In Vitro Investigation of Crosstalk between Fatty Acid and Polyketide Synthases in the Andrimid Biosynthetic Assembly Line.
Andrimid (Adm) synthase, which belongs to the type II system of enzymes, produces Adm in Pantoea agglomerans. The adm biosynthetic gene cluster lacks canonical acyltransferases (ATs) to load the malonyl group to acyl carrier proteins (ACPs), thus suggesting that a malonyl-CoA ACP transacylase (MCAT) from the fatty acid synthase (FAS) complex provides the essential AT activity in Adm biosynthesis. Here we report that an MCAT is essential for catalysis of the transacylation of malonate from malonyl-CoA to AdmA polyketide synthase (PKS) ACP in vitro. Catalytic self-malonylation of AdmA (PKS ACP) was not observed in reactions without MCAT, although many type II PKS ACPs are capable of catalyzing self-acylation. This lack of self-malonylation was explained by amino acid sequence analysis of the AdmA PKS ACP and the type II PKS ACPs. The results show that MCAT from the organism's FAS complex can provide the missing AT activity in trans, thus suggesting a protein-protein interaction between the fatty acid and polyketide synthases in the Adm assembly line.


Simplified protocol for pulsed-field gel electrophoresis analysis of Streptococcus pneumoniae.
A variety of pulsed-field gel electrophoresis (PFGE) protocols for the molecular subtyping of Streptococcus pneumoniae have been reported; most are time-consuming and complex. We sought to modify reference PFGE protocols to reduce the time required while creating high-quality gels. Only protocol modifications that resulted in high-quality banding patterns were considered. The following protocol components were modified. Lysis enzymes (lysozyme, mutanolysin, and RNase A) were deleted in a stepwise fashion, and then the lysis buffer was deleted. Lysis and digestion were accomplished in a single step with EDTA and N-lauroyl sarcosine (ES; pH 8.5 to 9.3) incubation at 50 degrees C in the absence of proteinase K. All enzymes except the restriction enzyme were omitted. A minimum incubation time of 6 h was required to achieve high-quality gels. All of the reactions were performed within 9 h, and the total protocol time from lysis to gel completion was reduced from 3 days to only 36 h. Combining lysis and digestion into a single step resulted in a substantial reduction in the time required to perform PFGE for S. pneumoniae. The ES solution may have caused cell lysis by activating N-acetylmuramyl-L-alanine amidase, the pneumococcal autolysin.


Growth of plants in solution culture containing low levels of chromium.
Chromium was not required for normal growth of romaine lettuce (Lactuca sativa L. subsp. longifolia), tomato (Lycopersicon esculentum Mill.), wheat (Triticum aestivum L.), or bean (Phaseolus vulgaris L.) in solution culture containing 3.8 x 10(-4) muM Cr. Plants grown on this purified nutrient solution contained an average of 22 ng Cr/g dry weight. Duckweed (Lemna sp.) grew and reproduced normally on a dilute nutrient solution containing 3.8 x 10(-5) muM Cr.


Modeling the importance of biota and black carbon as vectors of polybrominated diphenyl ethers (PBDEs) in the Baltic Sea ecosystem.
The POPCYCLING-Baltic model, a nonsteady state spatially resolved mass balance model of chemical transport in the Baltic Sea environment was modified to include black carbon (BC), dissolved organic carbon (DOC) and food-web bioaccumulation. The importance of these modifications to the transport of PBDE congeners BDE-47, -99, -153, and -209 was assessed by comparing time-series simulated with and without black carbon and biota between 1970 and 2005. Inclusion of black carbon improved the model fit to measurements from air, soil, and biota, and had a major effect on the mass balance. Modeled bulk concentrations of PBDEs in sediments and soils increased by a factor of 3 while concentrations in biota decreased by a factor of 2-5. Black carbon also doubled the recovery time of the system due to the limited availability of PBDEs for degradation. In comparison, the inclusion of biota had only a minor effect on the overall mass balance and recovery times. The modified model is constructed as a flexible matrix and can also be applied to persistent organic pollutants in other ecosystems besides the Baltic Sea.


Ethanol feeding alters death signaling in the pancreas.
Alcohol abuse is a major cause of pancreatitis, which is associated with death of parenchymal cells. The goal of this study was to explore the effects of ethanol on cell death pathways in the pancreas. Adult male Wistar rats were fed with ethanol diets using the Lieber-DeCarli method. Caspase-8, caspase-3, and cathepsin B expression and activity in the pancreas of these animals as well as the signals that regulate their expression were studied using Western blot analysis and specific assays for biochemical enzyme activity. In the pancreas from rats fed with ethanol, the protein expression and activity of caspase-8 decreased by 48% and 45%, respectively, and caspase-3 activity decreased by 39%. In contrast, cathepsin B protein expression and activity increased with ethanol feeding by 189% and 143%, respectively. Evaluation of the transcriptional regulatory system for caspase-8 and cathepsin B showed that the ethanol effects on these pathways were largely transcriptional. Our findings show effects of ethanol on the expression of several signals involved in cell death in the pancreas through alteration of transcriptional regulators. The decrease in caspase expression and increase in cathepsin B expression indicate that ethanol feeding may prevent apoptosis and promote necrosis of pancreatic tissue with stresses that cause pancreatitis.


Brief overview: cemiplimab for the treatment of advanced basal cell carcinoma: PD-1 strikes again.
Basal cell carcinoma (BCC) is the most common malignancy worldwide. Fortunately, most tumors are localized and easily amenable to surgical resection or locally destructive treatments. However, a subset of BCCs can become locally advanced or metastatic. The development of small-molecule inhibitors of smoothened, a protein in the hedgehog pathway, which is almost universally activated in BCCs, was a breakthrough in the treatment of patients with advanced BCC. However, these agents are associated with primary and secondary resistance and have a toxicity profile that makes long-term use difficult. The recent approval of cemiplimab for patients with advanced BCC who are resistant to or are intolerant of hedgehog inhibitor therapy fills a significant unmet need as these patients now have a viable, second-line systemic therapeutic option. This article summarizes the rationale and data leading to the approval for cemiplimab in advanced BCC.


[Interaction of rabbit IgG with anti-IgG: localization of epitopes and absence of immunoreactivity of the pFc'-fragment].
To localize essential epitopes of rabbit IgG, a series of proteolytic IgG fragments obtained by papain (Fab, Fc) or pepsin (pFc', F(ab')2) proteolysis have been prepared and their interaction with sheep antibodies against rabbit IgG has been studied. The data obtained suggest that essential immunoreactive epitopes of rabbit IgG are located in the CH2 domain and hinge region. This finding is in line with the results obtained by computing the antigenic sites of immunoglobulins. However, the deviation from the computed antigenic structure was deduced from the complete lack of immunoreactivity of the pFc fragment, it being a dimer of the terminal CH3 domain of the Fc fragment. The hinge region comparable in size with the dimensions of the epitope reveals high affinity binding to anti-IgG, thus testifying to the localization of the expressed epitope or its essential part in the hinge region. Proteolytic cleavage of this region leads to a significant decrease in the binding of the IgG fragment to anti-IgG. In addition to the CH2 domain and hinge region, a relatively low interaction of the antigen-binding antibody fragments with anti-IgG was found.


Dietary intake and nutritional status of Turkish pregnant women during Ramadan.
To determine the effects of Ramadan fasting on dietary intakes, nutritional status and biochemical parameters of Turkish pregnant women. We carried out this study at Dr. Zekai Tahir Burak Women's Health Care and Research Hospital, Ankara, Turkey from October 15th to November 13th 2004. Forty-nine fasting group and 49 non-fasting group (control group) voluntarily enrolled for the study. We analyzed the blood biochemical parameters of pregnant women and obtained the dietary intakes from the groups. Compared to the control group, weight gain and energy intake (p<0.05 for second and third trimesters) was less in the fasting group. The percentage of protein (p<0.05 for first and second trimesters) and carbohydrates (p<0.05 for all trimesters) from total energy was higher in the fasting group than in the control group. We noticed a slight increase in the fasting blood glucose, serum total cholesterol high-density lipoprotein-cholesterol and triglycerides (p<0.05 for first trimester) concentrations in the fasting group during Ramadan. However, we found decreased levels in the plasma urea, total cholesterol, triglycerides, low-density lipoprotein-cholesterol and total protein and albumin levels of the fasting group in comparison with the control group. Dietary mineral and vitamin intakes were lower than the recommended daily allowance, except vitamin A and vitamin C in both groups. Based on the results of the present study, Ramadan fasting had no significant adverse effect on the health of pregnant women.


[Effect of ketalar on the hypophyseo-adrenal system and glucose-insular homeostasis in children with malignant neoplasms].
The dynamics of content of the adreno-corticotropic and somatotropic hormones, cortisol , insulin and glucose in 30 children with malignant tumors operated upon under conditions of Ketalar anesthesia was analyzed. Direct influence of Ketalar upon the hypophysis-adrenal system and glucose-insular homeostasis was established. A possibility to prevent these changes by means of inclusion of Seduxen in the Ketalar anesthesia scheme was found.


Electric-field assisted immobilization and hybridization of DNA oligomers on thin-film microchips.
Single, square voltage pulses in the microsecond timescale result in selective 5'-end covalent bonding (immobilization) of thiolated single-stranded (ss) DNA probes to a modified silicon dioxide flat surface and in specific hybridization of ssDNA targets to the immobilized probe. Immobilization and hybridization rates using microsecond voltage pulses at or below 1 V are at least 10(8) times faster than in the passive control reactions performed without electric field (E), and can be achieved with at least three differently functionalized thin-film surfaces on plastic or glass substrates. The systematic study of the effect of DNA probe and target concentrations, of DNA probe and target length, and the application of asymmetric pulses on E-assisted DNA immobilization and hybridization showed that: (1) the rapidly rising edge of the pulse is most critical to the E-assisted processes, but the duration of the pulse is also important; (2) E-assisted immobilization and hybridization can be performed with micrometre-sized pixels, proving the potential for use on microelectronic length scales, and the applied voltage can be scaled down together with the electrode spacing to as low as 25 mV; and (3) longer DNA chains reduce the yield in the E-assisted immobilization and hybridization because the density of physisorbed single-stranded DNA is reduced. The results show that the E-induced reactions can be used as a general method in DNA microarrays to produce high-density DNA chips (E-immobilization) and speed the microarray-based analysis (E-hybridization).


