The smart approach: feasibility of lentigo maligna superficial margin assessment with hand-held reflectance confocal microscopy technology.

BACKGROUND: Lentigo maligna may be challenging to clear surgically. OBJECTIVE: To evaluate feasibility of using superficial skin cuts as RCM imaging anchors for attaining negative surgical margins in lentigo maligna. METHODS: Included patients presented with lentigo maligna near cosmetically sensitive facial structures. We evaluated, with hand-held-RCM, microscopic clearance of melanoma beyond its dermoscopically detected edges. Evaluated margins were annotated using shallow skin cuts. If a margin was positive at 'first-step' RCM evaluation, we sequentially advanced the margin radially outward at that segment by 2-mm intervals until an RCM-negative margin was identified. Prior to final surgical excision, we placed sutures at the outmost skin cuts to allow comparison of RCM and histopathological margin assessments. Primary outcome measure was histopathological verification that RCM-negative margins were clear of melanoma. RESULTS: The study included 126 first-step margin evaluations in 23 patients, median age 70 years (range: 43-91). Seventeen patients (74%) had primary in-situ melanoma and six (26%) invasive melanoma, mean thickness 0.3 mm (range 0.2-0.4 mm). Six cases (26%) showed complete negative RCM margins on 'first-step', 11 (48%) were negative at 'second-step', and four (17%) at 'third-step'. In two additional cases (9%), margins clearance could not be determined via RCM due to widespread dendritic cells proliferation. The RCM-negative margins in all 21 cases proved clear of melanoma on histopathology. Of the 15 cases that returned at 1-year follow-up, none showed any residual melanoma on dermoscopic and RCM examinations. Interobserver reproducibility showed fair agreement between bedside RCM reader and blinded remote-site reader, with Spearman's rho of 0.48 and Cohen's kappa of 0.43; using bedside reader as reference, the remote reader's sensitivity was 92% and specificity 57% in positive margin detection. CONCLUSIONS: Margin mapping of lentigo maligna with hand-held-RCM, using superficial skin cuts, appears feasible. This approach needs validation by larger studies.

Dermoscopic and reflectance confocal microscopy features of cutaneous squamous cell carcinoma.

BACKGROUND: Squamous cell carcinoma (SCC) of the skin is a highly prevalent neoplasm. The management and the prognosis of this tumour are dependent on its invasiveness and its grade of differentiation. OBJECTIVES: To evaluate whether specific dermoscopic and reflectance confocal microscopy (RCM) criteria can predict the diagnosis of invasive SCC vs. in situ SCC and poorly differentiated compared with well- and moderately differentiated SCC. METHODS: Dermoscopic and RCM images of SCC were retrospectively evaluated for the presence of predefined criteria. RESULTS: Among 143 SCCs, 121 cases had a complete set of images and thus were included in the study set. The head and neck area was the most frequently involved body site (74/121; 61.1%) followed by extremities (36/121, 29.7%) and trunk (11/121, 9.1%). Seventy tumours were in situ (57.8%), while 51 were invasive (42.1%), of these 11 were poorly differentiated (21.5%), 16 were moderately differentiated (31.3%), and 24 were well differentiated (47.0%). Chi-squared analysis demonstrated that invasive SCCs were characterized by polymorphic vessels, erosion/ulceration, architectural disarrangement, speckled nucleated cells in the dermis, irregularly dilated vessels and absence of hyperkeratosis. Buttonhole vessels, white structureless areas and dotted or glomerular vessels were significantly associated with in situ lesions. Poorly differentiated SCCs were typified by red areas, erosion/ulceration and architectural disarrangement. Well- or moderately differentiated SCCs were associated with white areas and speckled nucleated cells in the epidermis. CONCLUSION: Clinical, dermoscopic and RCM images provide useful information that should be integrated in order to achieve the optimal therapeutic management for the patient.

Dermoscopic difficult lesions: an objective evaluation of reflectance confocal microscopy impact for accurate diagnosis.

