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ObjectiveTo examine whether the pandemic in 2020 caused changes in psychiatric hospital cases, the percentage of patients exposed to coercive interventions, and aggressive incidents.MethodsWe used the case registry for coercive measures of the State of Baden-Wuerttemberg, comprising case-related data on mechanical restraint, seclusion, physical restraint, and forced medication in each of the State's 31 licensed hospitals treating adults, to compare data from 2019 and 2020.ResultsThe number of cases in adult psychiatry decreased by 7.6% from 105,782 to 97,761. The percentage of involuntary cases increased from 12.3 to 14.1%, and the absolute number of coercive measures increased by 4.7% from 26,269 to 27,514. The percentage of cases exposed to any kind of coercive measure increased by 24.6% from 6.5 to 8.1%, and the median cumulative duration per affected case increased by 13.1% from 12.2 to 13.8 hrs, where seclusion increased more than mechanical restraint. The percentage of patients with aggressive incidents, collected in 10 hospitals, remained unchanged.ConclusionsWhile voluntary cases decreased considerably during the pandemic, involuntary cases increased slightly. However, the increased percentage of patients exposed to coercion is not only due to a decreased percentage of voluntary patients, as the duration of coercive measures per case also increased. The changes that indicate deterioration in treatment quality were probably caused by the multitude of measures to manage the pandemic. The focus of attention and internal rules as well have shifted from prevention of coercion to prevention of infection.

IntroductionIn a meta-analysis of international studies, 17% of admitted patients in psychiatric hospitals had exhibited violent behavior toward others. Reported data from studies in Germany were considerably lower until recent years. However, studies examining only single hospitals, as well as the quality of the data itself, have raised questions as to the validity of these findings. Indeed, a debate currently exists as to whether there has, in fact, been an increase of violent incidents in German mental institutions.MethodsIn a group of 10 hospitals serving about half the population of the Federal State of Baden-Wuerttemberg with 11 million inhabitants, the Staff Observation Aggression Scale-Revised (SOAS-R) was introduced into patients' electronic charts as part of routine documentation. Data recording was strongly supported by staff councils and unions. A completed data set is now available for the year 2019. For one hospital, data are available since 2006. Due to some doubts with respect to fully covering self-directed aggression, we restricted the analysis to aggression toward others and toward objects.ResultsIn 2019, 17,599 aggressive incidents were recorded in 64,367 admissions (1,660 staying forensic psychiatric inpatients included). 5,084 (7.90%) of the admitted cases showed aggressive behavior toward others. Variation between hospitals was low to modest (SD = 1.50). The mean SOAS-R score was 11.8 (SD between hospitals 1.20%). 23% of the incidents resulted in bodily harm. The percentage of patients showing violent behavior was highest among patients with organic disorders (ICD-10 F0) and lowest among patients with addictive or affective disorders (F1, F3, F4). Forensic psychiatry had the highest proportion of cases with aggressive behavior (20.54%), but the number of incidents per bed was lower than in general adult psychiatry and child and adolescent psychiatry (indicating a lower risk for staff). In the hospital with longer-term recordings available, an increase could be observed since 2010, with considerable variation between years.ConclusionsThis is the most robust estimate of the frequency of violent incidents in German psychiatric hospitals thus far. The incidence is about half of what has been reported internationally, probably due to sample selection bias in previous studies and a relatively high number of hospital beds in Germany. Available data suggest an increase of violent incidents over the last ten years; however, it is unclear to which extent this is due to increased reporting.