Reconstitution of a Kv channel into lipid membranes for structural and functional studies.
To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.


[Current problems in prophylaxis and treatment of acute decompression disease of mild degree].
The authors discuss the causes leading to the rise of acute decompression illness in the amateur skin-divers. They include the insufficient training of most skin-drivers in the basis of diving physiology and medicine, the underwater dipping without consideration of individual sensitivity to decompression illness and nitrogen narcotic action, disorders in decompression regimens, non-observance of behavior rules after dipping, use of air transport immediately after dipping. The case of delayed treatment of decompression illness is described. The data concerning the possibility of chronic decompression illness formation due to the action of asymptomatic decompression gasformation and acute decompression illness of mild degree are presented. The authors propose the system of measures that would help to decrease the incidence of disease and to make the treatment of decompression illness more effective.


[Microwave assisted UV electrodeless discharge lamp photochemical degradation of 4-chlorophenol in aquatic solutions].
A microwave assisted UV electrodeless discharge lamp system (MW/UV) was used for photo-degradation of 4CP simulated wastewater. In order to evaluate the degradation efficiency of 4CP, UV spectrophotometry and ion chromatography were used for determination of 4CP and Cl- respectively. The degradation rate in MW/UV system was higher than that in the UV system within 120min, which were 52.40% and 21.56% respectively. The degradation efficiency was improved by increasing pH value of the solution, aerating O2 gas, enhancing light intensity, or adding H2O2 oxidant. The degradation of 4CP under MW/UV accords with the first order kinetics equation.


Conserved elements associated with ribosomal genes and their trans-splice acceptor sites in Caenorhabditis elegans.
The recent publication of the Caenorhabditis elegans cisRED database has provided an extensive catalog of upstream elements that are conserved between nematode genomes. We have performed a secondary analysis to determine which subsequences of the cisRED motifs are found in multiple locations throughout the C. elegans genome. We used the word-counting motif discovery algorithm DME to form the motifs into groups based on sequence similarity. We then examined the genes associated with each motif group using DAVID and Ontologizer to determine which groups are associated with genes that also have significant functional associations in the Gene Ontology and other gene annotation sources. Of the 3265 motif groups formed, 612 (19%) had significant functional associations with respect to GO terms. Eight of the first 20 motif groups based on frequent dodecamers among the cisRED motif sequences were specifically associated with ribosomal protein genes; two of these were similar to mouse EBP-45, rat HNF3-family and Drosophila Zeste transcription factor binding sites. Additionally, seven motif groups were extensions of the canonical C. elegans trans-splice acceptor site. One motif group was tested for regulatory function in a series of green fluorescent protein expression experiments and was shown to be involved in pharyngeal expression.


[Acute Mastoiditis in Children: a series of 38 Cases].
We reviewed retrospectively the clinical, radiological and therapeutic findings in 38 infants and children with acute mastoiditis. From 1988 to 2001, 38 children (mean age 36 months) were treated for acute mastoiditis and periostitis or retroauricular abscess. Diagnosis was usually made on the basis of typical retroauricular signs (60% of the cases), but was sometimes delayed owing to inappropriate antibiotics in 16 cases (42%) or atypical presentation in 3 (8%). Twenty-four children (70%) had no otolaryngological history. Pneumococci were isolated in most of the cases (n=15, 57%) with 13% having penicillin-resistant pneumococci. Lateral sinus thrombophlebitis was the most frequent complication, observed in 3 children (8%). Mastoiditis revealed congenital or acquired cholesteatoma in 4 children, who were all four infected with Gram-negative bacteria. Intravenous antibiotic therapy was associated with antroatticotomy in 36 children. Despite emergence of new antibiotic resistance, the annual rate of mastoiditis has remained unchanged, remaining a serious complication of middle ear acute otitis. Cholesteatoma should be suspected in case of Gram-negative bacteria or in older children.


Serum protein polymorphisms in a Liberian population.
Serum protein variations were studied in a Liberian population living in Buchanan town. Of the alpha 1-antitrypsin genes only M1 and M3 were polymorphic. The frequencies of the haptoglobin and Gc genes were in accordance with earlier known estimates in African populations. There was, however, a relatively low frequency of Hp 0 which may be related to the low malarial parasite prevalence in this group. The transferrin C2 gene was found in a significantly lower frequency among Liberians compared to European and Asiatic populations. A new transferrin variant was observed by isoelectric focusing. This variant could not be identified with conventional starch or polyacrylamide electrophoresis.


[Results of experimental radiobiological studies made within the 10-kilometer zone of the Chernobyl AES accident].
A study was made of the effect of high radioactive contamination on the animal organism (C57BL/6 mice) and HeLa cell culture within the ten-kilometer zone of the Chernobyl A.P.S. accident. The total radiation dose, as calculated by a gamma-component, was 0.09 to 2 Gy. A long-term exposure of mice within the zone (cumulative dose of 1.8 to 2 Gy) caused a significant decrease in bone marrow stem potencies and changes in the brain vascular system; subsequent acute exposure of animals increased interferon titres in the serum to a much greater extent than a single acute exposure did. As to HeLa cells, irradiation there of with doses of 0.09 to 0.4 Gy during 15-20 postirradiation generations caused a decrease in the proliferative activity, an emergence of cells with micronuclei and of giant cells, and remote cell death.


Gastric Emptying Is Not Delayed and Does Not Correlate With Attenuated Postprandial Blood Flow Increase in Medicated Patients With Early Parkinson's Disease.
We have recently used phase-contrast magnetic resonance imaging (PC-MRI) to demonstrate an attenuated postprandial blood flow response in the superior mesenteric artery (SMA) in 23 medicated patients with Parkinson's disease (PD) compared to 23 age- and sex-matched healthy controls. To investigate in a sub-sample of the original cohort whether the observed blood flow response in SMA after oral food intake is related to a delay in gastric emptying. We studied 15 patients with PD in an "ON-medication" state with a mean disease duration of 3.9 ± 2.2 years and 15 healthy age- and sex-matched individuals. Participants underwent dynamic gastric scintigraphy 0, 30, 60, 120, 180 and 240 minutes after the intake of a standardized radiolabeled test meal. Gastric emptying was compared between groups. 14 of the 15 PD patients and 12 of the 15 healthy control subjects had previously undergone serial postprandial PC-MRI measurements. In these individuals, we tested for a relationship between gastric emptying and postprandial blood flow response in the SMA. The dynamics of gastric emptying did not differ between groups (p = 0.68). There was substantial inter-subject variability of gastric emptying in PD patients and healthy participants. Only a single PD patient had delayed gastric emptying. In those participants who had undergone PC-MRI, postprandial increase in SMA blood flow was attenuated in PD compared to healthy controls as reported previously (p = 0.006). Gastric emptying did not correlate with the timing and amplitude of postprandial blood flow increase in SMA. Our preliminary results, obtained in a small group of early-stage PD patients who continued their usual dopamine replacement therapy, suggest that variations in gastric emptying after solid meal intake is within the normal range in the majority of cases. There is also no evidence for a tight relationship between the attenuated postprandial blood flow response in the SMA and normal variations in gastric emptying.


Genetic analysis of the relationship between interleukin production and worm rejection in Trichinella spiralis-infected inbred mice.
The production of interleukin 1 (IL-1), IL-2, and IL-3 by peritoneal macrophages, mesenteric lymph node (MLN), or spleen cells from inbred strains of mice infected with Trichinella spiralis was examined. The mice belonged to the worm rejection phenotypes previously characterized as strong (NFS), intermediate (C3H, BUB, DBA/1, SWR, CBA, etc.), or weak (B10.Q, B10.BR, etc.). Strong responder NFS mice produced approximately twice as much IL-1 as intermediate responder C3Heb/Fe or weak responder B10.BR mice. IL-3 production varied slightly among strains but did not show any relationship to the phenotype of rejection (highest: C3Heb/Fe, B10.BR; lowest: B10.Q). Of 16 strains of inbred mice and 6 F1 hybrid crosses assessed, marked variations occurred in IL-2 production from MLN cells in response to T. spiralis antigen challenge in vitro. When 16 mouse strains were compared IL-2 production ranged from 5.1 units/ml (A/J) to 29.8 (NFS). Variations in IL-2 production among mouse strains did not relate directly to MHC haplotype, and the capacity of an individual strain to release IL-2 or IL-3 did not correlate with adult worm rejection phenotype. Genetic linkage studies proved that the gene(s) regulating IL-2 production in T. spiralis infection were not linked to the gene(s) regulating adult worm rejection. Regression analysis showed a weak correlation of high IL-2 production with weak worm rejection suggesting that IL-2 production or an associated process is a negative factor in primary worm rejection.


Size-dependent electrochemical oxidation of silver nanoparticles.
Here we quantify the electrochemical oxidation of Ag nanoparticles (NPs) as a function of size by electrostatically attaching Ag NPs synthesized by seed-mediated growth in the presence of citrate (diameter = 8 to 50 nm) to amine-functionalized indium-tin oxide coated glass electrodes (Glass/ITO), obtaining a linear sweep voltammogram from 0.1 V, where Ag(0) is stable, up to 1.0 V, and observing the peak potential (E(p)) for oxidation of Ag(0) to Ag(+). Electrostatic attachment to the organic linker presumably removes direct interactions between Ag and ITO and allows control over the total Ag coverage by altering the soaking time. This is important as both metal-electrode interactions and overall Ag coverage can affect E(p). E(p) shifts positive from an average of 275 to 382 mV as the Ag NP diameter increases for a constant Ag coverage and under conditions of planar diffusion, suggesting a shift in E(p) due to a thermodynamic shift in E(0) for the Ag/Ag(+) redox couple with size. The negative shift in E(p) with decreasing Ag NP radius follows the general trend predicted by theory and agrees with previous qualitative experimental observations. A better understanding of metal nanostructure oxidation is crucial considering their potential use in many different applications and the importance of metal corrosion processes at the nanoscale.


Short peptides containing L-lysine and epsilon-aminocaproic acid as potential plasmin inhibitors.
Eight short peptides containing L-lysine and epsilon-aminocaproic acid were obtained and their effect on the amidolytic activities of plasmin, thrombin and trypsin was examined. Tripeptide amide Boc-EACA-L-Lys-EACA-NH2 was the most effective and specific plasmin inhibitor.


