Caution is required when using health facility-based data to evaluate the health impact of malaria control efforts in Africa.
The global health community is interested in the health impact of the billions of dollars invested to fight malaria in Africa. A recent publication used trends in malaria cases and deaths based on health facility records to evaluate the impact of malaria control efforts in Rwanda and Ethiopia. Although the authors demonstrate the use of facility-based data to estimate the impact of malaria control efforts, they also illustrate several pitfalls of such analyses that should be avoided, minimized, or actively acknowledged. A critique of this analysis is presented because many country programmes and donors are interested in evaluating programmatic impact with facility-based data. Key concerns related to: 1) clarifying the objective of the analysis; 2) data validity; 3) data representativeness; 4) the exploration of trends in factors that could influence malaria rates and thus confound the relationship between intervention scale-up and the observed changes in malaria outcomes; 5) the analytic approaches, including small numbers of patient outcomes, selective reporting of results, and choice of statistical and modeling methods; and 6) internal inconsistency on the strength and interpretation of the data. In conclusion, evaluations of malaria burden reduction using facility-based data could be very helpful, but those data should be collected, analysed, and interpreted with care, transparency, and a full recognition of their limitations.


An immunohistochemical study on cutaneous sensory receptors after chronic median nerve compression in man.
Carpal tunnel syndrome represents the most frequent chronic compressive neuropathy in man and hence may be investigated as a spontaneous model of peripheral nerve damage and repair. In the present report the fate of nerve fibers in the digital skin after long-lasting median nerve compression has been investigated immunohistochemically in comparison to normal digital skin, with special consideration to sensory endings and encapsulated receptors. The presence has been documented of the neurospecific marker PGP 9.5, the glia-associated protein S-100, and the neuropeptides CGRP and CPON which are mainly associated with the sensory and sympathetic nerve fibers respectively. The morphology and distribution of nerve fibers and corpuscles appeared comparable to that of normal digital skin; a reduction in the density of sensory receptors has, however, been observed, although not to the degree that was expected to explain the clinical deficits. It has been also demonstrated that at least part of the CGRP-containing sensory and CPON-containing sympathetic axons may survive unaltered even in patients with a long clinical history of profound sensorial impairment. An apparent discrepancy between the maintenance of nerve fibers and the sensory disturbances and the frequent observation of prompt postoperative recovery even after years of compression results from this investigation. The correlation of immunohistochemical observations and functional scores may not be considered conclusive. It must, however, be discussed if the sensorial impairment in this syndrome might have, at least in some cases, not only an anatomical but also an electrophysiological basis.


Inhalation of chlorine gas.
The clinical features of acute chlorine gas inhalation, and its management are reviewed. Current medical views on the chronic effects of an acute overwhelming exposure on lung function (reactive airways dysfunction syndrome), and the more controversial field of lung disease secondary to repeated inhalations of lower concentrations of chlorine gas are discussed.


Serum vitamin C concentration was inversely associated with subsequent 20-year incidence of stroke in a Japanese rural community. The Shibata study.
Epidemiological evidence suggests that vitamin C may decrease the risk of stroke. The purpose of the present study was to examine the association of serum vitamin C concentration with the subsequent incidence of stroke. In a Japanese rural community, a cohort of 880 men and 1241 women aged 40 years and older who were initially free of stroke was examined in 1977 and followed until 1997. The baseline examination included a measurement of serum vitamin C concentration. The incidence of stroke was determined by annual follow-up examinations and registry. During the 20-year observation period, 196 incident cases of all stroke, including 109 cerebral infarctions and 54 hemorrhagic strokes, were documented. Strong inverse associations were observed between serum vitamin C concentration and all stroke (sex- and age-adjusted hazard ratios were 0.93, 0.72, and 0.59, respectively, for the second, third, and fourth quartiles compared with the first quartile; P for trend=0.002), cerebral infarction (0.71, 0.59, and 0.51; P for trend=0.015), and hemorrhagic stroke (0.89, 0.75, and 0. 45; P for trend=0.013). Additional adjustments for blood pressure, serum total cholesterol, body mass index, physical activity, smoking, alcohol drinking, antihypertensive medication, atrial fibrillation, and history of ischemic heart disease did not attenuate these associations markedly. Serum vitamin C concentration was inversely related to the subsequent incidence of stroke. This relationship was significant for both cerebral infarction and hemorrhagic stroke. Additional mechanistic hypotheses may be required to explain our findings.


GRADE: Assessing the quality of evidence in environmental and occupational health.
There is high demand in environmental health for adoption of a structured process that evaluates and integrates evidence while making decisions and recommendations transparent. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) framework holds promise to address this demand. For over a decade, GRADE has been applied successfully to areas of clinical medicine, public health, and health policy, but experience with GRADE in environmental and occupational health is just beginning. Environmental and occupational health questions focus on understanding whether an exposure is a potential health hazard or risk, assessing the exposure to understand the extent and magnitude of risk, and exploring interventions to mitigate exposure or risk. Although GRADE offers many advantages, including its flexibility and methodological rigor, there are features of the different sources of evidence used in environmental and occupational health that will require further consideration to assess the need for method refinement. An issue that requires particular attention is the evaluation and integration of evidence from human, animal, in vitro, and in silico (computer modeling) studies when determining whether an environmental factor represents a potential health hazard or risk. Assessment of the hazard of exposures can produce analyses for use in the GRADE evidence-to-decision (EtD) framework to inform risk-management decisions about removing harmful exposures or mitigating risks. The EtD framework allows for grading the strength of the recommendations based on judgments of the certainty in the evidence (also known as quality of the evidence), as well as other factors that inform recommendations such as social values and preferences, resource implications, and benefits. GRADE represents an untapped opportunity for environmental and occupational health to make evidence-based recommendations in a systematic and transparent manner. The objectives of this article are to provide an overview of GRADE, discuss GRADE's applicability to environmental health, and identify priority areas for method assessment and development.


