Computer-Assisted Rehabilitation Shows Greater Efficacy Than Traditional in Visuospatial Skills and Cognition in Neglect Patients.

This study is crucial for improving unilateral spatial neglect (USN) treatments, focusing on comparing the effectiveness of computer-assisted cognitive rehabilitation (CACR) against conventional rehabilitation (CR) methods. It aimed to address a significant research gap and improve patient outcomes by evaluating the impact of CACR versus CR on visuospatial perception, visual field and attention, and visual memory in patients with USN. This study was a randomized controlled trial. Forty-five consecutive patients with USN from a university rehabilitation center were divided into two groups: 22 patients received CACR with Rehacom software, focusing on saccadic eye movement, visual field, and visual-motor coordination, while 23 underwent CR that combined hemispheric activation approach, mental imagery training, and vibration therapy. Assessments included the Motor-Free Visual Perception Test (MVPT), Line Bisection Test (LBT), Visual Span Test (VST), and Visual Recognition Test (VRT). The study employed ANCOVA and effect size calculations to evaluate the effectiveness of CACR compared to CR in treating patients with USN. Results indicated that CACR significantly outperformed CR in improving visuospatial perception, visual field, attention, and memory, showcasing its effectiveness in treating USN. These findings demonstrate the superiority of CACR over CR, particularly in enhancing visual memory and attention, as evidenced by the large effect size in VRT and moderate effects in LBT and VST. This suggests CACR's potential as a more effective approach for rehabilitation in patients with USN due to brain injuries.

Cullin-RING E3 ubiquitin ligase 4 regulates neurite morphogenesis during neurodevelopment.

Neuritogenesis is crucial for establishing proper neuronal connections during brain development; its failure causes neurodevelopmental defects. Cullin-RING E3 ubiquitin ligase complexes participate in various neurodevelopmental processes by regulating protein stability. We demonstrated the regulatory function of Cullin-RING E3 ubiquitin ligase 4 (CRL4) in neurite morphogenesis during early neurodevelopment. Cul4a and Cul4b, the core scaffold proteins of CRL4, exhibit high expression and activation within the cytosol of developing neurons, regulated by neuronal stimulation through N-methyl D-aspartate (NMDA) receptor signaling. CRL4 also interacts with cytoskeleton-regulating proteins involved in neurite morphogenesis. Notably, genetic depletion and inhibition of cytosolic CRL4 enhance neurite extension and branching in developing neurons. Conversely, Cul4a overexpression suppresses basal and NMDA-enhanced neuritogenesis. Furthermore, CRL4 and its substrate adaptor regulate the polyubiquitination and proteasomal degradation of doublecortin protein. Collectively, our findings suggest that CRL4 ensures proper neurite morphogenesis in developing neurons by regulating cytoskeleton-regulating proteins.

The Third Extracellular Loop of Mammalian Odorant Receptors Is Involved in Ligand Binding.

Mammals recognize chemicals in the air via G protein-coupled odorant receptors (ORs). In addition to their orthosteric binding site, other segments of these receptors modulate ligand recognition. Focusing on human hOR1A1, which is considered prototypical of class II ORs, we used a combination of molecular modeling, site-directed mutagenesis, and in vitro functional assays. We showed that the third extracellular loop of ORs (ECL3) contributes to ligand recognition and receptor activation. Indeed, site-directed mutations in ECL3 showed differential effects on the potency and efficacy of both carvones, citronellol, and 2-nonanone.

The mediating effect of caregiver burden on the caregivers' quality of life.