Role of glycosylation in cell surface expression and stability of HERG potassium channels.
The human ether-à-go-go-related gene (HERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel in the heart. We previously showed that HERG channel protein is modified by N-linked glycosylation. HERG protein sequence contains two extracellular consensus sites for N-linked glycosylation (N598, N629). In this study, we used the approaches of site-directed mutagenesis and biochemical modification to inhibit N-linked glycosylation and studied the role of glycosylation in the cell surface expression and turnover of HERG channels. Our results show that N598 is the only site for N-linked glycosylation and that glycosylation is not required for the cell surface expression of functional HERG channels. In contrast, N629 is not used for glycosylation, but mutation of this site (N629Q) causes a protein trafficking defect, which results in its intracellular retention. Pulse-chase experiments show that the turnover rate of nonglycosylated HERG channel is faster than that of the glycosylated form, suggesting that N-linked glycosylation plays an important role in HERG channel stability.


The prospective, randomized investigation of the safety and efficacy of telmisartan versus ramipril using ambulatory blood pressure monitoring (PRISMA I).
To compare the efficacy and safety of once-daily telmisartan and ramipril on blood pressure (BP) reductions during the last 6 h of the dosing interval. In a prospective, randomized, open-label, blinded-endpoint study using ambulatory BP monitoring, 801 patients with mild-to-moderate hypertension were randomly assigned to once-daily treatment with telmisartan 80 mg for 14 weeks or ramipril 5 mg for 8 weeks and then force titrated to ramipril 10 mg for the last 6 weeks. Primary endpoints were the reduction from baseline in the last 6-h mean ambulatory systolic BP (SBP) and diastolic BP (DBP). Secondary endpoints included changes in 24-h, morning, daytime and night-time mean ambulatory BP and ambulatory BP response rates. Telmisartan 80 mg produced greater reductions in the last 6-h mean ambulatory SBP and DBP compared with ramipril 5 mg (P < 0.0001) and 10 mg (P < 0.0001), and was superior to ramipril for all secondary ambulatory SBP and DBP endpoints (P < 0.05). Ambulatory BP response rates (24-h mean ambulatory SBP/DBP < 130/80 mmHg or reduction from baseline > or = 10 mmHg) were greater with telmisartan 80 mg (P < 0.01) than with ramipril 5 and 10 mg. Ramipril was associated with a higher incidence of treatment-related cough (5.7 versus 0.5% for telmisartan). Telmisartan was significantly more effective than ramipril in reducing BP throughout the 24-h dosing interval and particularly during the last 6 h, a time when patients appear to be at greatest risk of cerebro- and cardiovascular events. Both drugs were well tolerated, although ramipril was associated with a higher incidence of cough.


Schools opening and Covid-19 diffusion: Evidence from geolocalized microdata.
Are schools triggering the diffusion of the Covid-19? This question is at the core of an extensive debate about the social and long-run costs of stopping the economic activity and human capital accumulation from reducing the contagion. In principle, many confounding factors, such as climate, health system treatment, and other forms of restrictions, may impede disentangling the link between schooling and Covid-19 cases when focusing on a country or regional-level data. This work sheds light on the potential impact of school opening on the upsurge of contagion by combining a weekly panel of geocoded Covid-19 cases in Sicilian census areas with a unique set of school data. The identification of the effect takes advantage of both a spatial and time-variation in school opening, stemming from the flexibility in opening dates determined by a Regional Decree, and by the occurrence of a national referendum, which pulled a set of poll-station schools towards opening earlier or later September 24th. The analysis finds that census areas where schools opened earlier observed a significant and positive increase in the growth rate of Covid-19 cases between 2.5-3.7%. This result is consistent across several specifications, including accounting for several determinants of school opening, such as the number of temporary teachers, Covid-19 cases in August, and pupils with special needs. Finally, the analysis finds lower effects in more densely populated areas, on younger population, and on smaller class size. The results imply that school reopening generated an increase of one third in cases.


Paroxysmal nocturnal hemoglobinuria and complement-mediated erythrocyte damage.
The erythrocytes of paroxysmal nocturnal hemoglobinuria are abnormally sensitive to complement-mediated lysis because they are deficient in membrane proteins that regulate the functional activity of complement. All the deficient proteins in paroxysmal nocturnal hemoglobinuria share the common structural feature of being anchored to the cell surface by a glycosyl phosphatidylinositol moiety. Recent studies showed that the first intermediate in the pathway of the glycosyl phosphatidylinositol anchor synthesis is not formed in paroxysmal nocturnal hemoglobinuria cells. This observation suggests that the molecular basis of paroxysmal nocturnal hemoglobinuria is due to an abnormality involving a gene that encodes a protein essential for the normal biosynthesis of the first intermediate. By using expression cloning, the complementary DNA (called phosphatidylinositol glycan class A [PIG-A]) that corrects the abnormality in glycosyl phosphatidylinositol-anchor synthesis in paroxysmal nocturnal hemoglobinuria cells was identified. Subsequent studies showed that the PIG-A gene is located on the X chromosome. Together, these studies provided a molecular explanation for paroxysmal nocturnal hemoglobinuria.


Metabolic Engineering for Improved Curcumin Biosynthesis in Escherichia coli.
The biosynthetic efficiency of curcumin, a highly bioactive compound from the plant Curcuma longa, needs to be improved. In this study, we performed host cell and biosynthetic pathway engineering to improve curcumin biosynthesis. Using in vivo-directed evolution, the expression level of curcuminoid synthase (CUS), the rate-limiting enzyme in the curcumin biosynthetic pathway, was significantly improved. Furthermore, as curcumin is a highly hydrophobic compound, two cell membrane engineering strategies were applied to optimize the biosynthetic efficiency. Curcumin storage was increased by overexpression of monoglucosyldiacylglycerol synthase from Acholeplasma laidlawii, which optimized the cell membrane morphology. Furthermore, unsaturated fatty acid supplementation was used to enhance membrane fluidity, which greatly ameliorated the damaging effect of curcumin on the cell membrane. These two strategies enhanced curcumin biosynthesis and demonstrated an additive effect.


The proteomic landscape of the suprachiasmatic nucleus clock reveals large-scale coordination of key biological processes.
The suprachiasmatic nucleus (SCN) acts as the central clock to coordinate circadian oscillations in mammalian behavior, physiology and gene expression. Despite our knowledge of the circadian transcriptome of the SCN, how it impacts genome-wide protein expression is not well understood. Here, we interrogated the murine SCN proteome across the circadian cycle using SILAC-based quantitative mass spectrometry. Of the 2112 proteins that were accurately quantified, 20% (421 proteins) displayed a time-of-day-dependent expression profile. Within this time-of-day proteome, 11% (48 proteins) were further defined as circadian based on a sinusoidal expression pattern with a ∼24 h period. Nine circadianly expressed proteins exhibited 24 h rhythms at the transcript level, with an average time lag that exceeded 8 h. A substantial proportion of the time-of-day proteome exhibited abrupt fluctuations at the anticipated light-to-dark and dark-to-light transitions, and was enriched for proteins involved in several key biological pathways, most notably, mitochondrial oxidative phosphorylation. Additionally, predicted targets of miR-133ab were enriched in specific hierarchical clusters and were inversely correlated with miR133ab expression in the SCN. These insights into the proteomic landscape of the SCN will facilitate a more integrative understanding of cellular control within the SCN clock.


Overview of the Components of Cardiac Metabolism.
Metabolism in organs other than the liver and kidneys may play a significant role in how a specific organ responds to chemicals. The heart has metabolic capability for energy production and homeostasis. This homeostatic machinery can also process xenobiotics. Cardiac metabolism includes the expression of numerous organic anion transporters, organic cation transporters, organic carnitine (zwitterion) transporters, and ATP-binding cassette transporters. Expression and distribution of the transporters within the heart may vary, depending on the patient's age, disease, endocrine status, and various other factors. Several cytochrome P450 (P450) enzyme classes have been identified within the heart. The P450 hydroxylases and epoxygenases within the heart produce hydroxyeicosatetraneoic acids and epoxyeicosatrienoic acids, metabolites of arachidonic acid, which are critical in regulating homeostatic processes of the heart. The susceptibility of the cardiac P450 system to induction and inhibition from exogenous materials is an area of expanding knowledge, as are the metabolic processes of glucuronidation and sulfation in the heart. The susceptibility of various transcription factors and signaling pathways of the heart to disruption by xenobiotics is not fully characterized but is an area with implications for disruption of normal postnatal development, as well as modulation of adult cardiac health. There are knowledge gaps in the timelines of physiologic maturation and deterioration of cardiac metabolism. Cross-species characterization of cardiac-specific metabolism is needed for nonclinical work of optimum translational value to predict possible adverse effects, identify sensitive developmental windows for the design and conduct of informative nonclinical and clinical studies, and explore the possibilities of organ-specific therapeutics.


Immunologically-related or incidental coexistence of diabetes mellitus and Graves' disease; discrimination by anti-GAD antibody measurement.
Coexistence of diabetes mellitus and Graves' disease may be classified into either an immunologically-related or an incidental phenomenon. It has been reported that anti-GAD antibody (GAD-Ab) persists at high levels for longer duration in subjects with type 1 diabetes and Graves' disease, whereas the prevalence of positive GAD-Ab (1.5%) in 131 non-diabetic subjects with Graves' disease was comparable to that in normal subjects (0.3%, P=0.2012). Thus, GAD-Ab might be a marker of the immunologically-related coexistence of the two diseases. To test this hypothesis, we investigated characteristics of Japanese subjects having both diseases according to the presence or absence of GAD-Ab. Sixty-one patients having diabetes mellitus and Graves' disease (24 men, 37 women, aged 53+/-2 years old, mean +/- SE) were consecutively registered between 1993-1997. The patients were divided into two groups of 14 GAD-Ab positive and 47 negative subjects. In the GAD-Ab positive subjects, earlier (32+/-3 years old) and abrupt onset (86%) of diabetes and insulin dependency (64%) were documented, as would be expected from the features of type 1 diabetes. Graves' disease often preceded diabetes (57%), presenting typical manifestations (79%). In contrast, older (45+/-2 years old, P=0.0031) and gradual onset (87%, P<0.0001) of diabetes, non-insulin dependency (74%, P<0.0001), and masked manifestations of Graves' disease (57%, P=0.0214) were common in the negative subjects. Precedence of diabetes dominated in these subjects (43%, P=0.0109). Immunological studies showed less frequent HLA-DR 2 locus (0%, P<0.02) in the GAD-Ab positive subjects. There was also a trend of higher frequency of HLA-DQA1*03 allele and of lower frequency of DQA1*01 allele in these subjects. Allelic frequency of cytotoxic T lymphocyte antigen 4 (CTLA-4) differed between the positive and negative subjects (P=0.0432). There were distinct clinical and immunological differences between the GAD-Ab positive and negative subjects having both diabetes mellitus and Graves' disease. The present results indicate that GAD-Ab measurement could draw a distinction between the immunologically-related and incidental coexistence of the two diseases.