Shape-based regularization of electron tomographic reconstruction.
We introduce a tomographic reconstruction method implemented using a shape-based regularization technique. Spatial models of known features in the structure being reconstructed are integrated into the reconstruction process as regularizers. Our regularization scheme is driven locally through shape information obtained from segmentation and compared with a known spatial model. We demonstrated our method on tomography data from digital phantoms, simulated data, and experimental electron tomography (ET) data of virus complexes. Our reconstruction showed reduced blurring and an improvement in the resolution of the reconstructed volume was also measured. This method also produced improved demarcation of spike boundaries in viral membranes when compared with popular techniques like weighted back projection and the algebraic reconstruction technique. Improved ET reconstructions will provide better structure elucidation and improved feature visualization, which can aid in solving key biological issues. Our method can also be generalized to other tomographic modalities.


Remapping auditory-motor representations in voice production.
Evidence regarding visually guided limb movements suggests that the motor system learns and maintains neural maps between motor commands and sensory feedback. Such systems are hypothesized to be used in a feed-forward control strategy that permits precision and stability without the delays of direct feedback control. Human vocalizations involve precise control over vocal and respiratory muscles. However, little is known about the sensorimotor representations underlying speech production. Here, we manipulated the heard fundamental frequency of the voice during speech to demonstrate learning of auditory-motor maps. Mandarin speakers repeatedly produced words with specific pitch patterns (tone categories). On each successive utterance, the frequency of their auditory feedback was increased by 1/100 of a semitone until they heard their feedback one full semitone above their true pitch. Subjects automatically compensated for these changes by lowering their vocal pitch. When feedback was unexpectedly returned to normal, speakers significantly increased the pitch of their productions beyond their initial baseline frequency. This adaptation was found to generalize to the production of another tone category. However, results indicate that a more robust adaptation was produced for the tone that was spoken during feedback alteration. The immediate aftereffects suggest a global remapping of the auditory-motor relationship after an extremely brief training period. However, this learning does not represent a complete transformation of the mapping; rather, it is in part target dependent.


TCF21 hypermethylation regulates renal tumor cell clonogenic proliferation and migration.
We recently identified hypermethylation at the gene promoter of transcription factor 21 (TCF21) in clear cell sarcoma of the kidney (CCSK), a rare pediatric renal tumor. TCF21 is a transcription factor involved in tubular epithelial development of the kidney and is a candidate tumor suppressor. As there are no in vitro models of CCSK, we employed a well-established clear cell renal cell carcinoma (ccRCC) cell line, 786-O, which also manifests high methylation at the TCF21 promoter, with consequent low TCF21 expression. The tumor suppressor function of TCF21 has not been functionally addressed in ccRCC cells; we aimed to explore the functional potential of TCF21 expression in ccRCC cells in vitro. 786-O clones stably transfected with either pBABE-TCF21-HA construct or pBABE vector alone were functionally analyzed. We found that ectopic expression of TCF21 in 786-O cells results in a trend toward decreased cell proliferation (not significant) and significantly decreased migration compared with mock-transfected 786-O cells. Although the number of colonies established in colony formation assays was not different between 786-O clones, colony size was significantly reduced in 786-O cells expressing TCF21. To investigate whether the changes in migration were due to epithelial-to-mesenchymal transition changes, we interrogated the expression of selected epithelial and mesenchymal markers. Although we observed upregulation of mRNA and protein levels of epithelial marker E-cadherin in clones overexpressing TCF21, this did not result in surface expression of E-cadherin as measured by fluorescence-activated cell sorting and immunofluorescence. Furthermore, mRNA expression of the mesenchymal markers vimentin (VIM) and SNAI1 was not significantly decreased in TCF21-expressing 786-O cells, while protein levels of VIM were markedly decreased. We conclude that re-expression of TCF21 in renal cancer cells that have silenced their endogenous TCF21 locus through hypermethylation results in reduced clonogenic proliferation, reduced migration, and reduced mesenchymal-like characteristics, suggesting a tumor suppressor function for transcription factor 21.


Reducing Amygdala Activity and Phobic Fear through Cognitive Top-Down Regulation.
The amygdala is critically involved in emotional processing, including fear responses, and shows hyperactivity in anxiety disorders. Previous research in healthy participants has indicated that amygdala activity is down-regulated by cognitively demanding tasks that engage the PFC. It is unknown, however, if such an acute down-regulation of amygdala activity might correlate with reduced fear in anxious participants. In an fMRI study of 43 participants (11 men) with fear of snakes, we found reduced amygdala activity when visual stimuli were processed under high cognitive load, irrespective of whether the stimuli were of neutral or phobic content. Furthermore, dynamic causal modeling revealed that this general reduction in amygdala activity was partially mediated by a load-dependent increase in dorsolateral PFC activity. Importantly, high cognitive load also resulted in an acute decrease in perceived phobic fear while viewing the fearful stimuli. In conclusion, our data indicate that a cognitively demanding task results in a top-down regulation of amygdala activity and an acute reduction of fear in phobic participants. These findings may inspire the development of novel psychological intervention approaches aimed at reducing fear in anxiety disorders.


The best model for humans is human -- how to accelerate early drug development safely.
Traditionally, the choice of which candidate compounds to take forward into development has been based on pre-clinical data. However, lack of predictivity of the human clinical situation in the models used has led to poor decision-making, and the later in the development process that such mistakes are realised, the more costly and time-consuming it is to correct them. Furthermore, compounds that may have made perfectly good drugs, have been dropped due to poor pharmacokinetics in animal models. Accelerator mass spectrometry (AMS) is an ultra-sensitive detection technique that can be used to quantify carbon-14. By administering very small amounts of (14)C-labelled compounds, AMS can be used to obtain human clinical data very early in the drug development process. Such studies: a) can be helpful in understanding human pharmacokinetics using microdosing; b) can provide early human metabolism information, to validate the choice of animal species used in pre-clinical safety testing and identify unique or disproportionate human metabolites during Phase 1; and c) can provide fundamental human pharmacokinetic data, including absolute bioavailability, by facilitating a scientifically optimal and cost-effective study design. The provision of these clinical insights at the earliest possible opportunity can lead to improved decision-making, and therefore can reduce the time and cost involved in the drug development process.


