Quantification of inter-brain coupling: A review of current methods used in haemodynamic and electrophysiological hyperscanning studies.

Hyperscanning is a form of neuroimaging experiment where the brains of two or more participants are imaged simultaneously whilst they interact. Within the domain of social neuroscience, hyperscanning is increasingly used to measure inter-brain coupling (IBC) and explore how brain responses change in tandem during social interaction. In addition to cognitive research, some have suggested that quantification of the interplay between interacting participants can be used as a biomarker for a variety of cognitive mechanisms aswell as to investigate mental health and developmental conditions including schizophrenia, social anxiety and autism. However, many different methods have been used to quantify brain coupling and this can lead to questions about comparability across studies and reduce research reproducibility. Here, we review methods for quantifying IBC, and suggest some ways moving forward. Following the PRISMA guidelines, we reviewed 215 hyperscanning studies, across four different brain imaging modalities: functional near-infrared spectroscopy (fNIRS), functional magnetic resonance (fMRI), electroencephalography (EEG) and magnetoencephalography (MEG). Overall, the review identified a total of 27 different methods used to compute IBC. The most common hyperscanning modality is fNIRS, used by 119 studies, 89 of which adopted wavelet coherence. Based on the results of this literature survey, we first report summary statistics of the hyperscanning field, followed by a brief overview of each signal that is obtained from each neuroimaging modality used in hyperscanning. We then discuss the rationale, assumptions and suitability of each method to different modalities which can be used to investigate IBC. Finally, we discuss issues surrounding the interpretation of each method.

Effect of food additives on key bacterial taxa and the mucosa-associated microbiota in Crohn's disease. The ENIGMA study.

Food additives have been linked to the pro-inflammatory microbial dysbiosis associated with Crohn's disease (CD) but the underlying ecological dynamics are unknown. Here, we examine how selection of food additives affects the growth of multiple strains of a key beneficial bacterium (Faecalibacterium prausnitzii), axenic clinical isolates of proinflammatory bacteria from CD patients (Proteus, Morganella, and Klebsiella spp.), and the consortia of mucosa-associated microbiota recovered from multiple Crohn's disease patients. Bacterial growth of the axenic isolates was evaluated using a habitat-simulating medium supplemented with either sodium sulfite, aluminum silicate, carrageenan, carboxymethylcellulose, polysorbate 80, saccharin, sucralose, or aspartame, intended to approximate concentrations found in food. The microbial consortia recovered from post-operative CD patient mucosal biopsy samples were challenged with either carboxymethylcellulose and/or polysorbate 80, and the bacterial communities compared to unchallenged consortia by 16S rRNA gene amplicon profiling. Growth of all F. prausnitzii strains was arrested when either sodium sulfite or polysorbate 80 was added to cultures at baseline or mid-exponential phase of growth, and the inhibitory effects on the Gram-negative bacteria by sodium sulfite were conditional on oxygen availability. The effects from polysorbate 80, saccharin, carrageenan, and/or carboxymethylcellulose on these bacteria were strain-specific. In addition to their direct effects on bacterial growth, polysorbate 80 and/or carboxymethylcellulose can drive profound changes in the CD mucosa-associated microbiota via niche expansion of Proteus and/or Veillonellaceae - both implicated in early Crohn's disease recurrence. These studies on the interaction of food additives with the enteric microbiota provide a basis for dietary management in Crohn's disease.

Detecting monocyte trafficking in an animal model of glioblastoma using R2* and quantitative susceptibility mapping.

