Biological behaviors and longevity of ectothermic animals are remarkably influenced by ambient temperature. Development at 18°C significantly enhances the stress resistance of adult flies with more accumulation of nutrients (especially fat) in the body than development at 25°C. Gene expression analysis between the flies developed at 18°C and 25°C revealed that the Immune deficiency (Imd) pathway, including the downstream antimicrobial peptides (AMPs), is downregulated in the flies developed at 18°C. When hypomorphic imd mutant flies with reduced AMP expressions were developed at 25°C, they showed induced stress resistance with higher fat content in the body similar to the wild-type flies developed at 18°C. However, severe hypomorphic imd mutants could not enhance stress resistance due to the downregulation of another downstream JNK pathway that expresses stress tolerance genes. Interestingly, the downregulation of AMP genes, itself, extended lifespan with increased stress resistance. Especially, fat body-specific downregulation of Imd AMP genes exhibited a longer lifespan with higher heat resistance. The fat body is known to function in metabolic homeostasis, stress tolerance, growth, and longevity in Drosophila. Here, we provide the first evidence that mild downregulation of the Imd pathway with AMP genes increases fat content, stress resistance, and lifespan in adult flies.

Nanosizing represents a straight forward technique to unlock the biological activity of complex plant materials. The aim of this study was to develop herbal nanoparticles with medicinal value from dried leaves and stems of Loranthus micranthus with the aid of ball-milling, high speed stirring, and high-pressure homogenization techniques. The milled nanoparticles were characterized using laser diffraction analysis, photon correlation spectroscopy analysis, and light microscopy. The average size of leaf nanoparticles was around 245 nm and that of stem nanoparticles was around 180 nm. The nanoparticles were tested for their antimicrobial and nematicidal properties against a Gram-negative bacterium Escherichia coli, a Gram-positive bacterium Staphylococcus carnosus, fungi Candida albicans and Saccharomyces cerevisiae, and a nematode Steinernemafeltiae. The results show significant activities for both leaf and (particularly) stem nanoparticles of Loranthus micranthus on all organisms tested, even at a particle concentration as low as 0.01% (w/w). The results observed indicate that nanoparticles (especially of the stem) of Loranthus micranthus could serve as novel antimicrobial agents with wide-ranging biomedical applications.

Hepatitis C virus (HCV) enters human hepatocytes using four essential entry factors, one of which is human CD81 (hCD81). The tetraspanin hCD81 contains a large extracellular loop (LEL), which interacts with the E2 glycoprotein of HCV. The role of the non-LEL regions of hCD81 (intracellular tails, four transmembrane domains, small extracellular loop and intracellular loop) is poorly understood. Here, we studied the contribution of these domains to HCV susceptibility of hepatoma cells by generating chimeras of related tetraspanins with the hCD81 LEL. Our results show that non-LEL regions in addition to the LEL determine susceptibility of cells to HCV. While closely related tetraspanins (X. tropicalis CD81 and D. rerio CD81) functionally complement hCD81 non-LEL regions, distantly related tetraspanins (C. elegans TSP9 amd D. melanogaster TSP96F) do not and tetraspanins with intermediate homology (hCD9) show an intermediate phenotype. Tetraspanin homology and susceptibility to HCV correlate positively. For some chimeras, infectivity correlates with surface expression. In contrast, the hCD9 chimera is fully surface expressed, binds HCV E2 glycoprotein but is impaired in HCV receptor function. We demonstrate that a cholesterol-coordinating glutamate residue in CD81, which hCD9 lacks, promotes HCV infection. This work highlights the hCD81 non-LEL regions as additional HCV susceptibility-determining factors.