BACKGROUND:Activation of the AMP-activated protein kinase (AMPK) is an attractive approach for the treatment of type 2 diabetes. AMPK activation reduces glucose levels in animal models of type 2 diabetes by increasing glucose uptake in skeletal muscles and reducing hepatic glucose production. Furthermore, AMPK activation ameliorates hepatic steatosis in animal models. For the clinical development of AMPK activators it is essential to have a reliable target engagement marker for appropriate dose finding and to support proof of clinical principle. While the activation of AMPK by quantification of the phosphorylation of AMPK at Thr172 in target tissues can be assessed pre-clinically, this is not feasible in clinical studies. Therefore, we attempted to identify and translate a peripheral target engagement biomarker downstream of AMPK activation for clinical use in blood samples. METHODS:For pharmacological activation of AMPK, two AMPK activators were synthesized (compound 1 and 2). A compound with structural similarities but no pharmacological effect on AMPK phosphorylation was synthesized as negative control (compound 3). Whole blood from healthy volunteers was incubated with an AMPK activator for up to 6 hours and mRNA sequencing was performed. Additionally, human PBMCs were isolated to evaluate Thr172-phosphorylation of AMPK in Western blots. In order to enable identification of translatable biomarker candidates, blood samples from HanWistar rats treated for two weeks with an AMPK activator were also subjected to mRNA sequencing. Furthermore, concentration-response curves for four biomarker candidates were recorded in human blood samples using Nanostring nCounter technology. Finally, ZDF rats were treated with increasing doses of compound 2 for five weeks to investigate the glucose-lowering efficacy. To investigate changes of mRNA expression of two selected biomarker candidates in this ZDF rat study, qRT-PCR was performed. RESULTS:Pharmacological activation of AMPK in human PBMCs revealed an increase in Thr172-phosphorylation of AMPK, confirming target engagement in these blood cells. RNA sequencing of human blood samples identified 608 deregulated genes after AMPK activation. Additionally, AMPK activation led to deregulation of 367 genes in whole blood from HanWistar rats which mapped to the respective human genes. 22 genes out of the intersection of genes deregulated in both species are proposed as potential translatable target engagement biomarker candidates. The most prominent genes were transmembrane glycoprotein NMB (GPNMB, osteoactivin), calcium-binding protein A9 (S100A9), peptidoglycan recognition protein (PGLYRP1) and Ras homolog gene family, member B (RHOB). Specificity for AMPK was shown by testing inactive compound 3 in HanWistar rats. The exposure-effect relationship for GPNMB was investigated in a subchronic study in diabetic ZDF rats. GPNMB showed a dose-dependent up-regulation both acutely and after subchronic dosing. GPNMB up-regulation correlated with an increased Thr172-phosphorylation of AMPK in liver and quadriceps muscle in rats. CONCLUSION:GPNMB has been identified as a translatable target engagement biomarker for use in clinical studies.

The development of thrombocytopenia in sepsis is a poor prognostic indicator associated with a significantly increased mortality risk. Mechanisms underlying this phenomenon remain to be clearly elucidated. Matrix metalloproteinases (MMPs) are enzymes that regulate the turnover of the extra-cellular matrix. MMP-2 is recognised as a platelet agonist with MMP-9 proposed as an inhibitor of platelet activation. The existence of MMP-9 in platelets is a subject of debate. There is limited evidence thus far to suggest that toll-like receptor 4 (TLR-4) and platelet-leukocyte aggregate (PLA) formation may be implicated in the development of sepsis-associated thrombocytopenia.To investigate whether MMP -2/-9, toll-like receptor 4 (TLR-4) or platelet-leukocyte aggregate (PLA) formation are implicated in a decline in platelet numbers during septic shock.This was an observational study which recruited healthy controls, non-thrombocytopenic septic donors and thrombocytopenic septic donors. MMP-2, MMP-9 and TLR-4 platelet surface expression as well as PLA formation was examined using flow cytometry. In addition MMP-2 and MMP-9 were examined by gelatin zymography and enzyme-linked immunosorbent assay (ELISA) using a 3 compartment model (plasma, intraplatelet and platelet membrane).There was no difference found in MMP-2, MMP-9 or TLR-4 levels between non-thrombocytopenic and thrombocytopenic septic donors. PLA formation was increased in thrombocytopenic patients. MMP-9 was detected in platelets using flow cytometry, gelatin zymography and ELISA techniques.Platelet consumption into PLAs may account for the development of thrombocytopenia in septic shock. MMP-9 is found in platelets and it is upregulated during septic shock.