Dopamine sensor based on a boron-doped diamond electrode modified with a polyaniline/Au nanocomposites in the presence of ascorbic acid.
A selective dopamine (DA) sensor was developed using gold nanoparticles (Au-NPs) dispersed overoxidized-polyaniline (PANI(ox)) based on a boron-doped diamond (BDD) thin-film electrode. The concentration of the DA was determined using voltammetry as a non-enzymatic sensor. BDD thin film has a high signal-to-noise ratio, a long-term stability, a high sensitivity, and a good reproducibility. PANI nanocomposites were directly synthesized on the BDD electrode and overoxidized using 0.5 M H(2)SO(4) solution. The overoxidized PANI film enhances selectivity and sensitivity toward DA. The Au-NPs were dispersed on the PANI nanocomposite by electrochemical deposition. The nanometer-sized Au-NPs favor the sensing of DA in the presence of ascorbic acid (AA). The combination of the PANI with the Au-NPs and the BDD electrode can create synergetic effects for the performance of the biosensor, such as a fast response time, a lower detection limit, a wider linear range, enhanced selectivity, and higher sensitivity for the determination of DA.


Lamin Mutations Accelerate Aging via Defective Export of Mitochondrial mRNAs through Nuclear Envelope Budding.
Defective RNA metabolism and transport are implicated in aging and degeneration [1, 2], but the underlying mechanisms remain poorly understood. A prevalent feature of aging is mitochondrial deterioration [3]. Here, we link a novel mechanism for RNA export through nuclear envelope (NE) budding [4, 5] that requires A-type lamin, an inner nuclear membrane-associated protein, to accelerated aging observed in Drosophila LaminC (LamC) mutations. These LamC mutations were modeled after A-lamin (LMNA) mutations causing progeroid syndromes (PSs) in humans. We identified mitochondrial assembly regulatory factor (Marf), a mitochondrial fusion factor (mitofusin), as well as other transcripts required for mitochondrial integrity and function, in a screen for RNAs that exit the nucleus through NE budding. PS-modeled LamC mutations induced premature aging in adult flight muscles, including decreased levels of specific mitochondrial protein transcripts (RNA) and progressive mitochondrial degradation. PS-modeled LamC mutations also induced the accelerated appearance of other phenotypes associated with aging, including a progressive accumulation of polyubiquitin aggregates [6, 7] and myofibril disorganization [8, 9]. Consistent with these observations, the mutants had progressive jumping and flight defects. Downregulating marf alone induced the above aging defects. Nevertheless, restoring marf was insufficient for rescuing the aging phenotypes in PS-modeled LamC mutations, as other mitochondrial RNAs are affected by inhibition of NE budding. Analysis of NE budding in dominant and recessive PS-modeled LamC mutations suggests a mechanism by which abnormal lamina organization prevents the egress of these RNAs via NE budding. These studies connect defects in RNA export through NE budding to progressive loss of mitochondrial integrity and premature aging.


The interleukin 12 p40 gene promoter is primed by interferon gamma in monocytic cells.
Interleukin (IL) 12 is a proinflammatory cytokine produced by phagocytic cells, B cells, and other antigen-presenting cells that modulates adaptive immune responses by favoring the generation of T helper type 1 cells. IL-12 mediates some of its physiological activities by acting as a potent inducer of interferon (IFN) gamma production by T and natural killer cells. IFN-gamma enhances the ability of the phagocytic cells to produce IL-12 and other proinflammatory cytokines. Thus, IL-12-induced IFN-gamma acts in a positive feedback loop that represents an important amplifying mechanism in the inflammatory response to infections. We show here that IFN-gamma enhances IL-12 production mostly by priming phagocytic cells for lipopolysaccharide (LPS)-induced transcription of the IL-12 p40 gene, which encodes the heavy chain of the IL-12 heterodimer; furthermore, IFN-gamma directly induces transcription of the IL-12 p35 gene, which encodes the light chain of IL-12, and has at least an additive effect with LPS stimulation in inducing its transcription. The priming effect of IFN-gamma on the LPS-induced p40 gene transcription requires preincubation of the cells with IFN-gamma for at least 8 h to obtain a maximal effect. The priming effect of IFN-gamma for IL-12 production is predominantly at the transcriptional level for both the p40 and the p35 gene, and no evidence for a major role of posttranscriptional or translational mechanisms was found. A 3.3-kb human IL-12 p40 promoter construct transfected into cell lines recapitulated the tissue specificity of the endogenous gene, being silent in two human T cell lines, constitutively active in two human Epstein-Barr virus-positive B lymphoblastoid cell lines, and LPS inducible in the human THP-1 and mouse RAW264.7 monocytic cell lines. Because the RAW264.7 cell line is easily transfectable and regulates the endogenous IL-12 p40 gene in response to IFN-gamma or LPS similarly to human monocytes, it was used for analysis of the regulation of the cloned human IL-12 p40 promoter. A requirement for the region between -222 and -204 in both LPS responsiveness and IFN-gamma priming was established. This region contains an ets consensus sequence that was shown to mediate activation of the promoter by IFN-gamma and LPS, as well as by a cotransfected ets-2. The -222 construct was also regulated in a tissue-specific manner. Two other elements, IRF-1 located at -730 to -719, and NF-IL6 at -520 to -512, were also studied by deletion analysis, which did not result in decreased response to IFN-gamma and LPS stimulation.


Induced ATF-2 represses CDK4 transcription through dimerization with JunD inhibiting intestinal epithelial cell growth after polyamine depletion.
Intestinal epithelium is a rapidly self-renewing tissue in the body, and its homeostasis is tightly regulated by numerous factors including polyamines. Decreased levels of cellular polyamines increase activating transcription factor (ATF)-2, but the exact role and mechanism of induced ATF-2 in the regulation of intestinal epithelial cell (IEC) growth remain elusive. Cyclin-dependent kinase (CDK) 4 is necessary for the G1-to-S phase transition during the cell cycle, and its expression is predominantly controlled at the transcription level. Here, we reported that induced ATF-2 following polyamine depletion repressed CDK4 gene transcription in IECs by increasing formation of the ATF-2/JunD heterodimers. ATF-2 formed complexes with JunD as measured by immunoprecipitation using the ATF-2 and JunD antibodies and by glutathione S-transferase (GST) pull-down assays using GST-ATF-2 fusion proteins. Studies using various mutants of GST-ATF-2 revealed that formation of the ATF-2/JunD dimers depended on the COOH-terminal basic region-leucine zipper domain of ATF-2. Polyamine depletion increased ATF-2/JunD complex and inhibited CDK4 transcription as indicated by a decrease in the levels of CDK4-promoter activity and its mRNA. ATF-2 silencing not only prevented inhibition of CDK4 transcription in polyamine-deficient cells but also abolished repression of CDK4 expression induced by ectopic JunD overexpression. ATF-2 silencing also promoted IEC growth in polyamine-depleted cells. These results indicate that induced ATF-2/JunD association following polyamine depletion represses CDK4 transcription, thus contributing to the inhibition of IEC growth.


