A comparison of results from antihuman globulin-graded reactions with the monocyte monolayer assay.

Blood transfusions are a common medical treatment. Risks arise when compatible blood is not available. This study assesses the correlation between antibody reaction strength at the antihuman globulin (AHG) phase of testing and the antibody clinical significance as predicted using the monocyte monolayer assay (MMA). Multiple examples of anti-K donor plasma samples were selected to sensitize K+k+ red blood cells (RBCs). Reactivity was confirmed by testing the sensitized K+k+ RBCs at saline-AHG. Antibody titers were determined by serial dilution using neat plasma. Sixteen samples were selected for the study based on comparable graded reactions with neat plasma (1+, 2+, 3+, and 4+) and similar titration endpoints. Each sample was used to sensitize the same Kk donor and then tested by monocytes to evaluate the clinical significance using the MMA, an in vitro procedure that mimics in vivo extravascular hemolysis to predict the survivability of incompatible transfused RBCs. The monocyte index (MI), i.e., the percentage of RBCs adhered, ingested, or both versus free monocytes, was calculated for each sample. Regardless of the reaction strength, all examples of anti-K were predicted to be clinically significant. While anti-K is known to be clinically significant, the immunogenicity rate of K ensures ample supply of antibody samples for inclusion in this project. This study demonstrates that in vitro antibody strength is highly subjective and variable. These results show no correlation between graded reaction strength at AHG and the predicted clinical significance of an antibody as assessed using the MMA.

Contrasting Regional Carbon Cycle Responses to Seasonal Climate Anomalies Across the East-West Divide of Temperate North America.

Across temperate North America, interannual variability (IAV) in gross primary production (GPP) and net ecosystem exchange (NEE) and their relationship with environmental drivers are poorly understood. Here, we examine IAV in GPP and NEE and their relationship to environmental drivers using two state-of-the-science flux products: NEE constrained by surface and space-based atmospheric CO2 measurements over 2010-2015 and satellite up-scaled GPP from FluxSat over 2001-2017. We show that the arid western half of temperate North America provides a larger contribution to IAV in GPP (104% of east) and NEE (127% of east) than the eastern half, in spite of smaller magnitude of annual mean GPP and NEE. This occurs because anomalies in western ecosystems are temporally coherent across the growing season leading to an amplification of GPP and NEE. In contrast, IAV in GPP and NEE in eastern ecosystems is dominated by seasonal compensation effects, associated with opposite responses to temperature anomalies in spring and summer. Terrestrial biosphere models in the MsTMIP ensemble generally capture these differences between eastern and western temperate North America, although there is considerable spread between models.

Air Quality Response in China Linked to the 2019 Novel Coronavirus (COVID-19) Lockdown.

Efforts to stem the spread of COVID-19 in China hinged on severe restrictions to human movement starting 23 January 2020 in Wuhan and subsequently to other provinces. Here, we quantify the ancillary impacts on air pollution and human health using inverse emissions estimates based on multiple satellite observations. We find that Chinese NOx emissions were reduced by 36% from early January to mid-February, with more than 80% of reductions occurring after their respective lockdown in most provinces. The reduced precursor emissions increased surface ozone by up to 16 ppb over northern China but decreased PM2.5 by up to 23 µg m-3 nationwide. Changes in human exposure are associated with about 2,100 more ozone-related and at least 60,000 fewer PM2.5-related morbidity incidences, primarily from asthma cases, thereby augmenting efforts to reduce hospital admissions and alleviate negative impacts from potential delayed treatments.

Balance of Emission and Dynamical Controls on Ozone During the Korea-United States Air Quality Campaign From Multiconstituent Satellite Data Assimilation.