Evidence is emerging that systemic inflammation after trauma drives structural and functional impairment of cardiomyocytes and leads to cardiac dysfunction, thus worsening the outcome of polytrauma patients. This study investigates the structural and molecular changes in heart tissue 4 h after multiple injuries with additional hemorrhagic shock using a clinically relevant rodent model of polytrauma. We determined mediators of systemic inflammation (keratinocyte chemoattractant, macrophage chemotactic protein 1), activated complement component C3a and cardiac troponin I in plasma and assessed histological specimen of the mouse heart via standard histomorphology and immunohistochemistry for cellular and subcellular damage and ongoing apoptosis. Further we investigated spatial and quantitative changes of connexin 43 by immunohistochemistry and western blotting. Our results show significantly increased plasma levels of both keratinocyte chemoattractant and cardiac troponin I 4 h after polytrauma and 2 h after induction of hypovolemia. Although we could not detect any morphological changes, immunohistochemical evaluation showed increased level of tissue high-mobility group box 1, which is both a damage-associated molecule and actively released as a danger response signal. Additionally, there was marked lateralization of the cardiac gap-junction protein connexin 43 following combined polytrauma and hemorrhagic shock. These results demonstrate a molecular manifestation of remote injury of cardiac muscle cells in the early phase after polytrauma and hemorrhagic shock with marked disruption of the cardiac gap junction. This disruption of an important component of the electrical conduction system of the heart may lead to arrhythmia and consequently to cardiac dysfunction.

There are two major methodical approaches with which changes of status in stomatal pores are addressed: indirectly by measurement of leaf transpiration, and directly by measurement of stomatal apertures. Application of the former method requires special equipment, whereas microscopic images are utilized for the direct measurements. Due to obscure visualization of cell boundaries in intact leaves, a certain degree of invasive leaf manipulation is often required. Our aim was to develop a protocol based on the minimization of leaf manipulation and the reduction of analysis completion time, while still producing consistent results. We applied rhodamine 6G staining of Arabidopsis thaliana leaves for stomata visualization, which greatly simplifies the measurement of stomatal apertures. By using this staining protocol, we successfully conducted analyses of stomatal responses in Arabidopsis leaves to both closure and opening stimuli. We performed long-term monitoring of living stomata and were able to document the same leaf before and after treatment. Moreover, we developed a protocol for rapid-fixation of epidermal peels, which enables high throughput data analysis. The described method allows analysis of stomatal apertures with minimal leaf manipulation and usage of the same leaf for sequential measurements, and will facilitate the analysis of several lines in parallel.

MicroRNAs (miRNAs) are short non-coding RNA species which are important post-transcriptional regulators of gene expression and play an important role in the pathogenesis of diabetic nephropathy. miRNAs are present in urine in a remarkably stable form packaged in extracellular vesicles, predominantly exosomes. In the present study, urinary exosomal miRNA profiling was conducted in urinary exosomes obtained from 8 healthy controls (C), 8 patients with type II diabetes (T2D) and 8 patients with type II diabetic nephropathy (DN) using Agilent´s miRNA microarrays. In total, the expression of 16 miRNA species was deregulated (>2-fold) in DN patients compared to healthy donors and T2D patients: the expression of 14 miRNAs (miR-320c, miR-6068, miR-1234-5p, miR-6133, miR-4270, miR-4739, miR-371b-5p, miR-638, miR-572, miR-1227-5p, miR-6126, miR-1915-5p, miR-4778-5p and miR-2861) was up-regulated whereas the expression of 2 miRNAs (miR-30d-5p and miR-30e-5p) was down-regulated. Most of the deregulated miRNAs are involved in progression of renal diseases. Deregulation of urinary exosomal miRNAs occurred in micro-albuminuric DN patients but not in normo-albuminuric DN patients. We used qRT-PCR based analysis of the most strongly up-regulated miRNAs in urinary exosomes from DN patients, miRNAs miR-320c and miR-6068. The correlation of miRNA expression and micro-albuminuria levels could be replicated in a confirmation cohort. In conclusion, urinary exosomal miRNA content is altered in type II diabetic patients with DN. Deregulated miR-320c, which might have an impact on the TGF-β-signaling pathway via targeting thrombospondin 1 (TSP-1) shows promise as a novel candidate marker for disease progression in type II DN that should be evaluated in future studies.