BACKGROUND: The role of tumor-associated macrophages (TAMs) in glioblastoma (GBM) disease progression has received increasing attention. Recent advances have shown that TAMs can be re-programmed to exert a pro-inflammatory, anti-tumor effect to control GBMs. However, imaging methods capable of differentiating tumor progression from immunotherapy treatment effects have been lacking, making timely assessment of treatment response difficult. We showed that tracking monocytes using iron oxide nanoparticle (USPIO) with MRI can be a sensitive imaging method to detect therapy response directed at the innate immune system. METHODS: We implanted syngeneic mouse glioma stem cells into C57/BL6 mice and treated the animals with either niacin (a stimulator of innate immunity) or vehicle. Animals were imaged using an anatomical MRI sequence, R2* mapping, and quantitative susceptibility mapping (QSM) before and after USPIO injection. RESULTS: Compared to vehicles, niacin-treated animals showed significantly higher susceptibility and R2*, representing USPIO and monocyte infiltration into the tumor. We observed a significant reduction in tumor size in the niacin-treated group 7 days later. We validated our MRI results with flow cytometry and immunofluoresence, which showed that niacin decreased pro-inflammatory Ly6C high monocytes in the blood but increased CD16/32 pro-inflammatory macrophages within the tumor, consistent with migration of these pro-inflammatory innate immune cells from the blood to the tumor. CONCLUSION: MRI with USPIO injection can detect therapeutic responses of innate immune stimulating agents before changes in tumor size have occurred, providing a potential complementary imaging technique to monitor cancer immunotherapies. MANUSCRIPT HIGHLIGHT: We show that iron oxide nanoparticles (USPIOs) can be used to label innate immune cells and detect the trafficking of pro-inflammatory monocytes into the glioblastoma. This preceded changes in tumor size, making it a more sensitive imaging technique.

Non-perceptual Regions in the Left Inferior Parietal Lobe Support Phonological Short-term Memory: Evidence for a Buffer Account?

Buffer versus embedded processes accounts of short-term memory (STM) for phonological information were addressed by testing subjects' perception and memory for speech and non-speech auditory stimuli. Univariate and multivariate (MVPA) approaches were used to assess whether brain regions recruited in recognizing speech were involved in maintaining speech representations over a delay. As expected, a left superior temporal region was found to support speech perception. However, contrary to the embedded processes approach, this region failed to show a load effect, or any sustained activation, during a maintenance delay. Moreover, MVPA decoding during the maintenance stage was unsuccessful in this region by a perception classifier or an encoding classifier. In contrast, the left supramarginal gyrus showed both sustained activation and a load effect. Using MVPA, stimulus decoding was successful during the delay period. In addition, a functional connectivity analysis showed that, as memory load increased, the left temporal lobe involved in perception became more strongly connected with the parietal region involved in maintenance. Taken together, the findings provide greater support for a buffer than embedded processes account of phonological STM.

Twelve-year analysis of microbial keratitis trends at a UK tertiary hospital.

PurposeTo investigate the frequencies, trends, and in vitro drug susceptibilities of the causative pathogens in microbial keratitis in Manchester Royal Eye Hospital.Patients and methodsCorneal scrape results recorded by the microbiology service between 2004 and 2015 were extracted from an established database. A total of 4229 corneal scrape specimens were identified from an established database. First-line antibiotic treatment in our centre during the study period was ofloxacin and second line was cefuroxime and gentamicin.ResultsMean age was 45.9±21.0. A total of 1379 samples (32.6%) were culture positive. One hundred forty-eight (10.7%) specimens cultured multiple organisms. Of the 1539 organisms identified, 63.3% were Gram-positive bacteria, 27.3% Gram-negative bacteria, 7.1% fungi, and 2.3% Acanthamoebae. A decreasing trend in Gram-positive isolates was found together with a stable trend in Gram negatives and an increasing trend in Acanthamoeba and fungi. There appeared to be a significant increasing trend of Moraxella infection (P=0.001). In all, 83.1 and 90.8% of Gram-positive and -negative isolates tested were susceptible to ofloxacin, respectively. Cefuroxime covered 86.6% of Gram-positive and 61.4% of Gram-negative isolates, whereas gentamicin covered 88.8 and 96.5% of Gram-positive and -negative isolates, respectively.ConclusionWe found a change in the type of Gram-negative organisms isolated over time, with the Moraxella species on the rise. Reassuringly, no significant increase in resistance was observed in vitro for any of the commonly used antibiotics. Ofloxacin remains a good first-line antibiotic treatment but duo-therapy does have broader coverage and should be considered in non-responsive cases.