Global multiconstituent concentration and emission fields obtained from the assimilation of the satellite retrievals of ozone, CO, NO2, HNO3, and SO2 from the Ozone Monitoring Instrument (OMI), Global Ozone Monitoring Experiment 2, Measurements of Pollution in the Troposphere, Microwave Limb Sounder, and Atmospheric Infrared Sounder (AIRS)/OMI are used to understand the processes controlling air pollution during the Korea-United States Air Quality (KORUS-AQ) campaign. Estimated emissions in South Korea were 0.42 Tg N for NO x and 1.1 Tg CO for CO, which were 40% and 83% higher, respectively, than the a priori bottom-up inventories, and increased mean ozone concentration by up to 7.5 ± 1.6 ppbv. The observed boundary layer ozone exceeded 90 ppbv over Seoul under stagnant phases, whereas it was approximately 60 ppbv during dynamical conditions given equivalent emissions. Chemical reanalysis showed that mean ozone concentration was persistently higher over Seoul (75.10 ± 7.6 ppbv) than the broader KORUS-AQ domain (70.5 ± 9.2 ppbv) at 700 hPa. Large bias reductions (>75%) in the free tropospheric OH show that multiple-species assimilation is critical for balanced tropospheric chemistry analysis and emissions. The assimilation performance was dependent on the particular phase. While the evaluation of data assimilation fields shows an improved agreement with aircraft measurements in ozone (to less than 5 ppbv biases), CO, NO2, SO2, PAN, and OH profiles, lower tropospheric ozone analysis error was largest at stagnant conditions, whereas the model errors were mostly removed by data assimilation under dynamic weather conditions. Assimilation of new AIRS/OMI ozone profiles allowed for additional error reductions, especially under dynamic weather conditions. Our results show the important balance of dynamics and emissions both on pollution and the chemical assimilation system performance.

Fractionated radiation exposure amplifies the radioresistant nature of prostate cancer cells.

The risk of recurrence following radiation therapy remains high for a significant number of prostate cancer patients. The development of in vitro isogenic models of radioresistance through exposure to fractionated radiation is an increasingly used approach to investigate the mechanisms of radioresistance in cancer cells and help guide improvements in radiotherapy standards. We treated 22Rv1 prostate cancer cells with fractionated 2 Gy radiation to a cumulative total dose of 60 Gy. This process selected for 22Rv1-cells with increased clonogenic survival following subsequent radiation exposure but increased sensitivity to Docetaxel. This RR-22Rv1 cell line was enriched in S-phase cells, less susceptible to DNA damage, radiation-induced apoptosis and acquired enhanced migration potential, when compared to wild type and aged matched control 22Rv1 cells. The selection of radioresistant cancer cells during fractionated radiation therapy may have implications in the development and administration of future targeted therapy in conjunction with radiation therapy.

Carbonic anhydrase IX expression and outcome after radiotherapy for muscle-invasive bladder cancer.

AIMS: Carbonic anhydrase IX (CA IX) expression has been described as an endogenous marker of hypoxia in solid neoplasms. Furthermore, CA IX expression has been associated with an aggressive phenotype and resistance to radiotherapy. We assessed the prognostic significance of CA IX expression in patients with muscle-invasive bladder cancer treated with radiotherapy. MATERIALS AND METHODS: A standard immunohistochemistry technique was used to show CA IX expression in 110 muscle-invasive bladder tumours treated with radiotherapy. Clinicopathological data were obtained from medical case notes. RESULTS: CA IX immunostaining was detected in 89 ( approximately 81%) patients. Staining was predominantly membranous, with areas of concurrent cytoplasmic and nuclear staining and was abundant in luminal and perinecrotic areas. No significant correlation was shown between the overall CA IX status and the initial response to radiotherapy, 5-year bladder cancer-specific survival or the time to local recurrence. CONCLUSIONS: The distribution of CA IX expression in paraffin-embedded tissue sections seen in this series is consistent with previous studies in bladder cancer, but does not provide significant prognostic information with respect to the response to radiotherapy at 3 months and disease-specific survival after radical radiotherapy.

Measurements using the alkaline comet assay predict bladder cancer cell radiosensitivity.

