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Background It is known that cataract extraction is associated with a significant reduction in intraocular pressure, especially in narrow angled eyes; however, the modifications of anterior segment parameters associated with this phenomenon have still not been completely defined. The purpose of this study was to evaluate changes in anterior segment anatomy and intraocular pressure after cataract surgery in non-glaucomatous eyes. Methods and Material This retrospective case series study included 64 eyes of 64 consecutive patients who underwent phacoemulsification with intraocular lens implantation. Anterior segment parameters and intraocular pressure were assessed and compared before and 6 months after surgery. Anterior segment imaging was performed using Casia SS-1000 anterior segment optical coherence tomography (Tomey, Nagoya, Japan). Anterior segment measurements included anterior chamber depth, anterior chamber width, anterior chamber volume, angle opening distance at 500 µm anterior to the scleral spur, angle recess area 750 µm from the scleral spur, lens vault, trabecular iris space area at 500 µm from the scleral spur, and trabecular iris angle at 500 µm from the scleral spur. Intraocular pressure was measured using the Goldmann applanation tonometer (Model AT 900 C/M, Haag-Streit, Bern, Switzerland). Anterior segment parameters and the relationship of changes in intraocular pressure were also evaluated. Results All anterior segment parameters increased significantly after surgery (p Conclusion Cataract surgery led to significant widening of the anterior chamber angle and lowering of intraocular pressure. Further investigations are needed to better understand whether anterior chamber width may be a new independent predictive factor for reduction in postoperative intraocular pressure.

Purpose To evaluate and compare the long-term results after bilateral implantation of different multifocal intraocular lenses (MIOLs). Methods This retrospective comparative study included 42 patients who underwent cataract surgery with bilateral MIOL implantation. Patients were divided into 5 groups: Group 1 received a refractive ReZoom NGX1 IOL (AMO), Group 2 a diffractive Acrysof ReSTOR SA60D3 IOL (Alcon), and Group 3 a diffractive Tecnis ZM900 IOL (AMO). Group 4 and Group 5 were implanted using the mix and match approach with refractive ReZoom-diffractive ReSTOR IOL and refractive ReZoom-diffractive Tecnis ZM900 IOL, respectively. Primary outcome measures were distance, near, and intermediate distance visual acuity measured 6 months (T0) and 10 years (T1) after surgery. Secondary outcomes were defocus curves, contrast sensitivity, patientsʼ satisfaction, and spectacle independence. Results All patients achieved best-corrected distance visual acuity (BCDVA) greater than 0.11 logMAR and uncorrected distance visual acuity (UCDVA) greater than 0.14 logMAR at both time points. A decrease in contrast sensitivity was evident, particularly at high spatial frequencies; at T1, Group 4 reported statistically higher values than Group 2 at 12 cycles/degree and 18 cycles/degree and statistically higher values than Group 3 at 18 cycles/degree. Great overall satisfaction was reported even in the presence of dysphotopsia. Tecnis ZM900 IOL showed the lowest incidence of posterior capsular opacification. Conclusion MIOLs could provide adequate functional vision and patient satisfaction, despite the incidence of side effects, in carefully selected patients desiring spectacle independence.

A new system based on binary Deep Learning (DL) convolutional neural networks (CNNs) has been developed to recognize specific retinal abnormality signs on Optical Coherence Tomography (OCT) images useful for clinical practice. Images from the local hospital database were retrospectively selected from 2017 to 2022. Images were labeled by two retinal specialists and included central fovea cross-section OCTs. Nine models were developed using the Visual Geometry Group 16 (VGG-16) architecture to distinguish healthy versus abnormal retinas and to identify eight different retinal abnormality signs. A total of 21500 OCT images were screened, and 10770 central fovea cross-section OCTs were included in the study. The system achieved high accuracy in identifying healthy retinas and specific pathological signs, ranging from 93–99%. Accurately detecting abnormal retinal signs from OCT images is crucial for patient care. This study aimed to identify specific signs related to retinal pathologies, aiding ophthalmologists in diagnosis. The high-accuracy system identified healthy retinas and pathological signs, making it a useful diagnostic aid. Labelled OCT images remain a challenge, but our approach reduces dataset creation time and shows DL models' potential to improve ocular pathology diagnosis and clinical decision-making.

Artificial intelligence (AI)-based applications exhibit the potential to improve the quality and efficiency of patient care in different fields, including cataract management. A systematic review of the different applications of AI-based software on all aspects of a cataract patient's management, from diagnosis to follow-up, was carried out in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. All selected articles were analyzed to assess the level of evidence according to the Oxford Centre for Evidence-Based Medicine 2011 guidelines, and the quality of evidence according to the Grading of Recommendations Assessment, Development and Evaluation system. Of the articles analyzed, 49 met the inclusion criteria. No data synthesis was possible for the heterogeneity of available data and the design of the available studies. The AI-driven diagnosis seemed to be comparable and, in selected cases, to even exceed the accuracy of experienced clinicians in classifying disease, supporting the operating room scheduling, and intraoperative and postoperative management of complications. Considering the heterogeneity of data analyzed, however, further randomized controlled trials to assess the efficacy and safety of AI application in the management of cataract should be highly warranted. (c) 2021 The Authors. Published by Elsevier Inc.