BACKGROUND: Early detection of melanoma is the main objective to ensure a high survival rate. In some cases melanoma diagnosis still remain difficult and this leads to unnecessary excisions. OBJECTIVE: The aim of this study was to detect the most relevant Reflectance confocal microscopy (RCM) features for the detection of dermoscopic difficult melanomas. METHOD: A total of 322 lesions were selected from database and were evaluated on dermoscopy according to the 7-point checklist score, in blind from histological diagnosis. We classified the lesions into three categories: (i) 'featureless' lesions with score ranging between 0 and 2; (ii) 'positive-borderline' moles with score between 3 and 4 and (iii) 'positive-clear cut' lesions with score from 5 to 10. We evaluated confocal features of the 'featureless' lesions and of the 'positive-borderline' lesions. Evaluated confocal features were as follows: presence of pagetoid cells, cell shape (roundish or dendritic) and number (< 5 or >5 cells per mm(2) ), overall architecture (ringed, meshwork, clods and non-specific pattern); architectural disorder, presence of cytological atypia (>5 cells per mm(2) ) and cells arranged in nests. RESULTS: Among 322 lesions 70 were melanomas and 252 were nevi. According to the classification based on the 7-point checklist score, 130 'featureless lesions' (score 0-2) including six melanomas, and 102 'positive-borderline' moles (score 3-4) including 17 melanomas, were identified. Round pagetoid cells >5 cells per mm(2) and/or architectural disorder on RCM were found in all of six melanomas with featureless dermoscopy. Round pagetoid infiltration and five or more atypical cells at the DEJ were found in 16 positive 'borderline melanomas'. CONCLUSIONS: RCM represents a rapid non-invasive technique that can aid early diagnosis of dermoscopic difficult melanomas. Use of RCM on lesions with clinical and/or dermoscopic suspect of malignancy may reduce the number of unnecessary excision increasing the rate of accurate diagnoses.

Gene transfer of integration defective anti-HSV-1 meganuclease to human corneas ex vivo.

Corneal graft rejection is a major problem in chronic herpetic keratitis (HK) patients with latent infection. A new class of antiviral agents targeting latent and active forms of herpes simplex virus type 1 (HSV-1) is importantly required. Meganucleases are sequence-specific homing endonucleases capable of inducing DNA double-strand breaks. A proof-of-concept experiment has shown that tailor-made meganucleases are efficient against HSV-1 in vitro. To take this work a step forward, we hypothesized that the pre-treatment of human corneas in eye banks using meganuclease-encoding vectors will allow HK patients to receive a medicated cornea to resist the recurrence of the infection and the common graft rejection problem. However, this strategy requires efficient gene delivery to human corneal endothelium. Using recombinant adeno-associated virus, serotype 2/1 (rAAV2/1), efficient gene delivery of a reporter gene was demonstrated in human corneas ex vivo. The optimum viral dose was 3.7 × 10(11) VG with an exposure time of 1 day, followed by 6 days incubation in de-swelling medium. In addition, 12 days incubation can result in transgene expression in excess of 70%. Using similar transduction conditions, meganuclease transgene expression was detected in 39.4% of the endothelial cells after 2 weeks in culture. Reduction of the total viral load in the media and the endothelial cells of corneas infected with HSV-1 was shown. Collectively, this work provides information about the optimum conditions to deliver genetic material to the cornea, and demonstrates for the first time the expression of meganuclease in human corneas ex vivo and its antiviral activity. In conclusion, we demonstrate that the treatment of human corneas in eye banks before transplantation is a new approach to address the unmet clinical needs in corneal diseases.

Evaluation of tire tread particle toxicity to fish using rainbow trout cell lines.

