Fast label-free multilayered histology-like imaging of human breast cancer by photoacoustic microscopy

The goal of breast-conserving surgery is to completely remove all of the cancer. Currently, no intraoperative tools can microscopically analyze the entire lumpectomy specimen, which results in 20 to 60% of patients undergoing second surgeries to achieve clear margins. To address this critical need, we have laid the foundation for the development of a device that could allow accurate intraoperative margin assessment. We demonstrate that by taking advantage of the intrinsic optical contrast of breast tissue, photoacoustic microscopy (PAM) can achieve multilayered histology-like imaging of the tissue surface. The high correlation of the PAM images to the conventional histologic images allows rapid computations of diagnostic features such as nuclear size and packing density, potentially identifying small clusters of cancer cells. Because PAM does not require tissue processing or staining, it can be performed promptly and intraoperatively, enabling immediate directed re-excision and reducing the number of second surgeries

Bone matrix components activate the NLRP3 inflammasome and promote osteoclast differentiation

AbstractThe NLRP3 inflammasome senses a variety of signals referred to as danger associated molecular patterns (DAMPs), including those triggered by crystalline particulates or degradation products of extracellular matrix. Since some DAMPs confer tissue-specific activation of the inflammasomes, we tested the hypothesis that bone matrix components function as DAMPs for the NLRP3 inflammasome and regulate osteoclast differentiation. Indeed, bone particles cause exuberant osteoclastogenesis in the presence of RANKL, a response that correlates with NLRP3 abundance and the state of inflammasome activation. To determine the relevance of these findings to bone homeostasis, we studied the impact of Nlrp3 deficiency on bone using pre-clinical mouse models of high bone turnover, including estrogen deficiency and sustained exposure to parathyroid hormone or RANKL. Despite comparable baseline indices of bone mass, bone loss caused by hormonal or RANKL perturbations is significantly reduced in Nlrp3 deficient than in wild type mice. Consistent with the notion that osteolysis releases DAMPs from bone matrix, pharmacologic inhibition of bone resorption by zoledronate attenuates inflammasome activation in mice. Thus, signals originating from bone matrix activate the NLRP3 inflammasome in the osteoclast lineage, and may represent a bone-restricted positive feedback mechanism that amplifies bone resorption in pathologic conditions of accelerated bone turnover.</jats:p

Dynamic changes in the osteoclast cytoskeleton in response to growth factors and cell attachment are controlled by β3 integrin

The β3 integrin cytoplasmic domain, and specifically S752, is critical for integrin localization and osteoclast (OC) function. Because growth factors such as macrophage colony–stimulating factor and hepatocyte growth factor affect integrin activation and function via inside-out signaling, a process requiring the β integrin cytoplasmic tail, we examined the effect of these growth factors on OC precursors. To this end, we retrovirally expressed various β3 integrins with cytoplasmic tail mutations in β3-deficient OC precursors. We find that S752 in the β3 cytoplasmic tail is required for growth factor–induced integrin activation, cytoskeletal reorganization, and membrane protrusion, thereby affecting OC adhesion, migration, and bone resorption. The small GTPases Rho and Rac mediate cytoskeletal reorganization, and activation of each is defective in OC precursors lacking a functional β3 subunit. Activation of the upstream mediators c-Src and c-Cbl is also dependent on β3. Interestingly, although the FAK-related kinase Pyk2 interacts with c-Src and c-Cbl, its activation is not disrupted in the absence of functional β3. Instead, its activation is dependent upon intracellular calcium, and on the β2 integrin. Thus, the β3 cytoplasmic domain is responsible for activation of specific intracellular signals leading to cytoskeletal reorganization critical for OC function

Photoacoustic microscopy enables multilayered histological imaging of human breast cancer without staining