Purpose: To evaluate and compare the visual outcomes of an enhanced monofocal IOL with two different monofocal IOLs. Setting: Eye Clinic, Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy. Design: Prospective, single-centre, single-blind, randomized controlled clinical study. Methods: The study included patients undergoing phacoemulsification and IOL implantation. Patients were consecutively randomized by block randomization and assigned in a 1:1:1 allocation ratio to three study arms to bilaterally receive Tecnis® Eyhance (model ICB00) or Tecnis®1-piece (model ZCB00) or Alcon®1-piece (model Clareon, CNA0T0) respectively. Monocular and binocular (both corrected and uncorrected) visual acuities for far, intermediate and near were registered and compared among groups. To track changes in patient quality of life, the Catquest-9SF questionnaire was administered to each patient before and after cataract extraction. Results: A total of 90 patients (30 for each group) were enrolled. At 3 months follow-up, statistically significant differences for intermediate and near distance visual acuities were found between the three groups. Non-statistically significant differences were observed for distance visual acuities and the changes in Catquest-9SF scores. Conclusion: Tecnis® Eyhance ICB00 IOL provided better results in intermediate visual outcomes without negative effects on patients’ quality of life.

Spore formers are ubiquitous microorganisms commonly isolated from most environments, including the gastro-intestinal tract (GIT) of insects and animals. Spores ingested as food and water contaminants safely transit the stomach and reach the intestine, where some of them germinate and temporarily colonize that niche. In the lower part of the GIT, they re-sporulate and leave the body as spores, therefore passing through their entire life cycle in the animal body. In the intestine, both un-germinated spores and germination-derived cells interact with intestinal and immune cells and have health-beneficial effects, which include the production of useful compounds, protection against pathogenic microorganisms, contribution to the development of an efficient immune system and modulation of the gut microbial composition. We report a genomic and physiological characterization of SF106 and SF174, two aerobic spore former strains previously isolated from ileal biopsies of healthy human volunteers. SF106 and SF174 belong respectively to the B. subtilis and Alkalihalobacillus clausii (formerly Bacillus clausii) species, are unable to produce toxins or other metabolites with cytotoxic activity against cultured human cells, efficiently bind mucin and human epithelial cells in vitro and produce molecules with antimicrobial and antibiofilm activities.

Deep Learning methods have become dominant in various fields of medical imaging, including ophthalmology. In this preliminary study, we investigated a method based on Convolutional Neural Network for the identification of drusen and macular hole from Optical Coherence Tomography scans with the aim to assist ophthalmologists in diagnosing and assessing retinal diseases.

Purpose To evaluate the surface profile of a new-generation extended range-of-vision intraocular lens (IOL) and to compare it with that obtained for a monofocal IOL based on the same platform. Methods Prospective, experimental, laboratory study comparing the surface profile of the DFT015 (AcrySof IQ Vivity; Alcon Laboratories, Inc.), a new-generation presbyopia-correcting IOL, with the profile of the SN60WF (AcrySof IQ; Alcon Laboratories, Inc.), an aspheric monofocal IOL based on the same platform. Raw profiles were obtained using contact profilometry. The best-fit form was then subtracted from each raw profile to highlight potential differences. Results No significant differences were appreciated in raw profiles. On the contrary, after form removal, the new extended range-of-vision IOL showed a peculiar profile characterized by the presence of two altitudinal symmetrical changes in the order of 1 µm, localized in the central portion of the optic. Conclusions The new-generation extended range-of-vision IOL evaluated showed a smooth change of its surface compared to the same platform monofocal IOL. The altitudinal changes blended in the central design of the new presbyopia-correcting IOL, although micrometric, might play a crucial role in creating a continuous focal range while minimizing visual disturbances.

Background: The introduction of non-invasive diagnostic tools in ophthalmology has significantly reshaped current clinical practice in different settings. Recently, different anterior segment (AS) intraoperative optical coherence tomography (i-OCT) systems have been employed for different interventional procedures including cataract surgery. Materials and Methods: A review on the use of AS i-OCT in the management of cataract surgery, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines (PRISMA). The level of evidence according to the Oxford Centre for Evidence-Based Medicine (OCEM) 2011 guidelines, and the quality of evidence according to the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system were assessed for all included articles. Results: Out of 6302 articles initially extracted, 6302 abstracts were identified for screening and 32 of these met the inclusion/exclusion criteria for full-text review; 19 articles were excluded. Conclusions: The use of AS i-OCT in cataract surgery, even if only a few studies have a high level or grade of evidence, may represent a useful tool for novel surgeons approaching phacoemulsification but also for expert ones for teaching purposes and to plan and manage complicated cases.