Genome-wide association studies (GWASs) have identified several, mainly co-dominantly acting, single-nucleotide polymorphisms (SNPs) associated with Hodgkin lymphoma (HL). We searched for recessively acting disease loci by performing an analysis of runs of homozygosity (ROH) based on windows of homozygous SNP-blocks and by calculating genomic inbreeding coefficients on a SNP-wise basis. We used data from a previous GWAS with 906 cases and 1217 controls from a population with a long history of no matings between relatives. Ten recurrent ROHs were identified among 25 055 ROHs across all individuals but their association with HL was not genome-wide significant. All recurrent ROHs showed significant evidence for natural selection. As a novel finding genomic inbreeding among cases was significantly higher than among controls (P = 2.11*10-14) even after correcting for covariates. Higher inbreeding among the cases was mainly based on a group of individuals with a higher average length of ROHs per person. This result suggests a correlation of higher levels of inbreeding with higher cancer incidence and might reflect the existence of recessive alleles causing HL. Genomic inbreeding may result in a higher expression of deleterious recessive genes within a population.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

In the event of a dirty bomb scenario or an industrial nuclear accident, a significant dose of volatile radionuclides such as 137Cs and 90Sr may be dispersed into the atmosphere as a component of fallout and inhaled or ingested by hundreds and thousands of people. To study the effects of prolonged exposure to ingested radionuclides, we have performed long-term (30 day) internal-emitter mouse irradiations using soluble-injected 137CsCl and 90SrCl2 radioisotopes. The effect of ionizing radiation on the induction and repair of DNA double strand breaks (DSBs) in peripheral mouse lymphocytes in vivo was determined using the γ-H2AX biodosimetry marker. Using a serial sacrifice experimental design, whole-body radiation absorbed doses for 137Cs (0 to 10 Gy) and 90Sr (0 to 49 Gy) were delivered over 30 days following exposure to each radionuclide. The committed absorbed doses of the two internal emitters as a function of time post exposure were calculated based on their retention parameters and their derived dose coefficients for each specific sacrifice time. In order to measure the kinetic profile for γ-H2AX, peripheral blood samples were drawn at 5 specific timed dose points over the 30-day study period and the total γ-H2AX nuclear fluorescence per lymphocyte was determined using image analysis software. A key finding was that a significant γ-H2AX signal was observed in vivo several weeks after a single radionuclide exposure. A mechanistically-motivated model was used to analyze the temporal kinetics of γ-H2AX fluorescence. Exposure to either radionuclide showed two peaks of γ-H2AX: one within the first week, which may represent the death of mature, differentiated lymphocytes, and the second at approximately three weeks, which may represent the production of new lymphocytes from damaged progenitor cells. The complexity of the observed responses to internal irradiation is likely caused by the interplay between continual production and repair of DNA damage, cell cycle effects and apoptosis.

Immunotherapy targeting glioblastoma initiating cells (GIC) is considered a promising strategy. However, GIC are prone to evade immune response and there is a need for potent adjuvants. IFN-β might enhance the immune response and here we define its net effect on the innate immunogenicity of GIC. The transcriptomes of GIC treated with IFN-β and controls were assessed by microarray-based expression profiling for altered expression of immune regulatory genes. Several genes involved in adaptive and innate immune responses were regulated by IFN-β. We validated these results using reverse transcription (RT)-PCR and flow cytometry for corresponding protein levels. The up-regulation of the NK cell inhibitory molecules HLA-E and MHC class I was balanced by immune stimulating effects including the up-regulation of nectin-2. In 3 out of 5 GIC lines tested we found a net immune stimulating effect of IFN-β in cytotoxicity assays using NKL cells as effectors. IFN-β therefore warrants further investigation as an adjuvant for immunotherapy targeting GIC.