[Purpose] Quality of life (QoL) can be closely related to caregiver burden, which may be a potential mediating effect on the relationships among stroke patient caregivers. This study investigated the predictors of caregiver's QoL based on patient and caregiver characteristics, with caregiver burden as a mediator. [Methods] This study was conducted using surveys, a literature review, and interviews. Survey data were collected from 238 subjects, who were diagnosed with stroke, and their family caregivers from October 2013 to April 2014. [Results] Caregiver health status, income, spouses caring for patients, and duration of hospitalization were identified as significant predictors of caregivers' QoL with a mediating effect of caregiver burden. The time spent on caregiving per day and patient education level were the only direct predictors of caregivers' QoL. [Conclusion] The responsibility of caring for patients with stroke, in particular for a spouse, must be administered by means of a holistic family-centered rehabilitation program. In addition, financial support and availability of various health and social service programs must be comprehensively provided in order to maintain caregivers' well-being.

The Relationship between Rehabilitation and Changes in Depression in Stroke Patients.

[Purpose] This study investigated the primary factors behind changes in depressive symptoms among stroke patients after 8 weeks of rehabilitation (physical, occupational, and cognitive therapy). [Methods] This study was conducted using a literature review, and electronic medical records from January, 2008 to December, 2009. Data were collected for 120 subjects with chronic stroke. [Results] Cardiac disorder, left-brain lesion, early-stage depression, activities of daily living, and cognitive function were significant predictors of the changes in depression in chronic stroke patients. [Conclusion] Post-stroke depression can be controlled by rehabilitation. Also, clinicians should comprehend and share the psychological and physical affliction, develop back-up programs, and make them comprehensively available to support the psychological and physical health of subjects with chronic stroke.

Production of anteiso-branched fatty acids in Escherichia coli; next generation biofuels with improved cold-flow properties.

Microbial fermentation is emerging as an increasingly important resource for the production of fatty acids to serve as precursors for renewable diesel as well as detergents, lubricants and other industrial chemicals, as an alternative to traditional sources of reduced carbon such as petroleum. A major disadvantage of fuels derived from biological sources is their undesirable physical properties such as high cloud and pour points, and high viscosity. Here we report the development of an Escherichia coli strain that efficiently produces anteiso-branched fatty acids, which can be converted into downstream products with lower cloud and pour points than the mixtures of compounds produced via the native metabolism of the cell. This work addresses a serious limitation that must be overcome in order to produce renewable biodiesel and oleochemicals that perform as well as their petroleum-based counterparts.

Heterologous expression and manipulation of three tetracycline biosynthetic pathways.

A very accommodating host: Three tetracycline biosynthetic pathways were overexpressed and manipulated in the heterologous host Streptomyces lividans K4-114. Through the inactivation of various genes and characterization of the resulting biosynthetic intermediates, new tetracycline-modifying enzymes were identified (see scheme).

Heterologous production of polyketides by modular type I polyketide synthases in Escherichia coli.

Heterologous production of polyketide compounds, an important class of natural products with complex chemical structures, was first demonstrated with Streptomyces parvulus in 1984. Although Streptomyces strains are good first options for heterologous polyketide biosynthesis, their slow growth kinetics prompt other hosts to also be considered. Escherichia coli provides key elements of an ideal host in terms of the growth rate, culture conditions, and available recombinant DNA tools. Here we review the current status and potential for metabolic engineering of polyketides in E. coli.

Identification of three positive regulators in the geldanamycin PKS gene cluster of Streptomyces hygroscopicus JCM4427.

In the Streptomyces hygroscopicus JCM4427 geldanamycin biosynthetic gene cluster, Five putative regulatory genes were identified by protein homology searching. Among three of those genes, gel14, gel17, and gel19, are located downstream of polyketide synthase genes. Gel14 and Gel17 are members of the LAL family of transcriptional regulators, including an ATP/GTP-binding domain at the N-terminus and a DNA binding helix-turn-helix domain at the C-terminus. Gel19 is a member of the TetR family transcriptional regulators, which generally act to repress transcription. To verify the biological significance of the putative regulators in geldanamycin production, they were individually characterized by gene disruption, genetic complementation and transcriptional analyses. All three genes were confirmed as positive regulators of geldanamycin production. Specifically, Gel17 and Gel19 are required for gel14 as well as gelA gene expression.

Biochemical analysis of the biosynthetic pathway of an anticancer tetracycline SF2575.

