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Abstract Purpose: This paper describes the process developed at the University of Pittsburgh to increase the number of NIH-funded Diversity Supplements. Method: The authors formed a Diversity in Academia Workgroup where we created the infrastructure and process to increase the number of Diversity Supplements. Each year, the Office of Sponsored Programs provided a list of grants that would be eligible to submit a Diversity Supplement. We surveyed the Principal Investigators inquiring about their interest in working with a trainee on a Diversity Supplement. If yes, we included their information in a database we built so that trainees could search for eligible research studies. The Diversity Deans then identified underrepresented faculty and postdoctoral researchers. We invited Program Officers from NIH to participate in a panel presentation for trainees, which was well attended. Results: The number of Diversity Supplements awarded to Pitt researchers has significantly increased from 7 in 2020 to 10 in 2021 and to 15 in 2022. Six more have been awarded in the first half of 2023. Conclusions: We created the Diversity in Academia Workgroup with the goal to increase the number of Diversity Supplements at the University of Pittsburgh and in so doing, increase the diversity in the biomedical research workforce. While challenging, we know the critical importance and benefits of increased diversity at the University, and we have made significant strides toward this goal.

We report 2 fatal exacerbations of systemic capillary leak syndrome (SCLS), also known as Clarkson disease, associated with coronavirus disease (COVID-19) in the United States. One patient carried an established diagnosis of SCLS and the other sought treatment for new-onset hypotensive shock, hemoconcentration, and anasarca, classic symptoms indicative of an SCLS flare. Both patients had only mild-to-moderate symptoms of COVID-19. This clinical picture suggests that these patients succumbed to complications of SCLS induced by infection with severe acute respiratory syndrome coronavirus 2. Persons with known or suspected SCLS may be at increased risk for developing a disease flare in the setting of mild-to-moderate COVID-19 infection.

Abstract Immune thrombocytopenic purpura (ITP) is a rare complication associated with vaccines targeting various diseases, including influenza, measles‐mumps‐rubella, hepatitis B, and diphtheria‐tetanus‐pertussis. We report 2 cases of ITP in healthy 20‐year‐old and 21‐year‐old women presenting to Emory University in Atlanta, GA, 2 days after the second dose and 11 days after the first dose (respectively) of the Pfizer‐BioNTech messenger RNA severe acute respiratory syndrome coronavirus 2 vaccine. Both patients recovered quickly. With more than a billion doses of coronavirus disease 2019 vaccines safely administered worldwide as of May 2021, discussions with patients should put into perspective the low risks of vaccination against the enormous societal benefit of the coronavirus disease 2019 vaccine.

Abstract Background Two critical challenges in developing cell-transplantation therapies for injured or diseased tissues are to identify optimal cells and harmful side effects. This is of particular concern in the case of spinal cord injury, where recent studies have shown that transplanted neuroepithelial stem cells can generate pain syndromes. Results We have previously shown that astrocytes derived from glial-restricted precursor cells (GRPs) treated with bone morphogenetic protein-4 (BMP-4) can promote robust axon regeneration and functional recovery when transplanted into rat spinal cord injuries. In contrast, we now show that transplantation of GRP-derived astrocytes (GDAs) generated by exposure to the gp130 agonist ciliary neurotrophic factor (GDAsCNTF), the other major signaling pathway involved in astrogenesis, results in failure of axon regeneration and functional recovery. Moreover, transplantation of GDACNTF cells promoted the onset of mechanical allodynia and thermal hyperalgesia at 2 weeks after injury, an effect that persisted through 5 weeks post-injury. Delayed onset of similar neuropathic pain was also caused by transplantation of undifferentiated GRPs. In contrast, rats transplanted with GDAsBMP did not exhibit pain syndromes. Conclusion Our results show that not all astrocytes derived from embryonic precursors are equally beneficial for spinal cord repair and they provide the first identification of a differentiated neural cell type that can cause pain syndromes on transplantation into the damaged spinal cord, emphasizing the importance of evaluating the capacity of candidate cells to cause allodynia before initiating clinical trials. They also confirm the particular promise of GDAs treated with bone morphogenetic protein for spinal cord injury repair.

Abstract Background Cancer treatment with a variety of chemotherapeutic agents often is associated with delayed adverse neurological consequences. Despite their clinical importance, almost nothing is known about the basis for such effects. It is not even known whether the occurrence of delayed adverse effects requires exposure to multiple chemotherapeutic agents, the presence of both chemotherapeutic agents and the body's own response to cancer, prolonged damage to the blood-brain barrier, inflammation or other such changes. Nor are there any animal models that could enable the study of this important problem. Results We found that clinically relevant concentrations of 5-fluorouracil (5-FU; a widely used chemotherapeutic agent) were toxic for both central nervous system (CNS) progenitor cells and non-dividing oligodendrocytes in vitro and in vivo. Short-term systemic administration of 5-FU caused both acute CNS damage and a syndrome of progressively worsening delayed damage to myelinated tracts of the CNS associated with altered transcriptional regulation in oligodendrocytes and extensive myelin pathology. Functional analysis also provided the first demonstration of delayed effects of chemotherapy on the latency of impulse conduction in the auditory system, offering the possibility of non-invasive analysis of myelin damage associated with cancer treatment. Conclusions Our studies demonstrate that systemic treatment with a single chemotherapeutic agent, 5-FU, is sufficient to cause a syndrome of delayed CNS damage and provide the first animal model of delayed damage to white-matter tracts of individuals treated with systemic chemotherapy. Unlike that caused by local irradiation, the degeneration caused by 5-FU treatment did not correlate with either chronic inflammation or extensive vascular damage and appears to represent a new class of delayed degenerative damage in the CNS.