The increasing interest for environmentally friendly technologies is driving the transition from fossil-based economy to bioeconomy. A key enabler for circular bioeconomy is to valorize renewable biomasses as feedstock to extract high value-added chemicals. Within this transition the discovery and the use of robust biocatalysts to replace toxic chemical catalysts play a significant role as technology drivers. To meet both the demands, we performed microbial enrichments on two energy crops, used as low-cost feed for extremophilic consortia. A culture-dependent approach coupled to metagenomic analysis led to the discovery of more than 300 glycoside hydrolases and to characterize a new α-glucosidase from an unknown hyperthermophilic archaeon. Aglu1 demonstrated to be the most active archaeal GH31 on 4Np-α-Glc and it showed unexpected specificity vs. kojibiose, revealing to be a promising candidate for biotechnological applications such as the liquefaction/saccharification of starch.

The enzymes from hyperthermophilic microorganisms populating volcanic sites represent interesting cases of protein adaptation and biotransformations under conditions where conventional enzymes quickly denature. The difficulties in cultivating extremophiles severely limit access to this class of biocatalysts. To circumvent this problem, we embarked on the exploration of the biodiversity of the solfatara Pisciarelli, Agnano (Naples, Italy), to discover hyperthermophilic carbohydrate-active enzymes (CAZymes) and to characterize the entire set of such enzymes in this environment (CAZome). Here, we report the results of the metagenomic analysis of two mud/water pools that greatly differ in both temperature and pH (T = 85 °C and pH 5.5; T = 92 °C and pH 1.5, for Pool1 and Pool2, respectively). DNA deep sequencing and following in silico analysis led to 14 934 and 17 652 complete ORFs in Pool1 and Pool2, respectively. They exclusively belonged to archaeal cells and viruses with great genera variance within the phylum Crenarchaeota, which reflected the difference in temperature and pH of the two Pools. Surprisingly, 30% and 62% of all of the reads obtained from Pool1 and 2, respectively, had no match in nucleotide databanks. Genes associated with carbohydrate metabolism were 15% and 16% of the total in the two Pools, with 278 and 308 putative CAZymes in Pool1 and 2, corresponding to ~ 2.0% of all ORFs. Biochemical characterization of two CAZymes of a previously unknown archaeon revealed a novel subfamily GH5_19 β-mannanase/β-1,3-glucanase whose hemicellulose specificity correlates with the vegetation surrounding the sampling site, and a novel NAD+ -dependent GH109 with a previously unreported β-N-acetylglucosaminide/β-glucoside specificity. DATABASES: The sequencing reads are available in the NCBI Sequence Read Archive (SRA) database under the accession numbers SRR7545549 (Pool1) and SRR7545550 (Pool2). The sequences of GH5_Pool2 and GH109_Pool2 are available in GenBank database under the accession numbers MK869723 and MK86972, respectively. The environmental data relative to Pool1 and Pool2 (NCBI BioProject PRJNA481947) are available in the Biosamples database under the accession numbers SAMN09692669 (Pool1) and SAMN09692670 (Pool2).

Purpose: Implementation of the International Consortium for Health Outcomes Measurements (ICHOM) standard for cataract surgery into clinical practice at an Italian tertiary referral centre. Methods: Prospective, observational, descriptive study consisting of the registry and analysis of cataract surgeries performed during a 6-month enrolment period at the University Eye Clinic of Trieste, Trieste, Italy. Outcomes were recorded and analysed according to the ICHOM Cataract Standard Set version 2.0.1. Records included clinician-reported outcome measures (CROMs) – visual outcome and complications – and patient-reported outcome measures (PROMs) – self-assessed vision with the Catquest-9SF questionnaire. Correlations between PROMs and CROMs were evaluated. A multiple linear regression was used for predicting the change in PROMs with surgery. Results: A total of 218 eyes (of 218 patients) were analysed. Postoperative corrected distance visual acuity (CDVA) was ⩾0.3 in 89.0% (194/218) of eyes. There was a statistically significant improvement of the post-operative Catquest-9SF global average score. ( p 2: 0.527) based on preoperative Catquest-9SF total score, presence or not of macular degeneration, presence or not of intraoperative complications, age >75 years old, and preoperative CDVA. Conclusions: Cataract surgery improves the functional vision, with some factors limiting the outcomes such as comorbidities. Self-perceived improvement in intermediate vision significantly influenced the improvement in self-assessed vision.

