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Objectives: Falls in hospitals pose a significant safety risk, leading to injuries, prolonged hospitalization, and lasting complications. This study explores the potential of augmented reality (AR) technology in healthcare facility design to mitigate fall risk.Background: Few studies have investigated the impact of hospital room layouts on falls due to the high cost of building physical prototypes. This study introduces an innovative approach using AR technology to advance methods for healthcare facility design efficiently.Methods: Ten healthy participants enrolled in this study to examine different hospital room designs in AR. Factors of interest included room configuration, door type, exit side of the bed, toilet placement, and the presence of IV equipment. AR trackers captured trajectories of the body as participants navigated through these AR hospital layouts, providing insights into user behavior and preferences.Results: Door type influenced the degree of backward and sideways movement, with the presence of an IV pole intensifying the interaction between door and room type, leading to increased sideways and backward motion. Participants displayed varying patterns of backward and sideways travel depending on the specific room configurations they encountered.Conclusions: AR can be an efficient and cost-effective method to modify room configurations to identify important design factors before conducting physical testing. The results of this study provide valuable insights into the effect of environmental factors on movement patterns in simulated hospital rooms. These results highlight the importance of considering environmental factors, such as the type of door and bathroom location, when designing healthcare facilities.

Understanding how lithium-ion dynamics affect the (de)lithiation mechanisms of state-of-the-art nickel-rich layered oxide cathodes is crucial to improve electrochemical performance. Here, we directly observe two distinct kinetically induced lithium heterogeneities within single-crystal LiNixMnyCo(1−x−y)O2(NMC) particles using recently developed operandooptical microscopy, challenging the notion that uniform (de)lithiation occurs within individual particles. Upon delithiation, a rapid increase in lithium diffusivity at the beginning of charge results in particles with lithium-poor peripheries and lithium-rich cores. The slow ion diffusion at near-full lithiation states—and slow charge transfer kinetics—also leads to heterogeneity at the end of discharge, with a lithium-rich surface preventing complete lithiation. Finite-element modeling confirms that concentration-dependent diffusivity is necessary to reproduce these phenomena. Our results demonstrate how kinetic limitations cause significant first-cycle capacity losses in Ni-rich cathodes.

SUMMARYIt is highly likely that a psychiatrist will be called to an inquest at some point in their career. Our aim in this article is to educate psychiatrists in relation to the law and processes of a coroner's court in England and Wales and provide guidance on engaging with the system. To achieve this we review and discuss the relevant law and medico-legal aspects of inquests. Knowledge and preparation are key to negotiating any inquest and we would hope that the understanding and guidance offered in this article will reduce anxiety, make the situation manageable and aid professionalism, in often tragic circumstances.

To rationalize and improve the performance of newly developed high-rate battery electrode materials, it is crucial to understand the ion intercalation and degradation mechanisms occurring during realistic battery operation. Here we apply a laboratory-based operando optical scattering microscopy method to study micrometre-sized rod-like particles of the anode material Nb14W3O44during high-rate cycling. We directly visualize elongation of the particles, which, by comparison with ensemble X-ray diffraction, allows us to determine changes in the state of charge of individual particles. A continuous change in scattering intensity with state of charge enables the observation of non-equilibrium kinetic phase separations within individual particles. Phase field modelling (informed by pulsed-field-gradient nuclear magnetic resonance and electrochemical experiments) supports the kinetic origin of this separation, which arises from the state-of-charge dependence of the Li-ion diffusion coefficient. The non-equilibrium phase separations lead to particle cracking at high rates of delithiation, particularly in longer particles, with some of the resulting fragments becoming electrically disconnected on subsequent cycling. These results demonstrate the power of optical scattering microscopy to track rapid non-equilibrium processes that would be inaccessible with established characterization techniques.

The key to advancing lithium-ion battery technology—in particular, fast charging—is the ability to follow and understand the dynamic processes occurring in functioning materials under realistic conditions, in real time and on the nano- to mesoscale. Imaging of lithium-ion dynamics during battery operation (operando imaging) at present requires sophisticated synchrotron X-ray1–7or electron microscopy8,9techniques, which do not lend themselves to high-throughput material screening. This limits rapid and rational materials improvements. Here we introduce a simple laboratory-based, optical interferometric scattering microscope10–13to resolve nanoscopic lithium-ion dynamics in battery materials, and apply it to follow cycling of individual particles of the archetypal cathode material14,15, LixCoO2, within an electrode matrix. We visualize the insulator-to-metal, solid solution and lithium ordering phase transitions directly and determine rates of lithium diffusion at the single-particle level, identifying different mechanisms on charge and discharge. Finally, we capture the dynamic formation of domain boundaries between different crystal orientations associated with the monoclinic lattice distortion at the Li0.5CoO2composition16. The high-throughput nature of our methodology allows many particles to be sampled across the entire electrode and in future will enable exploration of the role of dislocations, morphologies and cycling rate on battery degradation. The generality of our imaging concept means that it can be applied to study any battery electrode, and more broadly, systems where the transport of ions is associated with electronic or structural changes. Such systems include nanoionic films, ionic conducting polymers, photocatalytic materials and memristors.