Enhancement of the magnetocaloric effect driven by changes in the crystal structure of Al-doped GGG, Gd3Ga5-xAlxO12 (0 ≤x ≤5).

The Gd3Ga5-xAlxO12 (0 ≤ x ≤ 5) solid solution has been prepared using ceramic synthesis routes and the structural and magnetic properties were investigated using x-ray diffraction, magnetic susceptibility, χ, and isothermal magnetisation, M(H), measurements. Our results indicate a contraction of the unit cell and more significant antiferromagnetic interactions as x increases. Despite the decrease in the magnetic polarisation on the application of a field and the corresponding decrease in the change in the magnetic entropy, ΔS, we find that Gd3Al5O12 has a significantly higher observed (17%) and theoretical (14%) ΔS per unit mass than Gd3Ga5O12. The theoretical increase in ΔS per unit volume (7%) is offset by the increased antiferromagnetic interactions in Gd3Al5O12. The differences in ΔS are driven by a decrease in both the mass and the density as Al ions replace Ga ions. These results highlight the importance of changes to the crystal structure when considering materials for solid state magnetic cooling.

Retina rejuvenation therapy for diabetic macular edema: a pilot study.

PURPOSE: To prospectively investigate the safety and efficacy of a novel frequency-doubled nanosecond-pulsed laser with discontinuous beam energy distribution (2RT, Ellex) for the treatment of diabetic macular edema. METHODS: Twenty-three consecutive patients (38 eyes) with newly diagnosed diabetic macular edema were recruited and assessed with logarithm of the minimum angle of resolution best-corrected visual acuity, central macular thickness measured with optical coherence tomography (OCT/scanning laser ophthalmoscope, OPKO/OTI), microperimetry, fundus photography, and fundus fluorescein angiography. Macular grid treatments were performed with 2RT laser system by 1 operator. Patients were examined with logarithm of the minimum angle of resolution best-corrected visual acuity, central macular thickness, microperimetry, and fundus photography at 3 weeks and 6 weeks and 3 months and 6 months. Fundus fluorescein angiography was repeated at 3 months and 6 months. RESULTS: Six months postoperatively, 17 patients (28 eyes) completed the study. No complications were identified after 2RT therapy. Intraoperative retinal discoloration was observed in 2 cases, fully resolved at 3 months with no permanent anatomical or functional changes. Mean logarithm of the minimum angle of resolution visual acuity improved from 20/44 at baseline to 20/27 at 6 months. The change in best-corrected visual acuity was significant (P = 0.0190). Central macular thickness in the central 1-mm subfield, retinal exudates and vascular leakage decreased in the majority of patients at 6 months (46, 41, and 55%, respectively), although the change from baseline was not statistically significant. Microperimetry confirmed photoreceptor integrity and showed a trend of improvement that correlated with decreased central macular thickness. CONCLUSION: For the first time, we achieved a beneficial effect on diabetic macular edema without the side effects of conventional laser therapy. The efficacy of this system in comparison with standard argon laser photocoagulation and in the treatment of other conditions affecting the retinal pigment epithelium needs further investigation.

Speech perception, rapid temporal processing, and the left hemisphere: a case study of unilateral pure word deafness.

