Pathogen infection and cholesterol deficiency activate the C. elegans p38 immune pathway through a TIR-1/SARM1 phase transition.

Intracellular signaling regulators can be concentrated into membrane-free, higher ordered protein assemblies to initiate protective responses during stress - a process known as phase transition. Here, we show that a phase transition of the Caenorhabditis elegans Toll/interleukin-1 receptor domain protein (TIR-1), an NAD+ glycohydrolase homologous to mammalian sterile alpha and TIR motif-containing 1 (SARM1), underlies p38 PMK-1 immune pathway activation in C. elegans intestinal epithelial cells. Through visualization of fluorescently labeled TIR-1/SARM1 protein, we demonstrate that physiologic stresses, both pathogen and non-pathogen, induce multimerization of TIR-1/SARM1 into visible puncta within intestinal epithelial cells. In vitro enzyme kinetic analyses revealed that, like mammalian SARM1, the NAD+ glycohydrolase activity of C. elegans TIR-1 is dramatically potentiated by protein oligomerization and a phase transition. Accordingly, C. elegans with genetic mutations that specifically block either multimerization or the NAD+ glycohydrolase activity of TIR-1/SARM1 fail to induce p38 PMK phosphorylation, are unable to increase immune effector expression, and are dramatically susceptible to bacterial infection. Finally, we demonstrate that a loss-of-function mutation in nhr-8, which alters cholesterol metabolism and is used to study conditions of sterol deficiency, causes TIR-1/SARM1 to oligomerize into puncta in intestinal epithelial cells. Cholesterol scarcity increases p38 PMK-1 phosphorylation, primes immune effector induction in a manner that requires TIR-1/SARM1 oligomerization and its intrinsic NAD+ glycohydrolase activity, and reduces pathogen accumulation in the intestine during a subsequent infection. These data reveal a new adaptive response that allows a metazoan host to anticipate pathogen threats during cholesterol deprivation, a time of relative susceptibility to infection. Thus, a phase transition of TIR-1/SARM1 as a prerequisite for its NAD+ glycohydrolase activity is strongly conserved across millions of years of evolution and is essential for diverse physiological processes in multiple cell types.

From worms to humans, animals have developed various strategies ­ including immune defences ­ to shield themselves from disease-causing microbes. A type of roundworm, called C. elegans, lives in environments rich in microbes, so it needs effective immune defences to protect itself. The roundworms share a key regulatory pathway with mammals that helps to control their immune responses. This so-called p38 pathway relies on proteins that interact with each other to activate protective immune defences. Proteins contain different regions or domains that can give them a certain function. For example, proteins with a region called TIR play important roles in immune defences in both animals and plants. One such protein, called SARM1, is unique among animal and plant proteins in that it is an enzyme, which cleaves an important metabolite in the cell. In C. elegans, the SARM1 homolog, TIR-1, controls the p38 pathway during infection, but how TIR-1 activates it is unclear. To find out more, Peterson, Icso et al. modified C. elegans to generate a fluorescent form of TIR-1 and infected the worms with bacteria. Imaging techniques revealed that infection caused TIR-1 in gut cells to cluster into organized structures, which increases the enzymatic activity of the protein to activate the p38 immune pathway. Moreover, stress situations, such as cholesterol nutrient withdrawal, activated the p38 pathway in the same way. This adaptive stress response allows the animal to defend itself against pathogen threats during times, when they are most susceptible to infections. Cells in the gut provide a primary line of defence against infectious bacteria and are important for maintaining a healthy gut immune system. When the mechanisms for pathogen sensing and immune maintenance are disrupted, it can lead to inflammation and higher risk of infection. Peterson, Icso et al. show how a key regulator of gut immunity, TIR-1, provides protection in C. elegans, which may suggest that SARM1 could have a similar role in mammals.

Synoptic assessment of coastal total alkalinity through community science.

Comprehensive sampling of the carbonate system in estuaries and coastal waters can be difficult and expensive because of the complex and heterogeneous nature of near-shore environments. We show that sample collection by community science programs is a viable strategy for expanding estuarine carbonate system monitoring and prioritizing regions for more targeted assessment. 'Shell Day' was a single-day regional water monitoring event coordinating coastal carbonate chemistry observations by 59 community science programs and seven research institutions in the northeastern United States, in which 410 total alkalinity (TA) samples from 86 stations were collected. Field replicates collected at both low and high tides had a mean standard deviation between replicates of 3.6 ± 0.3 µmol kg-1 (σ mean ± SE, n = 145) or 0.20 ± 0.02%. This level of precision demonstrates that with adequate protocols for sample collection, handling, storage, and analysis, community science programs are able to collect TA samples leading to high-quality analyses and data. Despite correlations between salinity, temperature, and TA observed at multiple spatial scales, empirical predictions of TA had relatively high root mean square error >48 µmol kg-1. Additionally, ten stations displayed tidal variability in TA that was not likely driven by low TA freshwater inputs. As such, TA cannot be predicted accurately from salinity using a single relationship across the northeastern US region, though predictions may be viable at more localized scales where consistent freshwater and seawater endmembers can be defined. There was a high degree of geographic heterogeneity in both mean and tidal variability in TA, and this single-day snapshot sampling identified three patterns driving variation in TA, with certain locations exhibiting increased risk of acidification. The success of Shell Day implies that similar community science based events could be conducted in other regions to not only expand understanding of the coastal carbonate system, but also provide a way to inventory monitoring assets, build partnerships with stakeholders, and expand education and outreach to a broader constituency.