This is a retrospective, multicenter study of consecutive patients with nAMD scheduled for a visit and/or a treatment with an intravitreal injection (IVI) during the 3 months before lockdown in the Ophthalmology Departments of six centers of Europe.The study was conducted on 546 patients, of which 55.13% were females, almost 100% of the patients were White/Caucasian race, and 71.53% of the patients presented a type 1 macular neovascularization (NVM). A total of 62.82% of patients (343 patients) that were on scheduled clinic visits and/or intravitreal injection treatment during the 3 months before the quarantine did not attend either to visit or for treatment during the lockdown. The mean number of injections during the lockdown was significantly reduced. This was followed by a significant reduction in the mean best-corrected visual acuity (BCVA) between the 3 months before the lockdown (mean BCVA of 60.68 ± 19.77 letters) and 6 months after lockdown (mean BCVA of 56.98 ± 22.59 letters). Patients with better BCVA before the lockdown and the ones showing neovascular activity were more likely to attend their scheduled visits and/or IVI treatments. The COVID-19 pandemic and the lockdown have led to a decrease in the number of IVI treatments in patients with nAMD, evidencing a significant vision loss at 6 months.

The progressive deterioration of the visual function in patients on waiting lists for cataract surgery has a negative impact on their quality of life, especially in the elderly population. Patient waiting times for cataract surgeries in many healthcare settings have increased recently due to the prolonged stop or slowdown of elective cataract surgery as a result of coronavirus disease 19 (COVID-19). The aim of this review is to highlight the impact of such a “de-prioritization” of cataract surgery and to summarize some critical issues and useful hints on how to reorganize cataract pathways, with a special focus on perioperative diagnostic tools during the recovery phase and beyond. The experiences of a group of surgeons originating from nine different countries, named the European COVID-19 Cataract Group (EUROCOVCAT), have been combined with the literature and recommendations from scientific ophthalmic societies and healthcare institutions. Key considerations for elective cataract surgery should include the reduction of the number of unnecessary visits and examinations, adoption of precautionary measures, and implementation of telemedicine instruments. New strategies should be adopted to provide an adequate level of assistance and to guarantee safety conditions. Flexibility will be the watchword and regular updates would be necessary following scientific insights and the development of the pandemic.

Background: Retinal nerve fiber layer (RNFL) and ganglion cell layer (GCL) measurements can be influenced by many factors including the presence of concomitant retinal diseases. The aim of this study it to assess the impact of epiretinal membrane (ERM) on RNFL and GCL assessment using optical coherence tomography (OCT). Methods: GCL, peripapillary RNFL (pRNFL), and Bruch’s Membrane Opening Minimum Rim Width (BMO-MRW) thicknesses were analysed using an SD-OCT (Spectralis OCT) in eyes with idiopathic ERM and compared with a control group. Results: 161 eyes were included, 73 eyes in the control group and 88 eyes with idiopathic ERM. The pRNFL analysis revealed a statistically significant difference between the two groups in overall and temporal sector thicknesses. For GCL thickness report, the percentage of scans in which the GCL was erroneously segmented by automatic segmentation was assessed for each eye. A statistically significant difference was found in all sectors (p < 0.001), with the exception of external nasal sector. A statistically significant difference (p < 0.001) in the GCL total volume report was found in ERM group compared to the control group. For MRW at BMO analysis, there was no statistically significant difference in MRW thickness in any sector. Conclusion: In eyes with ERM, the GCL and pRNFL analysis seemed affected by the morphological retinal layers’ modification. MRW-BMO did not appear to be directly affected by the presence of ERM.

Purpose: To evaluate the surface profile of different new-generation intraocular lenses (IOLs) designed to improve intermediate vision. Setting: Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy. Design: Prospective experimental laboratory study. Methods: One higher-order aspheric monofocal IOL (Tecnis Eyhance ICB00) and 2 extended depth-of-focus (EDOF) IOLs (Tecnis Symfony ZXR00, Mini Well Ready) were examined and compared with monofocal IOLs with the same platform (Sensar AAB00, Tecnis ZCB00, Mini 4 Ready) using contact profilometry. Raw profiles were obtained and compared. The best-fit circle was then subtracted from each raw profile to highlight differences. Results: The new higher-order aspheric IOL presented a peculiar profile, which appeared similar to the conventional monofocal IOL based on the same platform, with just minimal differences in the central design. One of the EDOF IOLs showed a diffractive design. The EDOF IOL based on spherical aberration presented a central steepening surrounded by a symmetric lateral change in depths. Conclusions: Improved intermediate vision can be achieved by means of different optic designs. Unlike the traditional EDOF and multifocal IOLs based on a diffractive design, the profiles of new-generation IOLs are characterized by smooth and progressive changes of the superficial geometry. New-generation IOLs can enhance the range of vision. Contact surface profilometry analysis of 2 IOLs showed minimal yet peculiar changes of their central optic designs.