Abstract Background Chemotherapy in cancer patients can be associated with serious short- and long-term adverse neurological effects, such as leukoencephalopathy and cognitive impairment, even when therapy is delivered systemically. The underlying cellular basis for these adverse effects is poorly understood. Results We found that three mainstream chemotherapeutic agents – carmustine (BCNU), cisplatin, and cytosine arabinoside (cytarabine), representing two DNA cross-linking agents and an antimetabolite, respectively – applied at clinically relevant exposure levels to cultured cells are more toxic for the progenitor cells of the CNS and for nondividing oligodendrocytes than they are for multiple cancer cell lines. Enhancement of cell death and suppression of cell division were seen in vitro and in vivo. When administered systemically in mice, these chemotherapeutic agents were associated with increased cell death and decreased cell division in the subventricular zone, in the dentate gyrus of the hippocampus and in the corpus callosum of the CNS. In some cases, cell division was reduced, and cell death increased, for weeks after drug administration ended. Conclusion Identifying neural populations at risk during any cancer treatment is of great importance in developing means of reducing neurotoxicity and preserving quality of life in long-term survivors. Thus, as well as providing possible explanations for the adverse neurological effects of systemic chemotherapy, the strong correlations between our in vitro and in vivo analyses indicate that the same approaches we used to identify the reported toxicities can also provide rapid in vitro screens for analyzing new therapies and discovering means of achieving selective protection or targeted killing.

Abstract Background Transplantation of embryonic stem or neural progenitor cells is an attractive strategy for repair of the injured central nervous system. Transplantation of these cells alone to acute spinal cord injuries has not, however, resulted in robust axon regeneration beyond the sites of injury. This may be due to progenitors differentiating to cell types that support axon growth poorly and/or their inability to modify the inhibitory environment of adult central nervous system (CNS) injuries. We reasoned therefore that pre-differentiation of embryonic neural precursors to astrocytes, which are thought to support axon growth in the injured immature CNS, would be more beneficial for CNS repair. Results Transplantation of astrocytes derived from embryonic glial-restricted precursors (GRPs) promoted robust axon growth and restoration of locomotor function after acute transection injuries of the adult rat spinal cord. Transplantation of GRP-derived astrocytes (GDAs) into dorsal column injuries promoted growth of over 60% of ascending dorsal column axons into the centers of the lesions, with 66% of these axons extending beyond the injury sites. Grid-walk analysis of GDA-transplanted rats with rubrospinal tract injuries revealed significant improvements in locomotor function. GDA transplantation also induced a striking realignment of injured tissue, suppressed initial scarring and rescued axotomized CNS neurons with cut axons from atrophy. In sharp contrast, undifferentiated GRPs failed to suppress scar formation or support axon growth and locomotor recovery. Conclusion Pre-differentiation of glial precursors into GDAs before transplantation into spinal cord injuries leads to significantly improved outcomes over precursor cell transplantation, providing both a novel strategy and a highly effective new cell type for repairing CNS injuries.

Human Accelerated Regions (HARs) are highly conserved across species but exhibit a significant excess of human-specific sequence changes, suggesting they may have gained novel functions in human evolution. HARs include transcriptional enhancers with human-specific activity and have been implicated in the evolution of the human brain. However, our understanding of how HARs contributed to uniquely human features of the brain is hindered by a lack of insight into the genes and pathways that HARs regulate. It is unclear whether HARs acted by altering the expression of gene targets conserved between HARs and their chimpanzee orthologs or by gaining new gene targets in human, a mechanism termed enhancer hijacking. We generated a high-resolution map of chromatin interactions for 1,590 HARs and their orthologs in human and chimpanzee neural stem cells (NSCs) to comprehensively identify gene targets in both species. HARs and their chimpanzee orthologs targeted a conserved set of 2,963 genes enriched for neurodevelopmental processes including neurogenesis and synaptic transmission. Changes in HAR enhancer activity were correlated with changes in conserved gene target expression. Conserved targets were enriched among genes differentially expressed between human and chimpanzee NSCs or between human and non-human primate developing and adult brain. Species-specific HAR gene targets did not converge on known biological functions and were not significantly enriched among differentially expressed genes, suggesting that HARs did not alter gene expression via enhancer hijacking. HAR gene targets, including differentially expressed targets, also showed cell type-specific expression patterns in the developing human brain, including outer radial glia, which are hypothesized to contribute to human cortical expansion. Our findings support that HARs influenced human brain evolution by altering the expression of conserved gene targets and provide the means to functionally link HARs with novel human brain features., Competing Interests: Competing Interests: The authors declare no competing financial interests.

