System identifiability in a time-evolving agent-based model.

Mathematical models are a valuable tool for studying and predicting the spread of infectious agents. The accuracy of model simulations and predictions invariably depends on the specification of model parameters. Estimation of these parameters is therefore extremely important; however, while some parameters can be derived from observational studies, the values of others are difficult to measure. Instead, models can be coupled with inference algorithms (i.e., data assimilation methods, or statistical filters), which fit model simulations to existing observations and estimate unobserved model state variables and parameters. Ideally, these inference algorithms should find the best fitting solution for a given model and set of observations; however, as those estimated quantities are unobserved, it is typically uncertain whether the correct parameters have been identified. Further, it is unclear what 'correct' really means for abstract parameters defined based on specific model forms. In this work, we explored the problem of non-identifiability in a stochastic system which, when overlooked, can significantly impede model prediction. We used a network, agent-based model to simulate the transmission of Methicillin-resistant staphylococcus aureus (MRSA) within hospital settings and attempted to infer key model parameters using the Ensemble Adjustment Kalman Filter, an efficient Bayesian inference algorithm. We show that even though the inference method converged and that simulations using the estimated parameters produced an agreement with observations, the true parameters are not fully identifiable. While the model-inference system can exclude a substantial area of parameter space that is unlikely to contain the true parameters, the estimated parameter range still included multiple parameter combinations that can fit observations equally well. We show that analyzing synthetic trajectories can support or contradict claims of identifiability. While we perform this on a specific model system, this approach can be generalized for a variety of stochastic representations of partially observable systems. We also suggest data manipulations intended to improve identifiability that might be applicable in many systems of interest.

Cav1.4 congenital stationary night blindness is associated with an increased rate of proteasomal degradation.

Pathogenic, generally loss-of-function, variants in CACNA1F, encoding the Cav1.4α1 calcium channel, underlie congenital stationary night blindness type 2 (CSNB2), a rare inherited retinal disorder associated with visual disability. To establish the underlying pathomechanism, we investigated 10 clinically derived CACNA1F missense variants located across pore-forming domains, connecting loops, and the carboxy-tail domain of the Cav1.4α subunit. Homology modeling showed that all variants cause steric clashes; informatics analysis correctly predicted pathogenicity for 7/10 variants. In vitro analyses demonstrated that all variants cause a decrease in current, global expression, and protein stability and act through a loss-of-function mechanism and suggested that the mutant Cav1.4α proteins were degraded by the proteasome. We showed that the reduced current for these variants could be significantly increased through treatment with clinical proteasome inhibitors. In addition to facilitating clinical interpretation, these studies suggest that proteasomal inhibition represents an avenue of potential therapeutic intervention for CSNB2.

Organ donation by children in Australia, 2000-2019: impact of the 2009 National Reform Program. A population-based registry data study.

OBJECTIVES: To assess the impact of the 2009 National Reform Program for organ donation in Australia on the number and characteristics of organ donors under 16 years of age. DESIGN, SETTING, PARTICIPANTS: Retrospective observational time series study; analysis of Australia and New Zealand Organ Donation (ANZOD) registry data for all consented potential deceased organ donors under 16 years of age during 2000-2019, and of numbers of donors aged 16 years or more reported in ANZOD annual reports. MAIN OUTCOME MEASURES: Difference between 2000-2008 (pre-reform) and 2009-2019 (reform period) in annual organ donor rates (donors per million population), by age group (under 16 years, 16 years or more), reported as incidence rate ratio (IRR). SECONDARY OUTCOMES: Differences in child donor characteristics during 2000-2008 and 2009-2019. RESULTS: During 2000-2019, 400 children under 16 years of age were consented potential deceased organ donors, of whom 374 were actual deceased donors (94%): 146 during 2000-2008, 228 during 2009-2019. The median annual rate was 3.3 (interquartile range [IQR], 3.0-4.3) actual donors per million population during 2000-2008 and 4.2 (IQR, 3.6-5.2) donors per million population during 2009-2019 (IRR, 1.15; 95% confidence interval [CI], 0.93-1.42). In contrast, the difference between the two periods was statistically significant for donors aged 16 years or more, rising from 11.7 (IQR, 11.2-11.8) to 19.9 (IQR, 18.3-24.4) actual donors per million population (IRR, 1.75; 95% CI, 1.66-1.85). The median age of actual organ donors under 16 was similar during 2000-2008 (11 years; IQR, 7-14 years) and 2009-2019 (10 years; IQR, 4-14 years), as was the proportion of donors in this age group under 10 kg (2000-2008: four of 146, 3%; 2009-2019: 14 of 228, 6%). CONCLUSIONS: Despite its overall effect on organ donation rates, the National Reform Program was not effective in increasing the numbers of donors under 16 years of age. Relying on broad initiatives for adult donors may not be appropriate for achieving this aim.