NOP53 is a tumor suppressor protein located in the nucleolus and is translocated to the cytoplasm during infection by vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1), as shown in our previous study. Cytoplasmic NOP53 interacts with the retinoic acid-inducible gene I (RIG-I) to remove its K63-linked ubiquitination, leading to attenuation of type I interferon IFN-β. In the present study, we found no obvious translocation of NOP53 in infection by a mutant virus lacking ICP4 (HSV-1/d120, replication inadequate). Blocking cytoplasmic translocation of NOP53 by the deletion of its nuclear export sequence (NES) abrogated its ability to support viral replication. These results demonstrated that NOP53 redistribution is related to viral replication. It is interesting that treatment with poly (I:C) or RIG-I-N (a constitutively-active variant) directly induced NOP53 cytoplasmic translocation. To better assess the function of cytoplasmic NOP53 in viral replication, the NOP53-derived protein N3-T, which contains a human immunodeficiency virus (HIV)-derived cell-penetrating Tat peptide at the C-terminal region of N3 (residues 330⁻432), was constructed and expressed. The recombinant N3-T protein formed trimers, attenuated the expression of IFN-β and IFN-stimulated genes, as well as decreased the phosphorylation level of interferon regulatory factor 3 (IRF3). Furthermore, N3-T promoted the efficient replication of enveloped and non-enveloped DNA and RNA viruses belonging to 5 families. Our findings expand the understanding of the mechanism by which viruses utilize the nucleolar protein NOP53 for optimal viral replication.

The cellular role of breast carcinoma-associated protein (BCA3), also known as A-kinase-interacting protein 1 (AKIP-1), is not fully understood. Recently, we reported that full-length, but not C-terminally truncated, BCA3 is incorporated into virions of Mason-Pfizer monkey virus, and that BCA3 enhances HIV-1 protease-induced apoptosis. In the present study, we report that BCA3 is associated with purified and subtilisin-treated HIV particles. Using a combination of immune-based methods and confocal microscopy, we show that the C-terminus of BCA3 is required for packaging into HIV-1 particles. However, we were unable to identify an HIV-1 binding domain for BCA3, and we did not observe any effect of incorporated BCA3 on HIV-1 infectivity. Interestingly, the BCA3 C-terminus was previously identified as a binding site for the catalytic subunit of protein kinase A (PKAc), a cellular protein that is specifically packaged into HIV-1 particles. Based on our analysis of PKAc⁻BCA3 interactions, we suggest that BCA3 incorporation into HIV-1 particles is mediated by its ability to interact with PKAc.

The Paced Auditory Serial Addition Test (PASAT) is widely used to evaluate processing speed and executive function in patients with multiple sclerosis, traumatic brain injury, and other neurological disorders. In the PASAT, subjects listen to sequences of digits while continuously reporting the sum of the last two digits presented. Four different stimulus onset asynchronies (SOAs) are usually tested, with difficulty increasing as SOAs are reduced. Ceiling effects are common at long SOAs, while the digit delivery rate often exceeds the subject's processing capacity at short SOAs, causing some subjects to stop performing altogether. In addition, subjects may adopt an "alternate answer" strategy at short SOAs, which reduces the test's demands on working-memory and processing speed. Consequently, studies have shown that the number of dyads (consecutive correct answers) is a more sensitive measure of PASAT performance than the overall number of correct sums. Here, we describe a 2.5-minute computerized test, the Dyad-Adaptive PASAT (DA-PASAT), where SOAs are adjusted with a 2:1 staircase, decreasing after each pair of correct responses and increasing after misses. Processing capacity is reflected in the minimum SOA (minSOA) achieved in 54 trials. Experiment 1 gathered normative data in two large populations: 1617 subjects in New Zealand ranging in age from 18 to 65 years, and 214 Californians ranging in age from 18 to 82 years. Minimum SOAs were influenced by age, education, and daily hours of computer-use. Minimum SOA z-scores, calculated after factoring out the influence of these factors, were virtually identical in the two control groups, as were response times (RTs) and dyad ratios (the proportion of hits occurring in dyads). Experiment 2 measured the test-retest reliability of the DA-PASAT in 44 young subjects who underwent three test sessions at weekly intervals. High intraclass correlation coefficients (ICCs) were found for minSOAs (0.87), response times (0.76), and dyad ratios (0.87). Performance improved across test sessions for all measures. Experiment 3 investigated the effects of simulated malingering in 50 subjects: 42% of simulated malingerers produced abnormal (p< 0.05) minSOA z-scores. Simulated malingerers with abnormal scores were distinguished with 87% sensitivity and 69% specificity from control subjects with abnormal scores by excessive differences between training performance and the actual test. Experiment 4 investigated patients with traumatic brain injury (TBI): patients with mild TBI performed within the normal range while patients with severe TBI showed deficits. The DA-PASAT reduces the time and stress of PASAT assessment while gathering sensitive measures of dyad processing that reveal the effects of aging, malingering, and traumatic brain injury on performance.