Alzheimer's disease (AD) is characterized by neurotoxic amyloid-ß plaque formation in brain parenchyma and cerebral blood vessels known as cerebral amyloid angiopathy (CAA). Besides CAA, AD is strongly related to vascular diseases such as stroke and atherosclerosis. Cerebrovascular dysfunction occurs in AD patients leading to alterations in blood flow that might play an important role in AD pathology with neuronal loss and memory deficits. Platelets are the major players in hemostasis and thrombosis, but are also involved in neuroinflammatory diseases like AD. For many years, platelets were accepted as peripheral model to study the pathophysiology of AD because platelets display the enzymatic activities to generate amyloid-ß (Aß) peptides. In addition, platelets are considered to be a biomarker for early diagnosis of AD. Effects of Aß peptides on platelets and the impact of platelets in the progression of AD remained, however, ill-defined. The present study explored the cellular mechanisms triggered by Aß in platelets. Treatment of platelets with Aß led to platelet activation and enhanced generation of reactive oxygen species (ROS) and membrane scrambling, suggesting enhanced platelet apoptosis. More important, platelets modulate soluble Aß into fibrillar structures that were absorbed by apoptotic but not vital platelets. This together with enhanced platelet adhesion under flow ex vivo and in vivo and platelet accumulation at amyloid deposits of cerebral vessels of AD transgenic mice suggested that platelets are major contributors of CAA inducing platelet thrombus formation at vascular amyloid plaques leading to vessel occlusion critical for cerebrovascular events like stroke.

Ricin, a member of the A-B family of ribosome-inactivating proteins, is classified as a Select Toxin by the Centers for Disease Control and Prevention because of its potential use as a biothreat agent. In an effort to engineer therapeutics for ricin, we recently produced a collection of alpaca-derived, heavy-chain only antibody VH domains (VHH or "nanobody") specific for ricin's enzymatic (RTA) and binding (RTB) subunits. We reported that one particular RTB-specific VHH, RTB-B7, when covalently linked via a peptide spacer to different RTA-specific VHHs, resulted in heterodimers like VHH D10/B7 that were capable of passively protecting mice against a lethal dose challenge with ricin. However, RTB-B7 itself, when mixed with ricin at a 1 ∶ 10 toxin:antibody ratio did not afford any protection in vivo, even though it had demonstrable toxin-neutralizing activity in vitro. To better define the specific attributes of antibodies associated with ricin neutralization in vitro and in vivo, we undertook a more thorough characterization of RTB-B7. We report that RTB-B7, even at 100-fold molar excess (toxin:antibody) was unable to alter the toxicity of ricin in a mouse model. On the other hand, in two well-established cytotoxicity assays, RTB-B7 neutralized ricin with a 50% inhibitory concentration (IC50) that was equivalent to that of 24B11, a well-characterized and potent RTB-specific murine monoclonal antibody. In fact, RTB-B7 and 24B11 were virtually identical when compared across a series of in vitro assays, including adherence to and neutralization of ricin after the toxin was pre-bound to cell surface receptors. RTB-B7 differed from both 24B11 and VHH D10/B7 in that it was relatively less effective at blocking ricin attachment to receptors on host cells and was not able to form high molecular weight toxin:antibody complexes in solution. Whether either of these activities is important in ricin toxin neutralizing activity in vivo remains to be determined.

When normal subjects fix their eyes upon a stationary target, their gaze is not perfectly still, due to small movements that prevent visual fading. Visual loss is known to cause greater instability of gaze, but reported comparisons with normal subjects using reliable measurement techniques are few. We measured binocular gaze using the magnetic search coil technique during attempted fixation (monocular or binocular viewing) of 4 individuals with childhood-onset of monocular visual loss, 2 individuals with late-onset monocular visual loss due to age-related macular degeneration, 2 individuals with bilateral visual loss, and 20 healthy control subjects. We also measured saccades to visual or somatosensory cues. We tested the hypothesis that gaze instability following visual impairment is caused by loss of inputs that normally optimize the performance of the neural network (integrator), which ensures both monocular and conjugate gaze stability. During binocular viewing, patients with early-onset monocular loss of vision showed greater instability of vertical gaze in the eye with visual loss and, to a lesser extent, in the normal eye, compared with control subjects. These vertical eye drifts were much more disjunctive than upward saccades. In individuals with late monocular visual loss, gaze stability was more similar to control subjects. Bilateral visual loss caused eye drifts that were larger than following monocular visual loss or in control subjects. Accurate saccades could be made to somatosensory cues by an individual with acquired blindness, but voluntary saccades were absent in an individual with congenital blindness. We conclude that the neural gaze-stabilizing network, which contains neurons with both binocular and monocular discharge preferences, is under adaptive visual control. Whereas monocular visual loss causes disjunctive gaze instability, binocular blindness causes both disjunctive and conjugate gaze instability (drifts and nystagmus). Inputs that bypass this neural network, such as projections to motoneurons for upward saccades, remain conjugate.