In the UK, the two main treatments of invasive bladder cancer are radiotherapy or cystectomy. However, approximately 50% of patients undergoing radiotherapy fail to respond. If tumour radiosensitivity could be predicted in advance, it may be possible to improve control rates significantly by selecting for radiotherapy those patients whose tumours are radiosensitive. Additionally, patients who would benefit from surgery would be identified earlier. The alkaline comet assay (ACA) is a sensitive method for the detection of DNA strand break damage in cells. In the present study, using six bladder cancer cell lines of differing radiosensitivities, cell survival was compared to the manifestation of radiogenic DNA damage as assessed by ACA. For all the cell lines, the extent of comet formation strongly correlates with cell killing (R2>0.96), with a greater response being noted in radiosensitive cells. In repair studies, measures of residual damage correlate with survival fraction at 2 Gy (R2>0.96), but for only five of the cell lines. Finally, cells from human bladder tumour biopsies reveal a wide range of predicted radiosensitivies as determined by ACA. Overall, these studies demonstrate ACA to be a good predictive measure of bladder cancer cell radiosensitivity at low dose, with potential clinical application.

Characterization of nuclease-resistant ribozymes directed against hepatitis B virus RNA.

Hepatitis B virus (HBV) is responsible for > 350 million cases of chronic hepatitis B worldwide and 1.2 million deaths each year. To explore the use of ribozymes as a novel therapy for HBV infection, nuclease-resistant ribozymes that target highly conserved regions of HBV RNA were screened in cell culture. These synthetic ribozymes have the potential to cleave all four major HBV RNA transcripts and to block the HBV lifecycle by cleavage of the pregenomic RNA. A number of the screened ribozymes demonstrate activity in cell culture systems, as measured by decreased levels of HBV surface antigen, HBV e antigen and HBV DNA. In addition, a lead anti-HBV ribozyme maintains activity against a lamivudine-resistant HBV variant in cell culture. Treatment of HBV transgenic mice with lead anti-HBV ribozymes significantly reduced viraemia compared with saline-treated animals and was as effective as treatment with lamivudine. In conclusion, the therapeutic use of a ribozyme alone or in combination with current therapies (lamivudine or interferons) may lead to improved HBV therapy.

Evaluation of phosphodiesterase I-based protocols for the detection of multiply damaged sites in DNA: the detection of abasic, oxidative and alkylative tandem damage in DNA oligonucleotides.

It has been proposed that DNA multiply damaged sites (MDS), where more than one moiety in a local region ( approximately 1 helical turn, 10 bp) of the DNA is damaged, are lesions of enhanced biological significance. However, other than indirect measures, there are few analytical techniques that allow direct detection of MDS in DNA. In the present study we demonstrate the potential of protocols incorporating an exonucleolytic snake venom phosphodiesterase (SVPD) digestion stage to permit the direct detection of certain tandem damage, in which two lesions are immediately adjacent to each other on the same DNA strand. A series of prepared oligonucleotides containing either single or pairs of tetrahydrofuran moieties (F), thymine glycol lesions (T(g)) or methylphosphotriester adducts (Me-PTE) were digested with SVPD and the digests examined by either (32)P-end-labelling or electrospray mass spectrometry. The unambiguous observation of SVPD-resistant 'trimer' species in the digests of oligonucleotides containing adjacent F, T(g) and Me-PTE demonstrates that the SVPD digestion strategy is capable of allowing direct detection of certain tandem damage. Furthermore, in studies to determine the specificity of SVPD in dealing with pairs of lesions on the same strand, it was found mandatory to have the two lesions immediately adjacent to each other in order to generate the trimer species; pairs of lesions separated by as few as one or two normal nucleotides behave principally as single lesions towards SVPD.

Chinese seniors' perspectives on end-of-life decisions.

Making end-of-life decisions is a painful and difficult process; one that can be intensified by cultural differences between physicians and their patients. The objective of this study was to examine attitudes of Chinese seniors towards end-of-life decisions. We conducted a qualitative survey in a Chinese community centre in Toronto, Canada. Face-to-face interviews, in Cantonese, were conducted with 40 Chinese seniors 65 years of age or older. Respondents based their end-of-life decision making on the following factors: hope, suffering and burden, the future, emotional harmony, the life cycle, respect for doctors, and the family. Respondents rejected advance directives. Respondents' attitudes toward end-of-life decision making can be understood through the lens of values from Confucianist, Buddhist and Taoist traditions. Health care workers can best achieve quality end-of-life care--and address the cultural differences that may arise--by focusing primarily on understanding the perspectives of patients and their families, and by continually striving for balanced and open communication at all stages of the caregiving process.

Biosynthesis of L-selectin ligands: sulfation of sialyl Lewis x-related oligosaccharides by a family of GlcNAc-6-sulfotransferases.