A Review of Genetic and Physiological Disease Mechanisms Associated With Cav1 Channels: Implications for Incomplete Congenital Stationary Night Blindness Treatment.

Calcium channels are crucial to a number of cellular functions. The high voltage-gated calcium channel family comprise four heteromeric channels (Cav1.1-1.4) that function in a similar manner, but that have distinct expression profiles. Three of the pore-forming α1 subunits are located on autosomes and the forth on the X chromosome, which has consequences for the type of pathogenic mutation and the disease mechanism associated with each gene. Mutations in this family of channels are associated with malignant hyperthermia (Cav1.1), various QT syndromes (Cav1.2), deafness (Cav1.3), and incomplete congenital stationary night blindness (iCSNB; Cav1.4). In this study we performed a bioinformatic analysis on reported mutations in all four Cav α1 subunits and correlated these with variant frequency in the general population, phenotype and the effect on channel conductance to produce a comprehensive composite Cav1 mutation analysis. We describe regions of mutation clustering, identify conserved residues that are mutated in multiple family members and regions likely to cause a loss- or gain-of-function in Cav1.4. Our research highlights that therapeutic treatments for each of the Cav1 channels will have to consider channel-specific mechanisms, especially for the treatment of X-linked iCSNB.

Small molecules restore the function of mutant CLC5 associated with Dent disease.

Dent disease type 1 is caused by mutations in the CLCN5 gene that encodes CLC5, a 2Cl- /H+ exchanger. The CLC5 mutants that have been functionally analysed constitute three major classes based on protein expression, cellular localization and channel function. We tested two small molecules, 4-phenylbutyrate (4PBA) and its analogue 2-naphthoxyacetic acid (2-NOAA), for their effect on mutant CLC5 function and expression by whole-cell patch-clamp and Western blot, respectively. The expression and function of non-Class I CLC5 mutants that have reduced function could be restored by either treatment. Cell viability was reduced in cells treated with 2-NOAA. 4PBA is a FDA-approved drug for the treatment of urea cycle disorders and offers a potential therapy for Dent disease.

Immediate and long-term consequences of vascular toxicity during zebrafish development.

Proper formation of the vascular system is necessary for embryogenesis, and chemical disruption of vascular development may be a key event driving developmental toxicity. In order to test the effect of environmental chemicals on this critical process, we evaluated a quantitative assay in transgenic zebrafish using angiogenesis inhibitors that target VEGFR2 (PTK787) or EGFR (AG1478). Both PTK787 and AG1478 exposure impaired intersegmental vessel (ISV) sprouting, while AG1478 also produced caudal and pectoral fin defects at concentrations below those necessary to blunt ISV morphogenesis. The functional consequences of vessel toxicity during early development included decreased body length and survival in juvenile cohorts developmentally exposed to inhibitor concentrations sufficient to completely block ISV sprouting angiogenesis. These data show that concentration-dependent disruption of the presumed targets for these inhibitors produce adverse outcomes at advanced life stages.

Designing Endocrine Disruption Out of the Next Generation of Chemicals.