Discovery of the first M5-selective and CNS penetrant negative allosteric modulator (NAM) of a muscarinic acetylcholine receptor: (S)-9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1H-imidazo[2,1-a]isoindol-5(9bH)-one (ML375).
A functional high throughput screen and subsequent multidimensional, iterative parallel synthesis effort identified the first muscarinic acetylcholine receptor (mAChR) negative allosteric modulator (NAM) selective for the M5 subtype. ML375 is a highly selective M5 NAM with submicromolar potency (human M5 IC50 = 300 nM, rat M5 IC50 = 790 nM, M1-M4 IC50 > 30 μM), excellent multispecies PK, high CNS penetration, and enantiospecific inhibition.


Fisetin improves lead-induced neuroinflammation, apoptosis and synaptic dysfunction in mice associated with the AMPK/SIRT1 and autophagy pathway.
Fisetin, a natural flavonoid found in plants, fruits and vegetables, exerts anti-cancer, anti-oxidant, anti-inflammatory and anti-mitotic effects. The current study instigates the protective effect of fisetin against lead-induced synaptic dysfunction, neuroinflammation and neurodegeneration in mice, and explores its underlying mechanisms. The results indicated fisetin can significantly ameliorated behavioral impairments in Pb-treated mice. Fisetin inhibited Pb-induced the apoptotic neurodegeneration, as indicated by the decreased levels of Bax and cleaved caspase-3. Fisetin suppressed activations of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), NF-κB and subsequently inactivate pro-inflammatory factor including interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). It can also decrease the accumulation of p-tau and amyloid-beta (Aβ) and increased the expression of the Aβ remover neprilysin (NEP) in brains of mice. Fisetin also reversed Pb-induced synaptic dysfunction by increasing the levels of synaptosomal associated protein-25 (SNAP-25), postsynaptic density-95 (PSD-95), cyclic-AMP-response element-binding protein (CREB) phosphorylation and calcium/calmodulin kinase II (CaMKII) phosphorylation. Fisetin promoted Pb-induced autophagy in the brains of mice. Moreover, fisetin can increase levels of the denosine 5'-monophosphate-activated protein kinase (AMPK) phosphorylation and SIRT1. Fisetin may be developed as a potential nutritional target for the prevention of Pb-induced neurotoxicity.


In vitro and in vivo antibiofilm activity of a coral associated actinomycete against drug resistant Staphylococcus aureus biofilms.
Staphylococcus aureus is now amongst the most important pathogenic bacteria responsible for bloodstream nosocomial infections and for biofilm formation on indwelling medical devices. Its increasing resistance to common antibiotics, partly attributed to its ability to form biofilms, is a challenge for the development of new antimicrobial agents. Accordingly, the goal of this study was to evaluate the effect of a coral associated actinomycete (CAA)-3 on S. aureus biofilms both in vitro and in vivo. Methanolic extracts of CAA-3 showed a reduction in in vitro biofilm formation by S. aureus ATCC 11632, methicillin resistant S. aureus ATCC 33591 and clinical isolates of S. aureus at the biofilm inhibitory concentration (BIC) of 0.1 mg ml(-1). Furthermore, confocal laser scanning microscope (CLSM) studies provide evidence of CAA-3 inhibiting intestinal colonisation of S. aureus in the nematode Caenorhabditis elegans. To conclude, this study for the first time, reports CAA as a promising source of anti-biofilm compounds, for developing novel drugs against highly resistant staphylococcal biofilms.


Monodisperse samarium and cerium orthovanadate nanocrystals and metal oxidation states on the nanocrystal surface.
A new solvothermal method has been developed for the synthesis of monodisperse SmVO4 and CeVO4 nanocrystals with controlled size and shape. The obtained materials were characterized by X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), transmission electron microscopy (TEM), and selected area electron diffraction (SAED) techniques. The results reveal that uniform nanocrystals and pure tetragonal phase of SmVO4 and CeVO4 can be achieved. To investigate the oxidation states of the metals on the mixed oxide nanocrystal surface, the XPS technique was employed. The results exhibit that only one oxidation state of samarium, cerium, and vanadium for each metal (e.g., Sm3+, Ce3+, V5+) was surprisingly well stable on the particle surface at the nanoscale, even after calcination, while the existence of two oxidation states of these metals is observed (e.g., Sm3+/Sm2+, Ce4+/Ce3+, V5+/V4+) in the corresponding single metal oxide nanocrystals.


Intracellular Recordings of Spectral Sensitivities in Stomatopods: a Comparison across Species.
Stomatopods (mantis shrimps) possess one of the most complex eyes in the world with photoreceptors detecting up to 12 different colors. It is not yet understood why stomatopods have almost four times the number of spectral photoreceptors compared with most other animals. It has, however, been suggested that these seemingly redundant photoreceptors could encode color through a new mechanism. Here we compare the spectral sensitivities across five species of stomatopods within the superfamily Gonodactyloidea using intracellular electrophysiological recordings. The results show that the spectral sensitivities across species of stomatopods are remarkably similar apart from some variation in the long-wavelength receptors. We relate these results to spectral sensitivity estimates previously obtained using microspectrophotometry and discuss the variation in the spectral sensitivity maxima (λmax) of the long-wavelength receptors in regard to the previous findings that stomatopods are able to tune their spectral sensitivities according to their respective light environment. We further discuss the similarities of the spectral sensitivities across species of stomatopods in regard to how color information might be processed by their visual systems.


Improved primate foamy virus vectors and packaging constructs.
Foamy virus (FV) vectors that have minimal cis-acting sequences and are devoid of residual viral gene expression were constructed and analyzed by using a packaging system based on transient cotransfection of vector and different packaging plasmids. Previous studies indicated (i) that FV gag gene expression requires the presence of the R region of the long terminal repeat and (ii) that RNA from packaging constructs is efficiently incorporated into vector particles. Mutants with changes in major 5' splice donor (SD) site located in the R region identified this sequence element as responsible for regulating gag gene expression by an unidentified mechanism. Replacement of the FV 5' SD with heterologous splice sites enabled expression of the gag and pol genes. The incorporation of nonvector RNA into vector particles could be reduced to barely detectable levels with constructs in which the human immunodeficiency virus 5' SD or an unrelated intron sequence was substituted for the FV 5' untranslated region and in which gag expression and pol expression were separated on two different plasmids. By this strategy, efficient vector transfer was achieved with constructs that have minimal genetic overlap.


125I-fibrin deposition in contact sensitivity reactions in the mouse. Sensitivity of the assay for quantitating reactions after active or passive sensitization.
We investigated the clotting associated with delayed hypersensitivity (DH) responses in the mouse by sensitizing the animals to the contactant oxazolone (Ox), and then administering 125I-guinea pig fibrinogen i.v. 10 to 30 min before antigen challenge 5 days later. Early (4 to 8 hr) contact sensitivity (CS) responses in immunized mice were barely detectable by three conventional measures of CS, but the total 125I-cpm in ears challenged with hapten was 3.6 to 4.5 X that in control ears challenged with vehicle alone; moreover, the amount of urea-insoluble cpm (cross-linked 125I-fibrin-associated cpm) in the reactions to Ox was 6.5-fold to 8.2-fold that present in the control reactions. In 24 hr reactions that were near peak intensity by measurements of ear swelling, ear weight ratios, and ratios of 125I-5-iodo-2-deoxyuridine-labeled leukocyte infiltration, the cpm in antigen-challenged ears exceeded that in control ears by 13-fold to 53-fold. In addition, antigen-challenged ears contained 27 to 300 X the urea-insoluble cpm present in control ears. 125I-Fibrin deposition was not a specific characteristic of CS reactions, because a small amount of urea-insoluble reactivity was also detected in some reactions to Ox in native mice. Nevertheless, the assay was exquisitely sensitive and readily detected quantitative differences between the immunologically specific and nonspecific reactions at very early intervals after challenge or with suboptimal doses of antigen. Furthermore, it was more sensitive than conventional tests of CS in detecting the reactions elicited in mice that had been passively sensitized to Ox by adoptive transfer of immune lymph node cells. Finally, we showed that the assay gave similar results when we tested CS reactions elicited in mast cell deficient WBB6F1-W/Wv and littermate normal (+/+) mice, demonstrating yet another similarity in the phenotype of DH reactions elicited in the presence or absence of mast cells.


Lack of Association of Locus Coeruleus Pathology with Orthostatic Hypotension in Parkinson's Disease.
Orthostatic hypotension (OH) is a common non-motor symptom in Parkinson's disease (PD) and is linked with increased mortality risk among the elderly. Although the locus coeruleus (LC) is the major source of noradrenaline (NA) modulation in the brain, its role in the pathogenesis of OH in PD remains largely elusive. Here we examined 44 well characterized postmortem brains of PD patients and available clinical data to explore the relationship between OH and LC pathology in PD. Our results failed to indicate that the LC pathology as well as the substantia nigra pathology were robustly associated with the presence of OH in PD patients, suggesting targeting LC norepinephrinergic system alone may not be sufficient to treat OH in PD.


[Efficacy and tolerance of disopyramide in arrhythmia associated with sinus node dysfunction].
The aim of this study was to assess the effect of delayed release Disopyramide 500 mg daily on sinus node function in 12 subjects with sinus node dysfunction and supraventricular and/or ventricular excitability. An ECG, Holter monitoring and electrophysiological studies of sinus node function with an oesophageal electrode were performed before and after 5 days treatment. The drug had to be discontinued in 1 patient because of urinary retention. The Disopyramide suppressed the clinical tachyarrhythmias in 8 out of the 11 subjects. The maximal sinus pause observed on Holter monitoring did not change significantly. It only increased in one subject. The mean sinus cycle length during electrophysiological study slightly decreased but there was a global increase in sinoatrial conduction time from 309 +/- 164 to 413 +/- 125 msec and of the corrected sinus mode recovery time from 470 +/- 168 to 605 +/- 303 msec. However, these changes were not significant. This study of delayed released Disopyramide shows a discordance between the tendency for sinus node function to deteriorate when assessed by electrophysiological studies and good clinical tolerance as assessed by Holter monitoring in the bradycardia-tachycardia syndrome.