The evolution of the human cerebral cortex involved modifications in the composition and proliferative potential of the neural stem cell (NSC) niche during brain development. Human Accelerated Regions (HARs) exhibit a significant excess of human-specific sequence changes and have been implicated in human brain evolution. Multiple studies support that HARs include neurodevelopmental enhancers with novel activities in humans, but their biological functions in NSCs have not been empirically assessed at scale. Here we conducted a direct-capture Perturb-seq screen repressing 180 neurodevelopmentally active HARs in human iPSC-derived NSCs with single-cell transcriptional readout. After profiling >188,000 NSCs, we identified a set of HAR perturbations with convergent transcriptional effects on gene networks involved in NSC apicobasal polarity, a cellular process whose precise regulation is critical to the developmental emergence of basal radial glia (bRG), a progenitor population that is expanded in humans. Across multiple HAR perturbations, we found convergent dysregulation of specific apicobasal polarity and adherens junction regulators, including PARD3, ABI2, SETD2 , and PCM1 . We found that the repression of one candidate from the screen, HAR181, as well as its target gene CADM1 , disrupted apical PARD3 localization and NSC rosette formation. Our findings reveal interconnected roles for HARs in NSC biology and cortical development and link specific HARs to processes implicated in human cortical expansion.

Water quality testing is crucial for protecting public health, especially considering the number of boil water advisories annually issued across Canada that impact daily life for residents in affected areas. To overcome these challenges, the development of drinking water safety plans and accessibility to regular testing using simple, rapid, and accurate materials are necessary. However, the significance of monitoring the accuracy of environmental microbiology testing laboratories cannot be overlooked. Participation in external quality assessment programs, such as those that include proficiency testing (PT), is a necessary risk management resource that ensures the effectiveness of these testing processes. Proficiency Testing Canada (PTC), in collaboration with the Canadian Microbiological Proficiency Testing (CMPT) program based at the University of British Columbia, have implemented a drinking-water microbiology PT program since 1996. Both PTC and CMPT are ISO/IEC 17043:2010-accredited EQA providers. The drinking water program provided PT challenges to subscribing testing laboratories twice per year. Each challenge consisted of four samples containing unknown concentrations of Escherichia coli (E. coli) and Enterobacter spp. Results from participants were assessed for accuracy based on the method of testing. This cross-sectional study evaluated 150 rural and metropolitan testing sites across Canada between 2016 and 2022. Multivariable logistic regression analysis was conducted to examine the impact of different testing methods and laboratory accreditation status on the proficiency scores. This approach enabled us to assess the association between multiple independent variables and the likelihood of achieving specific proficiency scores, providing insights into how testing methods and accreditation status affect overall performance. After adjusting for rural residence, testing time, and survey year, the membrane filtration method was positively associated with the likelihood of scoring satisfactory results compared to the enzyme-substrate method (OR: 1.75; CI: 1.37-2.24), as well as accreditation status (OR: 1.47; CI: 1.16-1.85). The potential for improvement in environmental laboratory testing performance through the implementation of regulated PT in drinking water safety plans is proposed, along with the need for reliable testing methods applicable to rapid drinking water microbiology testing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sreya, Alam, Daula, Lee, Restelli, Middlebrook, Noble and Perrone.)

Changes in gene regulation have been linked to the expansion of the human cerebral cortex and to neurodevelopmental disorders, potentially by altering neural progenitor proliferation. However, the effects of genetic variation within regulatory elements on neural progenitors remain obscure. We use sgRNA-Cas9 screens in human neural stem cells (hNSCs) to disrupt 10,674 genes and 26,385 conserved regions in 2,227 enhancers active in the developing human cortex and determine effects on proliferation. Genes with proliferation phenotypes are associated with neurodevelopmental disorders and show biased expression in specific fetal human brain neural progenitor populations. Although enhancer disruptions overall have weaker effects than gene disruptions, we identify enhancer disruptions that severely alter hNSC self-renewal. Disruptions in human accelerated regions, implicated in human brain evolution, also alter proliferation. Integrating proliferation phenotypes with chromatin interactions reveals regulatory relationships between enhancers and their target genes contributing to neurogenesis and potentially to human cortical evolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In response to the coronavirus pandemic (COVID-19) and scale up of diagnostic testing, the Canadian Microbiology Proficiency Testing program created a new proficiency testing (PT) program for the molecular and antigen detection of SARS-CoV-2. The program was geared to point of care testing (POCT) sites located in each of the eight provincial Health Authorities across British Columbia, Canada, with the intention to monitor testing quality. The PT program consisted of 6 shipments in a year, each containing a set of 4 samples either positive for SARS-CoV-2 virus or negative. The program began with initial 23 sites enrolling in March 2021, expanding to >100 participants by December 2021. After the first two surveys, it was observed that testing performance (accuracy) was consistently acceptable for sites using nucleic acid technology (NAT), however performance by sites using rapid antigen detection (RAD) methods was poor, especially when testing the weakly positive samples. A root cause investigation of poor testing performance revealed gaps in the execution of testing methods and also in results interpretation. These quality issues were most commonly associated with new testers who lacked experience with diagnostic testing. Tester training and mentoring was reinforced as was retraining of personnel; sample processing instructions were modified, and a training video was also created for testing sites. As a result of these interventions, sites improved their testing accuracy and the performance of POCT sites using RAD methods came to more closely match the performance of sites utilizing NAT. Overall, the PT program was highly successfully and improved quality of testing in the province. This work demonstrates the critical value of an external quality assessment (EQA) partner towards improving patient and public health and safety, especially when testing is conducted outside of an accredited medical laboratory setting., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Restelli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Incident reporting systems are useful tools to raise awareness of patient safety issues associated with healthcare error, including errors associated with the medical laboratory., Methods: Previously, we presented the analysis of data compiled by the British Columbia Patient Safety & Learning System over a 3-year period. A second comparable set was collected and analyzed to determine if reported error rates would tend to remain stable or change., Results: Compared to the original set, the second set presented changes that were both materially and statistically significant. Overall, the total number of reports increased by 297% with substantial changes between the pre-examination, examination and post-examination phases (χ2: 993.925, DF=20; p<0.00001). While the rate of change for pre-examination (clerical and collection) errors were not significantly different than the total year results, the rate of change for reporting examination errors rose by 998%. While the exact reason for dramatic change is not clear, possible explanations are provided., Conclusions: Longitudinal error rate tracking is a useful approach to monitor for laboratory quality improvement.