In 2016, an estimated ~250,000 new cases of invasive and non-invasive breast cancer were diagnosed in US women. About 60–75% of these cases were treated with breast conserving surgery (BCS) as the initial therapy. To reduce the local recurrence rate, the goal of BCS is to excise the tumor with a rim of normal surrounding tissue, so that no cancer cells remain at the cut margin, while preserving as much normal breast tissue as possible. Therefore, patients with remaining cancer cells at the cut margin commonly require a second surgical procedure to obtain clear margins. Different approaches have been used to decrease the positive margin rate to avoid re-excision. However, these techniques are variously ineffective in reducing the re-operative rate, difficult to master by surgeons, or time-consuming for large specimens. Thus, 20-60% of patients undergoing BCS still require second surgeries due to positive surgical margins. The ideal tool for margin assessment would provide the same information as histological analysis, without the need for processing specimens. To achieve this goal, we have developed and refined label-free photoacoustic microscopy (PAM) for breast specimens. Exploiting the intrinsic optical contrast of tissue, ultraviolet (UV) laser illumination can highlight cell nuclei, thus providing the same contrast as hematoxylin labeling used in conventional histology and measuring features related to the histological landscape without the need for labels. We demonstrate that our UV-PAM system can provide label-free, high-resolution, and histology-like imaging of fixed, unprocessed breast tissue

Bone loss and aggravated autoimmune arthritis in HLA-DRβ1-bearing humanized mice following oral challenge with Porphyromonas gingivalis

BACKGROUND: The linkage between periodontal disease and rheumatoid arthritis is well established. Commonalities among the two are that both are chronic inflammatory diseases characterized by bone loss, an association with the shared epitope susceptibility allele, and anti-citrullinated protein antibodies. METHODS: To explore immune mechanisms that may connect the two seemingly disparate disorders, we measured host immune responses including T-cell phenotype and anti-citrullinated protein antibody production in human leukocyte antigen (HLA)-DR1 humanized C57BL/6 mice following exposure to the Gram-negative anaerobic periodontal disease pathogen Porphyromonas gingivalis. We measured autoimmune arthritis disease expression in mice exposed to P. gingivalis, and also in arthritis-resistant mice by flow cytometry and multiplex cytokine-linked and enzyme-linked immunosorbent assays. We also measured femoral bone density by microcomputed tomography and systemic cytokine production. RESULTS: Exposure of the gingiva of DR1 mice to P. gingivalis results in a transient increase in the percentage of Th17 cells, both in peripheral blood and cervical lymph nodes, a burst of systemic cytokine activity, a loss in femoral bone density, and the generation of anti-citrullinated protein antibodies. Importantly, these antibodies are not produced in response to P. gingivalis treatment of wild-type C57BL/6 mice, and P. gingivalis exposure triggered expression of arthritis in arthritis-resistant mice. CONCLUSIONS: Exposure of gingival tissues to P. gingivalis has systemic effects that can result in disease pathology in tissues that are spatially removed from the initial site of infection, providing evidence for systemic effects of this periodontal pathogen. The elicitation of anti-citrullinated protein antibodies in an HLA-DR1-restricted fashion by mice exposed to P. gingivalis provides support for the role of the shared epitope in both periodontal disease and rheumatoid arthritis. The ability of P. gingivalis to induce disease expression in arthritis-resistant mice provides support for the idea that periodontal infection may be able to trigger autoimmunity if other disease-eliciting factors are already present