Combination of two regulatory elements in the Tetrahymena thermophila HSP70-1 gene controls heat shock activation.
The induction of heat shock genes (HSPs) is thought to be primarily regulated by heat shock transcription factors (HSFs), which bind target sequences on HSP promoters, called heat shock elements (HSEs). In this study, we investigated the 5' untranslated regions of the Tetrahymena thermophila HSP70-1 gene, and we found, in addition to the canonical and divergent HSEs, multiple sets of GATA elements that have not been reported previously in protozoa. By means of in vivo analysis of a green fluorescent protein reporter transgene driven by the HSP70-1 promoter, we demonstrate that HSEs do not represent the minimal regulatory elements for heat shock induction, since the HSP70-1 is tightly regulated by both HSE and GATA elements. Electrophoretic mobility shift assay also showed that HSFs are constitutively bound to the HSEs, whereas GATA elements are engaged only after heat shock. This is the first demonstration by in vivo analysis of functional HSE and GATA elements in protozoa. Furthermore, we provide evidence of a functional link between HSE and GATA elements in the activation of the heat shock response.


Cognitive-behavioral therapy for PTSD and depression symptoms reduces risk for future intimate partner violence among interpersonal trauma survivors.
Women who develop symptoms of posttraumatic stress disorder (PTSD) and depression subsequent to interpersonal trauma are at heightened risk for future intimate partner violence (IPV) victimization. Cognitive-behavioral therapy (CBT) is effective in reducing PTSD and depression symptoms, yet limited research has investigated the effectiveness of CBT in reducing risk for future IPV among interpersonal trauma survivors. This study examined the effect of CBT for PTSD and depressive symptoms on the risk of future IPV victimization in a sample of women survivors of interpersonal violence. The current sample included 150 women diagnosed with PTSD secondary to an array of interpersonal traumatic events; they were participating in a randomized clinical trial of different forms of cognitive processing therapy for the treatment of PTSD. Participants were assessed at 9 time points as part of the larger trial: pretreatment, 6 times during treatment, posttreatment, and 6-month follow-up. As hypothesized, reductions in PTSD and in depressive symptoms during treatment were associated with a decreased likelihood of IPV victimization at a 6-month follow-up even after controlling for recent IPV (i.e., IPV from a current partner within the year prior to beginning the study) and prior interpersonal traumas. These findings highlight the importance of identifying and treating PTSD and depressive symptoms among interpersonal trauma survivors as a method for reducing risk for future IPV.


Scanning electron microscopy of epidermal adherence and cavitation in murine candidiasis: a role for Candida acid proteinase.
Adherence of blastoconidia to epidermal corneocytes is an early event in Candida colonization and infection of the skin. Pathogenic species adhere more avidly than nonpathogenic species, transform to hyphal growth, and invade the stratum corneum of the skin. Adherence was studied by scanning electron microscopy of experimental murine cutaneous Candida infections, using six species of Candida. Candida albicans and C. stellatoidea blastoconidia, applied to newborn mouse skin, adhered to the stratum corneum in greater numbers than other species tested, acquired fibrils and strands of amorphous mucinlike material ("cohesin") between spores and the corneocyte cell surface, formed cavitations in the corneocyte surface, and invaded the corneocyte envelope by hyphal growth at sites distant to the point of blastoconidia attachment. Other species showed little or no adherence, colonization, or cavitation of the corneocyte surface, except C. tropicalis, which showed intermediate results. Pepstatin, an inhibitor of Candida acid proteinase, did not alter adherence or cohesion formation, but inhibited formation of corneocyte cavitations about adherent blastoconidia, suggesting that this enzyme may facilitate adherence/invasion events on skin. Depletion of surface lipids did not alter the formation of cohesin material or the adherence process. Adherence and invasion of epithelium by pathogenic Candida species include the interaction of blastoconidia with an epithelial surface cohesin material that participates in the adherence process. Candida acid proteinase, a keratinolytic enzyme, may participate in the cavitation process of the corneocyte surface by C. albicans.


Portable multispectral imaging system based on light-emitting diodes for spectral recovery from 370 to 1630 nm.
To expand and investigate the potential of spectral imaging, we developed a portable multispectral system using light-emitting diodes. This system recovers spectral information from the UV to the near IR over a large area using two different image sensors synchronized with 23 bands of illumination. The system was assessed for spectral reconstruction through simulations and experimental measurements by means of two methods of spectral reconstruction and three different evaluation metrics. The results over a Macbeth ColorChecker chart and other samples, including pigments usually employed in art paintings, are compared and discussed. The portable multispectral system using LEDs constitutes a cost-effective and versatile method for spectral imaging.


Synthesis of caffeic acid molecularly imprinted polymer microspheres and high-performance liquid chromatography evaluation of their sorption properties.
In the current work, a molecularly imprinted polymer (MIP) has been synthesised and used to enable the extraction of a naturally-occurring antioxidant from complex media. More specifically, we describe the first example of a caffeic acid (CA) MIP which has been synthesised in the form of well-defined polymer microspheres, and its use for the extraction of CA from fruit juice sample. The CA MIP was synthesised by precipitation polymerisation using 4-vinylpyridine as functional monomer, divinylbenzene-80 as crosslinker and acetonitrile:toluene (75/25, v/v) as porogen. The particle sizing and morphological characterisation of the polymers was carried out by means of scanning electron microscopy (narrow particle size distribution; ∼5 and 1.5 μm particle diameters for the MIP and NIP [non-imprinted polymer], respectively) and nitrogen sorption porosimetry (specific surface areas of 340 and 350 m(2)g(-1), and specific pore volumes of 0.17 and 0.19 cm(3)g(-1) for the MIP and NIP, respectively). The polymers were evaluated further by batch rebinding experiments, and from the derived isotherms their binding capacity and binding strength were determined (number of binding sites (N(K))=0.6 and 0.3 mmol g(-1) for the MIP and NIP, respectively, and apparent average adsorption constant (K(N))=10.0 and 1.6L mmol(-1) for the MIP and NIP, respectively). To evaluate the molecular recognition character of the MIP it was packed into a stainless steel column (50 mm × 4.6 mm i.d.) and evaluated as an HPLC-stationary phase. The mobile phase composition, flow rate, and the elution profile were then optimised in order to improve the peak shape without negatively affecting the imprinting factor (IF). Very interesting, promising properties were revealed. The imprinting factor (IF) under the optimised conditions was 11.9. Finally, when the imprinted LC column was used for the selective recognition of CA over eight related compounds, very good selectivity was obtained. This outcome enabled the direct extraction of CA in commercial apple juice samples with recoveries in excess of 81% and, rather significantly, without any need for a clean-up step prior to the extraction.