Background: This article reports on the findings of 12,278 laboratory related safety events that were reported through the British Columbia Patient Safety & Learning System Incident Reporting System., Methods: The reports were collected from 75 hospital-based laboratories over a 33-month period and represent approximately 4.9% of all incidents reported., Results: Consistent with previous studies 76% of reported incidents occurred during the pre-analytic phase of the laboratory cycle, with twice as many associated with collection problems as with clerical problems. Eighteen percent of incidents occurred during the post-analytic reporting phase. The remaining 6% of reported incidents occurred during the actual analytic phase. Examination of the results suggests substantial under-reporting in both the post-analytic and analytic phases. Of the reported events, 95.9% were reported as being associated with little or no harm, but 0.44% (55 events) were reported as having severe consequences., Conclusions: It is concluded that jurisdictional reporting systems can provide valuable information, but more work needs to be done to encourage more complete reporting of events.

The asymmetric dehydration of alcohols is an important process for the direct synthesis of alkenes. We report the structure and substrate specificity of the bifunctional linalool dehydratase isomerase (LinD) from the bacterium Castellaniella defragrans that catalyzes in nature the hydration of β-myrcene to linalool and the subsequent isomerization to geraniol. Enzymatic kinetic resolutions of truncated and elongated aromatic and aliphatic tertiary alcohols (C5-C15) that contain a specific signature motif demonstrate the broad substrate specificity of LinD. The three-dimensional structure of LinD from Castellaniella defragrans revealed a pentamer with active sites at the protomer interfaces. Furthermore, the structure of LinD in complex with the product geraniol provides initial mechanistic insights into this bifunctional enzyme. Site-directed mutagenesis confirmed active site amino acid residues essential for its dehydration and isomerization activity. These structural and mechanistic insights facilitate the development of hydrating catalysts, enriching the toolbox for novel bond-forming biocatalysis.

Background and Purpose: Endothelin-1 (ET-1) is present in murine and human skin and causes itch (pruritus) when injected in humans. This behavioural study examined the scratch reflex evoked by ET-1 in mice., Experimental Approach: An automated detector was used to determine whether ET-1 causes reflex scratching, the behavioural correlate of itching, in BALB/c mice. Selective agonists and antagonists were used to probe the ET receptor(s) involved., Key Results: ET-1 evoked dose-related reflex scratching lasting up to 20 min following intradermal injection (0.1-100 ng; 0.04-40 pmol). The ED(50) for ET-1 induced scratching was 2.1 ng and desensitization occurred with cumulative dosing. High doses of the ET(B) receptor agonist IRL1620 (10 microg; 5.5 nmol), also caused scratching (ED(50) 1.3 microg, 0.7 nmol). The ET(A) receptor antagonist BQ123 significantly reduced scratching evoked by ET-1 and IRL 1620, suggesting that both agonists caused scratching via an ET(A) receptor-dependent mechanism. The ET(B) receptor antagonist BQ788 significantly reduced scratching evoked by IRL1620 but had no effect on scratching evoked by ET-1. This indicated that activation of ET(B) receptors by high doses of ET(B) agonist, but not ET-1, can trigger scratching., Conclusion and Implications: ET-1 is a potent endogenous activator of reflex scratching (itch). Mechanisms for ET-induced scratching are considered, including direct action of ET-1 on pruriceptive nerve endings and indirect actions via release of endogenous mediators such as histamine from mast cells. ET-1 and ET(A) receptors, possibly also ET(B) receptors, are potential targets for developing specific anti-pruritic drugs to treat pruritic skin disorders such as atopic dermatitis.

Anemia frequently accompanies chronic diseases such as progressive renal failure, acquired immunodeficiency syndrome, and cancer. Patients are currently treated with erythropoietin (EPO) replacement therapy, using various recombinant human EPO protein formulations. Although this treatment is effective, gene therapy could be more economical and more convenient for the long-term management of the disease. The objective of this study was to develop a naked DNA-based gene therapy protocol that could fill this need. Hydrodynamic limb vein technology has been shown to be an effective and safe procedure for delivering naked plasmid DNA (pDNA) into the skeletal muscles of limbs. Using this method, we addressed the major challenge of an EPO-based gene therapy of anemia: maintaining stable, long-term expression at a level that sufficiently promotes erythropoiesis without leading to polycythemia. The results of our study, using a rat anemia model, provide proof of principle that repeated delivery of small pDNA doses has an additive effect and can gradually lead to the correction of anemia without triggering excessive hematopoiesis. This simple method provides an alternative approach for regulating EPO expression. EPO expression was also proportional to the injected pDNA dose in nonhuman primates. In addition, long-term (more than 450 days) expression was obtained after delivering rhesus EPO cDNA under the transcriptional control of the muscle-specific creatine kinase (MCK) promoter. In conclusion, these data suggest that the repeated delivery of small doses of EPO expressing pDNA into skeletal muscle is a promising, clinically viable approach to alleviate the symptoms of anemia.