Inhibition of CaMKK2 reverses age-associated decline in bone mass

Decline in bone formation is a major contributing factor to the loss of bone mass associated with aging. We previously showed that the genetic ablation of the tissue-restricted and multifunctional Ca(2+)/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) stimulates trabecular bone mass accrual, mainly by promoting anabolic pathways and inhibiting catabolic pathways of bone remodeling. In this study, we investigated whether inhibition of this kinase using its selective cell-permeable inhibitor STO-609 will stimulate bone formation in 32 week old male WT mice and reverse age-associated of decline in bone volume and strength. Tri-weekly intraperitoneal injections of saline or STO-609 (10 μM) were performed for six weeks followed by metabolic labeling with calcein and alizarin red. New bone formation was assessed by dynamic histomorphometry whereas micro-computed tomography was employed to measure trabecular bone volume, microarchitecture and femoral mid-shaft geometry. Cortical and trabecular bone biomechanical properties were assessed using three-point bending and punch compression methods respectively. Our results reveal that as they progress from 12 to 32 weeks of age, WT mice sustain a significant decline in trabecular bone volume, microarchitecture and strength as well as cortical bone strength. However, treatment of the 32 week old WT mice with STO-609 stimulated apposition of new bone and completely reversed the age-associated decrease in bone volume, quality, as well as trabecular and cortical bone strength. We also observed that regardless of age, male Camkk2(-/-) mice possessed significantly elevated trabecular bone volume, microarchitecture and compressive strength as well as cortical bone strength compared to age-matched WT mice, implying that the chronic loss of this kinase attenuates age-associated decline in bone mass. Further, whereas STO-609 treatment and/or the absence of CaMKK2 significantly enhanced the femoral mid-shaft geometry, the mid-shaft cortical wall thickness and material bending stress remained similar among the cohorts, implying that regardless of treatment, the material properties of the bone remain similar. Thus, our cumulative results provide evidence for the pharmacological inhibition of CaMKK2 as a bone anabolic strategy in combating age-associated osteoporosis

Association between ambient temperature, particulate air pollution and emergency room visits for conjunctivitis

BACKGROUND: Numerous studies have confirmed the association of ambient temperature and air pollution with a higher risk of morbidities, yet few have addressed their effect on the ocular system. The purpose of this study was to assess the association between temperature, air pollution, and emergency room visits for conjunctivitis. METHODS: In this case-crossover study, the records of all emergency room visits to Soroka University Medical Center (SUMC) from 2009 to 2014 were reviewed for patients with conjunctivitis. Daily exposure to fine and coarse particulate matter and temperature were determined by a hybrid model involving satellite sensors. Mean relative humidity was obtained from the Ministry of Environmental Protection meteorological monitoring station located in Beer-Sheva. RESULTS: Six hundred one patients were diagnosed with conjunctivitis in the SUMC emergency room. We discovered a positive association between temperature increments and incidence of conjunctivitis. The strongest effect was found during summer and autumn, with an immediate (lag0) incidence increase of 8.1% for each 1 degrees C increase in temperature (OR = 1.088, 95%CI: 1.046-1.132) between 24 and 28 degrees C in the summer and 7.2% for each 1 degrees C increase in temperature (OR = 1.072, 95%CI: 1.036-1.108) between 13 and 23 degrees C in the autumn. There was no statistically significant association between fine and coarse particulate matter and conjunctivitis incidence. CONCLUSION: Temperature increases during summer and autumn are significantly associated with an increased risk of conjunctivitis. Conjunctivitis is not associated with non-anthropogenic air pollution. These findings may help community clinics and hospital emergency rooms better predict conjunctivitis cases and will hopefully lead to improved prevention efforts that will lower the financial burden on both the individual and the public

LMWH in the prevention of preeclampsia and fetal growth restriction in women without thrombophilia. A systematic review and meta-analysis.

SummaryPlacental mediated pregnancy complications such as preeclampsia and fetal growth restriction (FGR) are common, serious, and associated with increased morbidity and mortality. We conducted a systematic review and meta-analysis to determine the effect of treatment with low-molecular-weight heparins (LMWHs) for secondary prevention of these complications in non thrombophilic women. We searched the electronic databases PubMed, Scopus, and Cochrane Library for randomised controlled trials addressing this question. Five studies including 403 patients met the inclusion criteria, 68 developed preeclampsia and 118 FGR. The studies were very heterogeneous in terms of inclusion criteria, LMWH preparation, and dosage. Meta-analyses were performed using random-effect models. The overall use of LMWHs was associated with a risk reduction for preeclampsia (Relative risk (RR) 0.366; 95 % confidence interval (CI), 0.219–0.614) and FGR (RR 0.409; 95 % CI, 0.195–0.932) vs. no treatment. From the data available for analysis it appears that the use of Dalteparin is associated with a risk reduction for preeclampsia (p=0.002) and FGR (p&lt;0.001); while Enoxaparin is associated with risk reduction for preeclampsia (p=0.013) but not for FGR (p=0.3). In spite of the small number of studies addressing the research question, and the high variability among them, our meta-analysis found a modest beneficial effect of LMWH for secondary prevention of preeclampsia and FGR. Further studies are needed to address these questions before a definite conclusion can be reached.Supplementary Material to this article is available online at www.thrombosis-online.com.</jats:p