The fatty acid oleate is required for innate immune activation and pathogen defense in Caenorhabditis elegans.

Fatty acids affect a number of physiological processes, in addition to forming the building blocks of membranes and body fat stores. In this study, we uncover a role for the monounsaturated fatty acid oleate in the innate immune response of the nematode Caenorhabditis elegans. From an RNAi screen for regulators of innate immune defense genes, we identified the two stearoyl-coenzyme A desaturases that synthesize oleate in C. elegans. We show that the synthesis of oleate is necessary for the pathogen-mediated induction of immune defense genes. Accordingly, C. elegans deficient in oleate production are hypersusceptible to infection with diverse human pathogens, which can be rescued by the addition of exogenous oleate. However, oleate is not sufficient to drive protective immune activation. Together, these data add to the known health-promoting effects of monounsaturated fatty acids, and suggest an ancient link between nutrient stores, metabolism, and host susceptibility to bacterial infection.

Computed Tomography Measures of Nutrition in Patients With End-Stage Liver Disease Provide a Novel Approach to Characterize Deficits.

BACKGROUND: Patients with cirrhosis and end-stage liver disease (ESLD) develop severe nutrition deficits that affect morbidity and mortality. Laboratory measures of nutrition fail to fully assess clinical deficits in muscle mass and fat stores. This study employs computed tomography imaging to assess muscle mass and subcutaneous and visceral fat stores in patients with ESLD. METHODS: This 1:1 case-control study design compares ESLD patients with healthy controls. Study patients were selected from a database of ESLD patients using a stratified method to assure a representative sample based on age, body mass index (BMI), sex, and model for end-stage liver disease score (MELD). Control patients were trauma patients with a low injury severity score (<10) who had a computed tomography scan during evaluation. Cases and controls were matched for age ± 5 years, sex, and BMI ± 2. RESULTS: There were 90 subjects and 90 controls. ESLD patients had lower albumin levels (P < .001), but similar total protein levels (P = .72). ESLD patients had a deficit in muscle mass (-19%, P < .001) and visceral fat (-13%, P < .001), but similar subcutaneous fat (-1%, P = .35). ESLD patients at highest risk for sarcopenia included those over age 60, BMI<25.0, and female sex. We found degree of sarcopenia to be independent of model for end-stage liver disease score. CONCLUSIONS: These results support previous research demonstrating substantial nutrition deficits in ESLD patients that are not adequately measured by laboratory testing. Patients with ESLD have significant deficits of muscle and visceral fat stores, but a similar amount of subcutaneous fat.

The mitotic kinase Aurora--a promotes distant metastases by inducing epithelial-to-mesenchymal transition in ERα(+) breast cancer cells.

In this study, we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces stabilization and accumulation of Aurora-A mitotic kinase that ultimately drives the transition from an epithelial to a highly invasive mesenchymal phenotype in estrogen receptor α-positive (ERα(+)) breast cancer cells. The transition from an epithelial- to a mesenchymal-like phenotype was characterized by reduced expression of ERα, HER-2/Neu overexpression and loss of CD24 surface receptor (CD24(-/low)). Importantly, expression of key epithelial-to-mesenchymal transition (EMT) markers and upregulation of the stemness gene SOX2 was linked to acquisition of stem cell-like properties such as the ability to form mammospheres in vitro and tumor self-renewal in vivo. Moreover, aberrant Aurora-A kinase activity induced phosphorylation and nuclear translocation of SMAD5, indicating a novel interplay between Aurora-A and SMAD5 signaling pathways in the development of EMT, stemness and ultimately tumor progression. Importantly, pharmacological and molecular inhibition of Aurora-A kinase activity restored a CD24(+) epithelial phenotype that was coupled to ERα expression, downregulation of HER-2/Neu, inhibition of EMT and impaired self-renewal ability, resulting in the suppression of distant metastases. Taken together, our findings show for the first time the causal role of Aurora-A kinase in the activation of EMT pathway responsible for the development of distant metastases in ERα(+) breast cancer cells. Moreover, this study has important translational implications because it highlights the mitotic kinase Aurora-A as a novel promising therapeutic target to selectively eliminate highly invasive cancer cells and improve the disease-free and overall survival of ERα(+) breast cancer patients resistant to conventional endocrine therapy.