SF2575 1 is a tetracycline polyketide produced by Streptomyces sp. SF2575 and displays exceptionally potent anticancer activity toward a broad range of cancer cell lines. The structure of SF2575 is characterized by a highly substituted tetracycline aglycon. The modifications include methylation of the C-6 and C-12a hydroxyl groups, acylation of the 4-(S)-hydroxyl with salicylic acid, C-glycosylation of the C-9 of the D-ring with D-olivose and further acylation of the C4'-hydroxyl of D-olivose with the unusual angelic acid. Understanding the biosynthesis of SF2575 can therefore expand the repertoire of enzymes that can modify tetracyclines, and facilitate engineered biosynthesis of SF2575 analogues. In this study, we identified, sequenced, and functionally analyzed the ssf biosynthetic gene cluster which contains 40 putative open reading frames. Genes encoding enzymes that can assemble the tetracycline aglycon, as well as installing these unique structural features, are found in the gene cluster. Biosynthetic intermediates were isolated from the SF2575 culture extract to suggest the order of pendant-group addition is C-9 glycosylation, C-4 salicylation, and O-4' angelylcylation. Using in vitro assays, two enzymes that are responsible for C-4 acylation of salicylic acid were identified. These enzymes include an ATP-dependent salicylyl-CoA ligase SsfL1 and a putative GDSL family acyltransferase SsfX3, both of which were shown to have relaxed substrate specificity toward substituted benzoic acids. Since the salicylic acid moiety is critically important for the anticancer properties of SF2575, verification of the activities of SsfL1 and SsfX3 sets the stage for biosynthetic modification of the C-4 group toward structure-activity relationship studies of SF2575. Using heterologous biosynthesis in Streptomyces lividans, we also determined that biosynthesis of the SF2575 tetracycline aglycon 8 parallels that of oxytetracycline 4 and diverges after the assembly of 4-keto-anhydrotetracycline 51. The minimal ssf polyketide synthase together with the amidotransferase SsfD produced the amidated decaketide backbone that is required for the formation of 2-naphthacenecarboxamide skeleton. Additional enzymes, such as cyclases C-6 methyltransferase and C-4/C-12a dihydroxylase, were functionally reconstituted.

Rational biosynthetic engineering for optimization of geldanamycin analogues.

Tailor made: We report the rational biosynthesis of C15 hydroxylated non-quinone geldanamycin analogues by site-directed mutagenesis of the geldanamycin polyketide synthase (PKS), together with a combination of post-PKS tailoring genes. Rational biosynthetic engineering allowed the generation of geldanamycin derivatives, such as DHQ3 illustrated in the figure, which had superior pharmacological properties in comparison to the parent compound. A rational biosynthetic engineering approach was applied to the optimization of the pharmacological properties of the benzoquinone ansamycin, geldanamycin. Geldanamycin and its natural or semisynthetic derivatives have the potential to serve as anticancer chemotherapeutic agents. However, these first-generation Hsp90 inhibitors share an unfavorable structural feature that causes both reduced efficacy and toxicity during clinical evaluation. We report the rationally designed biosynthesis of C15 hydroxylated non-quinone geldanamycin analogues by site-directed mutagenesis of the geldanamycin polyketide synthase (PKS), together with a combination of post-PKS tailoring genes. A 15-hydroxyl-17-demethoxy non-quinone analogue, DHQ3, exhibited stronger inhibition of Hsp90 ATPase activity (4.6-fold) than geldanamycin. Taken together, the results of the present study indicate that rational biosynthetic engineering allows the generation of derivatives of geldanamycin with superior pharmacological properties.

Characterization of tailoring genes involved in the modification of geldanamycin polyketide in Streptomyces hygroscopicus JCM4427.