Pulmonary hypertension (PH) is characterized by enhanced pulmonary vascular resistance, which causes right ventricle (RV) pressure overload and results in right sided heart failure and death. This work investigated the effectiveness of a combined therapy with PDE5 inhibitor (PDE5i) and a new adenosine A2A receptor (A2AR) agonist in mitigating monocrotaline (MCT) induced PH in rats.

Sweat includes active components and metabolites, which are needed to maintain skin homeostasis. Component changes in sweat derived from atopic dermatitis (AD) have been reported. To investigate the influence of sweat components on the pathogenesis of AD, we performed a multifaceted assessment, including nuclear magnetic resonance spectroscopy-based metabolomic analysis, and linked these features to clinical features of AD. Distinctive properties of AD sweat are the quite-variation in protein, anti-microbial peptides and glucose concentrations. pH, sodium, and other salt levels in sweat of AD were comparable to that of healthy subjects. Sweat from AD patients with acute inflammation had a more prominent increase in glucose concentration than sweat from healthy individuals or those with AD with chronic inflammation. Topical glucose application delayed recovery of transepidermal water loss in barrier-disrupted mice. Furthermore, the glucose transporter GLUT2 was highly expressed in the lumen of sweat glands from AD patients. AD patients with chronic inflammation had significantly increased GLUT2 mRNA expression and near normal sweat glucose levels. Despite the small sample size in our study, we speculate that the increased glucose levels might be affected by AD severity and phenotype. We hope that this report will bring novel insight into the impact of sweat components on the clinical manifestation of AD.

The molecular mechanisms underlying susceptibility to severe respiratory syncytial virus (RSV) infection remain poorly understood. Herein, we report on the role of osteopontin (OPN) in regulation of RSV infection in human epithelial cells and how interleukin-1 beta (IL-1β), a cytokine secreted soon after RSV infection, when persistently expressed can induce OPN expression leading to increased viral infection. We first compared OPN expression in two human epithelial cell lines: HEK-293 and HEp-2. In contrast to HEp-2, HEK-293 expresses low levels of pro-caspase-1 resulting in decreased IL-1β expression in response to RSV infection. We found a correlation between low IL-1β levels and a delay in induction of OPN expression in RSV-infected HEK-293 cells compared to HEp-2. This phenomenon could partially explain the high susceptibility of HEp-2 cells to RSV infection versus the moderate susceptibility of HEK-293 cells. Also, HEK-293 cells expressing low levels of pro-caspase-1 exhibit decreased IL-1β expression and delayed OPN expression in response to RSV infection. HEK-293 cells incubated with human rIL-1β showed a dose-dependent increase in OPN expression upon RSV infection. Also, incubation with rOPN increased RSV viral load. Moreover, HEp-2 cells or mice infected with a mucogenic RSV strain RSV-L19F showed elevated levels of OPN in contrast to mice infected with the laboratory RSV strain rA2. This correlated with elevated levels of OPN following infection with RSV-L19F compared to rA2. Together, these results demonstrate that increased OPN expression is regulated in part by IL-1β, and the interplay between IL-1β and OPN signaling may play a pivotal role in the spread of RSV infection.

HIV-1 replication normally requires Vif-mediated neutralization of APOBEC3 antiviral enzymes. Viruses lacking Vif succumb to deamination-dependent and -independent restriction processes. Here, HIV-1 adaptation studies were leveraged to ask whether viruses with an irreparable vif deletion could develop resistance to restrictive levels of APOBEC3G. Several resistant viruses were recovered with multiple amino acid substitutions in Env, and these changes alone are sufficient to protect Vif-null viruses from APOBEC3G-dependent restriction in T cell lines. Env adaptations cause decreased fusogenicity, which results in higher levels of Gag-Pol packaging. Increased concentrations of packaged Pol in turn enable faster virus DNA replication and protection from APOBEC3G-mediated hypermutation of viral replication intermediates. Taken together, these studies reveal that a moderate decrease in one essential viral activity, namely Env-mediated fusogenicity, enables the virus to change other activities, here, Gag-Pol packaging during particle production, and thereby escape restriction by the antiviral factor APOBEC3G. We propose a new paradigm in which alterations in viral homeostasis, through compensatory small changes, constitute a general mechanism used by HIV-1 and other viral pathogens to escape innate antiviral responses and other inhibitions including antiviral drugs.