A central goal of green chemistry is to avoid hazard in the design of new chemicals. This objective is best achieved when information about a chemical's potential hazardous effects is obtained as early in the design process as feasible. Endocrine disruption is a type of hazard that to date has been inadequately addressed by both industrial and regulatory science. To aid chemists in avoiding this hazard, we propose an endocrine disruption testing protocol for use by chemists in the design of new chemicals. The Tiered Protocol for Endocrine Disruption (TiPED) has been created under the oversight of a scientific advisory committee composed of leading representatives from both green chemistry and the environmental health sciences. TiPED is conceived as a tool for new chemical design, thus it starts with a chemist theoretically at "the drawing board." It consists of five testing tiers ranging from broad in silico evaluation up through specific cell- and whole organism-based assays. To be effective at detecting endocrine disruption, a testing protocol must be able to measure potential hormone-like or hormone-inhibiting effects of chemicals, as well as the many possible interactions and signaling sequellae such chemicals may have with cell-based receptors. Accordingly, we have designed this protocol to broadly interrogate the endocrine system. The proposed protocol will not detect all possible mechanisms of endocrine disruption, because scientific understanding of these phenomena is advancing rapidly. To ensure that the protocol remains current, we have established a plan for incorporating new assays into the protocol as the science advances. In this paper we present the principles that should guide the science of testing new chemicals for endocrine disruption, as well as principles by which to evaluate individual assays for applicability, and laboratories for reliability. In a 'proof-of-principle' test, we ran 6 endocrine disrupting chemicals (EDCs) that act via different endocrinological mechanisms through the protocol using published literature. Each was identified as endocrine active by one or more tiers. We believe that this voluntary testing protocol will be a dynamic tool to facilitate efficient and early identification of potentially problematic chemicals, while ultimately reducing the risks to public health.

Gene construction, expression and functional testing of an inotropic peptide from the venom of the black scorpion Hottentotta judaicus.

Anti-insect depressant toxins represent a subfamily of scorpion venom-derived ß-toxins that are polypeptides composed of 61-65 amino acid residues stabilized by four disulfide bridges. These toxins affect the activation of voltage-sensitive sodium channels (NaScTx) and exhibit the preferential ability to induce flaccid paralysis in insect larvae. Here we demonstrate the recombinant expression of the novel cardiac inotropic peptide (Bj-IP) that was classified as an anti-insect depressant ßNaScTx isolated from the venom of Hottentotta judaicus. By using "splicing by overlap extension" (SOE)-PCR, allowing for the first time one step de novo synthesis of long-chain scorpion toxin genes, we generated a codon-optimized DNA fragment of Bj-IP for cloning into the Escherichia coli vector pQE30. Moreover, the gene of interest was fused to a 6xHis coding DNA sequence. Subsequent recombinant expression was performed in E. coli KRX. The purification of the polypeptide was achieved by a combination of NiNTA agarose columns and RP (C(18)) high-performance liquid chromatography. The purified fusion protein was digested with factor Xa resulting in the elution of Bj-IP. The yield of recombinant Bj-IP expression was approximately 4.5 mg per liter of culture. Mass spectrometry confirmed the theoretical total mass of Bj-IP (6608 Da). Tag-free Bj-IP was refolded in guanidine chloride buffer with a glutathione redox system which was supplemented with different additives at 16 °C. Supplementation with 10% glycerol produced Bj-IP folding forms that exhibited reproducible biological activity in mouse cardiomyocytes. Cell contractility was increased by almost 3-fold and decay kinetics were hasten by 47% after administration of Bj-IP. Taken together, here we show the recombinant expression of the functionally active cardiac inotropic peptide Bj-IP, a new ßNaScTx from H. judaicus, for promising pharmacological applications. Furthermore, our data suggest that the use of SOE-PCR may help to facilitate in future the high throughput of cloning and/or modification of scorpion toxin genes.

Inhibition of protein tyrosine phosphatase activity mediates epidermal growth factor receptor signaling in human airway epithelial cells exposed to Zn2+.

Epidemiological studies have implicated zinc (Zn2+) in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads to Src-dependent activation of EGFR signaling in B82 and A431 cells. In order to elucidate the mechanism of Zn2+-induced EGFR activation in HAEC, we treated HAEC with 500 microM ZnSO4 for 5-20 min and measured the state of activation of EGFR, c-Src and PTPs. Western blots revealed that exposure to Zn2+ results in increased phosphorylation at both trans- and autophosphorylation sites in the EGFR. Zn2+-mediated EGFR phosphorylation did not require ligand binding and was ablated by the EGFR kinase inhibitor PD153035, but not by the Src kinase inhibitor PP2. Src activity was inhibited by Zn2+ treatment of HAEC, consistent with Src-independent EGFR transactivation in HAEC exposed to Zn2+. The rate of exogenous EGFR dephosphorylation in lysates of HAEC exposed to Zn2+ or V4+ was significantly diminished. Moreover, exposure of HAEC to Zn2+ also resulted in a significant impairment of dephosphorylation of endogenous EGFR. These data show that Zn2+-induced activation of EGFR in HAEC involves a loss of PTP activities whose function is to dephosphorylate EGFR in opposition to baseline EGFR kinase activity. These findings also suggest that there are marked cell-type-specific differences in the mechanism of EGFR activation induced by Zn2+ exposure.