The effect of apo-transferrin on bacterial adhesion to biomaterials.
Apo-transferrin (apo-Tf), the iron deficient form of Tf, has been identified previously as a potent inhibitor of Staphylococcus epidermidis adhesion to polyurethane surfaces. In this study, the ability of apo-Tf to suppress the adhesion of two other strains of bacteria, namely a Gram-positive Staphylococcus aureus and a Gram-negative Pseudomonas aeruginosa to several biomaterials, including polystyrene, polymethylmethacrylate, and silicone, is documented. The presence of apo-Tf in the medium at 20 microg/ml lowered bacterial adhesion to all tested biomaterials more than fourfold. Moreover, apo-Tf exerted its inhibitory activity even when protein coated surfaces were used as substrates for bacterial adhesion. To emphasize the importance of apo-Tf in the prevention of bacterial adhesion, human serum was depleted of Tf, employing affinity chromatography, and was shown to lose its inhibitory activity toward bacterial adhesion. Upon addition of apo-Tf to Tf-depleted serum, the activity was reestablished, resulting in a marked reduction in the number of bacteria adhered to the surfaces. Following the enzymatic deglycosylation, apo-Tf retained its ability to prevent bacterial adhesion. These results indicate that the carbohydrate moiety does not seem to play a role in this activity. The presented data provide the evidence that the inhibitory activity of apo-Tf is not bacterial strain specific and that the presence of apo-Tf in the medium results in a significant reduction of bacterial adhesion to a variety of neat and/or protein coated surfaces.


Physiologic adaptations to prolonged high-intensity exercise training in patients with coronary artery disease.
Exercise training elicits numerous beneficial adaptations in patients with coronary artery disease (CAD), but until 10 yr ago these adaptations were generally believed to be peripheral adaptations rather than improvements in myocardial blood supply. However, animal models of CAD have been shown to elicit improvements in myocardial blood flow and left ventricular performance with prolonged, intense exercise training. More recently we have provided electrocardiographic, echocardiographic hemodynamic, and radionuclide evidence of central cardiovascular adaptations in patients with CAD after a year-long program of 1 h of exercise at 70-90% of VO2max 5 d.wk(-1). These training-induced adaptations are consistent with an improvement in myocardial oxygenation and concomitantly an enhanced left ventricular function in these patients. This program also resulted in improvements in glucose tolerance, insulin sensitivity, and plasma lipoprotein-lipid profile. We have also shown that these beneficial cardiovascular and metabolic adaptations are maintained during six additional years of training in patients with CAD. These results do not imply that all patients with CAD should initiate such an intense training program, but rather that, in selected patients undergoing a training stimulus well in excess of that conventionally prescribed for coronary patients, the training-induced adaptations may be substantially greater than previously believed.


Deceleration Planning Algorithm Based on Classified Multi-Layer Perceptron Models for Smart Regenerative Braking of EV in Diverse Deceleration Conditions.
The smart regenerative braking system (SRS) is an autonomous version of one-pedal driving in electric vehicles. To implement SRS, a deceleration planning algorithm is necessary to generate the deceleration used in automatic regenerative control. To reduce the discomfort from the automatic regeneration, the deceleration should be similar to human driving. In this paper, a deceleration planning algorithm based on multi-layer perceptron (MLP) is proposed. The MLP models can mimic the human driving behavior by learning the driving data. In addition, the proposed deceleration planning algorithm has a classified structure to improve the planning performance in each deceleration condition. Therefore, the individual MLP models were designed according to three different deceleration conditions: car-following, speed bump, and intersection. The proposed algorithm was validated through driving simulations. Then, time to collision and similarity to human driving were analyzed. The results show that the minimum time to collision was 1.443 s and the velocity root-mean-square error (RMSE) with human driving was 0.302 m/s. Through the driving simulation, it was validated that the vehicle moves safely with desirable velocity when SRS is in operation, based on the proposed algorithm. Furthermore, the classified structure has more advantages than the integrated structure in terms of planning performance.


Surface-induced dissociation: an effective tool to probe structure, energetics and fragmentation mechanisms of protonated peptides.
The utility of surface-induced dissociation (SID) to probe the structure, energetics and fragmentation mechanisms of protonated peptides is discussed and demonstrated. High internal energy deposition provided by low-energy (eV range) ion-surface collisions yields extensive fragmentation of protonated peptides, allowing relatively uncomplicated and rapid sequence analysis of oligopeptides. SID of multiply protonated peptides is illustrated for peptides with molecular mass of up to approximately 5000 u. It is also illustrated that SID combined with electrospray ionization (ESI) provides a distinctive experimental technique to determine the energetics and mechanisms of peptide fragmentation. The relative position of ESI/SID fragmentation efficiency curves (plots of percentage fragmentation vs. laboratory collision energy) for peptides can be utilized to estimate relative energetics of peptide fragmentation and even to predict proton localization sites. The observed trends support the essential role of the mobile proton model in understanding peptide fragmentation by low-energy tandem mass spectrometry.


Predicting in vivo safety characteristics using physiochemical properties and in vitro assays.
There is increasing pressure on the pharmaceutical industry to deliver safer and more effective medicines while constraining research and development costs. In order to meet these demands, the industry is looking for basic design principles in terms of physicochemical properties as well as the use of higher throughput in vitro assays to select and evaluate new molecular entities for further development. Recent advances in understanding the relationships between a chemical's properties and its propensity for adverse events, as well as the development of new in vitro screening technologies, have enhanced our ability to potentially select molecules more likely to succeed in becoming drugs. However, these approaches are still limited by the availability of data and our lack of understanding of the mechanisms by which compounds can cause toxicity.


Solute diffusion in lipid bilayer membranes: an atomic level study by molecular dynamics simulation.
To elucidate the mechanism of solute diffusion through lipid bilayer membranes, nearly 4 ns of molecular dynamics simulations of solutes in phospholipid bilayers was conducted. The study, the first atomic level study of solute diffusion in a lipid bilayer, involved four simulations of an all-atom representation of a fully solvated dimyristoylphosphatidylcholine (DMPC) bilayer in the L alpha phase with benzene molecules as solutes, totaling over 7100 atoms. These simulations agree with experimental evidence that the presence of small solutes does not affect bilayer thickness but does result in slight perturbations in the ordering of the hydrocarbon chains. At room temperature, the benzene molecules have essentially isotropic motion and rotate freely. The rate of translational diffusion varies with position within the bilayer and is faster in the center than near the zwitterionic headgroups and is in excellent agreement with experimental values for the diffusion of small solutes in a bilayer. These simulations have elucidated the mechanism of diffusion in a bilayer to be similar to the "hopping" mechanism found for the diffusion of gases through soft polymers. Jumps of up to 8 A can occur in as little as 5 ps whereas average motions for that time period are only approximately 1.5 A. In many cases, the jumps are moderated by torsional changes in the hydrocarbon chains which serve as "gates" between voids through which the benzene molecules move. Comparison of these simulations with another 1000-ps simulation of benzene in a pure alkane provides evidence that lipid bilayers should not be treated as a homogeneous bulk hydrocarbon phase.


Common Questions About the Pharmacologic Management of Depression in Adults.
One in 11 U.S. adults currently meets diagnostic criteria for major depressive disorder, and a similar number report that they have taken an antidepressant medication in the past 30 days. In the primary care population, medications are modestly superior to placebo in achieving remission, with a number needed to treat of seven or eight for selective serotonin reuptake inhibitors and seven to 16 for tricyclic antidepressants. The benefit of antidepressants over placebo is more pronounced in patients with severe depression. Second-generation antidepressants are generally considered first-line therapy. Specific therapy choice should be based on cost, patient preference, and adverse effect profile. About two-thirds of patients receiving second-generation antidepressants experience at least one adverse effect during treatment. Nausea and vomiting are the most common reasons for discontinuation of therapy. The optimal treatment duration is unclear, but clinical guidelines suggest four to 12 months for an initial episode of major depression. Patients with recurrent depression may benefit from prolonged treatment. High-quality evidence is lacking on the benefits and harms of antidepressant use in pregnancy. It is unclear whether selective serotonin reuptake inhibitor use in breastfeeding mothers causes adverse effects in their infants, but sertraline and paroxetine transfer to breast milk in lower concentrations than other antidepressants. Consensus guidelines recommend a "start low, go slow" approach to antidepressant therapy in older persons; preferred medications include citalopram, escitalopram, sertraline, mirtazapine, and venlafaxine.


Interpreting changes in quality of life in atrial fibrillation: how much change is meaningful?
The Atrial Fibrillation Effect on QualiTy of Life (AFEQT) questionnaire is a novel quality of life (QOL) measure previously shown to be valid, reliable, and sensitive to clinical change in patients with atrial fibrillation (AF). The clinical relevance of a given change in the score is not known. The most useful "anchor" for a measure of meaningful change in QOL is patient-reported magnitude of change. The aim of this study was to define the interpretability of changes in the AFEQT score from the patients' perspective. With the use of the original validation study of AFEQT, in which 210 patients completed the questionnaire at baseline and at 3 months, we estimated the AFEQT score corresponding to a meaningful improvement in QOL using the patients' assessments of global change in QOL, AF symptoms from the Atrial Fibrillation Severity Scale (AFSS), and physicians' assessment of global QOL over the 3 months, as anchors. In patients with a moderate improvement in global QOL, the AFEQT scores increased from 51.9 ± 21.8 to 70.8 ± 17.4 (an increase of 18.9 ± 20.7), compared with an increase of 6.9 ± 16.9 units in patients with "unimportant change" in global QOL. Physicians' global assessment yielded a similar change in AFEQT score corresponding to a moderate change in global QOL (21.3 ± 20.2 units). Patients with moderate improvement in AF symptom severity using the AFSS scale had an increase of 17.9 ± 11.8 units on the AFEQT scale. A change in 19 units in the AFEQT score corresponded to a 0.9 SD unit change or greater than a minimal important difference from a distribution based method. A meaningful improvement in QOL in patients with AF can be measured from a change in the AFEQT score. These results can assist in monitoring patient progress and interpreting the effects of interventions in patients with AF.


