In vitro culture, ensuring rapid multiplication and production of plant material under aseptic conditions, represents an excellent tool for ex-situ conservation of tree species biodiversity and can be used for the conservation, among others, of endangered and rare crops. Among the Pyrus communis L. cultivars that have been abandoned over the years due to changed cultivation requirements, but which are still used today in breeding programs, there is the ‘Decana d’inverno’. Pear is generally considered a recalcitrant species for in vitro propagation due to weak multiplication rate, hyperhydricity, and susceptibility to phenolic oxidation. Therefore, the use of natural substances like neem oil  represents one of the options to improve the in vitro plant’s tissue culture. In this context, the aim of the present work was to evaluate the effect of adding neem oil (0.1 and 0.5 m L L−1) to the growth substrate in order to optimise the in vitro culture of the ancient pear tree cultivar ‘Decana d’inverno’. The neem oil addition resulted in an increase in the number of shoots produced especially at both concentrations used. On the contrary, an increase in length of proliferated shoots was observed only with the addition of 0.1 mL L−1. The neem oil addition did not affect the explants viability, fresh and dry weights. Therefore, the present study demonstrated for the first time the possibility of using neem oil to optimise the in vitro culture of an ancient pear tree cultivar. Pyrus is characterized by a high genetic variability with several species and thousands of cultivars that can be divided into two major groups, the Occidental (European) and the Oriental (Asian) pears [The genus n) pears . Regardin) pears .Valsa pyri are increasing [The world production of pears in 2021 was 25.6 Mt across a cultivated area of 1.4 million hectares (ha) . The lar® was obtained in 1994 from the INRA by crossing ‘Decana del Comizio’ × ‘Decana d’inverno’, while ‘Cepuna’ was obtained by crossing ‘Conference’ and ‘Decana d’inverno’ [in vitro culture [In vitro cell and tissue culture techniques do indeed ensure the rapid multiplication and production of plant material under aseptic conditions and can be used for the conservation among the others of endangered and rare crops [In vitro culture has been reported for several cultivars/rootstock of pear [in vitro propagation (micropropagation) [in vitro plant’s tissue culture [in vitro growing media there is neem oil. Previous researches [in vitro culture of other plants species. Neem oil is obtained from Azadirachta indica A. Juss seeds and thanks to its composition its widely used in agriculture and for human health [in vitro culture of an ancient pear tree cultivar (‘Decana d’inverno’).In addition, there is a lack of varietal innovation in all the growing countries. Indeed, in Europe, the mostly diffused pear cultivar is Conference, followed by Abbé Fétel, Williams, and Rocha, while in Italy, one of the world’s largest producers of pears, a large part of production is based on two main cultivars, Abbé Fétel and Williams . Among tinverno’ . In this culture ,8. In vi of pear ,14,15,16agation) . In this culture . Among tsearches  demonstrn health . Neem oin health . In partn health ,23. In t−1) (−1 (ON 0.1) and 0.5 mL L−1 (ON 0.5) of neem oil were added an increase of about 34% and 30% respect to the control (0 mL L−1 of neem oil) in the number of shoots was observed. On the contrary, a positive effect on length of shoots was detected only when neem oil was applied at 0.1 mL L−1. In the ON 0.1 substrate an increase in shoot length of about 30% was observed while at 0.5 mL L−1 neem oil did not induce an increase in shoot length.Neem oil does not affect proliferated explant’s viability. In all treatments, the shoot viability of cv ‘Decana d’inverno’ was equal to 100%. Adding neem oil to the culture medium allowed to obtain a better vegetative activity evaluated as number of shoots produced . The inc−1) . Indeed,−1 (ON 0.1) increased the callus fresh weight. On the contrary, the above-mentioned effect was not detected in the ON 0.5 culture media. The addition of neem oil did not affect green fresh weight and total dry weight of the proliferated explants ’ rootstock in vitro proliferation was obtained thorough a proper combination of plant growth regulators in the MS substrate: a combination of 2 mg L−1 6-Benzylaminopurine (BAP) and 1 mg L−1 Kinetin [−1 BAP [Pear tree is generally considered recalcitrant for micropropagation since during ion rate ,17. Howeexplants  and was explants . To the explants . Thanks explants . Improveexplants  while inexplants . On the  Kinetin . Evaluat [−1 BAP .in vitro Biotechnology of the Tree Science Research Unit of the Department of Agricultural Food and Environmental Sciences of the University of Perugia, where the experiment was conducted. The culture of ‘Decana d’inverno’ was stabilized aseptically, from meristematic apices taken from portions of vegetative resting branches, cut into pieces with a length of 3–4 cm (2–3 nodes), washed with running tap water, and immersed for 10 s in a solution of 70% ethyl alcohol with the addition of a few drops of surfactant (Tween 20) to remove surface impurities. The plant material was then immersed in a 2.5% sodium hypochlorite solution for 20 min and immediately transported under a laminar flow cabinet to operate under completely aseptic conditions. The explants were then washed twice in sterile distilled water to remove residues of the sterilizing agent. The meristematic apices of 0.3–0.5 mm length and approximately 0.1 mm in diameter were then removed under aseptic conditions using a stereomicroscope.The plant material used in the experiment was derived from shoots of the proliferating cultivar Decana d’inverno present at the Laboratory of Micropropagation and −1), hydrolyzed casein (100 mg L−1), agar provided by Duchefa (0.7 g L−1), pH of 5.5. Before exposing the apices to light, the tubes were covered with aluminium foil and kept in the dark in the growth chamber for 7 days to avoid photo-oxidation. The