Progranulin (Pgrn) is a 88 kDa secreted protein with pleiotropic functions including regulation of cell cycle progression, cell motility, wound repair and tumorigenesis. Using microarray based gene expression profiling we have recently demonstrated that the gene for Pgrn, granulin (GRN), is significantly higher expressed in aggressive CD38(+)ZAP-70(+) as compared to indolent CD38(-)ZAP-70(-) chronic lymphocytic leukemia (CLL) cases. Here, we measured Pgrn plasma concentrations by enzyme-linked immunosorbent assay (ELISA) in the Essen CLL cohort of 131 patients and examined Pgrn for association with established prognostic markers and clinical outcome. We found that high Pgrn plasma levels were strongly associated with adverse risk factors including unmutated IGHV status, expression of CD38 and ZAP-70, poor risk cytogenetics (11q-, 17p-) as detected by flourescence in situ hybridization (FISH) and high Binet stage. Pgrn as well as the aforementioned risk factors were prognostic for time to first treatment and overall survival in this series. Importantly, these results could be confirmed in the independent multicentric CLL1 cohort of untreated Binet stage A patients (n = 163). Here, multivariate analysis of time to first treatment revealed that high risk Pgrn (HR = 2.06, 95%-CI = 1.13-3.76, p = 0.018), unmutated IGHV status (HR = 5.63, 95%-CI = 3.05-10.38, p

The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 were produced recombinantly in E. coli BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 µkat(H-Ala-Pro-pNA) L(-1), which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in E. coli at high levels for the first time. The PepN activity reached 1,000 µkat(H-Ala-pNA) L(-1). After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn(2+)-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co(2+)>Zn(2+)>Mn(2+)>Ca(2+)>Mg(2+). PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50°C and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.

As some malaria control programs shift focus from disease control to transmission reduction, there is a need for transmission data to monitor progress. At lower levels of transmission, it becomes increasingly more difficult to measure precisely, for example through entomological studies. Many programs conduct regular cross sectional parasite prevalence surveys, and have access to malaria treatment data routinely collected by ministries of health, often in health management information systems. However, by themselves, these data are poor measures of transmission. In this paper, we propose an approach for combining annual parasite incidence and treatment data with cross-sectional parasite prevalence and treatment seeking survey data to estimate the incidence of new infections in the human population, also known as the force of infection. The approach is based on extension of a reversible catalytic model. The accuracy of the estimates from this model appears to be highly dependent on levels of detectability and treatment in the community, indicating the importance of information on private sector treatment seeking and access to effective and appropriate treatment.

Disruption of circadian rhythm is believed to play a critical role in cancer development. Cryptochrome 1 (CRY1) is a core component of the mammalian circadian clock and we have previously shown its deregulated expression in a subgroup of patients with chronic lymphocytic leukemia (CLL). Using real-time RT-PCR in a cohort of 76 CLL patients and 35 normal blood donors we now demonstrate that differential CRY1 mRNA expression in high-risk (HR) CD38+/immunoglobulin variable heavy chain gene (IgVH) unmutated patients as compared to low-risk (LR) CD38-/IgVH mutated patients can be attributed to down-modulation of CRY1 in LR CLL cases. Analysis of the DNA methylation profile of the CRY1 promoter in a subgroup of 57 patients revealed that CRY1 expression in LR CLL cells is silenced by aberrant promoter CpG island hypermethylation. The methylation pattern of the CRY1 promoter proved to have high prognostic impact in CLL where aberrant promoter methylation predicted a favourable outcome. CRY1 mRNA transcript levels did not change over time in the majority of patients where sequential samples were available for analysis. We also compared the CRY1 expression in CLL with other lymphoid malignancies and observed epigenetic silencing of CRY1 in a patient with B cell acute lymphoblastic leukemia (B-ALL).