Second trimester abortion by laminaria followed by vaginal misoprostol or intrauterine prostaglandin F2alpha: a randomized trial.

Intra-amniotic injection, as well as intravaginal application of prostaglandins, have been used to terminate second trimester pregnancies. There is as yet no consensus as to the most efficient protocol of such late abortions. Our goal was to compare the efficacy of intra-amniotic injection of prostaglandin F2 alpha (PGF2alpha) and intravaginal application of misoprostol in terminating second trimester pregnancies after pretreatment with intracervical laminaria. Women with live fetuses and requesting second trimester abortions were randomized into two groups. Eighteen hours following the insertion of intracervical laminaria, women were treated with either intra-amniotic injection of 40 mg PGF2alpha, or 12 hourly doses (to a maximum of 4 doses) of 200 mcg misoprostol. Fifty women were randomly assigned to each group. Failure to abort within 24 h of initiation of treatment occurred in 6 patients (12%) in the misoprostol group and 14 (28%) of the PGF2alpha group (p = 0.04). Mean time of induction of pharmacologic treatment to abortion was 13.6 h in the misoprostol group and 10.7 h in the PGF2alpha group (p = 0.03). The mean number of analgesic injections given were 0.8 in the misoprostol group and 1.6 in the PGF2alpha group (p = 0.0001). Only the method of abortion was predictive of abortion success and not other variables such as patient age, gestational age, gravidity, or parity. Following intracervical laminaria, vaginal misoprostol has been found to be more effective and less painful, compared with intra-amniotic PGF2alpha, for the termination of second trimester pregnancies with live fetuses.

Cationic proteins of neutrophil azurophilic granules: protein-protein interaction and blockade of NADPH oxidase activation.

We have previously reported inhibition of cell-free activation of the neutrophil superoxide-generating NADPH oxidase by a soluble cationic protein of neutrophil granules and by low concentrations of human defensin. Subcellular fractionation carried out in the current study indicated that the inhibitory substance was derived from azurophilic granules, was released into the medium on cell stimulation, and was resistant to phenylmethylsulfonyl fluoride (PMSF). Phorbol ester was the most effective stimulus for the release of the blocking activity. The possibility was raised that granule protein(s) act in vivo as negative modulators of superoxide production. Gel filtration of granule extract revealed a markedly retarded protein peak exhibiting oxidase-blocking activity and containing lysozyme as the main protein. Because lysozyme did not exert inhibitory effects on oxidase activation, association of the inhibitory protein with lysozyme was assumed. Indeed a column of immobilized lysozyme retained a fraction of the granule extract's oxidase-blocking activity. Elution with a low-pH buffer recovered a component capable of inhibition of the NADPH oxidase in stimulated neutrophils and in the cell-free system. The main 29-kDa protein band in the eluted fraction was identified as proteinase 3, a serine protease of azurophilic granules. Enzymatically active as well as PMSF-blocked conventionally purified proteinase 3 interfered with phorbol myristate acetate-induced superoxide release. These findings support the hypothesis that exocytosed granule constituents may prevent excessive activation of the NADPH oxidase.

Possible Role of Cbr, an Algal Early-Light-Induced Protein, in Nonphotochemical Quenching of Chlorophyll Fluorescence.