Elderly patient falls are expensive and may cause serious harm. Studies have identified the sit-to-stand-and-walk (STSW) task as the task where the greatest number of elderly patient falls occur. There is a great need to identify the particular movement and environmental conditions that lead to these elderly patient falls. This study begins to address this gap by evaluating the elderly patient during self-selected hospital bed egress. Using an observed fall risk episode (FRE) as a fall proxy, statistically significant parameters were identified which include bed height, pausing prior to initiating gait, level of fall risk, and Stand phase. Low bed height was identified as the least safe bed height. Patient-specific bed height (PSBH) using the patient’s lower leg length (LLL) is recommended. In addition, suggested guidelines are presented for clinical application in setting PSBH without measuring the patient’s LLL.

Objectives: This study proposes a computational model to evaluate patient room design layout and features that contribute to patient stability and mitigate the risk of fall.Background: While common fall risk assessment tools in nursing have an acceptable level of sensitivity and specificity, they focus on intrinsic factors and medications, making risk assessment limited in terms of how the physical environment contributes to fall risk.Methods: We use literature to inform a computational model (algorithm) to define the relationship between these factors and the risk of fall. We use a trajectory optimization approach for patient motion prediction.Results: Based on available data, the algorithm includes static factors of lighting, flooring, supportive objects, and bathroom doors and dynamic factors of patient movement. This preliminary model was tested using four room designs as examples of typical room configurations. Results show the capabilities of the proposed model to identify the risk associated with different room layouts and features.Conclusions: This innovative approach to room design evaluation and resulting estimation of patient fall risk show promise as a proactive evidence-based tool to evaluate the relationship of potential fall risk and room design. The development of the model highlights the challenge of heterogeneity in factors and reporting found in the studies of patient falls, which hinder our understanding of the role of the built environment in mitigating risk. A more comprehensive investigation comparing the model with actual patient falls data is needed to further refine model development.

Low back pain (LBP) is a common health problem and a major cause of lost productivity in workplaces. Manual materials handling (MMH) jobs have traditionally been regarded as risk factor for LBP. Compared to two-handed lifting, one-handed lifting has received little attention in both epidemiological and biomechanical research. In addition, one frequent complaint of the revised NIOSH lifting equation (RNLE) has been the lack of capability to directly evaluate one-handed lifting. Modifications have been proposed by the European Union, however their efficacy and influence have not yet been evaluated. This cross-sectional study provided objective survey of the MMH jobs, especially the one-handed lifting performed in manufacturing industry and investigated the outcomes of three proposed methods to address one-handed lifting using RNLE approach. Preliminary results suggest that workers with some one-handed lifting are associated with higher physical exposure. However, the increase was more significant among those who perform primarily one-handed lifting.

High-pressure level and sudden sound, especially during an elevated state of alertness can elicit a startle response. Startle response can induce sudden, intense muscle activations. Some studies have shown that increasing neck muscle activation during impact situations can reduce the risk of concussion and neck injury. This research aimed to study muscle coactivation patterns, contraction latency and the level of muscle activation in startle response compared to the voluntary response. To achieve this goal, a testbed capable of applying impacts to the head in four directions was created. Auditory (115 dB) startle stimulus was delivered and muscle activation measured using sEMG on neck muscles during startle and voluntary responses. We investigated a 1000 ms time period starting at the time that the sound is played to the time at impact. Results indicate that the first muscle activation in startle response is 2.1 times higher, 5.9 times faster and involved more muscles than in a voluntary response.

Low back pain (LBP) is a common health problem and a major cause of lost productivity in workplaces. Lifting and lowering (LL) activities have traditionally been regarded as risk factor for LBP. In the literature, very little data have been reported describing industrial workers’ exposures to measured job physical demands using comprehensive and quantitative method, such as the revised NIOSH lifting equation (RNLE). This study pooled physical exposure data for the RNLE commonly collected in three independent LBP prospective studies. In total, over one million subtasks (i.e., lifting/lowering activities) were included in this pooled study. Examination of the pooled dataset revealed an increased data distribution of the physical job demands quantified by the RNLE. Research using the pooled dataset will improve the overall statistical power and help address the weaknesses identified in previous studies.

Abstract

Neurological and psychiatric disorders account for an increasing proportion of the global disease burden. Correspondingly the neuropharmaceutical industry has experienced a significant contraction in recent years resulting in a poor variety of therapies available to treat an expanding range of conditions. Perhaps the greatest contributor to this failure in drug-discovery is the lack of understanding of the underlying biology of the nervous system and how molecular scale events translate into macroscale pathologies. Due to the unique nature of the human nervous system commonly used model organisms are often poorly representative of human pathologies resulting in a need for the development of advanced in vitro models that are capable of faithfully modeling complex structures within the brain. In this prospective, strategies for the generation of neuronal circuits and cultivation of complex three-dimensional (3D) cultures are explored. Frequently these constructs provide valuable insights into systems and processes that are difficult to explore in vivo due to the isolated and delicate nature of neuronal tissues. New developments are required to assess the physiological functions of 3D tissues in vitro.

Falls are the leading cause of fatal and non-fatal injuries among the elderly in the United States and throughout the world. Many studies have been done over the last few decades in an attempt to better understand how to prevent falls among the elderly population. Unfortunately, most of these studies are conducted by simulating falls in a laboratory with healthy adults which has been shown to be quite different from a real world fall. This pilot study defines corrective behaviors implemented by the elderly to maintain balance and looks specifically at measures of stability when the hand touch corrective behavior is used. These measures include base of support area, proximity of center of mass to base of support perimeter, time for center of mass to contact base of support perimeter, and jerk of the center of mass of the torso. Initial findings indicate that the hand touch corrective behavior is used to both maintain and regain stability. It is anticipated that, with the evaluation of the remaining trials, a model of fall-initiation of the frail elderly will be developed to provide key biomechanical stability measures for use as a proxy for a fall in laboratory studies, as well as to provide new insights in fall interventions.