It has been proposed that NADP+-specificity of isocitrate dehydrogenase (ICDH) evolved as an adaptation of microorganisms to grow on acetate as the sole source of carbon and energy. In Escherichia coli, changing the cofactor specificity of ICDH from NADP+ to NAD+ (cofactor swapping) decreases the growth rate on acetate. However, the metabolic basis of this phenotype has not been analyzed. In this work, we used constraint-based modeling to investigate the effect of the cofactor swapping of ICDH in terms of energy production, response of alternative sources of NADPH, and partitioning of fluxes between ICDH and isocitrate lyase (ICL) -a crucial bifurcation when the bacterium grows on acetate-. We generated E. coli strains expressing NAD+-specific ICDH instead of the native enzyme, and bearing the deletion of the NADPH-producing transhydrogenase PntAB. We measured their growth rate and acetate uptake rate, modeled the distribution of metabolic fluxes by Flux Balance Analysis (FBA), and quantified the specific activities of NADPH-producing dehydrogenases in central pathways. The cofactor swapping of ICDH led to one-third decrease in biomass yield, irrespective of the presence of PntAB. According to our simulations, the diminution in growth rate observed upon cofactor swapping could be explained by one-half decrease in the total production of NADPH and a lower availability of carbon for biosynthesis because of a change in the partition at the isocitrate bifurcation. Together with an increased total ATP production, this scenario resulted in a 10-fold increment in the flux of ATP not used for growing purposes. PntAB was identified as the primary NADPH balancing response, with the dehydrogenases of the oxidative branch of the Pentose Phosphate Pathway and the malic enzyme playing a role in its absence. We propose that in the context of E. coli growing on acetate, the NADP+-specificity of ICDH is a trait that impacts not only NADPH production, but also the efficient allocation of carbon and energy.

The strongest genetic association with Fuchs' endothelial corneal dystrophy (FECD) is the presence of an intronic (CTG·CAG)n trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene. Repeat-associated non-ATG (RAN) translation, an unconventional protein translation mechanism that does not require an initiating ATG, has been described in many TNR expansion diseases, including myotonic dystrophy type 1 (DM1). Given the similarities between DM1 and FECD, we wished to determine whether RAN translation occurs in FECD.

Retinoic acid (RA) is required for embryonic formation of the anterior segment of the eye and craniofacial structures. The present study further investigated the role of RA in maintaining the function of these neural crest-derived structures in adult zebrafish.

To study the effects of acute optic nerve damage on the reflectance of the retinal nerve fiber layer (RNFL) and to compare the time courses of changes of RNFL reflectance and thickness.

Understanding the physiology and genetics of human hypoxia tolerance has important medical implications, but this phenomenon has thus far only been investigated in high-altitude human populations. Another system, yet to be explored, is humans who engage in breath-hold diving. The indigenous Bajau people ("Sea Nomads") of Southeast Asia live a subsistence lifestyle based on breath-hold diving and are renowned for their extraordinary breath-holding abilities. However, it is unknown whether this has a genetic basis. Using a comparative genomic study, we show that natural selection on genetic variants in the PDE10A gene have increased spleen size in the Bajau, providing them with a larger reservoir of oxygenated red blood cells. We also find evidence of strong selection specific to the Bajau on BDKRB2, a gene affecting the human diving reflex. Thus, the Bajau, and possibly other diving populations, provide a new opportunity to study human adaptation to hypoxia tolerance. VIDEO ABSTRACT.

Teneurins (TENs) are cell-surface adhesion proteins with critical roles in tissue development and axon guidance. Here, we report the 3.1-Å cryoelectron microscopy structure of the human TEN2 extracellular region (ECR), revealing a striking similarity to bacterial Tc-toxins. The ECR includes a large β barrel that partially encapsulates a C-terminal domain, which emerges to the solvent through an opening in the mid-barrel region. An immunoglobulin (Ig)-like domain seals the bottom of the barrel while a β propeller is attached in a perpendicular orientation. We further show that an alternatively spliced region within the β propeller acts as a switch to regulate trans-cellular adhesion of TEN2 to latrophilin (LPHN), a transmembrane receptor known to mediate critical functions in the central nervous system. One splice variant activates trans-cellular signaling in a LPHN-dependent manner, whereas the other induces inhibitory postsynaptic differentiation. These results highlight the unusual structural organization of TENs giving rise to their multifarious functions.