healthy (uncontaminated) apices were kept in the growth chamber at 22 ± 2 °C and gradually accustomed to light intensities of 40 µE m−2 s−1 and a 16-h photoperiod of light. The stabilization phase was completed after approximately 30–35 days. The obtained proliferated shoots were then transferred in glass vessels (500 mL capacity), each containing ten explants and 100 mL of MS substrate enriched with vitamin mixture of Jacquiot instead of MS vitamin mixture [−1), naphthalenacetic acid (NAA) (0.1 mg L−1) and BAP (2 mg L−1), sucrose (30 g L−1) and agar (7 g L−1), pH 5.5.Finally, the apices were transferred into glass tubes each containing 5 mL of Murashige, and Skoog (MS) substrate , supplem mixture , gibbere−1 referred as ‘control’, 0.1 mL L−1 referred as ‘ON 0.1′, and 0.5 mL L−1 referred as ‘ON 0.5′) were added to the substrate before autoclaving at 115 °C for 20 min. In particular, a neem oil soluble in water provided by “Neem Italia company” composed by neem seed oil (from organic agriculture) and Polysorbate 85 was used. The placement of the explant inside the vessels occurred in aseptic conditions under a horizontal laminar flow cabinet. The explants were placed for 30 days in a growth chamber, characterized by a constant temperature of 22 ± 2 °C and a 16-h photoperiod of light with an intensity of 40 µE m−2 s−1.The initial explants for the experiment were represented by 10 mm about long uninodal explants deprived of the apical bud to maintain a high degree of uniformity. Glass vessels (500 mL capacity) were used in the experiment, each containing ten explants with two leaves and 100 mL of the above-mentioned substrate. Following previous experiences conducted on other species, three different neem oil concentrations  equal to six was foreseen: this allowed, at the end of the proliferation, to carry out the destructive measurements on the shoots of three pots and to assure the suitable number of explants for the next subculture.viability (%): incidence of green and viable explants;shoots (n): average number of shoots developed by each initial explant (multiplication rate);shoot length (mm): average length of developed shoots;callus (%): incidence of explants that produced basal callus;green fresh weight (mg): average fresh weight of developed vegetative organs ;callus fresh weight (mg): average fresh weight of callus masses that may have developed at the base of explants;total dry weight (mg): average dry weight of the vegetative organs and callus, obtained by keeping the plant material in an oven for three days at 105 °C.To evaluate the effect of n.o. treatments, after 30 days, the following parameters for each of the three subsequent subcultures were monitored:p = 0.05).The trial was organized according to a completely randomized design, with three treatments and 30 proliferated explants for each treatment and for each of the three subsequent subcultures. In particular, for each treatment and subculture, a number of vessels (replicates) equal to six was foreseen: at the end of the proliferation, this allowed to carry out the destructive measurements on the shoots of three pots (30 proliferated explants for each treatment) and to assure the suitable number of explants for the next subculture. Collected data were subjected to one-way variance analysis (ANOVA), and significant differences were assayed by Tukey HSD test (−1, allows to optimize the in vitro culture of ‘Decana d’inverno’ an ancient pear tree cultivar. The increase in the number of shoots produced and their length allow to increase the efficiency of the micropropagation process and to overcome some issues of the in vitro culture of a species considered recalcitrant.The obtained results suggest that the addition of neem oil, a natural substance, at 0.1 mL L


Definition of the microbiology of pleural space infection has been challenging due to the poor yield of conventional culture, a challenge that has led to recommendations for broad spectrum antimicrobial therapy for these infections. Since August 2019, we have offered a 16S ribosomal RNA gene PCR assay followed by Sanger or next generation sequencing, or reported as negative, depending on the PCR signal strength  in clinical practice at Mayo Clinic.Here, we describe the microbiologic and clinical findings of 57 pleural fluids testing positive by 16S rRNA gene PCR/sequencing from August 2020 to January 2023. Demographic data and clinical features, and results of pleural fluid and blood cultures were recorded. Analysis was performed using SAS software version 9.4 (SAS Inc).Fusobacterium nucleatum group and/or Streptococcus intermedius were detected in 31/57 (54%) cases [including 28/48 (58%) in the subset with community-acquired infection], with additional facultative and/or anaerobic species in various combinations also found in 17/31 (55%) of these cases.Median patient age was 63 years (range 5-83 years) and 75% were men. Pleural space infection was community-acquired in 84%. The most frequent symptom was dyspnea (80%), followed by chest pain (52%). 89% of patients had received antibiotics in the two weeks preceding specimen collection. Radiological consolidation was observed in 63% and pleural fluid was loculated in 58%. Pleural fluid and blood cultures were positive in 32 and 11%, respectively. The microbiology found with the sequencing assay is shown in the table. 23/57 (40%) detections were polymicrobial. Fusobacterium nucleatum group and/or Streptococcus intermedius, with or without other normal microbiota, a grouping for which we propose the term FuSion .Based on analysis of pleural fluid using a targeted metagenomic sequencing assay in clinical practice, the most frequent organism profile involved in community-acquired pleural space infection is a combination of Eva Carmona, MD, boehringer ingelheim: Advisor/Consultant Øyvind Kommedal, n/a, Pathogenomix inc.: Advisor/Consultant|Pathogenomix inc.: Stocks/Bonds Robin Patel, MD, Abbott Laboratories: Advisor/Consultant|Adaptive Phage Therapeutics: Grant/Research Support|Adaptive Phage Therapeutics: Mayo Clinic has a royalty-bearing know-how agreement and equity in Adaptive Phage Therapeutics.|BIOFIRE: Grant/Research Support|CARB-X: Advisor/Consultant|ContraFect: Grant/Research Support|Day Zero Diagnostics: Advisor/Consultant|HealthTrackRx: Advisor/Consultant|Mammoth Biosciences: Advisor/Consultant|Netflix: Advisor/Consultant|Oxford Nanopore Technologies: Advisor/Consultant|PhAST: Advisor/Consultant|See details: Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic|See details: continued, patent on an anti-biofilm substance issued|TenNor Therapeutics Limited: Grant/Research Support|Torus Biosystems: Advisor/Consultant|Trellis Bioscience, Inc.: Advisor/Consultant