The unicellular green alga Dunaliella bardawil exhibits typical responses to excessive light when starved for sulfate under normal light (60 [mu]E m-2 s-1) but not under low light (14 [mu]E m-2 s-1). Algae were analyzed during several days of sulfate starvation for nonphotochemical quenching of chlorophyll fluorescence in the absence or presence of the uncouplers SF-6847 (SF) or carbonyl cyanide p- trifluoromethoxyphenyl hydrazone. Parallel analyses followed two light-stress responses: (a) violaxanthin conversion to zeaxanthin and (b) accumulation of Cbr, a protein analogous to plant early-light-induced proteins and implicated in zeaxanthin binding. In cells starved under normal light SF inhibited nonphotochemical quenching during the first 24 h, but not from 40 h onward. In cells starved under low light SF inhibited nonphotochemical quenching throughout the starvation period. Under normal light accumulation of zeaxanthin was nearly maximal by 24 h, but Cbr was fully induced only by 40h. Under low light zeaxanthin accumulated slowly but no Cbr was evident. These results suggest that during exposure to excessive light, the initial pH gradient-dependent, Cbr-independent mode of nonphotochemical quenching is modified to become less dependent on pH gradient and requires Cbr.

Past and present contraceptive behavior of new Soviet immigrant women in Israel.

This exploratory research investigated past and current use of contraceptives among a purposive sample of 117 new immigrant women from the Commonwealth of Independent States (former Soviet Union). The findings confirm the widespread use of induced abortion (IA) as a method of birth control before immigration. Fifty-eight percent of the sample had had at least one IA, and the average was 2.7 IA. The most commonly used types of contraception before immigration were the pill, safe days, withdrawal, and the IUD. Currently used types of contraception were the IUD, safe days, withdrawal, and condoms; however only 45% of the sample were currently using any type of contraception. Of particular interest were the relatively high reported use of the pill before immigration and low current use, and the former low level of condom use and its increase in popularity in Israel. Despite the availability of more effective methods of birth control, safe days and withdrawal remain commonly used. The high cost of the pill was mentioned as a deterrent to its current use. Despite the high prior level of IA, the majority of women in this sample (84%) preferred today to use other birth control methods, and would like the opportunity to receive professional advice. These findings support the need for educational efforts directed toward new immigrant women from CIS.

PIP: In early 1992 in Israel, 117 recent Jewish immigrant women from Belarus, Russia, and the Ukraine completed a questionnaire designed to examine their past and current contraceptive behavior. The mean number of months in Israel was 2.7 months. The mean number of children among the 90 women with children was 1.74 (range 1-5). 58.1% of all 117 women had had at least one induced abortion. The mean number of induced abortions per woman was 2.7 (range 1-12). More than 40% of women who had had at least one induced abortion had at least 3 induced abortions. 104 women were or had been married, or had a steady partner. The most frequently used family planning methods in the past were oral contraceptives (OCs) (33.7%), safe days (24%), withdrawal (15.3%), and IUD (15.3%). Only 4% used condoms in the past. Current family planning methods used included IUD (23%), safe days (23%), withdrawal (18.3%), and condoms (13.5%). Only 2.9% currently used OCs. The mean number of methods currently used was 1.8. Reasons for not using contraception before immigration were: not safe (18%), reduced sexual pleasure (11%), not available (9%), no steady sexual partner (7%), and desire to conceive (5%). After immigration, reasons for not using contraception were too expensive (11%) (especially OCs) and not safe (9%). 84% preferred to use contraception rather than undergo an induced abortion. 62% wanted professional advise on contraception. These findings illustrate the need to provide new immigrant women from the Commonwealth of Independent States with accurate information about the health effects of various family planning methods.

Cbr, an algal homolog of plant early light-induced proteins, is a putative zeaxanthin binding protein.

The cbr gene, previously cloned from the unicellular green alga Dunaliella bardawil, is transcriptionally and translationally activated in parallel to accelerated carotenogenesis in response to light stress conditions. The product of cbr, structurally similar to Elips (early light-induced proteins of higher plants), is associated with a minor light harvesting complexes of photosystem II component (Levy, H., Gokhman, I., and Zamir, A. (1992) J. Biol. Chem. 267, 18831-18836). This study examines the relationship between the induction of Cbr and another plant response to light stress, the deepoxidation of violaxanthin to zeaxanthin. A parallel between the two processes was observed in cells exposed to high light, starved for sulfate, or treated with norflurazon, a herbicide inducing photooxidative damage by inhibiting de novo carotenoid biosynthesis. When highly illuminated cells were returned to normal light, Cbr decayed in parallel to the reepoxidation of zeaxanthin to violaxanthin. Evidence for the physical association of Cbr and zeaxanthin was provided by nondenaturing gel electrophoresis. In cells transferred from low to high light, zeaxanthin was associated with the faster migrating of two electrophoretically resolved fractions of light harvesting complexes of photosystem II that also contained Cbr. In cells growing under normal light, violaxanthin was bound equally to the two fractions. Based on these results we propose that Cbr/early light-induced proteins bind zeaxanthin to form photoprotective complexes within the light-harvesting antennae.