Introduction:There is a growing body of research connecting musculoskeletal diseases to both job physical exposures and psychosocial exposures. Associations between job physical exposures and psychosocial factors have not been well studied or measured. These exploratory analyses evaluate relationships between quantified exposures and psychosocial outcomes. Methods:Personalized measures of duration, repetition, force and composite scores of the Strain Index (SI) and the Threshold Limit Value for Hand Activity Level (TLV for HAL) were compared to 10 psychosocial measures. Associations and predicted probabilities were evaluated using ordered logistic regression. Results and Discussion:Among 1834 study participants there were numerous significant relationships. Overall, as job physical exposures increased, psychosocial measures worsened. However, both general health and mental exhaustion improved with higher job exposures. Depression was most strongly related with higher repetition, while physical exhaustion was most strongly related with higher force. SI and TLV for HAL were significantly related to numerous psychosocial factors. Relationships remained after adjustment for confounders of age, BMI and gender. Conclusion:This study measured multiple associations between job physical exposures and both occupational and non-occupational psychosocial factors. Further research is needed to quantify the impacts on occupational health outcomes.

A range of methodological challenges were encountered during a biomechanical study of 56 older adults ranging from ages 50-95 years of age. The sample included individuals with strength and gait impairments who were at risk for falls and fall-related injury. Methodological and participant-related issues encountered during data collection were 1) preventing falls and fall-related injuries, 2) difficulty with osteological landmark palpation and retroreflective marker placement due to obesity, 3) retroreflective marker obstruction by body fat and clothing, as well as the safety harness, hospital bed and other structures in the motion capture space, 4) marker loss during in-bed movements, 5) participant fatigue and instability necessitating trial modification. The development of a customized fall arrest system and other solutions will be discussed.

The article studies the way of combining double foot data into one gait cycle from data collected from only one force plate, and the reliability with comparing the Root Mean Square Error (RMSE) between combined and original trails is validated. The joint moment values, knee muscles value were also computed by OpenSim based on the dynamic model. Correlation coefficients and normalized RMSE were used as the compared factors between two trails. Correlation coefficients are all above 0.9 for joint moments, and all above 0.95 for knee muscles except for biceps femoris - long head. The technology would get higher reliability and improving the accuracy of simulation result by increasing the numbers of collection trails and selecting pairs trails which got higher correlation coefficients.

Understanding the pattern of gene expression during erythropoiesis is crucial for a synthesis of erythroid developmental biology. Here, we isolated 4 distinct populations at successive erythropoietin-dependent stages of erythropoiesis, including the terminal, pyknotic stage. The transcriptome was determined using Affymetrix arrays. First, we demonstrated the importance of using defined cell populations to identify lineage and temporally specific patterns of gene expression. Cells sorted by surface expression profile not only express significantly fewer genes than unsorted cells but also demonstrate significantly greater differences in the expression levels of particular genes between stages than unsorted cells. Second, using standard software, we identified more than 1000 transcripts not previously observed to be differentially expressed during erythroid maturation, 13 of which are highly significantly terminally regulated, including RFXAP and SMARCA4. Third, using matched filtering, we identified 12 transcripts not previously reported to be continuously up-regulated in maturing human primary erythroblasts. Finally, using transcription factor binding site analysis, we identified potential transcription factors that may regulate gene expression during terminal erythropoiesis. Our stringent lists of differentially regulated and continuously expressed transcripts containing many genes with undiscovered functions in erythroblasts are a resource for future functional studies of erythropoiesis. Our Human Erythroid Maturation database is available at https://cellline.molbiol.ox.ac.uk/eryth/index.html.

Hereditary hemochromatosis is an iron overload disorder that can lead to the impairment of multiple organs and is caused by mutations in one or more different genes. Type 1 hemochromatosis is the most common form of the disease and results from mutations in the HFEgene. Juvenile hemochromatosis (JH) is the most severe form, usually caused by mutations in hemojuvelin (HJV) or hepcidin (HAMP). The autosomal dominant form of the disease, type 4, is due to mutations in the SLC40A1gene, which encodes for ferroportin (FPN). Hereditary hemochromatosis is commonly found in populations of European origin. By contrast, hemochromatosis in Asia is rare and less well understood and can be masked by the presence of iron deficiency and secondary iron overload from thalassemia. Here, we provide a comprehensive report of hemochromatosis in a group of patients of Asian origin. We have identified novel mutations in HJV, HAMP, and SLC40A1in countries not normally associated with hereditary hemochromatosis (Pakistan, Bangladesh, Sri Lanka, and Thailand). Our family studies show a high degree of consanguinity, highlighting the increased risk of iron overload in many countries of the developing world and in countries in which there are large immigrant populations from these regions.