BackgroundHealthcare-related artificial intelligence (AI) is developing. The capacity of the system to carry out sophisticated cognitive processes, such as problem-solving, decision-making, reasoning, and perceiving, is referred to as higher cognitive thinking in AI. This kind of thinking requires more than just processing facts; it also entails comprehending and working with abstract ideas, evaluating and applying data relevant to the context, and producing new insights based on prior learning and experience. ChatGPT is an artificial intelligence-based conversational software that can engage with people to answer questions and uses natural language processing models. The platform has created a worldwide buzz and keeps setting an ongoing trend in solving many complex problems in various dimensions. Nevertheless, ChatGPT's capacity to correctly respond to queries requiring higher-level thinking in medical biochemistry has not yet been investigated. So, this research aimed to evaluate ChatGPT's aptitude for responding to higher-order questions on medical biochemistry.ObjectiveIn this study, our objective was to determine whether ChatGPT can address higher-order problems related to medical biochemistry.MethodsThis cross-sectional study was done online by conversing with the current version of ChatGPT . It was presented with 200 medical biochemistry reasoning questions that require higher-order thinking. These questions were randomly picked from the institution's question bank and classified according to the Competency-Based Medical Education (CBME) curriculum's competency modules. The responses were collected and archived for subsequent research. Two expert biochemistry academicians examined the replies on a zero to five scale. The score's accuracy was determined by a one-sample Wilcoxon signed rank test using hypothetical values.ResultThe AI software answered 200 questions requiring higher-order thinking with a median score of 4.0 . Using a single sample Wilcoxon signed rank test, the result was less than the hypothetical maximum of five (p=0.001) and comparable to four (p=0.16). There was no difference in the replies to questions from different CBME modules in medical biochemistry . The inter-rater reliability of the scores scored by two biochemistry faculty members was outstanding (ICC=0.926 (95% CI: 0.814-0.971); F=19; p=0.001)ConclusionThe results of this research indicate that ChatGPT has the potential to be a successful tool for answering questions requiring higher-order thinking in medical biochemistry, with a median score of four out of five. However, continuous training and development with data of recent advances are essential to improve performance and make it functional for the ever-growing field of academic medical usage. Artificial intelligence (AI) has various definitions. Still, none of them can likely adequately explain this complicated phenomenon, which is currently the focus of numerous research efforts across many scientific fields in addition to the realm of information technologies. Machines with artificial intelligence can recognize a problem and develop a workable solution using methods similar to human cognitive and psychological processes. The system learns how to accurately forecast a variable's value, classify a set of data, or carry out other challenging tasks using artificial intelligence techniques ,2.In recent years, numerous initiatives have been taken to use artificial intelligence and machine learning in molecular medicine, biochemistry, and cell biology ,4. Many AI systems' capacity for advanced cognitive functions, including problem-solving, decision-making, reasoning, and perceiving, is called higher cognitive thinking. This thinking requires more than just processing facts; it also entails comprehending and working with abstract ideas, interpreting and applying relevant information, and producing new insights based on prior learning and experience . HoweverIn the fields of biochemistry and laboratory medicine, AI's capacity to solve higher-order reasoning-type issues depends on the difficulty of the questions being asked and the quality of the training data the AI system has been presented with. AI systems can instantly and accurately respond to simple or uncomplicated inquiries . For insOne such AI-based conversational tool, ChatGPT, is being tested for biomedical writing and can produce replies that resemble human answers . For resStudy setting and ethical considerationThis cross-sectional study was conducted at the biochemistry department of Dr. B.C. Roy Multi-Speciality Medical Research Centre, Indian Institute of Technology Kharagpur, over the second and third week of March 2023. The information used in this research was gathered using the recent version of Chat GPT , presently an open-source free online tool for registered users. For data collection, we used laptops with institutional Wi-Fi broadband internet connection. There are no human research subjects in this study. As a result, the research is exempted from institutional ethical review under current norms.QuestionsAs per the competencies of biochemistry in the Competency-Based Medical Education (CBME) curriculum, as designed by the National Medical Commission for Indian Medical Graduates, we divided the entire curriculum into 11 sections. This topic-wise competency mapping is shown in Table Using the department's question bank, which is