Tire and road wear particles (TRWP) resulting from tire abrasion while driving raise concerns due to their potential contribution to aquatic toxicity. Our study aimed to assess cryogenically milled tire tread (CMTT) particle toxicity, used as a proxy for TRWP, and associated chemicals to fish using two Rainbow Trout (Oncorhynchus mykiss) cell lines representing the gill (RTgill-W1) and the intestinal (RTgutGC) epithelium. CMTT toxicity was evaluated through several exposure pathways, including direct contact, leaching, and digestion, while also assessing the impact of particle aging. Following OECD TG249, cell viability was assessed after 24 h acute exposure using a multiple-endpoint assay indicative of cell metabolic activity, membrane integrity and lysosome integrity. In vitro EC50 values for the fish cell lines exceeded river TRWP concentrations (2.02 g/L and 4.65 g/L for RTgill-W1 and RTgutGC cell lines, respectively), and were similar to in vivo LC50 values estimated at 6 g/L. Although toxicity was mainly driven by the leaching of tire-associated chemicals, the presence of the particles contributed to the overall toxicity by inducing a continuous leaching, highlighting the importance of considering combined exposure scenarios. Aging and digestion conditions were also found to mediate CMTT toxicity. Thermooxidation resulted in a decreased chemical leaching and toxicity, while in vitro digestion under mimicked gastrointestinal conditions increased leaching and toxicity. Specific chemicals, especially Zn, 2-mercaptobenzothiazole, 1,3-diphenylguanidine, and N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) were identified as contributors to the overall toxicity. Although 6PPD-quinone was detected in CMTT digestate, cytotoxicity assays with RTgill-W1 and RTgutGC cell lines showed no toxicity up to 6 mg/L, supporting the notion of a specific mode of action of this chemical. This study provides insights into the toxicological mechanisms induced by tire particles and their associated chemicals and can help in the evaluation of potential risks to aquatic life associated with TRWP.

Electrical stimulation as adjuvant treatment for chronic leg ulcers of different aetiology: an RCT.

OBJECTIVE: To investigate the effectiveness of an innovative electrical stimulation (ES) therapy as adjuvant treatment for chronic wounds of various aetiology, in terms of pain and ulcer healing. METHOD: Patients with chronic limb ulcers were enrolled for the study and randomised into the intervention or control group. The intervention group received conventional treatment plus ES therapy (FREMS; Lorenz Lifetech) while the control group received only conventional treatment. Each ES treatment cycle consisted of 12 sessions performed in 4 weeks (three sessions/week). All patients were treated until full wound healing occurred, or for a maximum of 9 ES cycles, with a 2-week rest between each cycle. RESULTS: A total of 60 patients were enrolled in the study and randomised into the two groups: the intervention group (n=30) and the control group (n=30). During follow-up, some patients terminated the protocol because they reached the ulcer closure before the maximum of 9 cycles. The analysis of the effect of ES on pain and ulcer healing was performed on all patients who underwent at least two consecutive clinical evaluations (two cycles), in order to reach a compatible sample size with the primary objective (one patient withdrew). In both groups, there was a significant reduction of pain compared with baseline (p < 0.05), starting from T6 visit in the first cycle. In particular, there was a significant reduction of pain in the intervention group compared with the control group after 14 days, and this reduction continued until the end of the second cycle. Similarly, there was a significant reduction of PUSH tool score in the intervention group compared with the control group after 14 days, and this reduction continued until the end of the second cycle. CONCLUSION: Data collected in this study support data in the literature. Analysis of longitudinal data analysed by simple models and complex models suggest that the ES therapy had a positive and significant effect on pain reduction (VAS) and on the improvement of ulcer healing process in terms of the PUSH tool total index compared with conventional treatment, and may have induced a significant acceleration of the wound-healing process.

Capturing greater 16S rRNA gene sequence diversity within the domain Bacteria.

A large proportion of "universal" 16S PCR primers lack sequence homology to many of the "candidate" divisions, severely limiting bacterial diversity assessments. We designed a primer set that offers a 50% increase in silico in coverage of the domain Bacteria over the commonly used primer combination 27F/519R. Comparisons using pyrosequencing on soil environments showed a significant increase in recovery of taxonomic diversity with around a 3-fold increase in recovery of sequences from candidate divisions.

First Report of Races 11 and 12 of Cercospora sojina, the Causal Agent of Soybean Frogeye Leaf Spot, in Argentina.