The leukocyte adhesion molecule L-selectin mediates lymphocyte homing to secondary lymphoid organs and to certain sites of inflammation. The cognate ligands for L-selectin possess the unusual sulfated tetrasaccharide epitope 6-sulfo sialyl Lewis x (Siaalpha2-->3Galbeta1-->4[Fucalpha1-->3][SO(3)-->6]GlcNAc). Sulfation of GlcNAc within sialyl Lewis x is a crucial modification for L-selectin binding, and thus, the underlying sulfotransferase may be a key modulator of lymphocyte trafficking. Four recently discovered GlcNAc-6-sulfotransferases are the first candidate contributors to the biosynthesis of 6-sulfo sLex in the context of L-selectin ligands. Here we report the in vitro activity of the four GlcNAc-6-sulfotransferases on a panel of synthetic oligosaccharide substrates that comprise structural motifs derived from sialyl Lewis x. Each enzyme preferred a terminal GlcNAc residue, and was impeded by the addition of a beta1,4-linked Gal residue (i.e., terminal LacNAc). Surprisingly, for three of the enzymes, significant activity was observed with sialylated LacNAc, and two of the enzymes were capable of detectable sulfation of GlcNAc in the context of sialyl Lewis x. On the basis of these results, we propose possible pathways for 6-sulfo sialyl Lewis x biosynthesis and suggest that sulfation may be an early committed step.

Hospital policy on appropriate use of life-sustaining treatment. University of Toronto Joint Centre for Bioethics/Critical Care Medicine Program Task Force.

OBJECTIVE: To describe the issues faced, and how they were addressed, by the University of Toronto Critical Care Medicine Program/Joint Centre for Bioethics Task Force on Appropriate Use of Life-Sustaining Treatment. The clinical problem addressed by the Task Force was dealing with requests by patients or substitute decision makers for life-sustaining treatment that their healthcare providers believe is inappropriate. DESIGN: Case study. SETTING: The University of Toronto Joint Centre for Bioethics/Critical Care Medicine Program Task Force on Appropriate Use of Life-Sustaining Treatment. PARTICIPANTS: The 24-member Task Force included physician and nursing leaders from five critical care units, bioethicists, a legal scholar, a health administration expert, a social worker, and a hospital public relations professional. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Our specific lessons learned include a) a policy focus on process; b) use of a negotiation and mediation model, rather than a hospital ethics committee model, for this process; and c) the policy development process is itself a negotiation, so we recommend equal involvement of interested groups including patients, families, and the public. CONCLUSIONS: This article describes the key issues faced by the Task Force while developing its policy. It will provide a useful starting point for other groups developing policy on appropriate use of life-sustaining treatment.

Differential effects of the poly (ADP-ribose) polymerase (PARP) inhibitor NU1025 on topoisomerase I and II inhibitor cytotoxicity in L1210 cells in vitro.

The potent novel poly(ADP-ribose) polymerase (PARP) inhibitor, NU1025, enhances the cytotoxicity of DNA-methylating agents and ionizing radiation by inhibiting DNA repair. We report here an investigation of the role of PARP in the cellular responses to inhibitors of topoisomerase I and II using NU1025. The cytotoxicity of the topoisomerase I inhibitor, camptothecin, was increased 2.6-fold in L1210 cells by co-incubation with NU1025. Camptothecin-induced DNA strand breaks were also increased 2.5-fold by NU1025 and exposure to camptothecin-activated PARP. In contrast, NU1025 did not increase the DNA strand breakage or cytotoxicity caused by the topoisomerase II inhibitor etoposide. Exposure to etoposide did not activate PARP even at concentrations that caused significant levels of apoptosis. Taken together, these data suggest that potentiation of camptothecin cytotoxicity by NU1025 is a direct result of increased DNA strand breakage, and that activation of PARP by camptothecin-induced DNA damage contributes to its repair and consequently cell survival. However, in L1210 cells at least, it would appear that PARP is not involved in the cellular response to etoposide-mediated DNA damage. On the basis of these data, PARP inhibitors may be potentially useful in combination with topoisomerase I inhibitor anticancer chemotherapy.