Interactions between a soluble form of microsomal cytochrome b(5) (b(5)) from Musca domestica (housefly) and Bacillus megaterium flavocytochrome P450 BM3 and its component reductase (CPR), heme (P450) and FAD/NADPH-binding (FAD) domains were analyzed by a combination of steady-state and stopped-flow kinetics methods, and optical spectroscopy techniques. The high affinity binding of b(5) to P450 BM3 induced a low-spin to high-spin transition in the P450 heme iron (K(d) for b(5) binding = 0.44 microM and 0.72 microM for the heme domain and intact flavocytochrome, respectively). The b(5) had modest inhibitory effects on steady-state turnover of P450 BM3 with fatty acids, and the ferrous-carbon monoxy P450 complex was substantially stabilized on binding b(5). Single turnover reduction of b(5) by BM3 using stopped-flow absorption spectroscopy (k(lim) = 116 s(-1)) was substantially faster than steady-state reduction of b(5) by P450 BM3 (or its CPR and FAD domains), indicating rate-limiting step(s) other than BM3 flavin-to-b(5) heme electron transfer in the steady-state reaction. Steady-state b(5) reduction by P450 BM3 was considerably accelerated at high ionic strength. Pre-reduction of P450 BM3 by NADPH decreased the k(lim) for b(5) reduction approximately 10-fold, and also resulted in a lag phase in steady-state b(5) reduction that was likely due to BM3 conformational perturbations sensitive to the reduction state of the flavocytochrome. Ferrous b(5) could not reduce the ferric P450 BM3 heme domain under anaerobic conditions, consistent with heme iron reduction potentials of the two proteins. However, rapid oxidation of both hemoproteins occurred on aeration of the ferrous protein mixture (and despite the much slower autoxidation rate of b(5) in isolation), consistent with electron transfer occurring from b(5) to the oxyferrous P450 BM3 in the complex. The results demonstrate that strong interactions occur between a eukaryotic b(5) and a model prokaryotic P450. Binding of b(5) perturbs BM3 heme iron spin-state equilibrium, as is seen in many physiologically relevant b(5) interactions with eukaryotic P450s. These results are consistent with the conservation of structure of P450s (particularly at the heme proximal face) between prokaryotes and eukaryotes, and may point to as yet undiscovered roles for b(5)-like proteins in the control of activities of certain prokaryotic P450s.

Laboratory accreditation and External Quality Assessment (also called proficiency testing) are mainstays of laboratory quality assessment and performance. Both practices are associated with examples of improved laboratory performance. The relationship between laboratory performance and improved patient safety is more difficult to assess because of the many variables that are involved with patient outcome. Despite this difficulty, the argument to continue external evaluation of laboratories is too compelling to consider the alternative.

Fecal indicator bacteria (FIB) were measured approximately 5 days a week in ankle-depth water at 19 surfzone stations along Huntington Beach and Newport Beach, California, from 1998 to the end of 2003. These sampling periods span the time before and after treated sewage effluent, discharged into the coastal ocean from the local outfall, was disinfected. Bacterial samples were also taken in the vicinity of the outfall during the pre- and post-disinfection periods. Our analysis of the results from both data sets suggest that land-based sources, rather than the local outfall, were the source of the FIB responsible for the frequent closures and postings of local beaches in the summers of 2001 and 2002. Because the annual cycle is the dominant frequency in the fecal and total coliform data sets at most sampling stations, we infer that sources associated with local runoff were responsible for the majority of coliform contamination along wide stretches of the beach. The dominant fortnightly cycle in enterococci at many surfzone sampling stations suggests that the source for these relatively frequent bacteria contamination events in summer is related to the wetting and draining of the land due to the large tidal excursions found during spring tides. Along the most frequently closed section of the beach at stations 3N-15N, the fortnightly cycle is dominant in all FIBs. The strikingly different spatial and spectral patterns found in coliform and in enterococci suggest the presence of different sources, at least for large sections of beach. The presence of a relatively large enterococci fortnightly cycle along the beaches near Newport Harbor indicates that contamination sources similar to those found off Huntington Beach are present, though not at high enough levels to close the Newport beaches.

Fecal indicator bacteria concentrations measured in the surf zone off Huntington Beach, CA from July 1998-December 2001 were analyzed with respect to their spatial patterns along 23 km of beach, and temporal variability on time scales from hourly to fortnightly. The majority of samples had bacterial concentrations less than, or equal to, the minimum detection limit, but a small percentage exceeded the California recreational water standards. Areas where coliform bacteria exceeded standards were more prevalent north of the Santa Ana River, whereas enterococci exceedances covered a broad area both north and south of the river. Higher concentrations of bacteria were associated with spring tides. No temporal correspondence was found between these bacterial events and either the timing of cold water pulses near shore due to internal tides, or the presence of southerly swell in the surface wave field. All three fecal indicator bacteria exhibited a diel cycle, but enterococci rebounded to high nighttime values almost as soon as the sun went down, whereas coliform levels were highest near the nighttime low tide, which was also the lower low tide.