Hereditary hemochromatosis is an iron overload disorder that can lead to the impairment of multiple organs and is caused by mutations in one or more different genes. Type 1 hemochromatosis is the most common form of the disease and results from mutations in the HFE gene. Juvenile hemochromatosis (JH) is the most severe form, usually caused by mutations in hemojuvelin (HJV) or hepcidin (HAMP). The autosomal dominant form of the disease, type 4, is due to mutations in the SLC40A1 gene, which encodes for ferroportin (FPN). Hereditary hemochromatosis is commonly found in populations of European origin. By contrast, hemochromatosis in Asia is rare and less well understood and can be masked by the presence of iron deficiency and secondary iron overload from thalassemia. Here, we provide a comprehensive report of hemochromatosis in a group of patients of Asian origin. We have identified novel mutations in HJV, HAMP, and SLC40A1 in countries not normally associated with hereditary hemochromatosis (Pakistan, Bangladesh, Sri Lanka, and Thailand). Our family studies show a high degree of consanguinity, highlighting the increased risk of iron overload in many countries of the developing world and in countries in which there are large immigrant populations from these regions.

The results from ignition tests conducted using the Lateral Ignition and Flame spread Test (LIFT) apparatus, Reduced scale Ignition and Flame spread Test (RIFT) apparatus, and ISO 5657 ignitability apparatus from nine different types of New Zealand wood-based products are used to obtain the critical ignition heat flux and thermal inertia using the procedure specified in ASTM E 1321-97a. It is found that vertically oriented samples in the LIFT and RIFT has a higher minimum ignition heat flux compared with the horizontal samples in the ISO 5657 apparatus. There is only a limited agreement between the three test methods for thermal inertia values.

Ferroportin (FPN) mediates iron export from cells; FPN mutations are associated with the iron overloading disorder hemochromatosis. Previously, we found that the A77D, V162del, and G490D mutations inhibited FPN activity, but that other disease-associated FPN variants retained full iron export capability. The peptide hormone hepcidin inhibits FPN as part of a homeostatic negative feedback loop. We measured surface expression and function of wild-type FPN and fully active FPN mutants in the presence of hepcidin. We found that the Y64N and C326Y mutants of FPN are completely resistant to hepcidin inhibition and that N144D and N144H are partially resistant. Hemochromatosis-associated FPN mutations, therefore, either reduce iron export ability or produce an FPN variant that is insensitive to hepcidin. The former mutation type is associated with Kupffer-cell iron deposition and normal transferrin saturation in vivo, whereas patients with the latter category of FPN mutation have high transferrin saturation and tend to deposit iron throughout the liver parenchyma. FPN-linked hemochromatosis may have a variable pathogenesis depending on the causative FPN mutant.

Ferroportin (FP141414148) mediates iron export from cells; FPN mutations are associated with the iron overloading disorder hemochromatosis. Previously, we found that the A77D, V162del, and G490D mutations inhibited FPN activity, but that other disease-associated FPN variants retained full iron export capability. The peptide hormone hepcidin inhibits FPN as part of a homeostatic negative feedback loop. We measured surface expression and function of wild-type FPN and fully active FPN mutants in the presence of hepcidin. We found that the Y64N and C326Y mutants of FPN are completely resistant to hepcidin inhibition and that N144D and N144H are partially resistant. Hemochromatosis-associated FPN mutations, therefore, either reduce iron export ability or produce an FPN variant that is insensitive to hepcidin. The former mutation type is associated with Kupffer-cell iron deposition and normal transferrin saturation in vivo, whereas patients with the latter category of FPN mutation have high transferrin saturation and tend to deposit iron throughout the liver parenchyma. FPN-linked hemochromatosis may have a variable pathogenesis depending on the causative FPN mutant.

Type IV hemochromatosis is associated with dominant mutations in the SLC40A1 gene encoding ferroportin (FPN). Known as the “ferroportin disease,” this condition is typically characterized by high serum ferritin, reduced transferrin saturation, and macrophage iron loading. Previously FPN expression in vitro has been shown to cause iron deficiency in human cell lines and mediate iron export from Xenopus oocytes. We confirm these findings by showing that expression of human FPN in a human cell line results in an iron deficiency because of a 3-fold increased export of iron. We show that FPN mutations A77D, V162Δ, and G490D that are associated with a typical pattern of disease in vivo cause a loss of iron export function in vitro but do not physically or functionally impede wild-type FPN. These mutants may, therefore, lead to disease by haploinsufficiency. By contrast the variants Y64N, N144D, N144H, Q248H, and C326Y, which can be associated with greater transferrin saturation and more prominent iron deposition in liver parenchyma in vivo, retained iron export function in vitro. Because FPN is a target for negative feedback in iron homeostasis, we postulate that the latter group of mutants may resist inhibition, resulting in a permanently “turned on” iron exporter.

Type IV hemochromatosis is associated with dominant mutations in the SLC40A1gene encoding ferroportin (FPN). Known as the “ferroportin disease,” this condition is typically characterized by high serum ferritin, reduced transferrin saturation, and macrophage iron loading. Previously FPN expression in vitro has been shown to cause iron deficiency in human cell lines and mediate iron export from Xenopusoocytes. We confirm these findings by showing that expression of human FPN in a human cell line results in an iron deficiency because of a 3-fold increased export of iron. We show that FPN mutations A77D, V162Δ, and G490D that are associated with a typical pattern of disease in vivo cause a loss of iron export function in vitro but do not physically or functionally impede wild-type FPN. These mutants may, therefore, lead to disease by haploinsufficiency. By contrast the variants Y64N, N144D, N144H, Q248H, and C326Y, which can be associated with greater transferrin saturation and more prominent iron deposition in liver parenchyma in vivo, retained iron export function in vitro. Because FPN is a target for negative feedback in iron homeostasis, we postulate that the latter group of mutants may resist inhibition, resulting in a permanently “turned on” iron exporter.

