) of disease. We determined the crystal structure of human laforin at 2.4 Å bound to oligosaccharides with a phospho‐glucan product at the active site. The structure reveals an integrated tertiary structure of the carbohydrate binding module and dual specificity phosphatase domains as well as an antiparallel dimer mediated by the phosphatase domain that results in a tetramodular architecture, positioning the two active sites ~31 Å from each other. We utilized the crystal structure and three solution‐based, biophysical techniques along with biochemical analyses of LD patient mutations and structured guided mutations to probe this unique tertiary and quaternary structure. We define a cooperative mechanism of action for laforin as well as establish the effect of LD disease mutations, thereby providing atomic level insights that connect basic glycogen metabolism to human neurodegenerative disease.These structures are the first of glucan phosphatases, and they provide new insights into the molecular basis of this medically‐, agriculturally‐, and industry‐relevant enzyme family as well as their unique mechanisms of catalysis, substrate specificity, and interaction with glucans.Support or Funding InformationNIH Grants R01NS070899 and P20GM103486; NSF grant MCB‐1252345; KSEF Grants KSEF‐2268‐RDE‐014 and KSEF‐2971‐RDE‐ 017; Mizutani Foundation for Glycoscience Award

(Invited) 300GHz CMOS Wireless Communication with 32 Quadrature-Amplitude-Modulation Capability

Over hundred years have passed since Marconi sent the first Atlantic wireless transmission in 1901. Over the years, frequency used for wireless communication has been increasing by ten folds every 20 years. According to the trend, frequency used for wireless communication will reach at terahertz band in 2020. In the research on terahertz wireless communication, transceivers with compound semiconductor were reported[1-2]. However, integration of large-scale digital circuits in a compound semiconductor is so difficult that complex modulation and demodulation for high-speed communication and frequency-domain equalization including propagation environment are unrealistic. On the other hand, although CMOS integrated circuits can realize complex modulation and frequency domain equalization, terahertz transceivers with CMOS are challenging since typical maximum operation frequency in advanced process is still around 300GHz. To overcome relatively low operation frequency with CMOS transistors, we have adopted a tripler in a transmitter, where the tripler utilizes cubic nonlinearity in a transistor[3]. Generally, when modulated signal is given to the tripler, its carrier frequency triples but original constellation is distorted. Therefore, in [4], the tripler was used only for QPSK (quadrature phase shift keying) where the amplitude of symbols is constant and the shape of constellation is not affected by tripler’s nonlinearity. Nevertheless, in order to realize higher communication speed with more bits per symbol such as 16 QAM or higher, how should the tripler be utilized? To solve the nonlinearity issue in the tripler, local oscillation signal (LO) as well as the modulated intermediate-frequency signal (IF) is given to the tripler simultaneously. When the two types of spectral components are given to the tripler, intermodulation between two signals occurs. As a result, spectral components of appear at the output. Since the second term maintains linearity for the IF, modulation information in the IF is preserved when only the second term is properly filtered. Adopting this technique, 300GHz-band CMOS transmitter is fabricated with 40nm process. Measured results show that 17.5Gbps per channel is realized over 6 channels with 32 QAM. As a result, total bit rate reaches at 105Gbps when all the 6 channels can be bonded. [1] H-J. Song et al., "50-Gb/s Direct Conversion QPSK Modulator and Demodulator MMICs for Terahertz Communications at 300 GHz," IEEE Trans. Microwave Theory & Tech., vol. 62, no. 3, pp. 600 - 609, March 2014. [2] C. Wang et al., "0.34-THz Wireless Link Based on High-Order Modulation for Future Wireless Local Area Network Applications ," IEEE Trans. Terahertz Science & Tech., vol. 4, no. 1, pp. 75 - 85, Jan. 2014. [3] K. Katayama et al., "A 300GHz 40nm CMOS Transmitter with 32-QAM 17.5Gb/s/ch Capability over 6 Channels," 2016 IEEE International Solid-State Circuits Conf. Dig. (in press). [4] S. Kang et al., "A 240GHz wideband QPSK transmitter in 65nm CMOS," 2014 IEEE Radio Frequency Integrated Circuits Symp., pp. 353 - 356, 1-3 June 2014.