Two Canadian urban areas received travelers with severe acute respiratory syndrome (SARS) before the World Health Organization issued its alert. By July 2003, Vancouver had identified 5 cases (4 imported); Toronto reported 247 cases (3 imported) and 43 deaths. Baseline preparedness for pandemic threats may account for the absence of sustained transmission and fewer cases of SARS in Vancouver.

ISO 15190:2003 is a new standard developed by the International Organization for Standardization to address the safety aspects of medical laboratories. It is directly linked to the ISO 15189:2003, the standard for quality and competence. It provides a framework for a safety program based upon the principles of quality management, including designation of responsibilities and authorities, regular audits, and continuous improvement.

The interaction of nitric oxide with cytochrome P450 BM3 from Bacillus megaterium has been analyzed by spectroscopic techniques and enzyme assays. Nitric oxide ligates tightly to the ferric heme iron, inducing large changes in each of the main visible bands of the heme and inhibiting the fatty acid hydroxylase function of the protein. However, the ferrous adduct is unstable under aerobic conditions, and activity recovers rapidly after addition of NADPH to the flavocytochrome due to reduction of the heme via the reductase domain and displacement of the ligand. The visible spectral properties revert to that of the oxidized resting form. Aerobic reduction of the nitrosyl complex of the BM3 holoenzyme or heme domain by sodium dithionite also displaces the ligand. A single electron reduction destabilizes the ferric-nitrosyl complex such that nitric oxide is released directly, as shown by the trapping of released nitric oxide. Aerobically and in the absence of exogenous reductant, nitric oxide dissociates completely from the P450 over periods of several minutes. However, recovery of the nativelike visible spectrum is accompanied by alterations in the catalytic activity of the enzyme and changes in the resonance Raman spectrum. Specifically, resonance Raman spectroscopy identifies the presence of internally located nitrated tyrosine residue(s) following treatment with nitric oxide. Analysis of a Y51F mutant indicates that this is the major nitration target under these conditions. While wild-type P450 BM3 does not form an aerobically stable ferrous-nitrosyl complex, a site-directed mutant of P450 BM3 (F393H) does form an isolatable ferrous-nitrosyl complex, providing strong evidence for the role of this residue in controlling the electronic properties of the heme iron. We report here the spectroscopic characterization of the ferric- and ferrous-nitrosyl complexes of P450 BM3 and describe the use of resonance Raman spectroscopy to identify nitrated tyrosine residue(s) in the enzyme. Nitration of tyrosine in P450 BM3 may exemplify a typical mechanism by which the ubiquitous messenger molecule nitric oxide exerts a regulatory function over the cytochromes P450.

The cyp102A2 and cyp102A3 genes encoding the two Bacillus subtilis homologues (CYP102A2 and CYP102A3) of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium have been cloned, expressed in Escherichia coli, purified, and characterized spectroscopically and enzymologically. Both enzymes contain heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors and bind a variety of fatty acid molecules, as demonstrated by conversion of the low-spin resting form of the heme iron to the high-spin form induced by substrate-binding. CYP102A2 and CYP102A3 catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduction of artificial electron acceptors at high rates. Binding of carbon monoxide to the reduced forms of both enzymes results in the shift of the heme Soret band to 450 nm, confirming the P450 nature of the enzymes. Reverse-phase high-performance liquid chromatography (HPLC) of products from the reaction of the enzymes with myristic acid demonstrates that both catalyze the subterminal hydroxylation of this substrate, though with different regioselectivity and catalytic rate. Both P450s 102A2 and 102A3 show kinetic and binding preferences for long-chain unsaturated and branched-chain fatty acids over saturated fatty acids, indicating that the former two molecule types may be the true substrates. P450s 102A2 and 102A3 exhibit differing substrate selectivity profiles from each other and from P450 BM3, indicating that they may fulfill subtly different cellular roles. Titration curves for binding and turnover kinetics of several fatty acid substrates with P450s 102A2 and 102A3 are better described by sigmoidal (rather than hyperbolic) functions, suggesting binding of more than one molecule of substrate to the P450s, or possibly cooperativity in substrate binding. Comparison of the amino acid sequences of the three flavocytochromes shows that several important amino acids in P450 BM3 are not conserved in the B. subtilis homologues, pointing to differences in the binding modes for the substrates that may explain the unusual sigmoidal kinetic and titration properties.