Geldanamycin and its analogs are important anticancer agents that inhibit the newly targeted, heat-shock protein (Hsp) 90, which is a chaperone protein in eukaryotic cells. To resolve which geldanamycin biosynthetic genes are responsible for particular post-polyketide synthase (PKS) processing steps and in which order the reactions occur, we individually inactivated candidate genes in Streptomyces hygroscopicus subsp. duamyceticus JCM4427, and isolated and elucidated the structures of intermediates from each mutant. The results indicated that gel7 governs at least one of the benzoquinone ring oxidation steps. In addition, gel16 was found to be involved in double-bond formation between C-4 and C-5 of 4,5-dihydrogeldanamycin, which confirmed our previous findings that this double bond reduced during the post-PKS modification of the polyketide assembly. In addition, pro-geldanamycin, which does not possess a double bond at C-4/5, was purified from the gel7 and 8 double-gene-inactivated mutant.

Synthesis and anticancer activity of geldanamycin derivatives derived from biosynthetically generated metabolites.

A new series of geldanamycin derivatives were synthesized using a semi-synthetic approach involving genetically engineered biosynthetic intermediates. These analogues were then evaluated for anti-proliferation activity in human cancer cell lines, SK-Br3 and SK-Ov3. Most of the synthesized compounds exhibited potent in vitro anti-proliferation activity toward both cell lines. Such compounds potently inhibited the expression of the Hsp90 client protein ErbB2.

Cytotoxic 6-alkylsalicylic acids from the endophytic Streptomyces laceyi.

Two new 6-alkylsalicylic acids, salaceyins A and B were isolated by bioassay-guided fractionation from the culture of the endophytic Streptomyces laceyi MS53 and their structures were determined on the basis of spectroscopic data. Salaceyins A and B exhibited modest cytotoxicity against a human breast cancer cell line (SKBR3) with IC50 values of 3.0 and 5.5 microg/ml, respectively.

Inactivation of the carbamoyltransferase gene refines post-polyketide synthase modification steps in the biosynthesis of the antitumor agent geldanamycin.

The post-polyketide synthase modification of geldanamycin (1) biosynthesis is of interest as a means of introducing structural diversity into the compound. From the inactivation of a gene encoding carbamoyltransferase, we demonstrated that the C-17 hydroxylation and the C-21 oxidation precede O-carbamoylation and that the hypothetical progeldanamycin does not possess a double bond at C-4 and C-5. More importantly, our result revealed new intermediates 4,5-dihydro-7-O-descarbamoyl-7-hydroxygeldanamycin (3) and 4,5-dihydrogeldanamycin (5), indicating that O-carbamoylation occurs prior to the C-4,5 cis double bond formation in geldanamycin biosynthesis.

Escherichia coli periplasmic thiol peroxidase acts as lipid hydroperoxide peroxidase and the principal antioxidative function during anaerobic growth.

To clarify the enzymatic property of Escherichia coli periplasmic thiol peroxidase (p20), the specific peroxidase activity toward peroxides was compared with other bacterial thiol peroxidases. p20 has the most substrate preference and peroxidase activity toward organic hydroperoxide. Furthermore, p20 exerted the most potent lipid peroxidase activity. Despite that the mutation of p20 caused the highest susceptibility toward organic hydroperoxide and heat stress, the cellular level of p20 did not respond to the exposure of oxidative stress. Expression level of p20 during anaerobic growth was sustained at the approximately 50% level compared with that of the aerobic growth. Viability of aerobic p20Delta without glucose was reduced to the approximately 65% level of isogenic strains, whereas viability of aerobic p20Delta with 0.5% glucose supplement was sustained. The deletion of p20 resulted in a gradual loss of the cell viability during anaerobic growth. At the stationary phase, the viability of p20Delta was down to approximately 10% level of parent strains. An analysis of the protein carbonyl contents of p20Delta as a marker for cellular oxidation indicates that severe reduction of viability of anaerobic p20Delta was caused by cumulative oxidative stress. P20Delta showed hypersensitivity toward membrane-soluble organic hydroperoxides. An analysis of protein carbonyl and lipid hydroperoxide contents in the membrane of the stress-imposed p20Delta demonstrates that the severe reduction of viability was caused by cumulative oxidative stress on the membrane. Taken together, present data uncover in vivo function for p20 as a lipid hydroperoxide peroxidase and demonstrate that, as the result, p20 acts as the principal antioxidant in the anaerobic habitats.