Robust Speech Recognition via Enhancing the Complex-Valued Acoustic Spectrum in Modulation Domain

The purpose of this paper is to develop a novel speech feature extraction framework for independently compensating the real and imaginary acoustic spectra of speech signals in the modulation domain with the techniques of histogram equalization (HEQ) and non-negative matrix factorization (NMF). By doing so, we can enhance not only the magnitude but also the phase components of the acoustic spectra, thereby creating noise-robust speech features. More specifically, the proposed framework makes the following three major contributions: First, via either of the HEQ and NMF operations, the long-term cross-frame correlation among the acoustic spectra at the same frequency can be captured to compensate for the spectral distortion caused by noise. Second, the noise effect can be handled in a high acoustic frequency resolution. Finally, the distortion dwelt in the acoustic spectra can be more extensively mitigated due to the independent processes for the respective real and imaginary parts. The evaluation experiments were carried out on the Aurora-2 and Aurora-4 benchmark tasks, and the corresponding results suggest that our proposed methods can achieve performance competitive to or better than many widely used noise robustness methods, including the well-known advanced front-end (AFE) extraction scheme, in speech recognition.

A Turnstile Mechanism for the Controlled Growth of Biosynthetic Intermediates on Assembly Line Polyketide Synthases.

Vectorial polyketide biosynthesis on an assembly line polyketide synthase is the most distinctive property of this family of biological machines, while providing the key conceptual tool for the bioinformatic decoding of new antibiotic pathways. We now show that the action of the entire assembly line is synchronized by a previously unrecognized turnstile mechanism that prevents the ketosynthase domain of each module from being acylated by a new polyketide chain until the product of the prior catalytic cycle has been passed to the downstream module from the corresponding acyl carrier protein domain. The turnstile is closed by virtue of tight coupling to the signature decarboxylative condensation reaction catalyzed by the ketosynthase domain of each polyketide synthase module. Reopening of the turnstile is coupled to the eventual chain translocation step that vacates the module. At the maximal rate of substrate turnover, one would expect the chain release step to initiate a cascade of chain translocation events that sequentially migrate back upstream, thereby repriming each module and setting up the assembly line for the next round of polyketide chain elongation.

Recent Developments in Speech Enhancement in the Short-Time Fourier Transform Domain

In this paper, we present an overview on the topic of noise reduction in the short-time Fourier transform (STFT) domain. First, we briefly review the conventional literature in the single- and multichannel cases separately. In the single-channel scenario, we focus on the spectral subtractive methods, Wiener filter based methods, speech amplitude estimators and estimators of the complex STFT coefficients. In the multi-channel scenario, we investigate in short a selection of key beamforming approaches as well as conventional post-filtering methods. Next, a detailed survey of the most recent advances in the STFT-based noise reduction methods is provided. This includes STSA estimators with super-Gaussian priors, noise power spectral density (PSD ) estimation, estimation methods in the modulation domain, estimation of spectral phase and noise PSD matrix estimation for multi-channel applications. Finally, we summarize the presented material and draw important conclusions on each of the investigated topics.

Lst4, the yeast Fnip1/2 orthologue, is a DENN-family protein.

The folliculin/Fnip complex has been demonstrated to play a crucial role in the mechanisms underlying Birt–Hogg–Dubé (BHD) syndrome, a rare inherited cancer syndrome. Lst4 has been previously proposed to be the Fnip1/2 orthologue in yeast and therefore a member of the DENN family. In order to confirm this, we solved the crystal structure of the N-terminal region of Lst4 from Kluyveromyces lactis and show it contains a longin domain, the first domain of the full DENN module. Furthermore, we demonstrate that Lst4 through its DENN domain interacts with Lst7, the yeast folliculin orthologue. Like its human counterpart, the Lst7/Lst4 complex relocates to the vacuolar membrane in response to nutrient starvation, most notably in carbon starvation. Finally, we express and purify the recombinant Lst7/Lst4 complex and show that it exists as a 1 : 1 heterodimer in solution. This work confirms the membership of Lst4 and the Fnip proteins in the DENN family, and provides a basis for using the Lst7/Lst4 complex to understand the molecular function of folliculin and its role in the pathogenesis of BHD syndrome.