Two sets of moorings were deployed along a cross-shelf transect in central Santa Monica bay for four months in the winter of 1998-1999. Both sites had an array of instruments attached to tripods set on the seafloor to monitor currents over the entire water column, surface waves, near-bed temperature, water clarity and suspended sediment. A companion mooring had temperature sensors spaced approximately 10 m apart to measure temperature profiles between the surface and the seafloor. One array was deployed in 70 m of water at a site adjacent to the shelf break, just northwest of a major ocean outfall. The other was deployed on the mid shelf in 35 m of water approximately 6 km from the shelf break site. The subtidal currents in the region flowed parallel to the isobaths with fluctuating time scales around 10 days, a typical coastal-ocean pattern. However, during the falling phase of the barotropic spring tide, sets of large-amplitude, sheared cross-shore current pulses with a duration of 2-5 h were observed at the shelf break site. Currents in these pulses flowed exclusively offshore in a thin layer near the bed with amplitudes reaching 30-40 cm/s. Simultaneously, currents with amplitudes around 15-20 cm/s flowed exclusively onshore in the thicker layer between the offshore flow layer and the sea surface. The net offshore transport was about half the onshore transport. Near-surface isotherms were depressed 30-40 m. These pulses were likely internal bores generated by tidal currents. Bed stresses associated with these events exceeded 3 dynes/cm(2). These amplitudes are large enough to resuspend and transport not only fine-grained material, but also medium to coarse sands from the shelf toward the slope. Consequently, the seafloor over the shelf break was swept clear of fine sediments. The data suggest that the internal bores dissipate and are reduced in amplitude as they propagate across this relatively narrow shelf. There is evidence that they reach the 35 m site, but other coastal ocean processes obscure their distinctive characteristics.

The acylation and deacylation stages of the hydrolysis of N -acetyl-Phe-Gly methyl thionoester catalysed by papain and actinidin were investigated by stopped-flow spectral analysis. Differences in the forms of pH-dependence of the steady-state and pre-steady-state kinetic parameters support the hypothesis that, whereas for papain, in accord with the traditional view, the rate-determining step is the base-catalysed reaction of the acyl-enzyme intermediate with water, for actinidin it is a post-acylation conformational change required to permit release of the alcohol product and its replacement in the catalytic site by the key water molecule. Possible assignments of the kinetically influential p K (a) values, guided by the results of modelling, including electrostatic-potential calculations, and of the mechanistic roles of the ionizing groups, are discussed. It is concluded that Asp(161) is the source of a key electrostatic modulator (p K (a) 5.0+/-0.1) in actinidin, analogous to Asp(158) in papain, whose influence is not detected kinetically; it is always in the 'on' state because of its low p K (a) value (2.8+/-0.06).

P450 BM3 and the nitric oxide synthases are related classes of flavocytochrome mono-oxygenase enzymes, containing NADPH-dependent FAD- and FMN-containing oxidoreductase modules fused to heme b-containing oxygenase domains. Domain-swap hybrids of these two multi-domain enzymes were created by genetic engineering of different segments of reductase and heme domains from neuronal nitric oxide synthase and P450 BM3, as a means of investigating the catalytic competence and substrate-binding properties of the fusions and the influence of tetrahydrpbiopterin and calmodulin binding regions on the electron transfer kinetics of the chimeras. Despite marked differences in hybrid stability and solubility, four catalytically functional chimeras were created that retained good reductase activity and which could be expressed successfully in Escherichia coli and purified. All of the BM3 reductase domain chimeras (chimeras I-III) exhibited inefficient flavin-to-heme inter-domain electron transfer, diminishing their oxygenase activity. However, the chimera containing the neuronal nitric oxide synthase reductase domain (chimera IV) showed good oxygenase domain activity, indicating that the flavin-to-heme electron transfer reaction is relatively efficient in this case. The data reinforce the importance of the nature of inter-domain linker constitution in multi-domain enzymes, and the difficulties posed in attempts to create chimeric enzymes with enhanced catalytic properties.

Post-transplant protocols for hepatitis B (HBV) prophylaxis using high-dose intravenous hepatitis B immune globulin (10,000 IU) with or without lamivudine are commonly reported. Our centre has previously reported a low-dose intramuscular (i.m.) protocol and lamivudine with excellent results. There have been, however, no pharmacokinetic studies of i.m. hepatitis B immune globulin (HBIG) in this setting. The objective of this study was to determine the pharmacokinetic profile of i.m. HBIG in long-term post-liver-transplant recipients to determine a rational dosing protocol. Six stable liver transplant recipients receiving monthly i.m. HBIG injections for greater than one year were enrolled in this study. All patients had no detectable HBV DNA levels. HBIG titers (anti-HBs) were measured predose, then three times weekly for four weeks and then twice weekly until the serum HBIG titers were 100 IU/L or less. The pharmacokinetic parameters were calculated using noncompartment methods. The mean time to maximum concentration was 10.5 d (range 4-20 d) and the mean half-life was 20 d (range 13.5-23.5 d). Based on these pharmacokinetic parameters in stable long-term post-transplant patients, a rational dosing protocol was developed that allows for more appropriate utility of HBIG and improved patient convenience.