High-fidelity, inexpensive surgical middle ear simulator.

HYPOTHESIS: A high-fidelity, inexpensive middle ear simulator could be created to enhance surgical training that would be rated as having high face validity by experts. BACKGROUND: With rapid prototyping using additive manufacturing technology (AMT), one can create high-resolution 3-dimensional replicas of the middle ear at low cost and high fidelity. Such a simulator could be of great benefit for surgical training, particularly in light of new resident training guidelines. METHODS: AMT was used to create surgical middle ear simulator (SMS) with 2 different materials simulating bone and soft tissue. The simulator is composed of an outer box with dimensions of an average adult external auditory canal without scutum and an inner cartridge based on an otosclerosis model. The simulator was then rated by otology experts in terms of face validity and fidelity as well as their opinion on the usefulness of such a device. RESULTS: Eighteen otologists from 6 tertiary academic centers rated the simulator; 83.3% agreed or highly agreed that SMS has accurate dimensions and 66.6% that it has accurate tactile feedback. When asked if performance of stapedotomy with the SMS improves with practice, 46% agreed. As to whether practicing stapedotomy with the SMS translates to improvement with live surgery, 78% agreed with this statement. Experts' average rating of the components of SMS (of possible 5) was as follows: middle ear dimensions, 3.9; malleus, 3.7; incus, 3.6; stapes, 3.6; chorda tympani, 3.7; tensor tympani, 4.1; stapedius, 3.8; facial nerve, 3.7; and promontory, 3.5. Overall, 83% found SMS to be at least "very useful" in training of novices, particularly for junior and senior residents. CONCLUSION: Most experts found the SMS to be accurate, but there was a large discrepancy in rating of individual components. Most found it to be very useful for training of novice surgeons. With these results, we are encouraged to proceed with further refinements that will strengthen the SMS as a training tool for otologic surgery.

Acquired epidermodysplasia verruciformis due to multiple and unusual HPV infection among vertically-infected, HIV-positive adolescents in Zimbabwe.

BACKGROUND: We have previously described the presentation of epidermodysplasia verruciformis (EV)-like eruptions in almost a quarter of hospitalized adolescents with vertically-acquired human immunodeficiency virus (HIV) infection in Harare, Zimbabwe, a region with a high prevalence of HIV infection. METHODS: We performed a clinical case note review and skin biopsy from affected sites in 4 HIV-infected adolescents with EV-like lesions in Harare. Biopsies were processed for histology and for human papillomavirus (HPV) typing. RESULTS: All patients had long-standing skin lesions that pre-dated the diagnosis of HIV by several years. The histology of skin biopsies from all patients was consistent with EV. In each biopsy, EV-associated ß-HPV type 5 was identified (additionally, type 19 was found in 1 biopsy). Cutaneous wart-associated HPV types 1 and 2 were detected in all biopsies, together with genital lesion-associated HPV types 6, 16, and 52, (as well as ≥3 other genital lesion-associated HPV types). Despite immune reconstitution with combination antiretroviral therapy (cART), there was no improvement in EV-like lesions in any patient. CONCLUSIONS: EV is a disfiguring and potentially stigmatizing condition among this patient group and is difficult to treat; cART appears to have no impact on the progression of skin disease. Among adolescents with longstanding HIV-induced immunosuppression and with high levels of sun exposure, close dermatological surveillance for potential skin malignancy is required.

Single-walled carbon nanotube-induced mitotic disruption.

Carbon nanotubes were among the earliest products of nanotechnology and have many potential applications in medicine, electronics, and manufacturing. The low density, small size, and biological persistence of carbon nanotubes create challenges for exposure control and monitoring and make respiratory exposures to workers likely. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to 24, 48 and 96 µg/cm(2) single-walled carbon nanotubes (SWCNT). To investigate mitotic spindle aberrations at concentrations anticipated in exposed workers, primary and immortalized human airway epithelial cells were exposed to SWCNT for 24-72 h at doses equivalent to 20 weeks of exposure at the Permissible Exposure Limit for particulates not otherwise regulated. We have now demonstrated fragmented centrosomes, disrupted mitotic spindles and aneuploid chromosome number at those doses. The data further demonstrated multipolar mitotic spindles comprised 95% of the disrupted mitoses. The increased multipolar mitotic spindles were associated with an increased number of cells in the G2 phase of mitosis, indicating a mitotic checkpoint response. Nanotubes were observed in association with mitotic spindle microtubules, the centrosomes and condensed chromatin in cells exposed to 0.024, 0.24, 2.4 and 24 µg/cm(2) SWCNT. Three-dimensional reconstructions showed carbon nanotubes within the centrosome structure. The lower doses did not cause cytotoxicity or reduction in colony formation after 24h; however, after three days, significant cytotoxicity was observed in the SWCNT-exposed cells. Colony formation assays showed an increased proliferation seven days after exposure. Our results show significant disruption of the mitotic spindle by SWCNT at occupationally relevant doses. The increased proliferation that was observed in carbon nanotube-exposed cells indicates a greater potential to pass the genetic damage to daughter cells. Disruption of the centrosome is common in many solid tumors including lung cancer. The resulting aneuploidy is an early event in the progression of many cancers, suggesting that it may play a role in both tumorigenesis and tumor progression. These results suggest caution should be used in the handling and processing of carbon nanotubes.