a compilation of first and second semester questions from various medical universities across India, we picked a total of 200 questions at random, covering the entire curriculum. Among them, 100 questions were reasoning or justification type of questions, which are typically set in first professional Bachelor of Medicine and Bachelor of Surgery (MBBS) examinations without any alternative options, and these are considered to be of higher order and an in-depth understanding of the topic is necessary to provide a solution. Instead of merely memorizing information, it focuses on fundamental ideas and principles. For instance, asking someone to apply a concept to a new circumstance requires analysis and synthesis of information as opposed in asking them to recollect a structure or description . We alsoData collectionThe process of gathering information occurred between March 14 and March 16, 2023. The questions were utilized to initiate the dialogue with ChatGPT. The response to reasoning type of questions given by the software was transcribed onto a notepad and stored on the computer to be examined later. Regarding the MCQs, we asked Chat GPT to provide the reasoning for choosing the answers. The first response was taken as final, and the option of "regenerate response" was not used. Thereafter the collected notes were printed out for evaluation. Scoring was done by two assessors on a scale of zero to five, with zero being incorrect and five being fully correct, based on a pre-selected answer key.Statistical analysisThe data were expressed as number, mean, median, standard deviation, and first and third quartiles using descriptive statistical tests. The Kolmogorov-Smirnov test was performed with the dataset, which showed the distribution was not normal. We conducted a one-sample Wilcoxon signed rank test using hypothetical anticipated values to assess the response's correctness. The intraclass correlation coefficient was used to evaluate the score between the two raters (ICC). For all of the statistical analyses, we utilized SPSS version 21 . A p-value of less than 0.05 was regarded as statistically significant. A brief outline of the study design has been highlighted in Figure The AI software answered a total of 200 questions requiring higher-order thinking with a mean score of 3.98 (± 0.70) and a median score of 4.0. . The overall and system-wise scores of the responses are shown in Table Using a single sample Wilcoxon signed rank test, the result was less than the hypothetical maximum of five (p=0.001) and comparable to four (p=0.16). The median score of BI 1 was near to five, and the median score of modules BI 10 and BI 11 was below four . There was no difference in the replies to questions from different CBME modules in medical biochemistry . The p-values from the post hoc test were not presented because there was no discernible difference between the two groups. The inter-rater reliability of the scores scored by two biochemistry faculty members was outstanding. The ICC was 0.926 with a 95% confidence interval of 0.814 to 0.971 . A few examples of Chat GPT responses are shown in the appendix. We tested ChatGPT's ability to solve complex biochemistry questions by giving it 200 random questions. Based on our findings, ChatGPT's responses scored at least four out of five marks on all the modules except the last two modules, where the score was lower. Numerous past investigations have evaluated the potential of ChatGPT for medical education purposes. The observation is similar to a recent study conducted by Sinha et al. demonstrating the utility of ChatGPT for solving higher-order problems in pathology . The stuOn the other hand, Huh's study discovered that ChatGPT's performance in parasitology is still lacking compared to that of a Korean student . The stuWe recommend that medical schools and colleges should not impose limitations on the utilization of AI. Instead, the students should be equipped with the necessary skills to use them optimally. This is of utmost importance that when a student is trying to solve MCQs, they can readily get the answer from ChatGPT with proper reasoning. Simply pasting an MCQ the same into an online search engine may not give any useful response. It is imperative to ensure that future AI systems are designed, developed, and validated meticulously to provide medical students with reliable and precise information. As AI continues to advance, there is a need for further development in health-related information to increase its potential to be incorporated into education and healthcare systems . It is aLimitationsThere are various limitations to this study. Firstly, we employed a scoring approach that ranged from zero to five. Despite preparing the answer keys beforehand, there may still have been a subjective bias in the assessment that was beyond our control. The questions used in our study were sourced from our question bank, and other institutions may have different questions. Therefore, future studies may need to be conducted multicentrically for a more generalizable outcome. Furthermore, even a slight modification to a question may result in a different response from ChatGPT, so this should be considered in future investigations.It can be concluded that ChatGPT helps in seeking answers for higher-order reasoning questions in medical biochemistry. The study showed a median score of four out of five in solving the questions with better performance in explaining traditional concepts rather than justifying recent advances due to its limitation in training databases in the constantly evolving medical research in cancer biochemistry, immunology, and clinical investigations. It can be an excellent tool for solving multiple-choice questions and getting the proper reasoning behind the solution. Although such cognitive ability in AI can benefit students and academicians seeking quick and functional responses to their inquiries, considering the evolution of AI programs worldwide, testing their capabilities in future studies in various medical disciplines is imperative.