BACKGROUND: In November 2010, Sierra Leone distributed over three million long-lasting insecticide-treated nets (LLINs) with the objective of providing protection from malaria to individuals in all households in the country. METHODS: We conducted a nationally representative survey six months after the mass distribution campaign to evaluate its impact on household insecticide-treated net (ITN) ownership and use. We examined factors associated with household ITN possession and use with logistic regression models. RESULTS: The survey included 4,620 households with equal representation in each of the 14 districts. Six months after the campaign, 87.6% of households own at least one ITN, which represents an increase of 137% over the most recent estimate of 37% in 2008. Thirty-six percent of households possess at least one ITN per two household members; rural households were more likely than urban households to have ≥ 1:2 ITN to household members, but there was no difference by socio-economic status or household head education. Among individuals in households possessing ≥ 1 ITN, 76.5% slept under an ITN the night preceding the survey. Individuals in households where the household head had heard malaria messaging, had correct knowledge of malaria transmission, and where at least one ITN was hanging, were more likely to have slept under an ITN. CONCLUSIONS: The mass distribution campaign was effective at achieving high coverage levels across the population, notably so among rural households where the malaria burden is higher. These important gains in equitable access to malaria prevention will need to be maintained to produce long-term reductions in the malaria burden.

Cyclic diguanosine monophosphate (c-di-GMP) and cyclic diadenosine monophosphate (c-di-AMP) are recently identified signaling molecules. c-di-GMP has been shown to play important roles in bacterial pathogenesis, whereas information about c-di-AMP remains very limited. Mycobacterium tuberculosis Rv3586 (DacA), which is an ortholog of Bacillus subtilis DisA, is a putative diadenylate cyclase. In this study, we determined the enzymatic activity of DacA in vitro using high-performance liquid chromatography (HPLC), mass spectrometry (MS) and thin layer chromatography (TLC). Our results showed that DacA was mainly a diadenylate cyclase, which resembles DisA. In addition, DacA also exhibited residual ATPase and ADPase in vitro. Among the potential substrates tested, DacA was able to utilize both ATP and ADP, but not AMP, pApA, c-di-AMP or GTP. By using gel filtration and analytical ultracentrifugation, we further demonstrated that DacA existed as an octamer, with the N-terminal domain contributing to tetramerization and the C-terminal domain providing additional dimerization. Both the N-terminal and the C-terminal domains were essential for the DacA's enzymatically active conformation. The diadenylate cyclase activity of DacA was dependent on divalent metal ions such as Mg(2+), Mn(2+) or Co(2+). DacA was more active at a basic pH rather than at an acidic pH. The conserved RHR motif in DacA was essential for interacting with ATP, and mutation of this motif to AAA completely abolished DacA's diadenylate cyclase activity. These results provide the molecular basis for designating DacA as a diadenylate cyclase. Our future studies will explore the biological function of this enzyme in M. tuberculosis.

Molecular studies of bacterial virulence are enhanced by expression of recombinant DNA during infection to allow complementation of mutants and expression of reporter proteins in vivo. For highly pathogenic bacteria, such as Yersinia pestis, these studies are currently limited because deliberate introduction of antibiotic resistance is restricted to those few which are not human treatment options. In this work, we report the development of alternatives to antibiotics as tools for host-pathogen research during Yersinia pestis infections focusing on the diaminopimelic acid (DAP) pathway, a requirement for cell wall synthesis in eubacteria. We generated a mutation in the dapA-nlpB(dapX) operon of Yersinia pestis KIM D27 and CO92 which eliminated the expression of both genes. The resulting strains were auxotrophic for diaminopimelic acid and this phenotype was complemented in trans by expressing dapA in single and multi-copy. In vivo, we found that plasmids derived from the p15a replicon were cured without selection, while selection for DAP enhanced stability without detectable loss of any of the three resident virulence plasmids. The dapAX mutation rendered Y. pestis avirulent in mouse models of bubonic and septicemic plague which could be complemented when dapAX was inserted in single or multi-copy, restoring development of disease that was indistinguishable from the wild type parent strain. We further identified a high level, constitutive promoter in Y. pestis that could be used to drive expression of fluorescent reporters in dapAX strains that had minimal impact to virulence in mouse models while enabling sensitive detection of bacteria during infection. Thus, diaminopimelic acid selection for single or multi-copy genetic systems in Yersinia pestis offers an improved alternative to antibiotics for in vivo studies that causes minimal disruption to virulence.