During the growing seasons of 2008 to 2009 and 2009 to 2010, severe outbreaks of soybean (Glycine max (L.) Merr.) frogeye leaf spot, a disease caused by Cercospora sojina Hara, occurred in several areas in Argentina (1). Two surveys were conducted in soybean fields, one in 2008 that included the provinces of Buenos Aires, Córdoba, and Santa Fe, and another that was performed in 2009 in the same provinces plus three others: Entre Ríos, Santiago del Estero, and Tucumán. In both surveys, plants presented circular lesions with reddish brown-to-gray spots and bordered by typical, narrow, reddish purple margins (3). To promote sporulation and to enable identification of the causal agent, leaves of diseased plants were collected and placed in a moist chamber for 24 h with a 12-h light cycle at 25°C. Conidia were plated on potato dextrose agar medium amended with streptomycin and were incubated at 25°C and 12 h of fluorescent light. Isolated cultures sporulated in 10 days and, on the basis of their morphology, were identified as C. sojina. A total of 147 isolates were deposited at the Culture Collection of CEREMIC (Centro de Referencia de Micología). They produced one- to nine-septate hyaline, elongate to fusiform conidia that measured 54.9 ± 16.2 × 5.7 ± 1.0 µm. Six isolates of C. sojina, each representing a province, were inoculated on a set of 12 differential soybean cultivars: Lee, Davis, Hood, Richland, Lincoln, Kent, Tracy, S 100, Palmetto, Peking, CNS, and Blackhawk (2). Fifteen plants of each differential were sprayed at V3 growth stage with a suspension of 6 × 104 conidia/ml. The test was conducted twice in a complete randomized design with three replicates. Control plants were sprayed with sterile distilled water. After inoculation, plants were placed in a greenhouse bench humidity chamber at 26 to 28°C for 72 h. Disease was rated 14 days after inoculation; plants with numerous lesions were considered susceptible and each of the 15 plants was given a score of 1. Plants with small or no lesions were classified as resistant and given a score of 0. Control plants remained healthy. The pathogen was reisolated from symptomatic plants and morphological characteristics were consistent with C. sojina. Based on the response of the differentials to each isolate and on the race designations, the isolates from Buenos Aires, Córdoba, Santa Fe, and Tucumán belong to race 11, while those from Santiago del Estero and Entre Ríos province to race 12. The finding of these two races threatening soybean cultivars in Argentina may be indicative of additional races. Thus, the incorporation of multiple resistance genes may reduce the impact of the disease on soybean. To our knowledge, this is the first report of the identification of races of C. sojina in Argentina. References: (1) M. A. Carmona et al. Plant Dis. 93:966, 2009. (2) M. A. R. Mian et al. Crop Sci. 48:14, 2008. (3) D.V. Phillips. Page 20 in: Compendium of Soybean Diseases. 4th ed. APS Press, St. Paul, MN, 1999.

Lombardy diagnostic and therapeutic network of thrombotic microangiopathy.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening thrombotic microangiopathy (TMA) requiring urgent treatment. Standardization of its diagnosis and optimal management is challenging. This study aimed to evaluate the role of centralized, rapid testing of ADAMTS13 in patients experiencing acute TMAs requiring plasma-exchange (PEX) and to estimate the incidence of TTP in a large Italian Region. METHODS: We perfomed a cohort study in the frame of the project "Set-up of a Lombardy network for the study and treatment of patients undergoing apheresis", including 11 transfusion centers in the Region. Consecutive patients referred from 2014 to 2016 with acute TMAs requiring PEX were enrolled. Centralized ADAMTS13 activity testing was performed at the Milan Hemophilia and Thrombosis Center within 24 h. RESULTS: Forty-three TMA patients (44 events) were enrolled, of whom 35 (81%) had severe ADAMTS13 deficiency. Patients with severe ADAMTS13 deficiency were younger, mainly women, with a higher prevalence of autoimmune disorders and a lower prevalence of cancer. Clinical and laboratory characteristics of patients with and without severe ADAMTS13 deficiency largely overlapped, with a lower platelet count being the only baseline marker that significantly differed between the two patient groups (ADAMTS13 activity < 10% vs ≥ 10%: median difference of -27 × 109/l, 95% CI - 37 to - 3). PEX treatment was initiated in all patients, but soon discontinued in cases without severe ADAMTS13 deficiency. In this group, the mortality rate was higher and no episode exacerbations or relapses within 6 months occured. The estimated average annual incidence of acute acquired TTP events was 1.17 [0.78-1.55] per million people. CONCLUSIONS: Severe ADAMTS13 deficiency distinguished two groups of patients with largely overlapping clinical features but different treatment and disease course. This study provides a feasible model implemented in a large Italian region for the practical clinical approach to TMAs and underlines the importance of urgent ADAMTS13 activity testing for an accurate differential diagnosis and therapeutic approach.