Interaction of DNA Aptamers with the ATP-Dependent Lon Protease from Escherichia coli

ATP-dependent Lon protease of E. coli (Ec-Lon) is a key enzyme of the quality control system of the cell proteome. Ec-Lon subunit comprises N-terminal non-catalytic region, ATPase module and proteolytic domain (serine-lysine endopeptidase). A distinctive feature of the Ec-Lon is its ability to interact with DNA, however either DNA binding site(s) or the role ofthe complex Ec-Lon · DNA have not yet been characterized. A promising tool for the study of molecular mechanisms of interaction between nucleic acids and protein ligands are known to be aptamers (small nucleic acids with high specificity to organic compounds of different nature). Ec-Lon-protease was found to form complexes with the previously obtained thrombin aptamers whose molecules comprise the duplex domains and G-quadruplex region. The aptamer affinities to the enzyme have been characterized. The synthesis of novel aptamers specific to Ec-Lon protease is planed for studying the mechanism of the enzyme-DNA complexation.

Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

Genome-based analysis of non-ribosomal peptide synthetase and type-I polyketide synthase gene clusters in all type strains of the genus Herbidospora.

BackgroundThe genus Herbidospora comprises actinomycetes belonging to the family Streptosporangiaceae and currently contains five recognized species. Although other genera of this family often produce bioactive secondary metabolites, Herbidospora strains have not yet been reported to produce secondary metabolites. In the present study, to assess their potential as secondary metabolite producers, we sequenced the whole genomes of the five type strains and searched for the presence of their non-ribosomal peptide synthetase (NRPS) and type-I polyketide synthase (PKS) gene clusters. These clusters are involved in the major secondary metabolite–synthetic pathways in actinomycetes.ResultsThe genome sizes of Herbidospora cretacea NBRC 15474T, Herbidospora mongoliensis NBRC 105882T, Herbidospora yilanensis NBRC 106371T, Herbidospora daliensis NBRC 106372T and Herbidospora sakaeratensis NBRC 102641T were 8.3, 9.0, 7.9, 8.5 and 8.6 Mb, respectively. They contained 15–18 modular NRPS and PKS gene clusters. Thirty-two NRPS and PKS pathways were identified, among which 9 pathways were conserved in all 5 strains, 8 were shared in 2–4 strains, and the remaining 15 were strain-specific. We predicted the chemical backbone structures of non-ribosomal peptides and polyketides synthesized by these gene clusters, based on module number and domain organization of NRPSs and PKSs. The relationship between 16S rRNA gene sequence-based phylogeny of the five strains and the distribution of their NRPS and PKS gene clusters were also discussed.ConclusionsThe genomes of Herbidospora strains carry as many NRPS and PKS gene clusters, whose products are yet to be isolated, as those of Streptomyces. Herbidospora members should synthesize large and diverse metabolites, many of whose chemical structures are yet to be reported. In addition to those conserved within this genus, each strain possesses many strain-specific gene clusters, suggesting the diversity of these pathways. This diversity could be accounted for by genus-level vertical inheritance and recent acquisition of these gene clusters during evolution. This genome analysis suggested that Herbidospora strains are an untapped and attractive source of novel secondary metabolites.