Roll Back Malaria set the goal of 100% of households in malaria endemic countries in Africa owning an insecticide-treated mosquito net (ITN) by 2010. Zambia has used mass free distribution campaigns and distribution through antenatal care (ANC) clinics to achieve high coverage.We conducted a probability survey of 801 households in 2008 to assess factors associated with households that lacked an ITN after mass distribution. Community perceptions of barriers to ITN access were also obtained from in-depth interviews with household heads that reported not owning an ITN. Nearly 74% of households in Luangwa district reported owning ≥1 ITN. Logistic regression showed households without a child

BACKGROUND AND METHODS:Malaria in Africa is most severe in young children and pregnant women, particularly in rural and poor households. In many countries, malaria intervention coverage rates have increased as a result of scale up; but this may mask limited coverage in these highest-risk populations. Reports were reviewed from nationally representative surveys in African malaria-endemic countries from 2006 through 2008 to understand how reported intervention coverage rates reflect access by the most at-risk populations. RESULTS:Reports were available from 27 Demographic and Health Surveys (DHSs), Multiple Indicator Cluster Surveys (MICSs), and Malaria Indicator Surveys (MISs) during this interval with data on household intervention coverage by urban or rural setting, wealth quintile, and sex. Household ownership of insecticide-treated mosquito nets (ITNs) varied from 5% to greater than 60%, and was equitable by urban/rural and wealth quintile status among 13 (52%) of 25 countries. Malaria treatment rates for febrile children under five years of age varied from less than 10% to greater than 70%, and while equitable coverage was achieved in 8 (30%) of 27 countries, rates were generally higher in urban and richest quintile households. Use of intermittent preventive treatment in pregnant women varied from 2% to more than 60%, and again tended to be higher in urban and richest quintile households. Across all countries, there were no significant male/female inequalities seen for children sleeping under ITNs or receiving antimalarial treatment for febrile illness. Parasitemia and anemia rates from eight national surveys showed predominance in poor and rural populations. CONCLUSIONS/SIGNIFICANCE:Recent efforts to scale up malaria intervention coverage have achieved equity in some countries (especially with ITNs), but delivery methods in other countries are not addressing the most at-risk populations. As countries seek universal malaria intervention coverage, their delivery systems must reach the rural and poor populations; this is not a small task, but it has been achieved in some countries.

Adenoid cystic carcinoma (ACC) is the second most common malignant neoplasm of the salivary glands. Most patients survive more than 5 years after surgery and postoperative radiation therapy. The 10 year survival rate, however, drops to 40%, due to locoregional recurrences and distant metastases. Improving long-term survival in ACC requires the development of more effective systemic therapies based on a better understanding of the biologic behavior of ACC. Much preclinical research in this field involves the use of cultured cells and, to date, several ACC cell lines have been established. Authentication of these cell lines, however, has not been reported. We performed DNA fingerprint analysis on six ACC cell lines using short tandem repeat (STR) examinations and found that all six cell lines had been contaminated with other cells. ACC2, ACC3, and ACCM were determined to be cervical cancer cells (HeLa cells), whereas the ACCS cell line was composed of T24 urinary bladder cancer cells. ACCNS and CAC2 cells were contaminated with cells derived from non-human mammalian species: the cells labeled ACCNS were mouse cells and the CAC2 cells were rat cells. These observations suggest that future studies using ACC cell lines should include cell line authentication to avoid the use of contaminated or non-human cells.