During the “omics era,” studies could provide significant, distinct, and exclusive insights into sarcoidosis phenotypes linking clinical, laboratory, imaging, and histologic characteristics with molecular signatures. In this context, the personalization of treatment for sarcoidosis patients might have reached its goal.Sarcoidosis is an inflammatory granulomatous disease of unknown etiology involving any organ or tissue along with any combination of active sites, even the most silent ones clinically. The unpredictable nature of the sites involved in sarcoidosis dictates the highly variable natural history of the disease and the necessity to cluster cases at diagnosis based on clinical and/or imaging common characteristics in an attempt to classify patients based on their more homogeneous phenotypes, possibly with similar clinical behavior, prognosis, outcome, and therefore with therapeutic requirements. In the course of the disease's history, this attempt relates to the availability of a means of detection of the sites involved, from the Karl Wurm and Guy Scadding's chest x-ray staging through the ACCESS, the WASOG Sarcoidosis Organ Assessment Instruments, and the GenPhenReSa study to the  Sarcoidosis is a systemic inflammatory disease of unknown etiology, which occurs in populations worldwide and involves the lungs and the intrathoracic lymph nodes –3. The eHowever, clinical compatibility for sarcoidosis in most cases—beyond the well-known and quite pathognomonic clinical syndromes (phenotypes), such as Löfgren's syndrome and Heerfordt's syndrome—constitutes primarily the unpredictable and “anarchic” tissue distribution of the disease in combination with some characteristic imaging features from the lungs, in addition to the asymptomatic or oligosymptomatic disease's first appearance , 10–12. 18F-fluoro-2-deoxyglucose (18F-FDG) positron emission tomography (PET) computed tomography (CT) scan phenotyping and far beyond to new technologies and/or the current “omics” availability  that is fairly distinct from tuberculosis, with good prognosis—an assumption that maintains its value till present –52. CrisIn the beginning, it was Wilhelm Conrad Röntgen (recipient of Nobel Prize in Physics in 1901) who was unaware of sarcoidosis, and by discovering the electromagnetic radiation known as Röntgen rays (X-rays), he offered the availability of chest roentgenogram to the pioneers of the disease  rendering them aware that the skin, eyes and joints disease in front of them was part of an internal organ, systemic disease involving almost always the lungs and the intrathoracic lymph nodes: the indispensable first encounter of every physician with any sarcoidosis patient to this day –56.HLA-DRB1*03 allele on the outcome of this phenotype—a new step in the effort to draw information about phenotypes from the science advancement adding to the clinical information the results of the “bench” (The first chest röentgenogram (x-rays) classification of sarcoidosis, which was designed by Karl Wrum, was divided into three stages  and was the first attempt to draw prognostic considerations of the disease, and accordingly arriving at therapeutic decisions for sarcoidosis patients , 32. It  “bench” , 57–61.  “bench” .To solve the problem of the elusive identification of the etiologically implicated antigen in sarcoidosis, the National Heart, Lung, and Blood Institute (NHLBI) set up the A Case Control Etiologic Study of Sarcoidosis (ACCESS) multicenter study –36. In pThe WASOG Sarcoidosis Organ Assessment Instrument was developed as an update of the previous ACCESS tool to establish reliable criteria for the probability of any organ being involved in the disease in a sarcoidosis patient, in the new technology era. The probability of organ involvement from sarcoidosis was based on two criteria: granulomatous inflammation and compatible clinical manifestation, excluding in both cases alternative etiologies. Based on the above findings, clinical manifestations were graded as  highly pThe GenPhenReSa consortium is the first attempt, entirely utilizing the WASOG Sarcoidosis Organ Assessment Instrument to identify almost all sites of involvement by the disease, in order firstly to identify reliable and homogeneous phenotypes of sarcoidosis appearance, useful cohorts for further biomedical studies and secondly to attempt possible genotype–phenotype associations were detected. The results of the phenotype module of the GenPhenReSa as evidenced by the cluster analysis of 2,163 Caucasian patients phenotyped in 31 different sarcoidosis expert study centers regard the identification of five “distinct” subgroups identified from organ involvement: (1) abdominal organ involvement, (2) ocular-cardiac-cutaneous-central nervous system disease involvement, (3) musculoskeletal-cutaneous involvement, (4) pulmonary and intrathoracic lymph node involvement, and (5) extrapulmonary involvement, defined by the authors as “homogeneous cohorts useful for further biomedical studies” . However18F-FDG PET/CT scan by unveiling glucose metabolism of inflammatory cells is able to identify, with high sensitivity, inflammatory active granulomas, the hallmark of sarcoidosis, even in clinically and physiologically silent sites, providing simultaneously whole-body PET and CT images  hilar–mediastinal nodal, (II) lungs and hilar–mediastinal nodal, (III) an extended nodal supraclavicular, thoracic, abdominal, inguinal, and (IV) all the above in addition to systemic organs and tissues such as muscles–bones–spleen and skin  scans and echocardiogram (ECHO). However, for the first time, the disease involvement was clearly identified in all possible extensions of the actual site, and