The nitric oxide synthases (NOSs) are dimeric flavocytochromes consisting of an oxygenase domain with cytochrome P450-like Cys-ligated haem, coupled to a diflavin reductase domain, which is related to cytochrome P450 reductase. The NOSs catalyse the sequential mono-oxygenation of arginine to N-hydroxyarginine and then to citrulline and NO. The constitutive NOS isoforms (cNOSs) are regulated by calmodulin (CaM), which binds at elevated concentrations of free Ca(2+), whereas the inducible isoform binds CaM irreversibly. One of the main structural differences between the constitutive and inducible isoforms is an insert of 40-50 amino acids in the FMN-binding domain of the cNOSs. Deletion of the insert in rat neuronal NOS (nNOS) led to a mutant enzyme which binds CaM at lower Ca(2+) concentrations and which retains activity in the absence of CaM. In order to resolve the mechanism of action of CaM activation we determined reduction potentials for the FMN and FAD cofactors of rat nNOS in the presence and absence of CaM using a recombinant form of the reductase domain. The results indicate that CaM binding does not modulate the reduction potentials of the flavins, but appears to control electron transfer primarily via a large structural rearrangement. We also report the creation of chimaeric enzymes in which the reductase domains of nNOS and flavocytochrome P450 BM3 (Bacillus megaterium III) have been exchanged. Despite its very different flavin redox potentials, the BM3 reductase domain was able to support low levels of CaM-dependent NO synthesis, whereas the NOS reductase domain did not effectively substitute for that of cytochrome P450 BM3.

Midpoint reduction potentials for the flavin cofactors in human NADPH-cytochrome P450 oxidoreductase were determined by anaerobic redox titration of the diflavin (FAD and FMN) enzyme and by separate titrations of its isolated FAD/NADPH and FMN domains. Flavin reduction potentials are similar in the isolated domains (FAD domain E(1) [oxidized/semiquinone] = -286 +/- 6 mV, E(2) [semiquinone/reduced] = -371 +/- 7 mV; FMN domain E(1) = -43 +/- 7 mV, E(2) = -280 +/- 8 mV) and the soluble diflavin reductase (E(1) [FMN] = -66 +/- 8 mV, E(2) [FMN] = -269 +/- 10 mV; E(1) [FAD] = -283 +/- 5 mV, E(2) [FAD] = -382 +/- 8 mV). The lack of perturbation of the individual flavin potentials in the FAD and FMN domains indicates that the flavins are located in discrete environments and that these environments are not significantly disrupted by genetic dissection of the domains. Each flavin titrates through a blue semiquinone state, with the FMN semiquinone being most intense due to larger separation (approximately 200 mV) of its two couples. Both the FMN domain and the soluble reductase are purified in partially reduced, colored form from the Escherichia coli expression system, either as a green reductase or a gray-blue FMN domain. In both cases, large amounts of the higher potential FMN are in the semiquinone form. The redox properties of human cytochrome P450 reductase (CPR) are similar to those reported for rabbit CPR and the reductase domain of neuronal nitric oxide synthase. However, they differ markedly from those of yeast and bacterial CPRs, pointing to an important evolutionary difference in electronic regulation of these enzymes.

The cytochromes P-450 are an immensely important superfamily of heme-containing enzymes. They catalyze the monooxygenation of an enormous range of substrates. In bacteria, cytochromes P-450 are known to catalyze the hydroxylation of environmentally significant substrates such as camphor, phenolic compounds and many herbicides. In eukaryotes, these enzymes perform key roles in the synthesis and interconversion of steroids, while in mammals hepatic cytochromes P-450 are vital for the detoxification of many drugs. As such, the cytochromes P-450 are of considerable interest in medicine and biotechnology and are obvious targets for protein engineering. The purpose of this article is to illustrate the ways in which protein engineering has been used to investigate and modify the properties of cytochromes P-450. Illustrative examples include: the manipulation of substrate selectivity and regiospecificity, the alteration of membrane binding properties, and probing the route of electron transfer.

The structure of the Escherichia coli flavodoxin NADP(+) oxidoreductase (FLDR) places three arginines (R144, R174 and R184) in the proposed NADPH-binding site. Mutant enzymes produced by site-directed mutagenesis, in which each arginine was replaced by neutral alanine, were characterized. All mutants exhibited decreased NADPH-dependent cytochrome c reductase activity (R144A, 241.6 min(-1); R174A, 132.1 min(-1); R184A, 305.5 min(-1) versus wild type, 338.9 min(-1)) and increased K(m) for NADPH (R144A, 5.3 microM; R174A, 20.2 microM; R184A, 54.4 microM versus wild type, 3.9 microM). The k(cat) value for NADH-dependent cytochrome c reduction was increased for R174A (42.3 min(-1)) and R184A (50.4 min(-1)) compared with the wild type (33.0 min(-1)), consistent with roles for R174 and R184 in discriminating between NADPH/NADH by interaction with the adenosine ribose 2'-phosphate. Stopped-flow studies indicated that affinity (K(d)) for NADPH was markedly reduced in mutants R144A (635 microM) and R184A (2.3 mM) compared with the wild type (<5 microM). Mutant R184A displays the greatest change in pyridine nucleotide preference, with the NADH/NADPH K(d) ratio >175-fold lower than for wild-type FLDR. The rate constant for hydride transfer from NADPH to flavin was lowest for R174A (k(red)=8.82 s(-1) versus 22.63 s(-1) for the wild type), which also exhibited tertiary structure perturbation, as evidenced by alterations in CD and fluorescence spectra. Molecular modelling indicated that movement of the C-terminal tryptophan (W248) of FLDR is necessary to permit close approach of the nicotinamide ring of NADPH to the flavin. The positions of NADPH phosphates in the modelled structure are consistent with the kinetic data, with R174 and R184 located close to the adenosine ribose 2'-phosphate group, and R144 likely to interact with the nicotinamide ribose 5'-phosphate group.