The mechanisms and functional anatomy underlying the early stages of speech perception are still not well understood. One way to investigate the cognitive and neural underpinnings of speech perception is by investigating patients with speech perception deficits but with preserved ability in other domains of language. One such case is reported here: patient NL shows highly impaired speech perception despite normal hearing ability and preserved semantic knowledge, speaking, and reading ability, and is thus classified as a case of pure word deafness (PWD). NL has a left temporoparietal lesion without right hemisphere damage and DTI imaging suggests that he has preserved cross-hemispheric connectivity, arguing against an account of PWD as a disconnection of left lateralized language areas from auditory input. Two experiments investigated whether NL's speech perception deficit could instead result from an underlying problem with rapid temporal processing. Experiment 1 showed that NL has particular difficulty discriminating sounds that differ in terms of rapid temporal changes, be they speech or non-speech sounds. Experiment 2 employed an intensive training program designed to improve rapid temporal processing in language impaired children (Fast ForWord; Scientific Learning Corporation, Oakland, CA) and found that NL was able to improve his ability to discriminate rapid temporal differences in non-speech sounds, but not in speech sounds. Overall, these data suggest that patients with unilateral PWD may, in fact, have a deficit in (left lateralized) temporal processing ability, however they also show that a rapid temporal processing deficit is, by itself, unable to account for this patient's speech perception deficit.

Clinical responses observed with imatinib or sorafenib in melanoma patients expressing mutations in KIT.

BACKGROUND: Mutations in KIT are more frequent in specific melanoma subtypes, and response to KIT inhibition is likely to depend on the identified mutation. METHODS: A total of 32 patients with metastatic acral or mucosal melanoma were screened for mutations in KIT exons 11, 13 and 17. RESULTS: KIT mutations were found in 38% of mucosal and in 6% of acral melanomas. Three patients were treated with imatinib and one with sorafenib. All four patients responded to treatment, but three have since progressed within the brain. CONCLUSION: The observed clinical responses support further investigation of KIT inhibitors in metastatic melanoma, selected according to KIT mutation status.

Validation of fixed sample size plans for monitoring lepidopteran pests of Brassica oleracea crops in North Korea.

The combined action of two lepidoteran pests, Plutella xylostella L. (Plutellidae) and Pieris rapae L. (Pieridae),causes significant yield losses in cabbage (Brassica oleracea variety capitata) crops in the Democratic People's Republic of Korea. Integrated pest management (IPM) strategies for these cropping systems are in their infancy, and sampling plans have not yet been developed. We used statistical resampling to assess the performance of fixed sample size plans (ranging from 10 to 50 plants). First, the precision (D = SE/mean) of the plans in estimating the population mean was assessed. There was substantial variation in achieved D for all sample sizes, and sample sizes of at least 20 and 45 plants were required to achieve the acceptable precision level of D < or = 0.3 at least 50 and 75% of the time, respectively. Second, the performance of the plans in classifying the population density relative to an economic threshold (ET) was assessed. To account for the different damage potentials of the two species the ETs were defined in terms of standard insects (SIs), where 1 SI = 1 P. rapae = 5 P. xylostella larvae. The plans were implemented using different economic thresholds (ETs) for the three growth stages of the crop: precupping (1 SI/plant), cupping (0.5 SI/plant), and heading (4 SI/plant). Improvement in the classification certainty with increasing sample sizes could be seen through the increasing steepness of operating characteristic curves. Rather than prescribe a particular plan, we suggest that the results of these analyses be used to inform practitioners of the relative merits of the different sample sizes.

Antimicrobial and brine shrimp toxicity of some plants used in traditional medicine in Bukoba District, north-western Tanzania.

Herbal medicines constitute a potentially important resource for new and safe drugs for the management of microbial infections and other diseases. In this study, dichloromethane, ethylacetate and ethanol extracts of Canarium schweinfurthii Engl., Dissotis brazzae Cong., Iboza urticifolia (Bak) E.A.Bruce, Isoglosa lacteal Lindau, Strombosia Scheffleri Engl., and Whitfieldia elongate T. Anders were tested for antimicrobial activity and brine shrimp toxicity. The objective was to validate claims that they are used to treat bacterial infections, diarrhoea and heal wounds among the Haya tribe of north-western Tanzania. At least one extract of each plant showed antibacterial activity. Dichloromethane extracts were the most active while ethanol extracts were the least active. Extracts of Whitfieldia elongate and Isoglossa lacteal were the most and least active with MICs in the range 0.08-0.62 mg/ml and 15.6-62.5 mg/ml, respectively. The dichloromethane extract of Whitfieldia elongate exhibited strong antifungal activity against Cryptococcus neoformans. Against brine shrimp larvae, the extracts from the six plants exhibited a low to very low toxicity with LC50 values ranging from 15.35-374.0 microg/ml. However, ethanol extracts of Dissotis brazzae and Strombosia scheffleri had LC50 values of >1000 microg/ml. The seemingly innocuous nature and relatively good antibacterial activity against skin infections and gastrointestinal pathogenic bacteria support the traditional uses of the plants and deserve more detailed studies.