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The aim of this study was to assess the central macular imaging captured with an optical biometer based on full-eye-length Swept-Source OCT (SS-OCT) scan as a screening strategy for identifying macular diseases in patients scheduled for cataract surgery. 1,114 eyes of 749 consecutive patients underwent a biometrical examination with IOLMaster 700 SS-OCT technology (Carl Zeiss) and conventional Spectral-Domain OCT (SD-OCT) (Spectralis OCT, Heidelberg) device analysis on the same day. Seven examiners graded the scans individually in a full-masked mode. Twenty-five eyes were excluded for media opacities. Among the 1,089 included eyes, statistical analysis revealed a mean Kendall’s Coefficient of 0.83 (range 0.76–0.89). A logistic regression model demonstrated a highly significant correlation (p

Content: Finished leather is a material purchased by surface measurement. Compared to other goods, which value is defined by a measure, for leather there is no certified reference material able to represent the numerous types of articles present on the market. Moreover, there is not a single type of machine, with the consequent difficulties in comparison of data obtained by different devices on different kind of leather. As far as the devices available are concerned, the pin-wheels machine was the most used device until the sixties, period after which the increasingly massive diffusion of electronic machines began. With regard to the tolerance allowed, in late nineties an agreement between the ICT and ICHSLTA was reached (International Contract N. 7): 2% and 3% for soft and elastic leathers. These limits were fixed without considering the possible deviation of results using different devices and without considering the reproducibility of measures in function of the effective flexibility and physical characteristics of leathers. Over the years, however, the pin-wheel machines have been completely substituted and, due to the high costs of maintenance and management of the pin-wheel machines, only a few of the institutes recognized by ICT, including the SSIP, are equipped with a fully functional pin-wheel machine. Despite this evidence, the Contract continues to consider pin-wheel machine as the reference one for disputes. For the above, an extensive data collection campaign has been carried out involving all the industrial chain of leather: tanneries, brands, users, laboratories, research institutes and devices manufacturers. 16 different kind of leather articles, representative different leather uses (gloves, apparel, footwear, leather goods, furniture, automotive), were measured using all the machines currently available on the market (roller, conveyor, scanner and digital image acquisition devices) and considering ISO 19076 and ISO 11646 measurement procedures as the reference ones. Thousands of data are now available to qualify the behaviour of the leather for each type of machine with its specific characteristics. This was done by statistical robust analysis of the data and by the comparison of the values obtained with the pin machine. The leather used were also mechanically characterized in order to be able to identify any correlations with the reproducibility of the measurements and with the imposed tolerances. Take-Away: The purpose is the possibility to overcome all the restrictions connected with the pin-wheel machine, the improvement of actual EN ISO methods of leather measurement and a better instrument to define tolerances considering the couple leather-machine.

Background: Digital retinal imaging is the gold standard technique for diabetic retinopathy (DR) and diabetic macular oedema (DME) assessment during DR screening. Objectives: To evaluate the diagnostic accuracy of digital retinal fundus image (DRFI) analysis in detecting DME using three manual grading systems (MGS) and comparing it with optical coherence tomography (OCT) findings. Method: A total of 287 DRFI of 287 eyes were analysed. Non-stereoscopic 45° images were acquired using a Kowa VX-20 camera and were graded according to three MGS: Early Treatment Diabetic Retinopathy Study (ETDRS), International Clinical Diabetic Retinopathy (ICDR), and United Kingdom National Screening Committee (UKNSC). The two graders were masked to the patient’s clinical DR status. DME characteristics were analysed using OCTs. Results: A very good agreement in detecting DME was found with Cohen’s κ = 0.83 (ICDR vs. ETDRS), κ = 0.83 (ICDR vs. UKNSC), and κ = 0.82 (ETDRS vs. UKNSC). Sensitivity and specificity of DRFI analysis in DME assessment were 70.0 and 69.6% for UKNSC, 71.9 and 67.4% for ETDRS, and 70.9 and 65.2% for ICDR, respectively. Positive and negative predictive values were 91.7 and 32.7% for UKNSC, 91.4 and 33.3% for ETDRS, and 90.7 and 31.9% for ICDR, respectively. On OCT scans, micro-architectural damages of both inner and outer retinal layers and mean ganglion cell layer thickness showed a significant association with the presence of DME detected with DRFI analysis. Conclusions: Despite the low negative predictive value, the good specificity and sensitivity of DRFI in detecting DME make it a useful tool in a routine clinical setting, and its potential in diabetic eye screening is yet to be realized.