Defensin interferes with the activation of neutrophil NADPH oxidase in a cell-free system.

HNP-1 [correction of NHP-1], a member of a family of microbicidal cationic, cystine-rich polypeptides abundant in the azurophilic granules of polymorphonuclear leukocytes (PMN) interfered with detergent-induced cell-free activation of superoxide-producing NADPH oxidase. IC50 of 3 microM has been evaluated for this dose dependent effect of defensin. Preactivated oxidase was not affected by HNP-1. The inhibitory effect could be relieved by raising the concentration of the cytosol or by the addition of a partially purified p47-phox. NADPH oxidase activated in the presence of HNP-1 exhibited an increased affinity for NADPH.

Establishment of cell lines from somatic cell hybrids between human monocytes and mouse myeloma cells.

To study human monocyte functions, we attempted to immortalize human monocytes by producing somatic cell hybrids between such monocytes and the mouse myeloma cell line NSI. In this study we report the successful establishment of eight hybrid cell lines that have been grown in culture for more than a year, and some of them retained part of the human chromosome complement, as well as monocyte markers and activities. Karyotype analysis of these hybrid lines revealed that cells of seven out of eight of the lines contained one to 16 human chromosomes and in four of them, more than nine human chromosomes were observed. Several of the cell lines expressed monocytic markers and functions. Thus, in two of the hybrid lines nonspecific esterase could be demonstrated in 10 to 29% of the cells, and Fc receptors were demonstrated in three of the hybrid cell lines. Significant levels of human ferritin were detected in one of the lines, and two other cell lines secreted interleukin 1-like substance into the culture medium. These results encourage us to use human-mouse somatic cell hybridization as an approach for the establishment of human monocyte cell lines, which will preserve their functions and produce monocyte-derived factors.

Constitutive secretion of interleukin 1 by human monocytes.

The constitutive and lipopolysaccharide (LPS)-induced secretion of interleukin 1 (IL 1) by cultured human monocytes and macrophages has been studied. Both freshly obtained monocytes and their culture-derived macrophages were induced by LPS to secrete similar amounts of IL 1. Such induction, however, was accompanied by the secretion of dialyzed inhibitory activity. Constitutive secretion of IL 1 was detected in concentrated supernatants of monocyte cultures. The factor obtained constitutively did not manifest significant inhibitory activity. A method is described for the recovery of IL 1-containing supernatants in serum- and other stimulant-free medium. The biological activities of the constitutively secreted IL 1 were similar to the LPS-induced activities. The constitutive secretion of IL 1 was not equally distributed in the entire monocyte population. We found that a small fraction of loosely adherent monocytes secreted higher amounts of IL 1 than the strongly adherent monocytes. However, the property of higher secretion of IL 1 was not stable and disappeared following monocyte cultivation. Thus, constitutive activity of IL 1 could be recovered either by concentrating the culture supernatants or by enriching a subset of monocytes with higher IL 1 activity.

Antigen presentation and regulatory functions of human monocytes in the in vitro response of lymphocytes against purified protein derivative of tuberculin (PPD).

Monocyte functions in the primary and secondary proliferative responses of human lymphocytes against purified protein derivative of tuberculin (PPD) have been studied. By applying the antigens either directly to the responding cells or on antigen-treated autologous monocytes, it was possible to distinguish between two different monocyte functions: antigen presentation and regulation. Their helper function in lymphocyte proliferation was found in cell-free supernatants of autologous and allogeneic monocytes and was expressed better in the secondary rather than in the primary responses. The antigen-presenting function of monocytes, however, was found to be more substantial in the primary response of lymphocytes. These results suggest that macrophage dependency of certain immune functions should be considered in regard to the various functions of macrophages, since at least quantitative differences for each of the macrophage functions were found in the primary and secondary proliferative responses.