Evaluation of ground information with respect to EPB tunnelling for the Thessaloniki metro, Greece

Ο Μητροπολιτικός σιδηρόδρομος της Θεσσαλονίκης αποτελείται από δύο παράλληλες σήραγγες διαμέτρου ~6 m και μήκους ~8 km η κάθε μία και περιλαμβάνει 13 σταθμούς. Η γεωλογία του πολεοδομικού συγκροτήματος της Θεσσαλονίκης χαρακτηρίζεται από την παρουσία νεογενών και τεταρτογενών αποθέσεων. Ο κύριος σχηματισμός της περιοχής του έργου είναι μία σειρά πολύ στιφρών έως σκληρών ερυθρών αργίλων ανωμειοκαινικής-πλειοκαινικής ηλικίας. Σχηματισμοί του Τεταρτογενούς που έχουν αποτεθεί πάνω σε αυτές τις αργίλους συνίστανται από αργιλώδεις-ιλυώδεις άμμους ή/και χάλικες. Το πρόγραμμα γεωερευνητικών εργασιών περιελάμβανε έναν σημαντικό αριθμό δειγματοληπτικών γεωτρήσεων, επί τόπου και εργαστηριακών δοκιμών. Τα στοιχεία του γεωερευνητικού προγράμματος αξιολογήθηκαν ώστε να κατανοηθεί καλύτερα το γεωλογικό προσομοίωμα της περιοχής του έργου και να διακριτοποιηθούν ζώνες με βάση τη συμπεριφορά των γεωυλικών κατά τη διάνοιξη της σήραγγας με μηχάνημα ολομέτωπης κοπής (ΤΒΜ). Όσον αφορά το μηχάνημα διάνοιξης, η επιλογή ενός μηχανήματος εδαφικής εξισορροπητικής πίεσης (ΕΡΒΜ) φαίνεται να είναι εύλογη τόσο από πλευράς ευστάθειας όσο και από πλευράς ρυθμού προχώρησης. Η επιλογή αυτή υπαγορεύεται από τα χαρακτηριστικά του εδάφους για την κάλυψη όλων των αντικειμενικών σκοπών όπως ο έλεγχος των καθιζήσεων και εδαφικών μετακινήσεων, η διατήρηση της στάθμης του υπόγειου νερού αλλά και η ικανοποιητική προχώρηση των σηράγγωνThe Thessaloniki Metropolitan Railway comprises two separate ~6 m diameter parallel tunnels with an ~8 km stretch each and 13 stations. The geology of the urban area of Thessaloniki is characterised by the presence of Neogene and Quaternary deposits. The base formation for the project area is a very stiff to hard red clay, dating to Upper Miocene-Pliocene. Upon this formation, Quaternary sediments have been deposited, most of which comprise sand and/or gravel in a clay-silt dominated matrix, covered in places by anthropogenic fill. Ground investigation campaigns incorporated a significant number of sampling boreholes and in situ and laboratory testing. This information was elaborated in order to obtain a better geological understanding and a geotechnical zonation of the ground with respect to mechanized tunnelling. EPB M appears to be the reasonable choice for the project in all aspects of tunnel safety and tunnelling performance. The characteristics and parameters of the soils and the hydrogeological regime directed towards this selection and it is expected that all the objectives, such as settlement and ground movements control, water table level maintenance and adequate performance, will be met by an EPBM provided it is properly operate