The field of thermophilic microbiology was born in the late 1970s with the pioneering work of Brock (Thermophiles biodiversity, ecology, and evolution. Springer, Boston, pp. 1–9, 2001) and dramatically expanded through the ’80s with the isolation of hyperthermophiles by Stetter (FEMS Microbiol Rev 18:149–158, 1996). The development of SSU rRNA phylogenetics revealed the complexity and diversity of prokaryotic phylotypes on biotopes widely differing in extreme conditions (e.g. spanning gradients of pH between 0 and 10 and temperatures from 60 °C to over 120 °C, respectively). Sites of volcanic activity all over the Earth’s surface and under the sea provide a variety of different environments for extremophilic microorganisms. Hot springs populated by hyperthermophiles (Topt > 65 °C), the majority of which belonging to the domain of Archaea, are very diverse and some of them show combinations of other extreme conditions, for example, acidic, alkaline, high pressure, and high concentrations of salts and heavy metals (Cowan et al. in Curr Opin Microbiol 25:97–102, 2015). Archaea inhabiting hot springs are considered to be the closest living descendants of the earliest living forms on Earth and their study provide insights into the origin and evolution of life (Woese et al. in Proc Natl Acad Sci USA 87:4576–4579, 1990; Olsen et al. in J Bacteriol 176:1–6, 1994). As with all studies of environmental microbiology, our understanding of the function of (hyper)thermophilic microbial consortia has lagged substantially behind. However, recent advances in ‘omics’ technologies, particularly within a system biology context, have made significant progresses into the prediction of in situ functionality (Cowan et al. in Curr Opin Microbiol 25:97–102, 2015). Most extremophilic microorganisms are recalcitrant to cultivation-based approaches (Amann et al. in Microbiol Rev 59:143–69, 1995; Lorenz et al. in Curr Opin Biotechnol 13:572–577, 2002); therefore, culture-independent metagenomic strategies are promising approaches to assess the phylogenetic composition and functional potential of microbial communities living in extreme environments (Lopez-Lopez et al. in Life 3:308–320, 2013). In addition, these approaches implement tremendously the access to enzymes from (hyper)thermophilic microorganisms that have important potential applications in several biotechnological processes. We report here on the state-of-the-art of the metagenomic surveys of different hot springs (T > 65 °C) (Table 5.1) and on the recent advance in the discovery of new hyperthermostable biocatalysts of biotechnological interest from metagenomic studies of these extreme environments.

Leber’s hereditary optic neuropathy (LHON) is typically characterized by vascular alterations in the acute phase. The aim of this study was to evaluate choroidal changes occurring in asymptomatic, acute and chronic stages of LHON. We enrolled 49 patients with LHON, 19 with Dominant Optic Atrophy (DOA) and 22 healthy controls. Spectral Domain-Optical Coherence Tomography (SD-OCT) scans of macular and peripapillary regions were performed in all subjects, to evaluate macular and peripapillary choroidal thickness, and retinal nerve fiber layer (RNFL) thicknes. Macular and peripapillary choroidal thicknesses were significantly increased in the acute LHON stage. On the contrary, macular choroidal thickness was significantly reduced in the chronic stage. Furthermore, peripapillary choroidal thickness was decreased in chronic LHON and in DOA. Both RNFL and choroid had the same trend (increased thickness, followed by thinning), but RNFL changes preceded those affecting the choroid. In conclusion, our study quantitatively demonstrated the involvement of the choroid in LHON pathology. The increase in choroidal thickness is a feature of the LHON acute stage, which follows the thickening of RNFL. Conversely, thinning of the choroid is the common outcome in chronic LHON and in DOA.

The study of the complex communities of hyperthermophiles, composed of Bacteria, Archaea, and viruses, populating volcanic sites is of interest to understand their adaptation and evolution to extreme conditions and as a rich source of biocatalysts [1, 2]. In fact, the intrinsic stability of their enzymes to common protein denaturants made them interesting tools for biotransformations in industrial applications. In the framework of the discovery of new enzymes for biocatalysis and biotransformations, we decided to explore the diversity of the solfataric field of Pisciarelli, Agnano (Naples, Italy), by performing a metagenomic analysis of the microbial community living in two mud/water pools that, although distant only 4.0 meters, greatly differ in both temperature and pH (T=85°C and pH 5.5; T=94°C and pH 1.5, for Pool1 and Pool2, respectively). In particular, DNA from Pool1 was extracted from the mud/water suspension while Pool2 DNA was obtained from the sediment. The deep sequencing by Illumina technology and the following in-silico analysis showed that the phylum Crenarcheaota was prevalent, but with great genera variance in the two pools according to the different environmental T and pH. The analysis of the functional categories revealed that genes involved in the carbohydrate metabolism were prevalent, followed by amino acid metabolism and by membrane transporters. The detailed phylogenetic and physiological analysis of the samples will be discussed together with the possible mechanisms of selection occurred in the two extremophilic environments. Bibliography 1. Cobucci-Ponzano B., Aurilia V., Riccio G., Henrissat B., Coutinho P.M., Strazzulli A., Padula A., Corsaro M.M., Pieretti G., Pocsfalvi G., Fiume I., Cannio R., Rossi M., and Moracci M.: J Biol Chem, 2010, 20691-20703. 2. Cobucci-Ponzano, B., Conte, F., Strazzulli, A., Capasso, C., Fiume, I., Pocsfalvi, G., Rossi, M., Moracci.: M. Biochimie, 2010, 92, 1895-907.