a lot of work must be done to identify its prognostic significance and therapeutic requirements (apparently necessitating none). Finally, the phenotype IV appears familial to the clinician-caring sarcoidosis patients since it appears to be satisfying to the current knowledge of the systemic nature of the disease, probably relating to the systemic spreading, from the lungs and hilar–mediastinal lymph nodes to the whole body, of the etiologically implicated antigen. Therefore, by clustering investigations of sarcoidosis through the 18F-FDG PET/CT scan, clinically logical phenotypes were identified. However, like all other clustering investigations, some patients may not respect the boundaries but also Scadding's staging by the simplest of the means of investigation, the chest roentgenogram, which clarifies that stages may not be “sharply demarcated,” and we feel right to confirm his elaboration concerning clustering. Based on hierarchical cluster analysis of the study population and implementing adequate preparation with low carbohydrate diet for 24 h followed by 18-h fasting to suppress radiotracer's uptake by normal myocardial cell, no significant difference was detected between the clusters regarding myocardial involvement, suggesting that heart disease could be detected and should be evaluated in any cluster. Given the important prognostic and treatment implications of cardiac involvement, the role of 18F-FDG PET/CT scan has been examined extensively. Although this modality requires a specific protocol including diet restriction and being more prone than cardiac magnetic resonance (CMR) to provide false positive results, it is a technique much more sensitive than transthoracic echocardiogram (70 vs. 25%) that should be used as complementary to CMR for the detection of myocardial inflammation and fibrosis, respectively , analysis and interpretation of all data, and wrote the manuscript. LK, NR, MS, GL, PG, and EC: interpretation of the data, wrote parts of the manuscript, and revised the work critically for very important intellectual content. AG and MK: produced the figures and revised the work critically for important intellectual content. AP, TR, VA, E-MA, EG, SC, and JG: major contribution in the interpretation of the data and revised the work critically for important intellectual content. EM: major contribution to the concept of the study, to the acquisition, analysis and interpretation of data, had access to all data, supervised the accuracy and integrity of any part of the work, and wrote the final version of the manuscript with SP. All authors read and approved the final version of the manuscript.


The generated photocurrent is altered under the polarization state of the input light, producing a 400% contrast between the maximum and minimum signals. This result is universally applicable to all on‐chip optoelectronic circuits.2D materials have manifested themselves as key components toward compact integrated circuits. Because of their capability to circumvent the diffraction limit, light manipulation using surface plasmon polaritons (SPPs) is highly‐valued. In this study, plasmonic photodetection using graphene as a 2D material is investigated. Non‐scattering near‐field detection of SPPs is implemented via monolayer graphene stacked under an SPP waveguide with a symmetric antenna. Energy conversion between radiation power and electrical signals is utilized for the photovoltaic and photoconductive processes of the gold‐graphene interface and biased electrodes, measuring a maximum photoresponsivity of 29.2 mA W −1, and the polarization state of input light produces a 400% contrast. This has potential applications in on‐chip optoelectronic circuits.This study explores plasmonic photodetection using graphene, achieving non‐scattering near‐field detection of surface plasmon polaritons. The maximum photoresponsivity is 29.2 mA W Optical communication technologies have been developed for global data traffic, cloud and data‐center optical interconnects, high‐performance computing, 5G and B6G, military applications, astronomy systems, and chip‐scale integrated circuits. Researchers have actively studied silicon photonics to comply with Moore's law. A system with optical interconnections between or within devices actualizes a broader bandwidth and faster data transfer.The unique properties of light, along with rapid advancements in quantum technology and next‐generation semiconductors, has driven researchers to explore innovative approaches for light manipulation and signal modulation. Specified by the photon energy, light has properties such as the primitive and orbital forms of angular momentum.1 Due  Light propagating in this form has significant field confinement. Owing to their ability to circumvent the diffraction limit, plasmonic nanostructures have attracted much interest, and a wide variety of designs have been reported in recent years. Plasmonic devices such as nanowaveguides, plasmonic nanolasers, circulators, and nanoantennas have been developed to advance further plasmonic circuitry technology. The conventional approach to detect SPPs is to break the homogeneity of the propagation route such that the waves couple out of the surface and collect the scattered light in the far field. Multiple optical elements and bulky systems are required to observe the SPPs radiation. Therefore, the compact and on‐chip SPP detection system is highly demanded.However, on‐chip silicon waveguides scale up to tens of micrometers, which is not sufficiently small compared to electronic components. Plasmonics uses the capability of metals to trap electromagnetic fields at deep‐subwavelength scale, allowing considerable scaling