Hydrophilic Astragalin Galactoside Induces T Helper Type 1-Mediated Immune Responses via Dendritic Cells.
A flavonoid Astragalin (kaempferol-3-O-β-d-glucopyranoside, Ast) has several biological activities including anti-oxidant, anti-HIV, and anti-allergic effects. Nonetheless, its insolubility in hydrophilic solvents imposes restrictions on its therapeutic applications. In this study, we investigated the effects of water-soluble astragalin-galactoside (kaempferol-3-O-β-d-isomaltotrioside, Ast-Gal) on murine bone marrow-derived dendritic cell (DC) maturation and T helper (Th) cell-mediated immune responses. Ast-Gal significantly increased maturation and activation of DCs through the upregulation of surface markers, such as cluster of differentiation (CD)80, CD86, and Major histocompatibility complex (MHC) II in a dose-dependent manner, while Ast had little effects. Additionally, Ast-Gal-treated DCs markedly secreted immune-stimulating cytokines such as interleukin (IL)-1β, IL-6, and IL-12. Importantly, Ast-Gal strongly increased expression of IL-12, a polarizing cytokine of Th1 cells. In a co-culture system of DCs and CD4⁺ T cells, Ast-Gal-treated DCs preferentially differentiates naïve CD4⁺ T cells into Th1 cells. The addition of neutralizing IL-12 monoclonal antibody (mAb) to cultures of Ast-Gal-treated DCs and CD4⁺ T cells significantly decreased interferon (IFN)-γ production, thereby indicating that Ast-Gal-stimulated DCs enhance the Th1 response through IL-12 production by DCs. Injection with Ast-Gal-treated DCs in mice increased IFN-γ-secreting Th1 cell population. Collectively, these findings indicate that hydrophilically modified astragalin can enhance Th1-mediated immune responses via DCs and point to a possible application of water-soluble astragalin-galactoside as an immune adjuvant.


Salivary Effector Sm9723 of Grain Aphid Sitobion miscanthi Suppresses Plant Defense and Is Essential for Aphid Survival on Wheat.
Aphid salivary effectors play important roles in modulating plant defense responses. The grain aphid Sitobion miscanthi is one of the most economically important cereal aphids worldwide. However, little information is available on the identification and functional analysis of salivary effectors of S. miscanthi. In this study, a candidate salivary effector Sm9723 was identified, which was specifically expressed in aphid salivary glands and highly induced during the aphid feeding phase. Transient overexpression of Sm9723 in Nicotiana benthamiana suppressed BAX and INF1-induced cell death. Further, Sm9723 overexpression inhibited N. benthamiana defense responses by reducing pattern-triggered immunity associated callose deposition and expression levels of jasmonic and salicylic acid-associated defense genes. In addition, the salivary effector Sm9723 of S. miscanthi was effectively silenced through nanocarrier-mediated dsRNA delivery system. After silencing Sm9723, fecundity and survival of S. miscanthi decreased significantly, and the aphid feeding behavior was also negatively affected. These results suggest salivary effector Sm9723 is involved in suppressing plant immunity and is essential in enabling aphid virulence, which could be applied as potential target gene for RNAi-mediated pest control of S. miscanthi.


Chiropractic: history and overview of theories and methods.
Chiropractic is one of the most controversial and poorly defined healthcare professions with recognition and licensure in the United States. Chiropractic was started by D. D. Palmer, a magnetic healer who formulated the vertebral subluxation theory. The profession was developed by his son, B. J. Palmer. Although the definition of chiropractic as a method of correcting vertebral subluxations to restore and maintain health is questionable, spinal manipulation is of value in the treatment of some types of back pain. The chiropractic profession is still based on the vertebral subluxation theory, and has the confusing image of a back specialty capable of treating a broad scope of health problems. Despite opposition to use of spinal manipulation as a method of treating a broad scope of health problems (as opposed to the generally accepted use of manipulation in the treatment of back pain), chiropractors seek support as primary care providers in alternative medicine. It is essential to understand the theories, philosophies, and methods of chiropractic for an objective evaluation.


A resource for improved predictions of Trypanosoma and Leishmania protein three-dimensional structure.
AlphaFold2 and RoseTTAfold represent a transformative advance for predicting protein structure. They are able to make very high-quality predictions given a high-quality alignment of the protein sequence with related proteins. These predictions are now readily available via the AlphaFold database of predicted structures and AlphaFold or RoseTTAfold Colaboratory notebooks for custom predictions. However, predictions for some species tend to be lower confidence than model organisms. Problematic species include Trypanosoma cruzi and Leishmania infantum: important unicellular eukaryotic human parasites in an early-branching eukaryotic lineage. The cause appears to be due to poor sampling of this branch of life (Discoba) in the protein sequences databases used for the AlphaFold database and ColabFold. Here, by comprehensively gathering openly available protein sequence data for Discoba species, significant improvements to AlphaFold2 protein structure prediction over the AlphaFold database and ColabFold are demonstrated. This is made available as an easy-to-use tool for the parasitology community in the form of Colaboratory notebooks for generating multiple sequence alignments and AlphaFold2 predictions of protein structure for Trypanosoma, Leishmania and related species.