none 12 si AIMS: To evaluate longitudinal retinal ganglion cell inner plexiform layer (GC-IPL) and macular retinal nerve fibre layer (mRNFL) thickness changes in acute Leber's hereditary optic neuropathy (LHON). METHODS: Six eyes of four patients with LHON underwent SD-OCT (optical coherence tomography) at month 1, 3, 6 and 12 after visual loss. In two eyes, the examination was carried out in the presymptomatic stage. The relationship and curves for area under the receiver operator characteristic (AUROC) were generated to assess the ability of each parameter to detect ganglion cell loss. RESULTS: Significant longitudinal thinning of GC-IPL and mRNFL was detected in LHON. GC-IPL thinning was detectable in the deviation map during the presymptomatic stage in the inner ring of the nasal sector and then it progressively extended following a centrifugal and spiral pattern. Similarly, mRNFL thinning began in the inferonasal sector and it progressively extended. No further statistically significant changes were detected after month 3. The highest level of AUROC values at 1 month were detected in the nasal sectors and inferonasal mRNFL thickness reached AUROC value=1. All the parameters were equally able to detect ganglion cell loss from month 2 to 12. CONCLUSIONS: The natural history of GC-IPL thinning follows a specific pattern of reduction, reflecting the anatomical course of papillomacular fibres. Month 6 represents the end of GC-IPL loss. GC-IPL and mRNFL thinning is detectable before onset of visual loss. These observations can help future therapeutic approaches for both LHON carriers at high risk of conversion and patients with acute early LHON. Fondazione Bietti is supported by the Italian Ministry of Health and Fondazione Roma; IRCCS Istituto di Scienze Neurologiche di Bologna is supported by the Ministry of Health. mixed Balducci, Nicole; Savini, Giacomo; Cascavilla, Maria Lucia; La Morgia, Chiara; Triolo, Giacinto; Giglio, Rosa; Carbonelli, Michele; Parisi, Vincenzo; Sadun, Alfredo A; Bandello, Francesco; Carelli, Valerio; Barboni, Piero Balducci, Nicole; Savini, Giacomo; Cascavilla, Maria Lucia; La Morgia, Chiara; Triolo, Giacinto; Giglio, Rosa; Carbonelli, Michele; Parisi, Vincenzo; Sadun, Alfredo A; Bandello, Francesco; Carelli, Valerio; Barboni, Piero

Metagenomic analysis applied to specialized environments offers the possibility to understand the evolution of complex biotypes. In particular, metagenomics of extreme environments, which might resemble those of primitive Earth, is of interest for the study of the origin, evolution and distribution of extremophiles (organisms thriving at these conditions) as primitive forms of life populating our planet and, in an astrobiological perspective, other bodies in the universe. The Pisciarelli solfatara, located in the Phelgrean Fields (Naples, Italy), is characterized by a remarkable diversity in terms of temperature and pHs of the different mud pools and solfataric vents [1]. Here several thermoacidophilic species, such as Acidianus, Archaeoglobus spp, Pyrobaculum, Sulfolobus, and several viruses have been isolated [1]. In order to map the composition of this biotope in terms of number and abundance of the different species, we decided to carry out a metagenomic deep sequencing project on soil and water/mud pools of Pisciarelly solfatara. We will show our preliminary results derived by the sampling in the main water pool. Total DNA was extracted from the pellets obtained by centrifuging water samples and analysed by using Illumina paired-end sequencing technology. In a single run, we produced~3.0 Gb total nucleotides. These reads were assembled into 1,543 contigs with an N50 of 15863 bp，and a total of 17,652 Open Reading Frames (ORFs) were identified. These ORFs were subjected to BLAST similarity searches and annotated with KEGG, eggNOG and NR database respectively. The clean reads rate was 99.7% and a total of 17,652 Open Reading Frames (ORFs) were identified. The majority (77%) of the species found belonged to the Crenarcheaota phylum, which branches deeply in the phylogenetic tree of life, followed by Proteobacteria (22%), and, in much lesser amounts, Euryarchaeota, Actinobacteria, and others. The number of species identified is much higher than those found by a conventional PCR-based approach [2]. Details on the species composition and on the realtive abundance of each ORF will be described together with the astrobiological implications.