Structure of zebrafish IRBP reveals fatty acid binding

Interphotoreceptor retinoid-binding protein (IRBP) has a remarkable role in targeting and protecting all-trans and 11-cis retinol, and 11-cis retinal during the rod and cone visual cycles. Little is known about how the correct retinoid is efficiently delivered and removed from the correct cell at the required time. It has been proposed that different fatty composition at that the outer-segments and retinal-pigmented epithelium have an important role is regulating the delivery and uptake of the visual cycle retinoids at the cell-interphotoreceptor-matrix interface. Although this suggests intriguing mechanisms for the role of local fatty acids in visual-cycle retinoid trafficking, nothing is known about the structural basis of IRBP-fatty acid interactions. Such regulation may be mediated through IRBP's unusual repeating homologous modules, each containing about 300 amino acids. We have been investigating structure-function relationships of Zebrafish IRBP (zIRBP), which has only two tandem modules (z1 and z2), as a model for the more complex four-module mammalian IRBP's. Here we report the first X-ray crystal structure of a teleost IRBP, and the only structure with a bound ligand. The X-ray structure of z1, determined at 1.90 Å resolution, reveals a two-domain organization of the module (domains A and B). A deep hydrophobic pocket with a single bound molecule of oleic acid was identified within the N-terminal domain A. In fluorescence titrations assays, oleic acid displaced all-trans retinol from zIRBP. Our study, which provides the first structure of an IRBP with bound ligand, supports a potential role for fatty acids in regulating retinoid binding.

Modeling attention-driven plasticity in auditory cortical receptive fields.

To navigate complex acoustic environments, listeners adapt neural processes to focus on behaviorally relevant sounds in the acoustic foreground while minimizing the impact of distractors in the background, an ability referred to as top-down selective attention. Particularly striking examples of attention-driven plasticity have been reported in primary auditory cortex via dynamic reshaping of spectro-temporal receptive fields (STRFs). By enhancing the neural response to features of the foreground while suppressing those to the background, STRFs can act as adaptive contrast matched filters that directly contribute to an improved cognitive segregation between behaviorally relevant and irrelevant sounds. In this study, we propose a novel discriminative framework for modeling attention-driven plasticity of STRFs in primary auditory cortex. The model describes a general strategy for cortical plasticity via an optimization that maximizes discriminability between the foreground and distractors while maintaining a degree of stability in the cortical representation. The first instantiation of the model describes a form of feature-based attention and yields STRF adaptation patterns consistent with a contrast matched filter previously reported in neurophysiological studies. An extension of the model captures a form of object-based attention, where top-down signals act on an abstracted representation of the sensory input characterized in the modulation domain. The object-based model makes explicit predictions in line with limited neurophysiological data currently available but can be readily evaluated experimentally. Finally, we draw parallels between the model and anatomical circuits reported to be engaged during active attention. The proposed model strongly suggests an interpretation of attention-driven plasticity as a discriminative adaptation operating at the level of sensory cortex, in line with similar strategies previously described across different sensory modalities.

Portability of the thiolation domain in recombinant pyoverdine non-ribosomal peptide synthetases.

BackgroundNon-ribosomal peptide synthetase (NRPS) enzymes govern the assembly of amino acids and related monomers into peptide-like natural products. A key goal of the field is to develop methods to effective recombine NRPS domains or modules, and thereby generate modified or entirely novel products. We previously showed that substitution of the condensation (C) and adenylation (A) domains in module 2 of the pyoverdine synthetase PvdD from Pseudomonas aeruginosa led to synthesis of modified pyoverdines in a minority of cases, but that more often the recombinant enzymes were non-functional. One possible explanation was that the majority of introduced C domains were unable to effectively communicate with the thiolation (T) domain immediately upstream, in the first module of PvdD.ResultsTo test this we first compared the effectiveness of C-A domain substitution relative to T-C-A domain substitution using three different paired sets of domains. Having previously demonstrated that the PvdD A/T domain interfaces are tolerant of domain substitution, we hypothesised that T-C-A domain substitution would lead to more functional recombinant enzymes, by maintaining native T/C domain interactions. Although we successfully generated two recombinant pyoverdines, having a serine or a N5-formyl-N5-hydroxyornithine residue in place of the terminal threonine of wild type pyoverdine, in neither case did the T-C-A domain substitution strategy lead to substantially higher product yield. To more comprehensively examine the abilities of non-native T domains to communicate effectively with the C domain of PvdD module 2 we then substituted the module 1 T domain with 18 different T domains sourced from other pyoverdine NRPS enzymes. In 15/18 cases the recombinant NRPS was functional, including 6/6 cases where the introduced T domain was located upstream of a C domain in its native context.ConclusionsOur data indicate that T domains are generally able to interact effectively with non-native C domains, contrasting with previous findings that they are not generally portable upstream of epimerisation (E) or thioesterase (TE) domains. This offers promise for NRPS recombination efforts, but also raises the possibility that some C domains are unable to efficiently accept non-native peptides at their donor site due to steric constraints or other limitations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0496-3) contains supplementary material, which is available to authorized users.