down of devices. This reduction in size aids in welding optical and electronic elements of the same size, introducing optoelectronic integrated circuits. SPP is the delocalized oscillation of electrons at the metal–dielectric interface when light couples with plasmon frequency under required conditions.4 Ligh germanium, perovskite, and III‐V materials have progressed remarkably due to their capability to bridge the gap between optical and electrical signals. However, the conventional semiconductor photodetectors are with the thickness in µm scale. Furthermore, the band gaps of these semiconductors facilitate a narrow range of practical applications. Graphene is a one‐atom‐thick carbon sheet packed in a hexagonal lattice and is known for its excellent optical, mechanical and electrical properties. Because of its transparency and flexibility, it has been applied in wearable sensors. Graphene has a gapless band structure that allows the absorption of light from 300 to 2500 nm and an accessible interband transition. The high carrier mobility in graphene guarantees an ultrafast response of these photogenerated carriers, which is promising for photodetector applications and integrated photonics.Semiconductor‐based photodetectors, including silicon,17 ger However, this design has limitations in its application spectrum because the propagation direction is fixed upon fabrication. Alternatively, SPP steering can be realized using symmetric nanostructures under different excitation conditions. This approach enables multiport signal transfer and is presumably applicable to all plasmonic‐integrated circuits. The proposed SPP waveguide was fabricated on a single‐crystalline gold flake to minimize unnecessary propagation loss due to the inhomogeneous lattice orientation and surface roughness of the evaporated films. A graphene sheet was placed under the waveguide to collect excited SPPs and convert them into electrical signals. The mechanism of converting photoenergy into electrical signals in graphene can be broadly classified into two categories, namely, carrier generation and thermal effects. The former involves carrier generation, including the photovoltaic effect (PV), photoconductive effect (PC) and photogating effect (PG). The latter include the photothermoelectric effect (PTE) and bolometric effect (BOL). The PV effect entails the generation of photo‐induced carriers in the graphene channel. Once photons are absorbed by the graphene sheet, electron‐hole pairs (EHPs) separate to form free carriers that diffuse by introducing a usable electromotive force (EMF), usually proportional to the irradiance power. The diffusion force originates from the divergence of the material band structure, also known as the p‐n junction in semiconductors. Graphene can be modified as p‐ or n‐type by applying a gate voltage or placing materials with different work functions underneath the sheet. However, the recombination length based on the internal electric field of the free carriers barely exceeds 200 nm. Therefore, incorporating an external electric field could facilitate carrier collection efficiency at the source‐drain terminals. The photoconductive effect occurs when the radiation energy is adequately high to excite the electrons in graphene from the valence band to the conduction band. This interband transition enables the free movement of electrons, thereby enhancing the conductivity of the sheet. The photogating process occurs when a particular type of charged carriers are trapped in graphene, providing an ultrahigh gain in responsivity. This trap occurs in gated field effect transistors (FETs) and graphene heterostructures. The photo‐thermoelectric process depends on the temperature gradient of the graphene channel induced by light illumination. The majority type of carriers (n‐type or p‐type) are driven from the hot side toward the cold side of the channel, and the net carrier migration leads to a detectable photocurrent response. In highly doped graphene photodetectors, the bolometric process dominates the detection response. The resistivity of the graphene channel is modulated by the thermal disruption of local carriers caused by the incident light. This retards the charged‐carrier mobility characterized by the bolometric coefficient β=dσdT, contributing to a bolometric photocurrent response that is opposite in polarity to the given bias current (IDS).+− Regarding multiple mechanisms, our proposed polarization‐dependent photodetector was based on a fusion of the PV and PC effects, which introduced a device with low power consumption and fast response. The PTE effect can be neglected owing to the large channel length and outstanding thermal conductivity of graphene. The conditions for the BOL effect are investigated further in this manuscript. It is difficult to decouple these effects; however, with careful design, engineering these mechanisms constitutes a crucial step in the optoelectronic industry.This study investigates a direction‐tunable SPP graphene photodetector. Pioneering the use of graphene for detecting polarization steered SPP propagation integrated with a subwavelength nanoantenna and plasmonic waveguide, creates a new paradigm in reconfigurable plasmonic photodetection systems. Previous studies have demonstrated that asymmetric nanostructures can realize directional launching of SPPs.30 How22.1Figure 2 was deposited on a silicon substrate. To form the graphene channel, graphene was prepared by chemical vapor deposition (CVD), transferred to the substrate, and etched by oxygen plasma for 40 s. A shadow mask was deposited by thermal evaporation on 200 nm gold electrodes. Finally, a single‐crystal gold flake was