AbstractObjectives:To test the feasibility of adopting and evaluating a systematic case-finding approach to the early diagnosis of hereditary haemochromatosis (HHC) in primary care, and to estimate the prevalence of presenting conditions for which HHC testing could be offered. Methods:Systematic identification of, and genotyping for, C282Y and H63D mutations in patients presenting in primary care with possible symptoms of HHC during a 4-week period to 1 of 14 doctors in Oxfordshire. Results:From a total of 4,022 consultations, 169 (4.2%; 95% CI: 3.6–4.8) adult patients had possible symptoms of HHC. Of these, 88 (2.2%; 95% CI: 1.7–2.6) were aged 25–70 and were offered genotyping for HHC, of whom 60 agreed to be tested. There were no C282Y homozygotes (0%; 95% CI: 0–6.0), no C282Y/H63D compound heterozygotes (0%; 95% CI: 0–6.0), 2 H63D homozygotes with normal iron indices (3.3%%; 95% CI: 0.4–11.5) and 3 C282Y heterozygotes (5.0%; 95% CI: 1.0–13.9). Conclusions:This study raises doubts about a case-finding approach to early diagnosis of HHC in primary care. The non-specific nature and high prevalence of possible symptoms of HHC in primary care mean that many patients would require testing to identify a single case. Whether this offers a more cost-effective alternative to population screening requires further study.Copyright © 2002 S. Karger AG, Basel

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder causing inappropriate dietary iron absorption that affects North Europeans. HH is associated with the C282Y mutation of the HFE gene, and the H63D mutation to a lesser degree. Both mutations are abundant in Europe, with H63D also appearing in North Africa, the Middle East, and Asia. Emigration from Europe over the past 500 years has introduced C282Y and H63D to America, Australia, New Zealand, and South Africa in an essentially predictable fashion. The distinctive characteristics of the population genetics of HH are the confined racial distribution and high frequency in North European peoples. C282Y frequencies in North Europeans are typically between 5% and 10%, with homozygotes accounting for between 1/100 and 1/400 of these populations. The scarcity of the C282Y mutation in other populations accounts for the lack of HH in non-Europeans.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism. Iron absorption from the gut is inappropriately high, resulting in increasing iron overload. The hemochromatosis gene (HFE) was identified in 1996 by extensive positional cloning by many groups over a period of about 20 years. Two missense mutations were identified. Homozygosity for one of these, a substitution of a tyrosine for a conserved cysteine (C282Y), has now clearly been shown to be associated with HH in 60-100% of patients. The role of the second mutation, the substitution of an aspartic acid for a histidine (H63D), is not so clear but compound heterozygotes for both these mutations have a significant risk of developing HH. Here we review other putative mutations in the HFE gene and document a number of diallelic polymorphisms in HFE introns.

The gene for hemochromatosis was identified in 1996 and two mutations were found. Homozygosity for one of these, C282Y, is associated with hemochromatosis in a high percentage of patients. Genetic analysis of patient DNA is, therefore, a very useful tool to aid and confirm diagnosis and to screen asymptomatic relatives of patients to identify those at risk of developing this common, easily treated disease.

Membrane type 4 matrix metalloproteinase (MT4-MMP) shows the least sequence homology to the other MT-MMPs, suggesting a distinct function for this protein. We have isolated a complete cDNA corresponding to the mouse homologue which includes the signal peptide and a complete pro-domain, features that were lacking from the human form originally isolated. Mouse MT4-MMP (mMT4-MMP) expressed in COS-7 cells is located at the cell surface but does not show ability to activate pro-MMP2. The pro-catalytic domain was expressed in Escherichia coli as insoluble inclusions and active enzyme recovered after refolding. Activity of the isolated catalytic domain against synthetic peptides commonly used for MMP enzyme assays could be inhibited by TIMP1, -2, and -3. The recombinant mMT4-MMP catalytic domain was also unable to activate pro-MMP2 and was very poor at hydrolyzing components of the extracellular matrix with the exception of fibrinogen and fibrin. mMT4-MMP was able to hydrolyze efficiently a peptide consisting of the pro-tumor necrosis factor alpha (TNFalpha) cleavage site, a glutathione S-transferase-pro-TNFalpha fusion protein, and was found to shed pro-TNFalpha when co-transfected in COS-7 cells. MT4-MMP was detected by Western blot in monocyte/macrophage cell lines which in combination with its fibrinolytic and TNFalpha-converting activity suggests a role in inflammation.

Membrane type 4 matrix metalloproteinase (MT4-MMP) shows the least sequence homology to the other MT-MMPs, suggesting a distinct function for this protein. We have isolated a complete cDNA corresponding to the mouse homologue which includes the signal peptide and a complete pro-domain, features that were lacking from the human form originally isolated. Mouse MT4-MMP (mMT4-MMP) expressed in COS-7 cells is located at the cell surface but does not show ability to activate pro-MMP2. The pro-catalytic domain was expressed in Escherichia colias insoluble inclusions and active enzyme recovered after refolding. Activity of the isolated catalytic domain against synthetic peptides commonly used for MMP enzyme assays could be inhibited by TIMP1, -2, and -3. The recombinant mMT4-MMP catalytic domain was also unable to activate pro-MMP2 and was very poor at hydrolyzing components of the extracellular matrix with the exception of fibrinogen and fibrin. mMT4-MMP was able to hydrolyze efficiently a peptide consisting of the pro-tumor necrosis factor α (TNFα) cleavage site, a glutathioneS-transferase-pro-TNFα fusion protein, and was found to shed pro-TNFα when co-transfected in COS-7 cells. MT4-MMP was detected by Western blot in monocyte/macrophage cell lines which in combination with its fibrinolytic and TNFα-converting activity suggests a role in inflammation.