Healthcare professionals' perspectives on lung cancer screening in the UK: a qualitative study.
Lung cancer screening with low-dose CT has been shown to decrease mortality. Low lung cancer survival rates in the UK, driven primarily by late-stage presentation, provide the impetus for implementing screening. Nascent guidance on screening in the UK recommends primary care case-finding. However, the potential impact and acceptability on primary care, and the opportunistic utilisation of other case-finding routes such as pharmacies, smoking cessation services and respiratory clinics, have not been fully explored. To explore healthcare professionals' views and perspectives about lung cancer screening and their preparedness and willingness to be involved in its implementation. A qualitative study was carried out with semi-structured interviews conducted with GPs, pharmacists and staff from smoking cessation services within Southwark and Lambeth and from respiratory clinics in Guy's and St. Thomas' NHS Trust in London in 2018. Sixteen participants were interviewed, and the interview transcripts were analysed thematically. Participants described lung cancer screening as an important diagnostic tool for capturing lung cancer at an earlier stage and in increasing survivorship. However, the majority expressed a lack of awareness and understanding, uncertainty and concerns about the validity of screening, its misuse and the potential impact on their patients and workload. Study participants had mixed opinions about lung cancer screening and expressed their concerns about its implementation. Addressing these concerns by providing resources and effective and detailed guidelines for their use may lead to greater engagement and willingness to be involved in lung cancer screening.


Factors maintaining the intraurethral pressure in women.
To determine the components of the intraurethral pressure at rest, five continent women were studied with simultaneous urethrocystometry, including urethral pressure profile measurements, before, during and after curarization, as well as after clamping of the arterial blood supply to the urethra. The results showed that the striated muscle component in the urethra and pelvic floor is responsible for one-third of the total intraurethral pressure. Another one-third is exerted by the urethral vascular bed. The remaining one-third is most probably attributable to the smooth musculature and connective tissues in the urethra and the periurethral tissues.


Tumor Heterogeneity on FDG PET/CT and Immunotherapy: An Imaging Biomarker for Predicting Treatment Response in Patients With Metastatic Melanoma.
OBJECTIVE. The purpose of this study is to evaluate the ability of quantitative 18F-FDG PET parameters to predict outcomes of patients with malignant melanoma who have been treated with immune modulation therapy. MATERIALS AND METHODS. We retrospectively investigated 34 patients with malignant melanoma. Twenty-three patients received immunotherapy as first-line therapy, and 11 patients received it as second-line therapy. The maximum standardized uptake value (SUVmax), metabolic tumor volume, tumor lesion glycolysis, and intratumoral metabolic heterogeneity (as measured by the tumor heterogeneity [TH] index) were measured for the primary tumors and metastatic sites associated with up to five of the most FDG-avid lesions per patient. The TH index was calculated as the AUC value of a cumulative SUV volume histogram curve for all patients. The median follow-up was 29.5 months (range, 3-288 months). Outcome endpoints were progression-free survival and overall survival. Kaplan-Meier survival plots were used, and Cox regression analysis was performed for predictors of survival. RESULTS. A total of 101 lesions were analyzed. Five lesions were analyzed in 12 patients, four lesions in three patients, three lesions in three patients, two lesions in four patients, and one lesion in 12 patients. Of the 34 patients included in the study, 15 (44.1%) had disease progression and 11 (32.3%) had died by the time the last follow-up occurred. The mean (± SD) SUVmax, peak SUV, metabolic tumor volume, tumor lesion glycolysis, and TH values for all lesions were 9.68 ± 6.6, 7.82 ± 5.83, 81.96 ± 146.87 mL, 543.65 ± 1022.92 g, and 5841.36 ± 1249.85, respectively. TH had a negative correlation with SUVmax, peak SUV, and tumor lesion glycolysis (p < 0.0001 for all). CONCLUSION. The TH index is significantly associated with overall survival in patients with metastatic melanoma treated with immune modulation therapy as first-line or second-line therapy.


Long-term hepatic consequences of chemotherapy-related HBV reactivation in lymphoma patients.
To investigate the long-term consequences of chemotherapy-related HBV reactivation in patients with lymphoma. This study was based on the database of published prospective study evaluating HBV reactivation in HBV lymphoma patients during chemotherapy. Deteriorated liver reserve (DLR) was defined as development of either one of the following conditions during follow-up: (1) newly onset parenchyma liver disease, splenomegaly or ascites without evidence of lymphoma involvement; (2) decrease of the ratio (albumin/globulin ratio) to less than 0.8 or increase of the ratio of INR of prothrombin time to larger than 1.2 without evidence of malnutrition or infection. Liver cirrhosis was diagnosed by imaging studies. A total of 49 patients were included. The median follow-up was 6.2 years (range, 3.9-8.1 years). There were 31 patients with and 18 patients without HBV reactivation. Although there was no difference of overall survival (OS) and chemotherapy response rate between the two groups, DLR developed more frequently in patients with HBV reactivation (48.4% vs 16.7%; P = 0.0342). Among the HBV reactivators, HBV genotype C was associated with a higher risk of developing DLR (P = 0.0768) and liver cirrhosis (P = 0.003). Four of five patients with sustained high titer of HBV DNA and two of three patients with multiple HBV reactivation developed DLR. Further, patients with a sustained high titer of HBV DNA had the shortest OS among the HBV reactivators (P = 0.0000). No patients in the non-HBV reactivation group developed hepatic failure or liver cirrhosis. Chemotherapy-related HBV reactivation is associated with the long-term effect of deterioration of hepatic function.


Insect transformation with piggyBac: getting the number of injections just right.
The insertion of exogenous genetic cargo into insects using transposable elements is a powerful research tool with potential applications in meeting food security and public health challenges facing humanity. piggyBac is the transposable element most commonly utilized for insect germline transformation. The described efficiency of this process is variable in the published literature, and a comprehensive review of transformation efficiency in insects is lacking. This study compared and contrasted all available published data with a comprehensive data set provided by a biotechnology group specializing in insect transformation. Based on analysis of these data, with particular focus on the more complete observational data from the biotechnology group, we designed a decision tool to aid researchers' decision-making when using piggyBac to transform insects by microinjection. A combination of statistical techniques was used to define appropriate summary statistics of piggyBac transformation efficiency by species and insect order. Publication bias was assessed by comparing the data sets. The bias was assessed using strategies co-opted from the medical literature. The work culminated in building the Goldilocks decision tool, a Markov-Chain Monte-Carlo simulation operated via a graphical interface and providing guidance on best practice for those seeking to transform insects using piggyBac.