Benefits of Tendon Transfer Surgery in Cerebral Palsy

Research Article| August 01 2015 Benefits of Tendon Transfer Surgery in Cerebral Palsy AAP Grand Rounds (2015) 34 (2): 17. https://doi.org/10.1542/gr.34-2-17 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation Benefits of Tendon Transfer Surgery in Cerebral Palsy. AAP Grand Rounds August 2015; 34 (2): 17. https://doi.org/10.1542/gr.34-2-17 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search toolbar search search input Search input auto suggest filter your search All PublicationsAll JournalsAAP Grand RoundsPediatricsHospital PediatricsPediatrics In ReviewNeoReviewsAAP NewsAll AAP Sites Search Advanced Search Topics: arm, botulinum toxins, cerebral palsy, tendon transfer procedure Source: Van Heest A, Bagley A, Molitor F, et al. Tendon transfer surgery in upper-extremity cerebral palsy is more effective than botulinum toxin injections or regular, ongoing therapy. J Bone Joint Surg. 2015; 97(7): 529– 536; doi: https://doi.org/10.2106/JBJS.M.01577Google Scholar Investigators from 7 Shriners Hospitals sought to determine the most effective treatment for upper extremity muscle contractures in children with spastic, hemiplegic cerebral palsy (CP). Patients with upper extremity CP, aged 4 to 16 years, were prospectively assigned randomly or by patient/family preference to 1 of 3 treatment groups: surgery (Group 1), botulinum toxin injections (Group 2), or regular ongoing therapy (Group 3). All 3 patient groups also received standard supervised occupational therapy (OT) followed by home therapy. Standardized validated outcome tools were used for assessment. Outcomes measurements included active range of motion, pinch and grip strength, stereognosis, and scores from 8 other functional or patient-orientated validated outcome tools including dynamic positional analysis (DPA), 5 domains in the Pediatric Quality of Life Inventory (PedsQL), and the Canadian Occupational Performance Measure (COPM-satisfaction score). Change from baseline for each of these outcomes was compared among children in the 3 treatment groups. Thirty-nine patients were enrolled in the study, 29 by randomization and 10 by patient/family preference. Thirty-four patients (25 randomized and 9 patient/family-preference) were evaluated 12 months post-treatment. This included 16 in Group 1, 9 in Group 2, and 9 in Group 3. Significantly greater improvement in DPA was seen among those in Group 1 (surgery) compared to the other 2 groups (P < .001). These changes reflected improvement in supination and wrist extension during functional activities after surgical treatment. Participants in Group 1 also showed more improvement in the PedsQL-CP module domain of movement compared to those in the other groups (P = .002). The mean increase in Group 1 was 26.4% while Group 2 and Group 3 both had a mean decrease of 3.3%. For the other 4 domains of the PedsQL-CP module – school, fatigue, eating, and speech – there were no significant differences among treatment groups. Group 1 also showed more improvement in the COPM score than Groups 2 and 3 (P = .002). Children in groups 1 and 3 showed significantly more improvement in pinch strength than those from Group 2. The authors conclude that for children with upper extremity CP, surgical treatment results in significantly greater functional improvement and satisfaction than botulinum toxin injections or regular therapy. Dr Hennrikus has disclosed no financial relationship relevant to this commentary. This commentary does not contain a discussion of an unapproved/investigative use of a commercial product/device. In children with hemiplegic CP, the abnormal muscle tone patterns cause the arm and hand to be positioned in elbow flexion, forearm pronation, wrist flexion, and thumb in palm. These contractures limit function. Nonsurgical and surgical treatments exist to reduce the deformities and improve function. Nonsurgical treatments include botulinum toxin injections and OT.1–3 Botulinum toxin... You do not currently have access to this content.