Communication, negotiation, and mediation: dealing with conflict in end-of-life decisions.

In recent years, it has become possible for the end of life to be a negotiated event, particularly in the intensive care unit. A multitude of often unidentified and poorly understood factors affect such negotiations. These include, family dynamics, ever-changing health care teams, inconsistent opinions about prognosis, and cultural differences between physicians, and patients and their families. When these factors converge, conflict may erupt. This article explores the nature, antecedents, and cost of such conflict. Arguments for the importance of balanced communication, negotiation, and mediation in end-of-life care are put forward.

A randomized comparison of all-polyethylene and metal-backed tibial components.

Clinical failures of all-polyethylene tibial components in total knee arthroplasty generally have been failures of design, not materials. The current study was designed to compare a modern congruent all-polyethylene tibial component with a metal-backed tibial component with the same articular design and geometry. All patients older than 60 years of age requiring total knee arthroplasty were randomized prospectively to receive either a cemented posterior cruciate ligament-retaining all-polyethylene component or a metal-backed tibial component with identical articular surfaces. All patients received identical cemented femoral and all-polyethylene patellar implants. The mean age of the patients was 69 years, and the mean American Society of Anesthesiology score was 3. The diagnosis was osteoarthritis in 92% of this population. Three hundred twenty-four total knee arthroplasties in 296 patients were performed; 213 joints (111 all-polyethylene tibias and 102 metal-backed tibias) with a minimum of 3 years followup (mean, 49 months) are reported. The preoperative Knee Society knee score in the group of patients who received an all-polyethylene tibial component was 38 points, improving to 84 points at latest followup, whereas in the group of patients who received a metal-backed tibial component, the score improved from 35 to 85 points. Functional scores increased from preoperative values of 56 to 74 points in the patients who received all-polyethylene tibial components, and 57 to 72 points in the patients who received metal-backed tibial components. Range of motion measured at latest followup averaged 106 degrees in patients who received an all-polyethylene tibial component and 107 degrees in the patients who received a metal-backed component, and postoperative tibiofemoral alignment averaged 6 degrees valgus for both groups. There were 13 reoperations for instability, patellofemoral problems, or deep infection, but none for aseptic loosening or wear in either group. These differences were not statistically significant, nor were any measures of patient satisfaction or clinical outcome between the two groups in this period. Total knee arthroplasty with a well-designed, contemporary congruent all-polyethylene tibial component functions equivalently to its metal-backed tibial counterpart at 3- to 5-year followup in this patient population, and is less costly ($675).

Expression, purification, and characterization of deglycosylated human pro-prostate-specific antigen.

Wild-type and deglycosylated forms of human prostate-specific antigen were expressed in Chinese hamster ovary (CHO) cells as zymogens. ProPSA was collected from conditioned medium and purified using a single cation-exchange chromatographic step for the deglycosylated form and cation-exchange followed by gel filtration chromatography for the wild-type form. Recombinant wild-type proPSA produced in CHO cells has an average MW of 34.5 kDa, whereas the deglycosylated proPSA has a MW of 32.4 kDa. Both forms of proPSA were activated in vitro and the kinetic properties measured for the deglycosylated PSA are very similar to those of the wild-type recombinant PSA and the native PSA isolated from seminal fluid. These results suggest that deglycosylated PSA is likely to be very similar to native PSA with respect to its three-dimensional structure and will provide a homogeneous protein preparation necessary for X-ray crystallographic analysis.

Crystal structure of human cathepsin V.

Cathepsin V is a lysosomal cysteine protease that is expressed in the thymus, testis and corneal epithelium. We have determined the 1.6 A resolution crystal structure of human cathepsin V associated with an irreversible vinyl sulfone inhibitor. The fold of this enzyme is similar to the fold adopted by other members of the papain superfamily of cysteine proteases. This study provides a framework for understanding the structural basis for cathepsin V's activity and will aid in the design of inhibitors of this enzyme. A comparison of cathepsin V's active site with the active sites of related proteases revealed a number of differences, especially in the S2 and S3 subsites, that could be exploited in identifying specific cathepsin V inhibitors or in identifying inhibitors of other cysteine proteases that would be selective against cathepsin V.