Comparison of the effects of cetylpyridinium chloride with an essential oil mouth rinse on dental plaque and gingivitis - a six-month randomized controlled clinical trial.

OBJECTIVE: To compare the effects of an experimental mouth rinse containing 0.07% cetylpyridinium chloride (CPC) (Crest Pro-Health) with those provided by a commercially available mouth rinse containing essential oils (EOs) (Listerine) on dental plaque accumulation and prevention of gingivitis in an unsupervised 6-month randomized clinical trial. MATERIAL AND METHODS: This double-blind, 6-month, parallel group, positively controlled study involved 151 subjects balanced and randomly assigned to either positive control (EO) or experimental (CPC) mouth rinse treatment groups. At baseline, subjects received a dental prophylaxis procedure and began unsupervised rinsing twice a day with 20 ml of their assigned mouthwash for 30 s after brushing their teeth for 1 min. Subjects were assessed for gingivitis and gingival bleeding by the Gingival index (GI) of Löe & Silness (1963) and plaque by the Silness & Löe (1964) Plaque index at baseline and after 3 and 6 months of rinsing. At 3 and 6 months, oral soft tissue health was assessed. Microbiological samples were also taken for community profiling by the DNA checkerboard method. RESULTS: Results show that after 3 and 6 months of rinsing, there were no significant differences (p=0.05) between the experimental (CPC) and the positive control mouth rinse treatment groups for overall gingivitis status, gingival bleeding, and plaque accumulation. At 6 months, the covariant (baseline) adjusted mean GI and bleeding sites percentages for the CPC and the EO rinses were 0.52 and 0.53 and 8.7 and 9.3, respectively. Both mouth rinses were well tolerated by the subjects. Microbiological community profiles were similar for the two treatment groups. Statistically, a significant greater reduction in bleeding sites was observed for the CPC rinse versus the EO rinse. CONCLUSION: The essential findings of this study indicated that there was no statistically significant difference in the anti-plaque and anti-gingivitis benefits between the experimental CPC mouth rinse and the positive control EO mouth rinse over a 6-month period.

Progress in silicon-based quantum computing.

We review progress at the Australian Centre for Quantum Computer Technology towards the fabrication and demonstration of spin qubits and charge qubits based on phosphorus donor atoms embedded in intrinsic silicon. Fabrication is being pursued via two complementary pathways: a 'top-down' approach for near-term production of few-qubit demonstration devices and a 'bottom-up' approach for large-scale qubit arrays with sub-nanometre precision. The 'top-down' approach employs a low-energy (keV) ion beam to implant the phosphorus atoms. Single-atom control during implantation is achieved by monitoring on-chip detector electrodes, integrated within the device structure. In contrast, the 'bottom-up' approach uses scanning tunnelling microscope lithography and epitaxial silicon overgrowth to construct devices at an atomic scale. In both cases, surface electrodes control the qubit using voltage pulses, and dual single-electron transistors operating near the quantum limit provide fast read-out with spurious-signal rejection.

Replicating potato spindle tuber viroid RNA is accompanied by short RNA fragments that are characteristic of post-transcriptional gene silencing.