Intranuclear interactomic inhibition of NF-κB suppresses LPS-induced severe sepsis

Suppression of nuclear factor-κB (NF-κB) activation, which is best known as a major regulator of innate and adaptive immune responses, is a potent strategy for the treatment of endotoxic sepsis. To inhibit NF-κB functions, we designed the intra-nuclear transducible form of transcription modulation domain (TMD) of RelA (p65), called nt-p65-TMD, which can be delivered effectively into the nucleus without influencing the cell viability, and work as interactomic inhibitors via disruption of the endogenous p65-mediated transcription complex. nt-p65-TMD effectively inhibited the secretion of pro-inflammatory cytokines, including TNF-α, IL-1β, or IL-6 from BV2 microglia cells stimulated by lipopolysaccharide (LPS). nt-p65-TMD did not inhibit tyrosine phosphorylation of signaling mediators such as ZAP-70, p38, JNK, or ERK involved in T cell activation, but was capable of suppressing the transcriptional activity of NF-κB without the functional effect on that of NFAT upon T-cell receptor (TCR) stimulation. The transduced nt-p65-TMD in T cell did not affect the expression of CD69, however significantly inhibited the secretion of T cell-specific cytokines such as IL-2, IFN-γ, IL-4, IL-17A, or IL-10. Systemic administration of nt-p65-TMD showed a significant therapeutic effect on LPS-induced sepsis model by inhibiting pro-inflammatory cytokines secretion. Therefore, nt-p65-TMD can be a novel therapeutics for the treatment of various inflammatory diseases, including sepsis, where a transcription factor has a key role in pathogenesis, and further allows us to discover new functions of p65 under normal physiological condition without genetic alteration.

Spring loading a pre-cleavage intermediate for hairpin telomere formation.

The Borrelia telomere resolvase, ResT, forms the unusual hairpin telomeres of the linear Borrelia replicons in a process referred to as telomere resolution. Telomere resolution is a DNA cleavage and rejoining reaction that proceeds from a replicated telomere intermediate in a reaction with mechanistic similarities to that catalyzed by type IB topoisomerases. Previous reports have implicated the hairpin-binding module, at the end of the N-terminal domain of ResT, in distorting the DNA between the scissile phosphates so as to promote DNA cleavage and hairpin formation by the catalytic domain. We report that unwinding the DNA between the scissile phosphates, prior to DNA cleavage, is a key cold-sensitive step in telomere resolution. Through the analysis of ResT mutants, rescued by substrate modifications that mimic DNA unwinding between the cleavage sites, we show that formation and/or stabilization of an underwound pre-cleavage intermediate depends upon cooperation of the hairpin-binding module and catalytic domain. The phenotype of the mutants argues that the pre-cleavage intermediate promotes strand ejection to favor the forward reaction and that subsequent hairpin capture is a reversible reaction step. These reaction features are proposed to promote hairpin formation over strand resealing while allowing reversal back to substrate of aborted reactions.

Linker length and flexibility induces new cellobiohydrolase activity of PoCel6A from Penicillium oxalicum.

In a previous study, a novel cellobiohydrolase, PoCel6A, with new enzymatic activity against p-nitrophenyl-β-D-cellobioside (pNPC), was purified from Penicillium oxalicum. The cellulose-binding module and catalytic domain of PoCel6A showed a high degree of sequence similarity with other fungal Cel6As. However, PoCel6A had 11 more amino acids in the linker region than other Cel6As. To evaluate the relationship between the longer linker of PoCel6A and its enzymatic activity, 11 amino acids were deleted from the linker region of PoCel6A. The shortened PoCel6A linker nullified the enzymatic activity against pNPC but dramatically increased the enzyme's capacity for crystalline cellulose degradation. The shortened linker segment appeared to have no effect on the secondary structural conformation of PoCel6A. Another variant (PoCel6A-6pro) with six consecutive proline residues in the interdomain linker had a higher rigid linker, and no enzymatic activity was observed against soluble and insoluble substrate. The flexibility of the linker had an important function in the formation of active cellulase. The length and flexibility of the linker is clearly able to modify the function of PoCel6A and induce new characteristics of Cel6A.