[The effect of 18β-sodium glycyrrhetinic acid on the nasal mucosa epithelial cilia in rat models of allergic rhinitis].
To investigate 18β-sodium glycyrrhetinic acid impact on nasal mucosa epithelial cilia in rat models of allergic rhinitis (AR). AR models were established by ovalbumin-induction. Wister rats were randomly divided into groups as normal group, model group, budesonide (0.2 mg/kg) group and sodium glycyrrhetinic acid (20 mg/kg and 40 mg/kg) group after the success of AR models. At 2 weeks and 4 weeks after treatment, the behavioral changes of rats were observed and recorded, and nasal septum mucosae were collected after 2 week and 4 week intervention, and the morphological changes of nasal mucosae were observed by electron microscope. Model group developed typical AR symptoms, the total score in all animals was > 5. With budesonide and sodium glycyrrhetinic acid treatment, the AR symptoms were relieved, and the total scores were reduced significantly (P < 0.01). Compared with the model group: after 2 weeks' intervention, thick mucous secretions on the top of columnar epithelium cilia in rat nasal mucosa was significantly reduced, and cilia adhesion, lodging, shedding were relieved in budesonide group and sodium glycyrrhetinic acid group, the relieve in budesonide group was slightly better than that in sodium glycyrrhetinic acid group; after 4 week intervention, Cilia adhesion, lodging, shedding were completely vanished, and the cilia were ranged in regular direction in budesonide group and sodium glycyrrhetinic acid group. Cilia in sodium glycyrrhetinic acid (20 mg/kg) group was more orderly, smooth than that in budesonide group and sodium glycyrrhetinic acid group (40 mg/kg), and the condition of cilia in sodium glycyrrhetinic acid group (20 mg/kg) was similar to the normal group. 18β-sodium glycyrrhetinic acid is effective to restrain the pathological changes of nasal mucosa cilia in rat models of AR.


Protective effect of Enterococcus faecalis DAPTO 512 on the intestinal tract and gut mucosa: milk allergy application.
The allergenicity of β-lactoglobulin (β-Lg) was studied by using Ussing chamber in a murine model of β-Lg allergy supplemented with hydrolysates obtained after fermentation of milk for 48 h at 37 (°)C with Enterococcus faecalis DAPTO 512, isolated from cow milk and identified by 16S rDNA sequence analysis. Balb/c mice were sensitised intraperitoneally with β-Lg. Three groups of mice were formed: group 1, composed of naive mice used as control received only NaCl; group 2, positive control composed of mice sensitised intraperitoneally with β-Lg; group 3, formed by mice which were given hydrolysates of 48 h then sensitised with β-Lg. After 48 h of fermentation β-casein and β-Lg were degraded by E. faecalis DAPTO 512. β-Lg immunisation was associated with strong IgG and IgE production in case of positive controls and a significant increase in short current circuit (Isc) and high conductance (G) responses were observed. The control and the hydrolysate groups showed a significant decrease in the production of IgG and IgE anti β-Lg compared to the positive control. The allergenic potential of β-Lg was markedly reduced in the group that received hydrolysates (Isc and G remained unchanged after intestine challenge with β-Lg). The histological scrutiny showed villi atrophy, lymphocyte hyperplasia and a significant chorion detachment in the positive control group. In the group administered with hydrolysates of fermented milk, inflammatory signs were lower, the villi were long and thin and lymphocytes were less dense. The results showed that feeding of milk fermented with E. faecalis DAPTO 512 during 18 days prior to β-Lg allergy induction exerts a protecting effect on the murine intestine and induces a significant decrease in the β-Lg allergenicity.


Breaking Down Barriers to Physical Activity in Patients with Nonalcoholic Fatty Liver Disease.
Lifestyle changes, including physical activity, are the cornerstones of the treatment of nonalcoholic fatty liver disease (NAFLD). For unclear reasons, most NAFLD patients do not achieve the recommended amount of weekly activity. Our aim was to measure perceived barriers to physical activity and enablers to exercise intervention. Consecutive subjects aged 18-70 with NAFLD were prospectively enrolled. An exercise motivation questionnaire was administered to assess current behaviors and perceived barriers. Eighty-seven subjects (60% female) were enrolled with mean age 52 years and mean body mass index (BMI) 34.5 kg/m2. Metabolic comorbidities were common: 49% had hyperlipidemia, 42% hypertension, and 40% diabetes. The majority (75%) did not achieve ≥ 150 min/week of physical activity. Ninety-one percent agreed that activity was important in improving NAFLD; 88% desired to be more active. Lack of exercise resources and education from treating provider (47%), physical discomfort during exercise (44%), and time constraints (32%) were the most common barriers. Rates of fitness tracker (34%), gym (33%), exercise program (33%), and personal trainer (17%) use were low. While nearly all subjects with NAFLD identify physical activity to be important and desire to be more active, only a few meet activity recommendations. This discordance is due to a perceived lack of resources and education, physical discomfort, and time constraints. Better understanding of these barriers and behaviors are important to improve morbidity and mortality in NAFLD. Future behavioral research removing the identified barriers is of great importance to global public health and should be prioritized.


Dysregulation of methionine metabolism in multiple sclerosis.
We report a significant reduction in plasma methionine concentrations in relapse remitting multiple sclerosis (MS) patients compared to controls. In vivo studies demonstrate that changes in peripheral methionine levels in mice can regulate histone H3 methylation and expression of DNA methyltransferase 3A (DNMT3A) centrally, in the cerebral cortex. Therefore, we propose that decreases in circulating methionine represent one of the earliest manifestations of dysregulated methionine metabolism in MS with potential impacts on both histone H3 and DNA methylation in the central nervous system.


The ECOTOXicology Knowledgebase: A Curated Database of Ecologically Relevant Toxicity Tests to Support Environmental Research and Risk Assessment.
The need for assembled existing and new toxicity data has accelerated as the amount of chemicals introduced into commerce continues to grow and regulatory mandates require safety assessments for a greater number of chemicals. To address this evolving need, the ECOTOXicology Knowledgebase (ECOTOX) was developed starting in the 1980s and is currently the world's largest compilation of curated ecotoxicity data, providing support for assessments of chemical safety and ecological research through systematic and transparent literature review procedures. The recently released version of ECOTOX (Ver 5, www.epa.gov/ecotox) provides single-chemical ecotoxicity data for over 12,000 chemicals and ecological species with over one million test results from over 50,000 references. Presented is an overview of ECOTOX, detailing the literature review and data curation processes within the context of current systematic review practices and discussing how recent updates improve the accessibility and reusability of data to support the assessment, management, and research of environmental chemicals. Relevant and acceptable toxicity results are identified from studies in the scientific literature, with pertinent methodological details and results extracted following well-established controlled vocabularies and newly extracted toxicity data added quarterly to the public website. Release of ECOTOX, Ver 5, included an entirely redesigned user interface with enhanced data queries and retrieval options, visualizations to aid in data exploration, customizable outputs for export and use in external applications, and interoperability with chemical and toxicity databases and tools. This is a reliable source of curated ecological toxicity data for chemical assessments and research and continues to evolve with accessible and transparent state-of-the-art practices in literature data curation and increased interoperability to other relevant resources. Environ Toxicol Chem 2022;41:1520-1539. © 2022 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.


LC-MS Analysis Revealed the Significantly Different Metabolic Profiles in Spent Culture Media of Human Embryos with Distinct Morphology, Karyotype and Implantation Outcomes.
In this study we evaluated possible differences in metabolomic profiles of spent embryo culture media (SECM) of human embryos with distinct morphology, karyotype, and implantation outcomes. A total of 153 samples from embryos of patients undergoing in vitro fertilization (IVF) programs were collected and analyzed by HPLC-MS. Metabolomic profiling and statistical analysis revealed clear clustering of day five SECM from embryos with different morphological classes and karyotype. Profiling of day five SECM from embryos with different implantation outcomes showed 241 significantly changed molecular ions in SECM of successfully implanted embryos. Separate analysis of paired SECM samples on days three and five revealed 46 and 29 molecular signatures respectively, significantly differing in culture media of embryos with a successful outcome. Pathway enrichment analysis suggests certain amino acids, vitamins, and lipid metabolic pathways to be crucial for embryo implantation. Differences between embryos with distinct implantation potential are detectable on the third and fifth day of cultivation that may allow the application of culture medium analysis in different transfer protocols for both fresh and cryopreserved embryos. A combination of traditional morphological criteria with metabolic profiling of SECM may increase implantation rates in assisted reproductive technology programs as well as improve our knowledge of the human embryo metabolism in the early stages of development.


In-water truly monodisperse aggregation of gear-shaped amphiphiles based on hydrophobic surface engineering.
Exactly six gear-shaped amphiphiles self-assemble into a highly stable, water-soluble, box-shaped capsule, in which indented hydrophobic surfaces of the components mesh with each other like gears. A water-soluble, tetrahedron-shaped capsule was also constructed from four gear-shaped amphiphiles with a template guest. These findings provide a guideline for creating aggregates with a given number of amphiphiles based on hydrophobic surface engineering.


Diagnostic value of Tl-201 lung uptake is dependent on measurement method.
Increased lung uptake during exercise thallium 201 single photon emission computed tomography is related to left ventricular dysfunction and extent of coronary artery disease (CAD). We studied the influence of the lung region of interest (ROI), used to quantify Tl-201 lung uptake, on the diagnostic value of the lung-to-heart uptake ratio (LHR) in detecting CAD with Tl-201 myocardial single photon emission computed tomography. We retrospectively studied 152 consecutive patients referred to our center for stress Tl-201 scanning. Of these, 116 had proven multivessel CAD and 36 had either normal findings on coronary angiogram or a low likelihood (<5%) of CAD. Poststress quantitative analysis was performed from a 4 x 4-pixel ROI over the hottest myocardial region, an 8 x 8-pixel left lung ROI, an 8 x 8-pixel right lung ROI, and a manual ROI encompassing the whole right lung. The LHR was calculated for each lung ROI. Right LHR (R-LHR) provided the best interobserver and intraobserver reproducibility. R-LHR and total R-LHR values were significantly higher in patients with CAD. Only history of myocardial infarction significantly influenced the R-LHR measurement. The methodology of LHR measurement significantly influences the clinical contribution of Tl-201 lung uptake evaluation. Optimal reproducibility and diagnostic accuracy are provided by a right lung ROI.