The low molecular weight fraction of tomato plants inoculated with potato spindle tuber viroid (PSTVd) contains a population of short PSTVd-specific RNAs of either polarity. The main constituents were RNAs of 22 and 23 nt representing different domains of the viroid genome. The occurrence of such distinct RNA species indicated that the nuclear replicating PSTVd RNA induces post-transcriptional gene silencing. The short RNAs were slightly more abundant at 30 days post-inoculation than at later stages and were present in plants infected with a mild, severe or lethal isolate of PSTVD: There was no apparent correlation between the quantity of small PSTVd-specific RNAs and the degree of virulence of the viroid isolate.

HER2. a 'predictive factor' ready to use in the daily management of breast cancer patients?

The past few years have witnessed an exponential increase in studies trying to identify molecular markers in patients with breast tumours that might predict for the success or failure of hormonal therapy or chemotherapy. HER2, a tyrosine kinase membrane receptor of the epidermal growth factor receptor family, has been the most widely studied marker in this respect. This paper attempts to critically review to what extent HER2 may improve 'treatment individualisation' for the breast cancer patient.

Production and characterization of recombinant Aspergillus fumigatus Cu,Zn superoxide dismutase and its recognition by immune human sera.

The Cu,Zn superoxide dismutase (SOD) of Aspergillus fumigatus has previously been purified and shown to be immunoreactive to the sera of patients with aspergillosis; however, the purification of large quantities of the enzyme for expanded immunological analysis is both difficult and time-consuming. Accordingly, a lambdaEMBL3 A. fumigatus genomic library was screened with degenerate oligonucleotides based on N-terminal amino acid sequence data; from this initial screen a 1,400-bp fragment was identified, labelled, and used to screen an A. fumigatus lambdagt11 cDNA library. A full-length cDNA encoding Cu,Zn SOD was subsequently identified and cloned. The cDNA encodes a protein of 154 amino acids, which does not have a signal peptide. The A. fumigatus Cu,Zn SOD possesses the typical metal binding ligands of fungal Cu,Zn SODs (six histidines and one aspartic acid) and has significant overall homology with Cu, Zn SODs in general. A recombinant A. fumigatus Cu,Zn SOD has been expressed in Pichia pastoris, is enzymatically active, and has biochemical and biophysical properties that are similar to those of the native enzyme. A sheep polyclonal antibody raised against purified native A. fumigatus Cu,Zn SOD was reactive to the recombinant enzyme by immunoenzyme development of Western blots. Sixty percent of serum samples from patients with A. fumigatus infections were reactive against the recombinant Cu,Zn SOD via immunoenzyme development of Western blots, indicating that the recombinant protein may be useful in the serodiagnostic identification of A. fumigatus infections.

Inhibition of Ras and related guanosine triphosphate-dependent proteins as a therapeutic strategy for blocking malignant glioma growth: II--preclinical studies in a nude mouse model.