The kinetics of formation and dissociation of the horse metmyoglobin/azide complex has been investigated between pH 3.5 and 11.5. The ionic strength dependence of the reaction has been determined at integral pH values between 5 and 10. Hydrazoic acid, HN3, binds to metmyoglobin with a rate constant of (3.8 ± 1.0) × 105M−1s−1. Protonation of a group with an apparent pKaof 4.0 ± 0.3 increases the rate of HN3binding 6.5-fold to (2.5 ± 0.8) × 106M−1s−1. The ionizable group is attributed to the distal histidine, His-64. The azide anion, N−3, binds to metmyoglobin with a rate constant of (4.7 ± 0.3) × 103M−1s−1, about two orders of magnitude slower than HN3. Conversion of aquometmyoglobin to hydroxymetmyoglobin slows azide binding significantly. Binding of HN3to hydroxymetmyoglobin cannot be detected, while N−3binds to hydroxymetmyoglobin with a rate of 5.7 ± 3.2 M−1s−1, almost three orders of magnitude slower than N−3binding to aquometmyoglobin. Protonation of the distal histidine facilitates HN3dissociation from the complex. HN3dissociates from the metmyoglobin/azide complex with a rate constant of 18 ± 6 s−1, while the azide anion dissociates with a rate constant of 0.16 ± 0.02 s−1, about 100 times slower. The apparent pKafor His-64 is essentially the same in metmyoglobin and the metmyoglobin/azide complex, 4.0 ± 0.3 and 4.4 ± 0.2, respectively. The ionic strength dependence of the observed association rate constant is influenced by both primary and secondary kinetic salt effects. The primary kinetic salt effect is anomalous, with the rate of N−3binding decreasing with increasing ionic strength above the isoelectric point of metmyoglobin where the protein has a net negative charge. The ionic strength dependence of the dissociation rate constant can be described solely in terms of the ionic strength dependence of the acid dissociation constant for His-64 in the metmyoglobin/azide complex, a secondary kinetic salt effect.

The sog gene of the conjugative plasmid ColIb‐P9 specifies two sequence‐related polypeptides with the N‐terminal third of the larger product having DNA primase activity. To resolve the function of the C‐terminal portion of the polypeptides, we constructed a ColIb mutant containing a Tn5 insertion in the 3′ region of sog. The mutation truncated sog gene products without inactivating DNA primase and rendered the plasmid defective in conjugation. Tests for the presence of conjugative pili, for complementation by a sog+ recombinant, and for mobilization of small origin of transfer (oriT) recombinant plasmids indicated that the mutant ColIb allows conjugative aggregation of cells but it is defective in DNA transfer at some stage subsequent to its initiation at oriT. Physical evidence is given that normal sog polypeptides are among a group of proteins transferred selectively from the donor to the recipient cell by a conjugation‐specific process. No transfer of the mutant sog proteins was detected. It is proposed that the C‐terminal region of sog polypeptides facilitates transfer of single‐stranded ColIb DNA between conjugating cells following initiation of transfer at the oriT site, and that in this role the proteins are transmitted to the recipient cell.

We have investigated the efficacy of using the Escherichia coli lac operator‐repressor system to control plant gene expression. The lacI gene was modified to allow optimal expression in plant cells and then placed downstream of the cauliflower mosaic virus (CaMV) 35S RNA promoter. This construct was introduced into tobacco plants by leaf disc transformation. Transgenic tobacco plants synthesized significant quantities of LacI protein (up to 0.06% of total soluble protein). We have used the E.coli beta‐glucuronidase gene (gus) as the reporter gene by placing it downstream of the maize chlorophyll a/b binding protein (CAB) gene promoter. Lac operators were introduced into several positions within the CAB promoter and operator‐free plasmid was used as control. Repression was assessed by comparing the transient expression from CAB‐operator‐gus reporter constructs in protoplasts expressing lac protein, with that in control cells not expressing the repressor. Repression varied between 10 and 90% with different operator positions. Transient assays were also performed in the presence of the inducer, isopropyl‐beta‐D‐thiogalactoside (IPTG). In lacI protoplasts the presence of IPTG manifested itself in a 4.2‐fold relief of repression. The study was extended to show regulation of expression in stable transformants. Tobacco transformants harbouring a CAB‐operator‐gus reporter construct and the lacI gene were shown to have repressed GUS levels, but in the presence of IPTG, repression was relieved 15‐fold. We conclude that the lac repressor can enter the plant cell nucleus, find its cognate operator sequence in the chromatin to form a repressor‐‐operator complex and effectively block transcription of a downstream gene.

myb-related transcription factors contain highly conserved DNA-binding domains. Using a mixture of degenerate oligonucleotides derived from the highly conserved region as probe, 14 myb-related clones were isolated from a cDNA library constructed using tomato hypocoyl mRNA. The expression of these clones was studied by northern blot analysis using poly(A)+ RNA from 7 tissue types (hypocotyl, leaf, root, green and red fruit, immature and mature flower). This study has revealed a wide range of expression patterns which include multiple and single transcripts, some of which show marked tissue specificity. Two clones showing different expression patterns have been fully sequenced. The DNA-binding domains of these two tomato myb clones are compared with myb genes from other plant species and organisms. Of the three clones analysed so far by Southern hybridization, two are single-copy genes and one has multiple genomic copies.