Nuclear thiol peroxidase as a functional alkyl-hydroperoxide reductase necessary for stationary phase growth of Saccharomyces cerevisiae.

Yeast nucleus-localized thiol peroxidase (nTPx) was characterized as a functional peroxidase. There are two cysteine residues in nTPx. Replacement of Cys-106 or Cys-111 with serine resulted in a complete loss of thioredoxin-linked peroxidase activity. However, when their activities were measured in terms of the ability to inhibit oxidation of glutamine synthetase, C111S showed the same antioxidant activity as the wild type protein. SDS-PAGE gel analysis revealed that only C111S existed as the dimer form. In addition to the identification of Cys-106 as the primary catalytic site, these data suggest the formation of the intradisulfide bond as a part of the catalytic cycle between nTPx and thioredoxin. nTPx preferentially reduced alkyl-hydroperoxides rather than H2O2. Furthermore, a nTPx mutant strain showed higher sensitivity toward alkyl-hydroperoxide than hydrogen peroxide. Also, reduction of the viability of nTPx mutant strain against various oxidants supports an in vivo antioxidant role for nTPx. nTPx transcriptional activity was not significantly detectable in log phase yeast, but the activity was exponentially increased after the diauxic shift. The transcriptional activity was highly induced even in the log phase yeast grown in nonfermentable carbon source. Deletion of Tor1p, Ras1p, and Ras2p resulted in considerable induction when compared with their parent strains, demonstrating the activation of the transcription of nTPx gene at the diauxic shift. Transcription of nTPx gene was induced in response to oxidative stress. Viability of a stationary phase nTPx mutant was considerably reduced compared with the isogenic strain. Collectively, these data demonstrate that nTPx is a thiol peroxidase family acting as alkyl-hydroperoxide reductase in the nucleus during post-diauxic growth.

Msn2p/Msn4p act as a key transcriptional activator of yeast cytoplasmic thiol peroxidase II.

We observed that the transcription of Saccharomyces cerevisiae cytoplasmic thiol peroxidase type II (cTPx II) (YDR453C) is regulated in response to various stresses (e.g. oxidative stress, carbon starvation, and heat-shock). It has been suggested that both transcription-activating proteins, Yap1p and Skn7p, regulate the transcription of cTPx II upon exposure to oxidative stress. However, a dramatic loss of transcriptional response to various stresses in yeast mutant strains lacking both Msn2p and Msn4p suggests that the transcription factors act as a principal transcriptional activator. In addition to two Yap1p response elements (YREs), TTACTAA and TTAGTAA, the presence of two stress response elements (STREs) (CCCCT) in the upstream sequence of cTPx II also suggests that Msn2p/Msn4p could control stress-induced expression of cTPx II. Analysis of the transcriptional activity of site-directed mutagenesis of the putative STREs (STRE1 and STRE2) and YREs (TRE1 and YRE2) in terms of the activity of a lacZ reporter gene under control of the cTPx II promoter indicates that STRE2 acts as a principal binding element essential for transactivation of the cTPx II promoter. The transcriptional activity of the cTPx II promoter was exponentially increased after postdiauxic growth. The transcriptional activity of the cTPx II promoter is greatly increased by rapamycin. Deletion of Tor1, Tor2, Ras1, and Ras2 resulted in a considerable induction when compared with their parent strains, suggesting that the transcription of cTPx II is under negative control of the Ras/cAMP and target of rapamycin signaling pathways. Taken together, these results suggest that cTPx II is a target of Msn2p/Msn4p transcription factors under negative control of the Ras-protein kinase A and target of rapamycin signaling pathways. Furthermore, the accumulation of cTPx II upon exposure to oxidative stress and during the postdiauxic shift suggests an important antioxidant role in stationary phase yeast cells.