Environmental DNA metabarcoding for benthic monitoring: A review of sediment sampling and DNA extraction methods.

Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring.

Depression and anxiety disorders in patients with multiple sclerosis: association with neurodegeneration and neurofilaments.

There is increasing evidence that neurofilament light chain (NF-L) can be considered as a biomarker for neuro-axonal damage. This polypeptide can be released into the cerebrospinal fluid (CSF) and the blood, where it can be quantified. The concentration of NF-L is elevated in patients with multiple sclerosis (MS) and psychiatric disorders. We aimed to investigate the NF-L levels in the CSF from treated MS patients and the relationship with depression or anxiety. The study involved three groups: control group (individuals without inflammation), the relapse-remitting multiple sclerosis (RRMS)-untreated group, and the RRMS-Fingo group (RRMS patients who were treated with fingolimod). MS disability was assessed by the Expanded Disability Status Scale, and depression and anxiety were evaluated by a neuropsychologist, using the Hospital Anxiety and Depression Scale, the Beck Depression Inventory-II, and the Beck Anxiety Inventory. Individual CSF samples were collected to measure NF-L levels. The results of the statistical analysis on levels of NF-L in the CSF of control subjects, RRMS-untreated patients, and RRMS-Fingo patients were significant. The relationship between depression and anxiety in RRMS-Fingo patients and NF-L levels was not statistically significant. In conclusion, MS events such as anxiety and depression appear to contribute to the onset of clinical relapses, subclinical cases, and neurodegeneration.

A novel real-world ecotoxicological dataset of pelagic microbial community responses to wastewater.

Real-world observational datasets that record and quantify pressure-stressor-response linkages between effluent discharges and natural aquatic systems are rare. With global wastewater volumes increasing at unprecedented rates, it is urgent that the present dataset is available to provide the necessary information about microbial community structure and functioning. Field studies were performed at two time-points in the Austral summer. Single-species and microbial community whole effluent toxicity (WET) testing was performed at a complete range of effluent concentrations and two salinities, with accompanying environmental data to provide new insights into nutrient and organic matter cycling, and to identify ecotoxicological tipping points. The two salinity regimes were chosen to investigate future scenarios based on a predicted salinity increase at the study site, typical of coastal regions with rising sea levels globally. Flow cytometry, amplicon sequencing of 16S and 18S rRNA genes and micro-fluidic quantitative polymerase-chain reactions (MFQPCR) were used to determine chlorophyll-a and total bacterial cell numbers and size, as well as taxonomic and functional diversity of pelagic microbial communities. This strong pilot dataset could be replicated in other regions globally and would be of high value to scientists and engineers to support the next advances in microbial ecotoxicology, environmental biomonitoring and estuarine water quality modelling.

Cultivation of fastidious bacteria by viability staining and micromanipulation in a soil substrate membrane system.

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.

Development of a two-color fluorescence in situ hybridization technique for species-level identification of human-infectious Cryptosporidium spp.

A two-color fluorescence in situ hybridization assay that allows for the simultaneous identification of Cryptosporidium parvum and C. hominis was developed. The assay is a simple, rapid, and cost-effective tool for the detection of the major Cryptosporidium species of concern to public health.