The kinetics of formation and dissociation of the horse metmyoglobin/fluoride complex has been investigated between pH 3.4 and 11. The ionic strength dependence of the reaction has been measured at integral pH values between pH 5 and 10. Hydrofluoric acid, HF, binds to metmyoglobin with a rate constant of (4.7 ± 0.7) × 104M−1s−1. An apparent ionization in metmyoglobin with a pKaof 4.4 ± 0.5 influences the rate of HF binding and is attributed to the distal histidine, His-64. Protonation of His-64 increases the HF binding rate by a factor of 2.6. The fluoride anion, F−, binds to metmyoglobin with a rate constant of (5.6 ± 1.4) × 10−2M−1s−1, about 106times slower than HF. Binding of either HF or F−to hydroxymetmyoglobin cannot be detected. Protonation of the distal histidine facilitates HF dissociation from the metmyoglobin/fluoride complex. HF dissociates with a rate constant of 1.9 ± 0.3 s−1. The fluoride anion dissociates 2000 times more slowly, with a rate constant of (8.7 ± 1.6) × 10−4s−1. The apparent pKafor His-64 ionization in the fluorometmyoglobin complex is 5.7 ± 0.1. The association and dissociation rate constants are relatively independent of ionic strength with secondary kinetic salt effects sufficient to account for the ionic strength variation of both, consistent with the idea that association and dissociation of neutral HF dominate the kinetics of fluoride binding to metmyoglobin.

The sog gene of the conjugative plasmid ColIb‐P9 specifies two sequence‐related polypeptides with the N‐terminal third of the larger product having DNA primase activity. To resolve the function of the C‐terminal portion of the polypeptides, we constructed a ColIb mutant containing a Tn5 insertion in the 3′ region of sog. The mutation truncated sog gene products without inactivating DNA primase and rendered the plasmid defective in conjugation. Tests for the presence of conjugative pili, for complementation by a sog+ recombinant, and for mobilization of small origin of transfer (oriT) recombinant plasmids indicated that the mutant ColIb allows conjugative aggregation of cells but it is defective in DNA transfer at some stage subsequent to its initiation at oriT. Physical evidence is given that normal sog polypeptides are among a group of proteins transferred selectively from the donor to the recipient cell by a conjugation‐specific process. No transfer of the mutant sog proteins was detected. It is proposed that the C‐terminal region of sog polypeptides facilitates transfer of single‐stranded ColIb DNA between conjugating cells following initiation of transfer at the oriT site, and that in this role the proteins are transmitted to the recipient cell.

Insecticidal effects of three plant-derived genes, those encoding snowdrop lectin (GNA), bean (Phaseolus vulgaris) chitinase (BCH) and wheat α-amylase (WAI), were investigated and compared with effects of the cowpea trypsin inhibitor gene (CpTI). Transgenic potato plants containing each of the three genes singly, and in pairwise combinations were produced. All the introduced genes were driven by the CaMV 35S promoter; expression was readily detectable at the RNA level in transformants, but not detectable accumulation of WAI could be detected in transgenic potatoes containing its encoding gene. GNA and BCH were accumulated at levels up to 2.0% of total soluble protein; both proteins were expressed in a functional form, and GNA was shown to undergo 'correct' N-terminal processing. Accumulation levels of individual proteins were higher in plants containing a single foreign gene than in plants containing two foreign genes. Resistance of the transgenic plants to insect attack was assayed by exposing the plants to larvae of the tomato moth, Lacanobia oleracea. All the plants tested which were expressing GNA showed an enhanced level of resistance. Leaf damage was reduced by more than 50% compared to controls; total insect biomass per plant was reduced by 45-65%, but larval survival was only slightly reduced (20%). These results support the hypothesis that GNA has a significant antifeedant effect on insects. Expression of BCH had no protective effect against this insect. Expression of CpTI in transgenic potatoes had similar effects to expression of GNA on total insect biomass and survival, but did not afford protection against insect damage to the plant.

We have investigated the efficacy of using the Escherichia coli lac operator‐repressor system to control plant gene expression. The lacI gene was modified to allow optimal expression in plant cells and then placed downstream of the cauliflower mosaic virus (CaMV) 35S RNA promoter. This construct was introduced into tobacco plants by leaf disc transformation. Transgenic tobacco plants synthesized significant quantities of LacI protein (up to 0.06% of total soluble protein). We have used the E.coli beta‐glucuronidase gene (gus) as the reporter gene by placing it downstream of the maize chlorophyll a/b binding protein (CAB) gene promoter. Lac operators were introduced into several positions within the CAB promoter and operator‐free plasmid was used as control. Repression was assessed by comparing the transient expression from CAB‐operator‐gus reporter constructs in protoplasts expressing lac protein, with that in control cells not expressing the repressor. Repression varied between 10 and 90% with different operator positions. Transient assays were also performed in the presence of the inducer, isopropyl‐beta‐D‐thiogalactoside (IPTG). In lacI protoplasts the presence of IPTG manifested itself in a 4.2‐fold relief of repression. The study was extended to show regulation of expression in stable transformants. Tobacco transformants harbouring a CAB‐operator‐gus reporter construct and the lacI gene were shown to have repressed GUS levels, but in the presence of IPTG, repression was relieved 15‐fold. We conclude that the lac repressor can enter the plant cell nucleus, find its cognate operator sequence in the chromatin to form a repressor‐‐operator complex and effectively block transcription of a downstream gene.