A virtual surgical environment for rehearsal of tympanomastoidectomy.

This article presents a virtual surgical environment whose purpose is to assist the surgeon in preparation for individual cases. The system constructs interactive anatomical models from patient-specific, multi-modal preoperative image data, and incorporates new methods for visually and haptically rendering the volumetric data. Evaluation of the system's ability to replicate temporal bone dissections for tympanomastoidectomy, using intraoperative video of the same patients as guides, showed strong correlations between virtual and intraoperative anatomy. The result is a portable and cost-effective tool that may prove highly beneficial for the purposes of surgical planning and rehearsal.

In vivo micro-image mosaicing.

Recent advances in optical imaging have led to the development of miniature microscopes that can be brought to the patient for visualizing tissue structures in vivo. These devices have the potential to revolutionize health care by replacing tissue biopsy with in vivo pathology. One of the primary limitations of these microscopes, however, is that the constrained field of view can make image interpretation and navigation difficult. In this paper, we show that image mosaicing can be a powerful tool for widening the field of view and creating image maps of microanatomical structures. First, we present an efficient algorithm for pairwise image mosaicing that can be implemented in real time. Then, we address two of the main challenges associated with image mosaicing in medical applications: cumulative image registration errors and scene deformation. To deal with cumulative errors, we present a global alignment algorithm that draws upon techniques commonly used in probabilistic robotics. To accommodate scene deformation, we present a local alignment algorithm that incorporates deformable surface models into the mosaicing framework. These algorithms are demonstrated on image sequences acquired in vivo with various imaging devices including a hand-held dual-axes confocal microscope, a miniature two-photon microscope, and a commercially available confocal microendoscope.

What you can't feel won't hurt you: Evaluating haptic hardware using a haptic contrast sensitivity function.

In this paper, we extend the concept of the contrast sensitivity function - used to evaluate video projectors - to the evaluation of haptic devices. We propose using human observers to determine if vibrations rendered using a given haptic device are accompanied by artifacts detectable to humans. This determination produces a performance measure that carries particular relevance to applications involving texture rendering. For cases in which a device produces detectable artifacts, we have developed a protocol that localizes deficiencies in device design and/or hardware implementation. In this paper, we present results from human vibration detection experiments carried out using three commercial haptic devices and one high performance voice coil motor. We found that all three commercial devices produced perceptible artifacts when rendering vibrations near human detection thresholds. Our protocol allowed us to pinpoint the deficiencies, however, and we were able to show that minor modifications to the haptic hardware were sufficient to make these devices well suited for rendering vibrations, and by extension, the vibratory components of textures. We generalize our findings to provide quantitative design guidelines that ensure the ability of haptic devices to proficiently render the vibratory components of textures.

Induction of aneuploidy by single-walled carbon nanotubes.

Engineered carbon nanotubes are newly emerging manufactured particles with potential applications in electronics, computers, aerospace, and medicine. The low density and small size of these biologically persistent particles makes respiratory exposures to workers likely during the production or use of commercial products. The narrow diameter and great length of single-walled carbon nanotubes (SWCNT) suggest the potential to interact with critical biological structures. To examine the potential of nanotubes to induce genetic damage in normal lung cells, cultured primary and immortalized human airway epithelial cells were exposed to SWCNT or a positive control, vanadium pentoxide. After 24 hr of exposure to either SWCNT or vanadium pentoxide, fragmented centrosomes, multiple mitotic spindle poles, anaphase bridges, and aneuploid chromosome number were observed. Confocal microscopy demonstrated nanotubes within the nucleus that were in association with cellular and mitotic tubulin as well as the chromatin. Our results are the first to report disruption of the mitotic spindle by SWCNT. The nanotube bundles are similar to the size of microtubules that form the mitotic spindle and may be incorporated into the mitotic spindle apparatus.