Inflammatory mediators increase Nav1.9 current and excitability in nociceptors through a coincident detection mechanism.
Altered function of Na+ channels is responsible for increased hyperexcitability of primary afferent neurons that may underlie pathological pain states. Recent evidence suggests that the Nav1.9 subunit is implicated in inflammatory but not acute pain. However, the contribution of Nav1.9 channels to the cellular events underlying nociceptor hyperexcitability is still unknown, and there remains much uncertainty as to the biophysical properties of Nav1.9 current and its modulation by inflammatory mediators. Here, we use gene targeting strategy and computer modeling to identify Nav1.9 channel current signature and its impact on nociceptors' firing patterns. Recordings using internal fluoride in small DRG neurons from wild-type and Nav1.9-null mutant mice demonstrated that Nav1.9 subunits carry the TTX-resistant "persistent" Na+ current called NaN. Nav1.9(-/-) nociceptors showed no significant change in the properties of the slowly inactivating TTX-resistant SNS/Nav1.8 current. The loss in Nav1.9-mediated Na+ currents was associated with the inability of small DRG neurons to generate a large variety of electrophysiological behaviors, including subthreshold regenerative depolarizations, plateau potentials, active hyperpolarizing responses, oscillatory bursting discharges, and bistable membrane behaviors. We further investigated, using CsCl- and KCl-based pipette solutions, whether G-protein signaling pathways and inflammatory mediators upregulate the NaN/Nav1.9 current. Bradykinin, ATP, histamine, prostaglandin-E2, and norepinephrine, applied separately at maximal concentrations, all failed to modulate the Nav1.9 current. However, when applied conjointly as a soup of inflammatory mediators they rapidly potentiated Nav1.9 channel activity, generating subthreshold amplification and increased excitability. We conclude that Nav1.9 channel, the molecular correlate of the NaN current, is potentiated by the concerted action of inflammatory mediators that may contribute to nociceptors' hyperexcitability during peripheral inflammation.


Immunodominance of the VH1-46 antibody gene segment in the primary repertoire of human rotavirus-specific B cells is reduced in the memory compartment through somatic mutation of nondominant clones.
Detailed characterization of Ag-specific naive and memory B cell Ab repertoires elucidates the molecular basis for the generation of Ab diversity and the optimization of Ab structures that bind microbial Ags. In this study, we analyzed the immunophenotype and VH gene repertoire of rotavirus (RV) VP6-specific B cells in three circulating naive or memory B cell subsets (CD19+IgD+CD27-, CD19+IgD+CD27+, or CD19+IgD-CD27+) at the single-cell level. We aimed to investigate the influence of antigenic exposure on the molecular features of the two RV-specific memory B cell subsets. We found an increased frequency of CD19+IgD+CD27+ unclass-switched memory B cells and a low frequency of somatic mutations in CD19+IgD-CD27+ class-switched memory B cells in RV-specific memory B cells, suggesting a reduced frequency of isotype switching and somatic mutation in RV VP6-specific memory B cells compared with other memory B cells. Furthermore, we found that dominance of the VH1-46 gene segment was a prominent feature in the VH gene repertoire of RV VP6-specific naive B cells, but this dominance was reduced in memory B cells. Increased diversity in the VH gene repertoire of the two memory B cell groups derived from broader usage of VH gene segments, increased junctional diversity that was introduced by differential TdT activities, and somatic hypermutation.


Effects of backbone substitutions on the conformational behavior of Shigella flexneri O-antigens: implications for vaccine strategy.
The O-antigen (O-Ag), the polysaccharide part of the lipopolysaccharide, is the major target of the serotype-specific protective humoral response elicited upon host infection by Shigella flexneri, the main causal agent of the endemic form of bacillary dysentery. The O-Ag repeat units (RUs) of 12 S. flexneri serotypes share the tetrasaccharide backbone →2)-α-l-Rhap-(1 → 2)-α-l-Rhap-(1 → 3)-α-l-Rhap-(1 → 3)-β-d-GlcpNAc-(1→, with site-selective glucosylation(s) and/or O-acetylation defining the serotypes. To investigate the conformational basis of serotype specificity, we sampled conformational behaviors during 60 ns of molecular dynamic simulations for oligosaccharides representing three RUs of each one of the O-Ags corresponding to serotypes 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, X and Y, respectively. The calculated trajectories were checked by nuclear magnetic resonance (NMR) for 1a, 2a, 3a and 5a O-Ags. The simulations predict that in all O-Ags, but 1a and 1b, serotype-specific substitutions of the backbone do not induce any new backbone conformations compared with the linear type O-Ag Y, although they restrain locally the accessible conformational space. Moreover, the influence of any given substituent on the backbone is independent of the eventual presence of other substituents. Finally, only slight differences in conformational behavior between terminal and inner RUs were observed. These results suggest that the reported serotype-specificity of the protective immune response is not due to recognition of distinct backbone conformations, but to binding of the serotype-defining substituents in the O-Ag context. The gained knowledge is discussed in terms of impact on the development of a broad-serotype coverage vaccine.


Differential impact of transplantation on peripheral and tissue-associated viral reservoirs: Implications for HIV gene therapy.
Autologous transplantation and engraftment of HIV-resistant cells in sufficient numbers should recapitulate the functional cure of the Berlin Patient, with applicability to a greater number of infected individuals and with a superior safety profile. A robust preclinical model of suppressed HIV infection is critical in order to test such gene therapy-based cure strategies, both alone and in combination with other cure strategies. Here, we present a nonhuman primate (NHP) model of latent infection using simian/human immunodeficiency virus (SHIV) and combination antiretroviral therapy (cART) in pigtail macaques. We demonstrate that transplantation of CCR5 gene-edited hematopoietic stem/progenitor cells (HSPCs) persist in infected and suppressed animals, and that protected cells expand through virus-dependent positive selection. CCR5 gene-edited cells are readily detectable in tissues, namely those closely associated with viral reservoirs such as lymph nodes and gastrointestinal tract. Following autologous transplantation, tissue-associated SHIV DNA and RNA levels in suppressed animals are significantly reduced (p ≤ 0.05), relative to suppressed, untransplanted control animals. In contrast, the size of the peripheral reservoir, measured by QVOA, is variably impacted by transplantation. Our studies demonstrate that CCR5 gene editing is equally feasible in infected and uninfected animals, that edited cells persist, traffic to, and engraft in tissue reservoirs, and that this approach significantly reduces secondary lymphoid tissue viral reservoir size. Our robust NHP model of HIV gene therapy and viral persistence can be immediately applied to the investigation of combinatorial approaches that incorporate anti-HIV gene therapy, immune modulators, therapeutic vaccination, and latency reversing agents.