Actin as a potential target for decavanadate.
ATP prevents G-actin cysteine oxidation and vanadyl formation specifically induced by decavanadate, suggesting that the oxometalate-protein interaction is affected by the nucleotide. The ATP exchange rate is increased by 2-fold due to the presence of decavanadate when compared with control actin (3.1×10(-3) s(-1)), and an apparent dissociation constant (k(dapp)) of 227.4±25.7 μM and 112.3±8.7 μM was obtained in absence or presence of 20 μM V(10), respectively. Moreover, concentrations as low as 50 μM of decameric vanadate species (V(10)) increases the relative G-actin intrinsic fluorescence intensity by approximately 80% whereas for a 10-fold concentration of monomeric vanadate (V(1)) no effects were observed. Upon decavanadate titration, it was observed a linear increase in G-actin hydrophobic surface (2.6-fold), while no changes were detected for V(1) (0-200 μM). Taken together, three major ideas arise: i) ATP prevents decavanadate-induced G-actin cysteine oxidation and vanadate reduction; ii) decavanadate promotes actin conformational changes resulting on its inactivation, iii) decavanadate has an effect on actin ATP binding site. Once it is demonstrated that actin is a new potential target for decavanadate, being the ATP binding site a suitable site for decavanadate binding, it is proposed that some of the biological effects of vanadate can be, at least in part, explained by decavanadate interactions with actin.


Mycobacterium tuberculosis H37Rv parietal and cellular lipoarabinomannans. Characterization of the acyl- and glyco-forms.
Mannosylated lipoarabinomannans are multifaceted molecules. They have been shown to exert an immunosuppressive role in the immunopathogenesis of tuberculosis. They are also described as antigens of host double negative alphabeta T-cells. Delimitation of ManLAMs epitopes require knowledge of the precise structure of these molecules. The two major functional domains (the cap motifs and the phosphatidylinositol anchor) of the parietal and cellular ManLAMs of Mycobacterium tuberculosis H37Rv were investigated here. Using capillary electrophoresis, we established that parietal and cellular ManLAMs share the same capping motifs, mono-, di-, and trimannosyl units with the same relative abundance. By (31)P NMR analysis of the native LAMs in Me(2)SO-d(6), the major acyl-form of both parietal and cellular H37Rv ManLAM anchors, typified by the P3 phosphorus resonance, comprised a diacylglycerol unit. Three other acyl-forms were characterized in the cellular ManLAMs. Comparative analysis of the cellular Mycobacterium bovis BCG and M. tuberculosis ManLAM acyl-forms revealed the presence of the same populations, but with different relative abundance. The biological importance of the H37Rv ManLAM acyl-form characterization is discussed, particularly concerning the molecular mechanisms of binding of ManLAMs to the CD1 proteins involved in the presentation of ManLAMs to T-cell receptors.


Identification of three highly confused marine Loxophyllum (Ciliophora: Pleurostomatida) with a key to seven congeners from the China Sea.
Three highly confused Loxophyllum helus-like morphotypes (i.e. Loxophyllum rostratum Cohn, 1866, Loxophyllum sinicum n. sp., and Loxophyllum simplex Kahl, 1933) found in mariculture waters near the coast of Qingdao, China, were investigated with emphasis on their live morphology and infraciliature. Comparative descriptions of these three organisms are presented and synonyms are critically discussed. The validity of L. sinicum n. sp. is confirmed mainly by the combination of the distribution of extrusomes, features of general living morphology, morphometric data, and the characters of the somatic ciliature. Two previously reported organisms under the name of L. helus by Dragesco (1966, variety a) and by Dragesco and Dragesco-Kernéis (1986) are discussed and are believed to be synonyms of L. sinicum. Furthermore, two isolates described by Dragesco (1960) and Ozaki and Yagiu (1943) under the name of L. helus are very likely, in our opinion, misidentifications, and might be two unknown forms. In the light of the current study, a key is presented to the seven clearly defined marine Loxophyllum species found in the coastal areas of north China Sea.


The Effect of pH, O(2), and Temperature on the CO(2) Compensation Point of Isolated Asparagus Mesophyll Cells.
The effect of pH, O(2) concentration, and temperature on the CO(2) compensation point (capital GHE, Cyrillic[CO(2)]) of isolated Asparagus sprengeri Regel mesophyll cells has been determined in a closed, aqueous environment by a sensitive gas-chromatographic technique. Measured values range between 10 and 100 microliters per liter CO(2) depending upon experimental conditions. The capital GHE, Cyrillic(CO(2)) increases with increasing temperature. The rate of increase is dependent upon the O(2) concentration and is more rapid at high (250-300 micromolar), than at low (30-60 micromolar), O(2) concentrations. The differential effect of temperature on capital GHE, Cyrillic(CO(2)) is more pronounced at pH 6.2 than at pH 8.0, but this pH-dependence is not attributable to a direct, differential effect of pH on the relative rates of photosynthesis and photorespiration, as the O(2)-sensitive component of capital GHE, Cyrillic(CO(2)) remains constant over this range. The capital GHE, Cyrillic(CO(2)) of Asparagus cells at 25 degrees C decreases by 50 microliters per liter when the pH is raised from 6.2 to 8.0, regardless of the prevailing O(2) concentration. It is suggested that the pH-dependence of capital GHE, Cyrillic(CO(2)) is related to the ability of the cell to take up CO(2) from the aqueous environment. The correlation between high HCO(3) (-) concentrations and low capital GHE, Cyrillic(CO(2)) at alkaline pH indicates that extracellular HCO(3) (-) facilitates the uptake of CO(2), possibly by increasing the flux of inorganic carbon from the bulk medium to the cell surface. The strong O(2)- and temperature-dependence of capital GHE, Cyrillic(CO(2)) indicates that isolated Asparagus mesophyll cells lack an efficient means for concentrating intracellular CO(2) to a level sufficient to reduce or suppress photorespiration.


Differential chromatin condensation of female and male pronuclei in mouse zygotes.
Mouse zygotes or halves of zygotes, containing either a female or a male pronucleus, were fused with ovulated metaphase II oocytes. In 59.7% of the resulting hybrid cells, the pronuclei underwent premature chromosome condensation (PCC). In some of these heterokaryons the 2 pronuclei differed in the dynamics of condensation. Detectability of differential PCC of pronuclei (dPCC) depended on the type of preparation. In hybrids with PCC, produced by fusion of intact zygotes with metaphase II oocytes and processed for whole-mount preparations, one pronucleus was more advanced in the condensation process in 47% of cases. In air-dried preparations dPCC was detected in as many as 94% of hybrids. Experiments with the fusion of halves of zygotes with metaphase II oocytes have shown that the differential reaction of pronuclei to condensation factor depended on their parental origin. Maternal chromatin responded faster to the condensation factor and attained more advanced stages of PCC than paternal chromatin. Different responses of the maternal and paternal pronucleus to the condensation factor suggests that the 2 pronuclei are not identical with regard to the organization of chromatin and/or the lamin composition of the nuclear envelope.


A portrait of the radiographer of the future.
This article describes the collection of data used by a community college in developing a plan to identify and meet the educational needs of radiographers for the year 2000 and beyond. Radiography program directors in the Midwest were asked to rank seven assumptions regarding the radiographer of the future. Respondents assigned a numerical ranking to each assumption, indicated the extent of agreement or disagreement and rated the importance of that assumption to teaching, the program and the institution.


Self-reported physical functional health predicts future bone mineral density in EPIC-Norfolk cohort.
Using a large population sample from the UK, we found that self-reported physical functional health may be used to predict future bone mineral density especially in women. It may be a useful and inexpensive way to identify individuals before further decline in bone mineral density and the risk of fracture. Self-reported physical functional health may predict bone mineral density (BMD) and thus provide a method to identify people at risk of low BMD. In this study, the association between the 36-item short-form questionnaire (SF-36) physical component summary (PCS) score and future BMD in participants aged 40-79 years enrolled in the European Prospective Investigation of Cancer-Norfolk study was investigated. Associations between a participant's SF-36 PCS score, measured 18 months after baseline health check, and broadband ultrasound attenuation (BUA-a measure of BMD), measured 2-5 years after baseline, were examined using sex-specific linear and logistic regression analyses adjusting for age, BMI, medical co-morbidities, lifestyle and socioeconomic factors. Data from 10,203 participants, mean (standard deviation (SD)) age 61.5 (8.9) years (57.4% women), were analysed from 1993 to 2000. For every five points lower PCS score in men and women, there was approximately a 0.5 dB/MHz lower mean BUA. In women, a PCS score of less than one standard deviation (1SD) below the sex-specific mean was associated with having a low BUA (< 1SD below sex-specific mean) and very low BUA (< 2.5SD below the sex specific mean); odds ratio (OR) (95% confidence interval) 1.53 (1.24, 1.88) and 8.28 (2.67, 25.69), respectively. The relationship was lesser so in men; corresponding OR (95% CI) were 1.34 (0.91, 1.98) and 2.57 (0.72, 9.20), respectively. Self-reported physical functioning predicts BMD in an apparently healthy population, particularly in women. This could potentially provide an inexpensive, simple screening tool to identify individuals at risk of osteoporosis.


Functional Importance of Hydrophobic Patches on the Ebola Virus VP35 IFN-Inhibitory Domain.
Viral protein 35 (VP35) of Ebola virus (EBOV) is a multifunctional protein that mainly acts as a viral polymerase cofactor and an interferon antagonist. VP35 interacts with the viral nucleoprotein (NP) and double-stranded RNA for viral RNA transcription/replication and inhibition of type I interferon (IFN) production, respectively. The C-terminal portion of VP35, which is termed the IFN-inhibitory domain (IID), is important for both functions. To further identify critical regions in this domain, we analyzed the physical properties of the surface of VP35 IID, focusing on hydrophobic patches, which are expected to be functional sites that are involved in interactions with other molecules. Based on the known structural information of VP35 IID, three hydrophobic patches were identified on its surface and their biological importance was investigated using minigenome and IFN-β promoter-reporter assays. Site-directed mutagenesis revealed that some of the amino acid substitutions that were predicted to disrupt the hydrophobicity of the patches significantly decreased the efficiency of viral genome replication/transcription due to reduced interaction with NP, suggesting that the hydrophobic patches might be critical for the formation of a replication complex through the interaction with NP. It was also found that the hydrophobic patches were involved in the IFN-inhibitory function of VP35. These results highlight the importance of hydrophobic patches on the surface of EBOV VP35 IID and also indicate that patch analysis is useful for the identification of amino acid residues that directly contribute to protein functions.