Mitochondrial quality is under surveillance by autophagy, the cell recycling process which degrades and removes damaged mitochondria. Inadequate autophagy results in deterioration in mitochondrial quality, bioenergetic dysfunction, and metabolic stress. Here we describe in an integrated work-flow to assess parameters of mitochondrial morphology, function, mtDNA and protein damage, metabolism and autophagy regulation to provide the framework for a practical assessment of mitochondrial quality. This protocol has been tested with cell cultures, is highly reproducible, and is adaptable to studies when cell numbers are limited, and thus will be of interest to researchers studying diverse physiological and pathological phenomena in which decreased mitochondrial quality is a contributory factor.

The presence of arsenic (As) in groundwater is a major problem in several parts of Latin America. In the present work, non-destructive approaches to monitor the effects of As on plants of Cichorium intybus, an herbaceous Asteraceae, were explored. In this sense, the effects of As at different levels of water and radiation were evaluated on these crops. Plants were grown in a greenhouse, watered daily with As solutions and exposed to different water and/or light conditions for four months, using a three-factor (As, water, radiation) and two-level resource (As vs non As, field capacity vs half-field capacity condition, light vs shade condition) factorial design. The parameters most affected by this treatment were the area under the first derivative of the reflectance spectrum in the blue region, chlorophyll concentration, the Fred/Ffar-red fluorescence ratio and the quantum yield for the photophysical decay. These changes indicated the ability of this plant species to be a biomonitor for the presence of arsenic in irrigation water. Interestingly, it was further proved in this work that the biomonitoring capacity was enhanced in the presence of sunlight.

The toxicity towards the algal species Pseudokirchneriella subcapitata of 425 organic chemical substances was tested in a growth inhibition test. Precautions were taken to prevent loss of the compounds from the water phase and the test system (closed test system, low biomass, shorter test duration, silanized glass) and to keep pH constant by applying a higher alkalinity. Chemical phase distribution was modelled taking ionization, volatilisation, and adsorption to glass and biomass into consideration. If the modelled water concentration was below 90% of the nominal concentration the calculated EC values were corrected accordingly. The model helped to identify substances, where the calculated water concentration was too uncertain. Substances covering a wide range of physical-chemical properties and different modes of action were tested. Median effect concentrations (EC50) lower than 1000 mg/L were found for 310 substances; 216 of these were in the range from 1 to 1000 mg/L and 94 substances had EC50s below 1 mg/L and should be classified as "Very toxic". 36 substances fell in the group with EC50 > 1000 mg/L. An EC50 could not be established for 79 substances. These 425 different organic substances were tested under uniform conditions and thus considered a valuable source of information for administrators, industry, risk assessors and QSAR modellers.

A biological system is more than the sum of its parts - it accomplishes many functions via synergy. Deconstructing the system down to the molecular mechanism level necessitates the complement of reconstructing functions on all levels, i.e., in our conceptualization of biology and its perturbations, our experimental models and computer modelling. Toxicology contains the somewhat arbitrary subclass "systemic toxicities"; however, there is no relevant toxic insult or general disease that is not systemic. At least inflammation and repair are involved that require coordinated signaling mechanisms across the organism. However, the more body components involved, the greater the challenge to reca-pitulate such toxicities using non-animal models. Here, the shortcomings of current systemic testing and the development of alternative approaches are summarized. We argue that we need a systematic approach to integrating existing knowledge as exemplified by systematic reviews and other evidence-based approaches. Such knowledge can guide us in modelling these systems using bioengineering and virtual computer models, i.e., via systems biology or systems toxicology approaches. Experimental multi-organ-on-chip and microphysiological systems (MPS) provide a more physiological view of the organism, facilitating more comprehensive coverage of systemic toxicities, i.e., the perturbation on organism level, without using substitute organisms (animals). The next challenge is to establish disease models, i.e., micropathophysiological systems (MPPS), to expand their utility to encompass biomedicine. Combining computational and experimental systems approaches and the chal-lenges of validating them are discussed. The suggested 3S approach promises to leverage 21st century technology and systematic thinking to achieve a paradigm change in studying systemic effects.