Terminal restriction fragment length polymorphism for identification of Cryptosporidium species in human feces.

Effective management of human cryptosporidiosis requires efficient methods for detection and identification of the species of Cryptosporidium isolates. Identification of isolates to the species level is not routine for diagnostic assessment of cryptosporidiosis, which leads to uncertainty about the epidemiology of the Cryptosporidium species that cause human disease. We developed a rapid and reliable method for species identification of Cryptosporidium oocysts from human fecal samples using terminal restriction fragment polymorphism (T-RFLP) analysis of the 18S rRNA gene. This method generated diagnostic fragments unique to the species of interest. A panel of previously identified isolates of species was blind tested to validate the method, which determined the correct species identity in every case. The T-RFLP profiles obtained for samples spiked with known amounts of Cryptosporidium hominis and Cryptosporidium parvum oocysts generated the two expected diagnostic peaks. The detection limit for an individual species was 1% of the total DNA. This is the first application of T-RFLP to protozoa, and the method which we developed is a rapid, repeatable, and cost-effective method for species identification.

Glycoprotein 60 diversity in C. hominis and C. parvum causing human cryptosporidiosis in NSW, Australia.

Management and control of cryptosporidiosis in human requires knowledge of Cryptosporidium species contributing to human disease. Markers that are able to provide information below the species level have become important tools for source tracking. Using the hypervariable surface antigen, glycoprotein 60 (GP60), C. hominis (n=37) and C. parvum (n=32) isolates from cryptosporidiosis cases in New South Wales, Australia, were characterised. Extensive variation was observed within this locus and the isolates could be divided into 8 families and 24 different subtypes. The subtypes identified have global distributions and indicate that anthroponotic and zoonotic transmission routes contribute to sporadic human cryptosporidiosis in NSW.

Development of biocompatible and fully bioabsorbable PLA/Mg films for tissue regeneration applications.

During recent years, Mg reinforced polylactic acid (PLA) composites have emerged as potential biocompatible and bioabsorbable materials for biomedical applications. It has been shown that Mg particles added to a matrix based on a biodegradable polymer can address the lack of bioactivity and the low mechanical properties of the polymers and, furthermore, it can counteract the detrimental effects associated to the high degradation rate of Mg, as alkalinization and elevated H2 release. Additionally, the polymer can protect the Mg particles, by tailoring their degradation rate. Former processing of these composites performed by extrusion, compression and injection molding employed Mg contents up to 10 wt%. Higher amounts of Mg resulted in heterogeneous materials and thermally degraded matrices, with the corresponding higher degradation rate. In the present work, Mg reinforced PLA films with Mg content as high as 50 wt% were obtained without compromising the thermal stability of the polymer. Firstly, a successful dispersion of Mg microparticles was achieved by a breakthrough in processing introducing a colloidal step where organic additives were added to modify the Mg particle surface and promote a chemically stable suspension. The resulting colloidal suspension was then used as feedstock to obtain composite films by tape casting. The films show advantageous in vitro behaviour in terms of degradation, hydrogen release and oxygen permeability. In addition, the viability with fibroblast cells (MEF) opens a window of opportunity for these composite films as bioabsorbable material for tissue engineering and wound dressing applications. STATEMENT OF SIGNIFICANCE: Magnesium materials have extraordinary biodegradable properties and bioactive behavior due to release of Mg2+ ions, which offer a promising opportunity for their applicability as biomaterials for tissue regeneration. However, Mg is one of the most reactive metals with a high degradation rate. In contact with water produces H2, associated with a risk of failure of the implant. One alternative to minimize this drawback is the use of Mg particles surrounded by a biodegradable biocompatible polymer such as polylactic acid (PLA) to obtain PLA/Mg composites. In this work we processed Mg reinforced PLA in the shape of films that would be suitable for tissue regeneration. In vitro behavior of PLA/Mg films demonstrated that Mg2+ ions increase the fibroblast cells growth.