Most local barrier systems are designed primarily either to protect animals from airborne contamination (exclusion) or to ensure the safety of personnel (hazard containment). Few, other than isolators, are able to cope with the often conflicting demands to do both. The Positive Individually Ventilated system (PIV), which provides pressure adjustable, individual supply and exhaust ventilation to each cage has been tested with this in mind as well as the need to ensure that environmental conditions comply with the requirements of the Home Office Code of Practice (CoP).The results indicate that when compared with traditional open racking the system can reduce both the risk of animals becoming contaminated by airborne infection from the room and the risk of aeroallergens escaping from the cages into the room air. At the same time, environmental conditions within the cages are both less variable and less stressful to the occupants. Conditions of air temperature, relative humidity, ventilation rates, light intensity and (with appropriate air handling) sound levels, all comply with or are better than those required by the CoP.Even with the room air change rate set at only 8/h (8 ac/h) the air distribution system results in draught-free cage ventilation rates of around 50-120 ac/h. This means that with at least the PIV exhaust(s) linked directly into the air conditioning system of the building, the size of the latter and its associated running costs can be reduced by around 50%; this energy saving concept has been approved in principle by the Home Office. Additional benefits include the fact that bedding is kept much drier allowing further cost savings in bedding and associated labour costs.The system is thus beneficial to the animals in protecting them from airborne infection and other stresses. By providing a less variable environment it also helps to minimise the sort of interference with experiments which can arise from that source. Similarly, in accordance with the aims of 'The Control of Substances Hazardous to Health Regulations' (COSHH 1988) and the Health & Safety Executive (HSE 1990), by reducing dust levels in the room air, including allergens, it is also beneficial to the personnel working in the animal rooms.

The kinetics of the synthesis of the components of polyribosomes was investigated in the uterus of the immature rat responding to the administration of oestradiol-17 beta. The hormone brings about a rapid stimulation of the association of newly synthesized mRNA with uterine ribosomes, which is maximal 2-4 h after oestradiol administration and causes the aggregation of pre-existing ribosomes into polyribosomes. Despite the striking stimulation of rRNA synthesis 2-4 h after hormone treatment [Knowler & Smellie (1971) Biochem. J. 125, 605-614], the accumulation of new rRNA into ribosomes does not reach a peak until 12 h after administration. At this time, the incorporation of new ribosomal protein is also maximal. A second peak of incorporation of newly synthesized mRNA into polyribosomes follows the peak of ribosome synthesis and coincides with the oestrogen-activated synthesis of DNA.

The successful expression of animal or human virus epitopes on the surface of plant viruses has recently been demonstrated. These chimeric virus particles (CVPs) could represent a cost-effective and safe alternative to conventional animal cell-based vaccines. We report the insertion of oligonucleotides coding for a short linear epitope from the VP2 capsid protein of mink enteritis virus (MEV) into an infectious cDNA clone of cowpea mosaic virus and the successful expression of the epitope on the surface of CVPs when propagated in the black-eyed bean, Vigna unguiculata. The efficacy of the CVPs was established by the demonstration that one subcutaneous injection of 1 mg of the CVPs in mink conferred protection against clinical disease and virtually abolished shedding of virus after challenge with virulent MEV, demonstrating the potential utility of plant CVPs as the basis for vaccine development. The epitope used occurs in three different virus species—MEV, canine parvovirus, and feline panleukopenia virus—and thus the same vaccine could be used in three economically important viral hosts—mink, dogs, and cats, respectively.

Abstract:  Early-season colonisation of new roots of Hyacinthoides non-scripta (L.) Chouard ex Rothm. was investigated to determine how arbuscular mycorrhizal symbiosis is re-established after the annual root system is shed. During the rootless phase in summer, colonies of bulbs were removed and replanted after the soil around and below the bulb had been mixed (major disturbance) so as to disrupt the external mycelium of arbuscular mycorrhizal (AM) fungi. As a minor disturbance treatment, top soil was removed, bulbs were turned or not in their growth position with as little other disturbance as possible, and the top soil replaced. Control plants were left undisturbed. Half of the plants were harvested 3–4 weeks after the onset of root emergence. Populations of all AM fungi in roots were greatly reduced by major disturbance, whilst those in other treatments and controls were unaffected. At the second harvest, in spring, when shoots had emerged, root colonisation by fine endophytes and Scutellospora morphotypes developed in all treatments, whereas that of Acaulospora morphotypes remained low after major disturbance. Disturbance treatments delayed the appearance, at the second harvest, of mycorrhizas with degenerate arbuscules. Leaf phosphorus concentration was unaffected by soil disturbance, possibly due to partial recovery of AM fungal populations or buffering by resources stored in the bulb.

The RNA genome of human hepatitis A virus (HAV) was molecularly cloned. Recombinant DNA clones representing the entire HAV RNA were used to determine the primary structure of the viral genome. The length of the viral genome is 7478 nucleotides. An open reading frame starting at nucleotide 734 and terminating at nucleotide 7415 encodes a polyprotein of Mr 251,940. Comparison of the HAV nucleotide sequence with that of other picornaviruses has failed to reveal detectable areas of homology. However, a computer analysis of the putative amino acid sequence of HAV and poliovirus demonstrated the existence of short areas of homology in virion protein 3 (VP3) and throughout the carboxyl-terminal portion of the polyproteins. In addition, extensive protein structural homologies with poliovirus were detected.