The biotransformation of lignocellulose biomasses into fermentable sugars is a very complex procedure including, as one of the most critical steps, the (hemi) cellulose hydrolysis by specific enzymatic cocktails. We explored here, the potential of stable glycoside hydrolases from thermophilic organisms, so far not used in commercial enzymatic preparations, for the conversion of glucuronoxylan, the major hemicellulose of several energy crops. Searches in the genomes of thermophilic bacteria led to the identification, efficient production, and detailed characterization of novel xylanase and a-glucuronidase from Alicyclobacillus acidocaldarius (G1-110-XA and GH67-GA, respectively) and a alpha-glucuronidase from Caldicellulosiruptor saccharolyticus (GH67-GC). Remarkably, GH10-XA, if compared to other thermophilic xylanases from this family, coupled good specificity on beechwood xylan and the best stability at 65 degrees C (3.5 days). In addition, GH67-GC was the most stable alpha-glucuronidases from this family and the first able to hydrolyse both aldouronic acid and aryl-alpha-glucuronic acid substrates. These enzymes, led to the very efficient hydrolysis of beechwood xylan by using 7- to 9-fold less protein (concentrations

Bacterial spores are surrounded by the coat, a multilayered shell that contributes in protecting the genome during stress conditions. In Bacillus subtilis, the model organism for spore formers, the coat is composed by about seventy different proteins, organized into four layers by the action of several regulatory proteins. A major component of this regulatory network, CotE, is needed to assemble the outer coat and develop spores fully resistant to lysozyme and able to germinate efficiently. Another regulator, CotH, is controlled by CotE and is present in low amounts both during sporulation and in mature spores. In spite of this CotH controls the assembly of at least nine outer coat proteins and cooperates with CotE in producing fully resistant and efficiently germinating spores. In order to improve our understanding of CotH role in spore formation, we over-produced CotH by placing its coding region under the control of a promoter stronger than its own promoter but with a similar timing of activity during sporulation. Over-production of CotH in an otherwise wild type strain did not cause any major effect, whereas in a cotE null background a partial recovery of the phenotypes associated to the cotE null mutation was observed. Western blot, fluorescence microscopy and Surface-Enhanced Raman Scattering spectroscopy data indicate that, in the absence of CotE, over-production of CotH allowed the formation of spores overall resembling wild type spores and carrying in their coat some CotE-/CotH-dependant proteins. Our results suggest that the B. subtilis spore differentiation programme is flexible, and that an increase in the amount of a regulatory protein can replace a missing partner and partially substitute its function in the assembly of the spore coat.

There was an error in the title of the article. The correct version of the title is: Flexibility of the Programme of Spore Coat Formation in Bacillus subtilis: Bypass of CotE Requirement by Over-Production of CotH The correct citation is: Isticato R, Sirec T, Giglio R, Baccigalupi L, Rusciano G, et al. (2013) Flexibility of the Programme of Spore Coat Formation in Bacillus subtilis: Bypass of CotE Requirement by Over-Production of CotH. PLoS ONE 8(9): e74949. doi:10.1371/journal.pone.0074949

The second generation bioethanol represents a main challenge in global efforts to utilize renewable resources rather than fossil fuels. However, the close association of cellulose and hemicelluloses to lignin in the plant cell wall makes it difficult to degrade lignocellulose into fermentable sugars. Consequently, pretreatments are necessary to make the polysaccharides more accessible to the enzymes, but the high temperature and extreme pH conditions required give rise to problems when using conventional enzymes in the saccharification step (Galbe and Zacchi 2002). Microorganisms thriving in habitats characterized by harsh conditions, and the enzymes derived therein, represent a helpful tool in the development of bioethanol production processes. In fact, they allow bioconversions at non-conventional conditions under which common biocatalysts are denatured. The use of high operational temperatures allows energy savings by reducing the cooling cost after high temperature pretreatments, and, in ethanol production, thermophilic conditions permit ethanol evaporation allowing harvest during fermentation.

The cotG and cotH genes of Bacillus subtilis encode two previously characterized spore coat proteins. The two genes are adjacent on the chromosome and divergently transcribed by σ K , a sporulation-specific σ factor of the RNA polymerase. We report evidence that the cotH promoter maps 812 bp upstream of the beginning of its coding region and that the divergent cotG gene is entirely contained between the promoter and the coding part of cotH . A bioinformatic analysis of all entirely sequenced prokaryotic genomes showed that such chromosomal organization is not common in spore-forming bacilli. Indeed, CotG is present only in B. subtilis , B. amyloliquefaciens , and B. atrophaeus and in two Geobacillus strains. When present, cotG always encodes a modular protein composed of tandem repeats and is always close to but divergently transcribed with respect to cotH . Bioinformatic and phylogenic data suggest that such genomic organizations have a common evolutionary origin and that the modular structure of the extant cotG genes is the outcome of multiple rounds of gene elongation events of an ancestral minigene.