Phenotype of Coats disease in females.
To determine whether the clinical presentation of Coats disease differs between males and females. Records of patients diagnosed with Coats disease at a single institution were retrospectively reviewed. Demographic data, main reason for initial consultation, comprehensive ocular examination at diagnosis and modalities of treatments during the follow-up were recorded. Records from 114 patients with Coats disease were analysed. Ninety-eight patients (86%) were male and 16 (14%) female. Mean age at diagnosis was 6.2 years±6.1 in males and 7.4 years±4.7 in females. The main initial reason for consultation was strabismus in males and decreased visual acuity in females. Stage severity at diagnosis was similar in the two groups with half of the patients presenting with stage 2B2 or lower. The extension of peripheral retinal telangiectasia was also similar (mean: 6.2±3.4 and 5.8±4.0, respectively), as was the extension of intraretinal exudation (mean: 5.0±4.5 and 5.8±4.4) and the frequency of a subfoveal nodule at diagnosis (40% vs 30%, respectively). There was no distinction between the number of laser photocoagulation or cryotherapy sessions required for both groups during the follow-up. Coats disease presentation does not differ between genders despite being much rarer in females. We propose a pathogenic mechanism accounting for the gender-dependent incidence combined with gender-independent expressivity of Coats disease.


Assessing monoclonal antibody product quality attribute criticality through clinical studies.
Recombinant therapeutic proteins, including antibodies, contain a variety of chemical and physical modifications. Great effort is expended during process and formulation development in controlling and minimizing this heterogeneity, which may not affect safety or efficacy, and, therefore, may not need to be controlled. Many of the chemical conversions also occur in vivo, and knowledge about the alterations can be applied to assessment of the potential impact on characteristics and the biological activity of therapeutic proteins. Other attributes may affect the drug clearance and thereby alter drug efficacy. In this review article, we describe attribute studies conducted using clinical samples and how information gleaned from them is applied to attribute criticality assessment. In general, how fast attributes change in vivo compared to the rate of mAb elimination is the key parameter used in these evaluations. An attribute with more rapidly changing levels may have greater potential to affect safety or efficacy and thereby reach the status of a Critical Quality Attribute (CQA) that should be controlled during production and storage, but the effect will depend on whether compositional changes are due to chemical conversion or differential clearance.


Concomitant delineation of surface Ig, B-cell differentiation antigens, and HLADR on lymphoid proliferations using three-color immunocytometry.
Accurate and consistent enumeration of B-cell subpopulations in lymphoid tissue was achieved through multiparameter three-color immunofluorescence and flow cytometric analysis (FCM). Phycoerythrin (PE)-anti-CD19 (Leu12) and biotinylated anti-HLADr/streptavidin-Duochrome (PE/Texas Red), used in conjunction with polyclonal fluorescein isothiocyanate (FITC) conjugated anti-surface immunoglobulin (SIg) antibodies, effectively separated non-specific binding and background fluorescence from true B-cell surface FITC immunofluorescence, while concomitantly analyzing for HLADr and CD19 phenotypic expression/deletion. Autofluorescence was measured to establish a fluorescence threshold. A second control measured non-specific binding of isotypic control mouse Ig and non-immune rabbit IgG. Cell suspensions from 128 samples of various lymphoid proliferations were studied. In 116 of the 128 samples, kappa/lambda ratios determined by flow cytometry correlated well with immunocytology results obtained using cytospins from the same cell suspension and with histopathologically established diagnosis. Clonality and lineage as defined immunotypically by flow cytometry was concordant with genotypic results in 64 of the 67 cases evaluated. SIg, HLADr, and CD19 deletions were demonstrated by flow cytometry in 8, 4, and 1 case(s), respectively. Discordance was usually attributable to selective loss of large neoplastic cells in flow cytometry specimens or absent expression of SIg by some cytoplasmic Ig (CIg+) lymphomas.


Caring for critically ill adults in paediatric intensive care units in England during the COVID-19 pandemic: planning, implementation and lessons for the future.
To describe the experience of paediatric intensive care units (PICUs) in England that repurposed their units, equipment and staff to care for critically ill adults during the first wave of the COVID-19 pandemic. Descriptive study. Seven PICUs in England. (1) Modelling using historical Paediatric Intensive Care Audit Network data; (2) space, staff, equipment, clinical care, communication and governance considerations during repurposing of PICUs; (3) characteristics, interventions and outcomes of adults cared for in repurposed PICUs. Seven English PICUs, accounting for 137 beds, repurposed their space, staff and equipment to admit critically ill adults. Neighbouring PICUs increased their bed capacity to maintain overall bed numbers for children, which was informed by historical data modelling (median 280-307 PICU beds were required in England from March to June). A total of 145 adult patients (median age 50-62 years) were cared for in repurposed PICUs (1553 bed-days). The vast majority of patients had COVID-19 (109/145, 75%); the majority required invasive ventilation (91/109, 85%). Nearly, a third of patients (42/145, 29%) underwent a tracheostomy. Renal replacement therapy was provided in 20/145 (14%) patients. Twenty adults died in PICU (14%). In a rapid and unprecedented effort during the first wave of the COVID-19 pandemic, seven PICUs in England were repurposed to care for adult patients. The success of this effort was underpinned by extensive local preparation, close collaboration with adult intensivists and careful national planning to safeguard paediatric critical care capacity.


Preliminary report of infection in dogs related to Ehrlichia equi: description of three cases.
The clinical course of naturally infected dogs with E. equi are described. Fever (up to 41 degrees C), depression, dysorexia and ascites were observed. Laboratory findings revealed mild anemia, inclusion bodies within neutrophils in one dog, thrombocytopenia, hypoproteinemia, hypoalbuminemia and in two dogs hyperglobulinemia. In acute and convalescent dogs sera antibody titers from 1:60 to 1:480 to E. equi using IFA test were detected. Clinical resolution was obtained with long-term doxycycline monohydrate therapy.


Brain tryptophan/serotonin perturbations in metabolic encephalopathy and the hazards involved in the use of psychoactive drugs.
Several combined pathogenetic factors such as hyperammonemia, different brain tryptophan metabolic disturbances and serotonin physiological/pharmacological alterations not yet defined in all details, will often give rise to the clinical neuropsychiatric condition known as hepatic encephalopathy (HE). Indeed, to this the probable exposure to novel potent CNS-monoamine acting drugs today may put such patients at certain risk for other pharmacodynamic (PD) responses than usually are expected from these "safe" drugs. Moreover, with a compromised liver function in HE, also pharmacokinetic (PK) features for the drugs are likely changed in these patients. Thus, the ultimate clinical outcome by this probable but unknown PD/PK-deviation for such psychoactive drugs when given to HE-patients needs further clarification. Accordingly, delineation of both PD- and PK-effects in experimental HE should shed light on this issue of relevance for monoamine-active drug safety as well as on some further details in the complex tryptophan/monoamine-related pathophysiology that comes into play in HE.


Coherent 1-microm lidar measurements of atmospheric-turbulence-induced spatial decorrelation using a multielement heterodyne detector array.
We have employed a coherent 1-microm Nd:YAG lidar system to measure directly, for the first time to our knowledge, the reduced spatial coherence length, p(0), of the lidar returns caused by atmospheric turbulence. Our experiments were conducted by using a 2 x 2 heterodyne detector array, which permitted real-time spatial correlation measurements of the lidar returns at two different detector spacings. The spatial correlation coefficients and spatial coherence length of the lidar returns from a hard target weremeasured during a day-to-night time period when the atmospheric turbulence parameter, C(n)(2), was measured to vary from 2 x 10(-13) to 2 x 10(-14) m(-1/3). These directly measured values of p(0) as a function of C(n)(2) were found to be in good agreement with theoretical predictions.


Strategy of restraining ripple error on surface for optical fabrication.
The influence from the ripple error to the high imaging quality is effectively reduced by restraining the ripple height. A method based on the process parameters and the surface error distribution is designed to suppress the ripple height in this paper. The generating mechanism of the ripple error is analyzed by polishing theory with uniform removal character. The relation between the processing parameters (removal functions, pitch of path, and dwell time) and the ripple error is discussed through simulations. With these, the strategy for diminishing the error is presented. A final process is designed and demonstrated on K9 work-pieces using the optimizing strategy with magnetorheological jet polishing. The form error on the surface is decreased from 0.216λ PV (λ=632.8 nm) and 0.039λ RMS to 0.03λ PV and 0.004λ RMS. And the ripple error is restrained well at the same time, because the ripple height is less than 6 nm on the final surface. Results indicate that these strategies are suitable for high-precision optical manufacturing.


Monitoring of chronic wasting disease (CWD) (IV).
The European Commission requested an analysis of the Chronic Wasting Disease (CWD) monitoring programme in Norway, Sweden, Finland, Iceland, Estonia, Latvia, Lithuania and Poland (9 January 2017-28 February 2022). Thirteen cases were detected in reindeer, 15 in moose and 3 in red deer. They showed two phenotypes, distinguished by the presence or absence of detectable disease-associated normal cellular prion protein (PrP) in lymphoreticular tissues. CWD was detected for the first time in Finland, Sweden and in other areas of Norway. In countries where the disease was not detected, the evidence was insufficient to rule out its presence altogether. Where cases were detected, the prevalence was below 1%. The data also suggest that the high-risk target groups for surveillance should be revised, and 'road kill' removed. Data show that, in addition to differences in age and sex, there are differences in the prion protein gene (PRNP) genotypes between positive and negative wild reindeer. A stepwise framework has been proposed with expanded minimum background surveillance to be implemented in European countries with relevant cervid species. Additional surveillance may include ad hoc surveys for four different objectives, specific to countries with/without cases, focusing on parallel testing of obex and lymph nodes from adult cervids in high-risk target groups, sustained over time, using sampling units and a data-driven design prevalence. Criteria for assessing the probability of CWD presence have been outlined, based on the definition of the geographical area, an annual assessment of risk of introduction, sustained minimum background surveillance, training and engagement of stakeholders and a surveillance programme based on data-driven parameters. All positive cases should be genotyped. Sample sizes for negative samples have been proposed to detect and estimate the frequency of PRNP polymorphisms. Double-strand sequencing of the entire PRNP open reading frame should be undertaken for all selected samples, with data collated in a centralised collection system at EU level.