Intravenous nesiritide, a natriuretic peptide, in the treatment of decompensated congestive heart failure. Nesiritide Study Group.
Intravenous infusion of nesiritide, a brain (B-type) natriuretic peptide, has beneficial hemodynamic effects in patients with decompensated congestive heart failure. We investigated the clinical use of nesiritide in such patients. Patients hospitalized because of symptomatic congestive heart failure were enrolled in either an efficacy trial or a comparative trial. In the efficacy trial, which required the placement of a Swan-Ganz catheter, 127 patients with a pulmonary-capillary wedge pressure of 18 mm Hg or higher and a cardiac index of 2.7 liters per minute per square meter of body-surface area or less were randomly assigned to double-blind treatment with placebo or nesiritide (infused at a rate of 0.015 or 0.030 microg per kilogram of body weight per minute) for six hours. In the comparative trial, which did not require hemodynamic monitoring, 305 patients were randomly assigned to open-label therapy with standard agents or nesiritide for up to seven days. In the efficacy trial, at six hours, nesiritide infusion at rates of 0.015 and 0.030 microg per kilogram per minute decreased pulmonary-capillary wedge pressure by 6.0 and 9.6 mm Hg, respectively (as compared with an increase of 2.0 mm Hg with placebo, P<0.001), resulted in improvements in global clinical status in 60 percent and 67 percent of the patients (as compared with 14 percent of those receiving placebo, P<0.001), reduced dyspnea in 57 percent and 53 percent of the patients (as compared with 12 percent of those receiving placebo, P<0.001), and reduced fatigue in 32 percent and 38 percent of the patients (as compared with 5 percent of those receiving placebo, P<0.001). In the comparative trial, the improvements in global clinical status, dyspnea, and fatigue were sustained with nesiritide therapy for up to seven days and were similar to those observed with standard intravenous therapy for heart failure. The most common side effect was dose-related hypotension, which was usually asymptomatic. In patients hospitalized with decompensated congestive heart failure, nesiritide improves hemodynamic function and clinical status. Nesiritide is useful for the treatment of decompensated congestive heart failure.


Recombinant human BMP-2 enhances the effects of materials used for reconstruction of large cranial defects.
Cranial defect reconstruction presents 2 challenges: induction of new bone formation, and providing structural support during the healing process. This study compares quantity and quality of new bone formation based on various materials and support frameworks. Eighteen dogs underwent surgical removal of a significant portion of their cranial vault. Demineralized bone matrix was used to fill the defect in all animals. In 9 dogs, recombinant human bone morphogenetic protein-2 (rhBMP-2) was added, while the other 9 served as the non-rhBMP-2 group. In each group, 3 animals were fixed with cobalt chrome plates, 3 with adding platelet-rich plasma, and 3 fixed with a Lactosorb (Walter Lorenz Surgical, Inc, Jacksonville, FL) resorbable mesh. Necropsy was done at 12 weeks postoperative. Histomorphometry, density, and mechanical properties of the regenerate were analyzed. The non-rhBMP-2 groups showed minimal substitution of demineralized bone matrix with new bone, while only sporadic remnants of demineralized bone matrix were present in the rhBMP-2 groups. The defect showed more new bone formation (P < .001) and density (P < .001) in the rhBMP-2 groups by Kruskal-Wallis test. The area of new bone was not significantly different among the rhBMP-2 subgroups. The resorbable mesh struts showed no sign of bone invasion or substitution. In the non-rhBMP-2 resorbable mesh group, demineralized bone matrix almost totally disintegrated without replacement by new bone. The addition of rhBMP-2 to demineralized bone matrix accelerated new bone formation in large cranial defects, regardless of the supporting framework or the addition of platelet-rich plasma. The use of a resorbable mesh in such defects is advisable only if rhBMP-2 is added.


Pathology and heredity of breast cancer in younger women.
The pathology of early-age onset breast cancer is considered here from three perspectives: 1) benign proliferative disease, 2) the cancers themselves, and 3) familial and hereditary breast cancer. Hereditary breast cancer, a subset of familial breast cancer featuring a strong autosomal dominant pedigree pattern and multiple primary cancers, has a strong predilection for younger women, accounting for about one half of breast cancers under age 30. With respect to benign proliferative disease, the increased relative risk of breast cancer associated with proliferative disease with atypia, about fourfold to fivefold for all ages, is doubled by the presence of a family history of breast cancer and amplified by young age. With respect to the carcinomas, the relative incidences of medullary carcinoma and ductal carcinoma in situ are increased in young women, while lobular and tubular carcinomas are decreased. Invasive breast cancer is higher grade and more proliferative in younger women, as measured by thymidine-labeling index, DNA flow cytometric S-phase fraction, and proliferation-associated proteins. The increased fraction of ductal carcinoma in situ and higher grade invasive cancers may help to account, respectively, for increased recurrence rates with conservative therapy, and more aggressive natural history in younger women. Familial breast cancers show trends for increased medullary type, but the effect is not independent of age. Weak associations of family history with tubular carcinoma have been reported, but data for associations with lobular carcinoma in situ and invasive lobular carcinoma are conflicting. Hereditary breast cancer as a class has higher tumor proliferation rates, an effect independent of age. Knowledge of the pathology and biomarker characteristics of BRCA1 gene-linked hereditary breast cancers, which account for a substantial fraction of breast cancers in younger women, should shed light on the nature of the responsible gene(s) and guide approaches to therapy and prophylaxis.