OBJECTIVE: Preliminary studies have demonstrated that the Ras family and related guanosine triphosphate-dependent proteins are overactivated in malignant gliomas and that inhibition of the activation of such proteins, by blockade of their post-translational processing, reduces tumor cell growth in vitro. The current study evaluates the utility of this therapeutic strategy in vivo, using preclinical glioma model systems. METHODS: We examined the efficacy against U-87 human malignant glioma cells, in both subcutaneous and intracranial nude mouse models, of selective peptidomimetic inhibitors of farnesyltransferase (FTI-276) and geranylgeranyltransferase (GGTI-297), which are involved in critical steps in the post-translational processing of Ras and related guanosine triphosphate-dependent proteins. For the subcutaneous model, 2 x 10(5) U-87 cells were implanted; after measurable tumors were detected on Day 7, animals were treated with either FTI-276, GGTI-297, or vehicle, administered by continuous infusion for 7 days. Differences in tumor volumes among the treatment groups were examined for significance using a Student's t test. For the intracranial model, 2 x 10(5) U-87 cells were implanted in the right frontal lobe and treatment was initiated on Day 7. In initial studies, animals received a 7-day course of either FTI-276, GGTI-297, or vehicle. In subsequent studies, a 28-day treatment period was used. Comparisons of survival times among treatment groups were performed using a rank-sum test. RESULTS: Although the two agents exhibited comparable antiproliferative activities in previous in vitro studies, an obvious difference in efficacy was apparent in this study. Whereas the geranylgeranyltransferase inhibitor failed to improve survival rates, compared with those observed for control animals, in either the subcutaneous or intracranial model, the farnesyltransferase inhibitor produced objective regression of tumor growth in the subcutaneous model and significant prolongation of survival times in the intracranial model, without apparent toxicity. In the subcutaneous model, tumor volumes for the control, GGTI-297-treated, and FTI-276-treated animals on Day 28 after implantation were 621+/-420, 107+/-104, and 18.5+/-12.7 mm3, respectively (P < 0.05). In the 7-day-treated intracranial model, survival times for the control, GGTI-297-treated, and FTI-276-treated groups were 27.7+/-2.9, 29.8+/-2.1, and 43.6+/-2.7 days, respectively (P < 0.001). In the 28-day-treated intracranial model, survival times for the control, GGTI-297-treated, and FTI-276-treated groups were 29.2+/-3.7, 28.3+/-3.9, and 58.7+/-6.2 days, respectively, with five of six animals in the latter group surviving more than 55 days after tumor implantation (P < 0.001). CONCLUSION: These studies demonstrate that farnesyltransferase inhibition is effective in diminishing the growth of human glioma cells in vivo. Evaluation of this treatment approach in clinical trials is warranted.

Expression and characterization of three tomato 1-aminocyclopropane-1-carboxylate oxidase cDNAs in yeast.

Heterologous expression in yeast has previously shown that the tomato cDNA LE-ACO1 encodes a functional 1-aminocyclopropane-1-carboxylate (ACC) oxidase (ACO) protein [Hamilton, A. J., Bouzayen, M. & Grierson, D. (1991) Proc. Natl Acad. Sci. USA 88, 7434-7437]. In the present work, full-length cDNAs encoding the two other members of the tomato ACO family (LE-ACO2 and LE-ACO3) were isolated and expressed in Saccharomyces cerevisiae. Analysis of the predicted amino acid sequences showed that the ACO1 and ACO3 proteins are highly similar (95%) while ACO2 is more divergent (89%). Yeast strains transformed with each of the three cDNAs were able to convert exogenous ACC to ethylene, the ACO1 strain exhibiting the highest activity in vivo and the ACO3 and ACO2 strains reaching 65% and 45% of ACO1 maximum activity, respectively. None of the ACO activities expressed in yeast required addition of ascorbate in vivo. ACO activities assayed in vitro revealed no significant differences between the three isoforms with regards to optimum temperature (29 degrees C), optimum pH (6.8-7.2), absolute dependence for ascorbate, Fe2+ and carbon dioxide, and inhibition by iron-chelating agents (1,10-phenanthroline and EDTA), Co2+ and free-radical scavengers (n-propyl gallate). However, differences were detected in the apparent Km values for ACC, the pI and the specific activity. The biochemical features that might explain the differences between the isoenzyme activities are discussed.

Protein surface recognition by synthetic agents: design and structural requirements of a family of artificial receptors that bind to cytochrome c.

The design, synthesis, and evaluation of a novel series of receptors for protein surface recognition are described. The design of these agents is based around the attachment of four constrained peptide loops onto a central calix[4]arene scaffold. This arrangement mimics the role of the hypervariable loops in antibody combining regions and defines a large surface area for binding to a complementary region of the exterior of a target protein. Using affinity and gel filtration chromatographies we show that one particular receptor binds strongly to the surface of cytochrome c. The site of binding is presumably close to the heme edge region, which contains several charged lysine residues. This is supported by the observation that the receptor inhibits the reduction of Fe(III) cytochrome c to its Fe(II) form. We also show that binding is strongly dependent on the nature of the substituents on the lower rim of the calixarene. The nmr and computational studies suggest that this effect may be due to conformational differences among the differently substituted receptors.