grown on a silicon substrate and patterned via focused ion beam (FIB). The flake with the plasmonic structure was flipped upside down and partially transferred onto the channel, as shown in the inset of Figure A schematic diagram of the proposed photodetector (Device A) is shown in  were engineered through superposition. In this work, a 650 nm continuous wave (CW) laser was used because of its accessibility.The dimer antenna shown in Figure 10 wer The parameters of this plasmonic waveguide designated by finite‐difference time‐domain (FDTD) simulation are briefly introduced in Figure arm, the length of the waveguide arm, was set to 4 µm. It is not a deciding factor for the wavelength or propagation mode but essential for keeping the confined electric fields in plane with the graphene sheet and preventing scattering loss. With the optimal design of the dimer antenna , SPPs can be launched at the target wavelength effectively. The upper, lower, and grooves  of the waveguide preserved the targeted wavelengths and granted propagation in the waveguide. The waveguide radius was optimized to R = 3 µm by FDTD simulation, which was mainly determined by the propagation length and bending loss of the SPP wave. The gap (Wgap = 110 nm) between the two SPP channels provides the spatial phase distribution required to interfere with the field profiles generated by the dimer antennas.The main purpose of this study was to demonstrate the near‐field detection of SPPs through graphene. Therefore, the plasmonic structure could also be replaced by other designs with directional launching capability. The directional launching of SPPs has been reported and adequately studied.44 The2.2Figure hsc) was grown and transferred via poly (methyl methacrylate) (PMMA) onto a 100‐nm gold film evaporated on a glass substrate. The gold stack with a total thickness of 300 nm was patterned with the designed plasmonic waveguide using FIB. The waveguide groove depth (d = 120 nm) was designated by simulation, and further analysis of additional etching depths is provided in the Figure Before fabricating the proposed photodetector, its ability to steer SPPs with linearly polarized light was tested. Device B was fabricated using the designed SPP waveguide structure shown in Figure RSC) and left (LSC) scattering ports were obtained under an excitation of 45° using a spectrometer. Figure RSC/LSC.2.3Figure PC = 9 nA. SPPs under this scenario were launched toward and confined in the waveguide, where the graphene channel was positioned. The local EHPs in the graphene channel is separated owing to the thermal effects caused by the SPPs in the waveguide. At an excitation angle of 60°, the SPPs would propagate along the opposite channel lacking graphene; therefore, it could be inferred that small or no photocurrent could be generated. As illustrated in Figure  The excitation energies are proportional to the number of photons responsible for the SPP generation intensity. In contrast, SPPs generate hot carriers that excite free electrons and holes driven by externally applied electric fields (VDS = −0.3 V), thereby contributing to the photocurrent.After the proposed plasmonic waveguide exhibited positive results in the optical far‐field characterization, a near‐field photodetector device was fabricated. The result in n Figure 46 TheDS| < 2 V. In Figure To further study the effects of photocurrent in graphene, a bias‐dependent experiment was conducted. The measured photocurrent exhibited a polarity equivalent to the externally applied current when |V3Optically integrated circuits have been developed to meet the ever‐growing demands of global communication technology. Photodetectors are the solutions to optical and electrical signal conversion. However, owing to size incompatibility, on‐chip integration remains an issue for conventional photodetectors. Miniaturized photodetectors have attracted significant interest in recent years.−1, respectively, introducing a 400% electrical signal contrast for further applications in integrated circuits. In addition, the photocurrent generation mechanism of graphene in the device was investigated. The device was subjected to different VDS values to measure the PV, PC, and BOL effects of graphene. The different effects of photocurrent generation can be employed under careful design and particular conditions to accommodate the vast applications of the optoelectronic device industry. For future improvement of the device performance, the integrated transition‐metal dichalcogenides (TMDCs) 2D heterostructures, hybrid plasmonic waveguides (HPWGs), and decreasing the drain to source channel length could be considered. Near‐field SPP detection, introduced by the fusion of photovoltaic and photoconductive effects, saves energy and avoids complicated and expensive fabrication procedures. The proposed device can be used in nanolaser‐integrated plasmonic circuits for ultracompact nanophotonic communication.A polarization‐dependent SPP near‐field photodetector based on graphene was developed in this work. By illuminating the backside of the groove waveguide, the antenna served as a nanoscale light source, exciting waves that propagated through the desired channel. Subsequently, the SPP waves are absorbed directly and converted into electrical signals in the near field, offering a promising near‐optical field detector based on graphene. Different photocurrents were observed under alternating incident polarization. The responsivities of the device under 150° and 60° excitation were 29.2 and 6.2 mA Wructures,47 hyb4 using a reactive ion etching system to obtain a channel with uniform edges. Subsequently, the designed plasmonic waveguide was milled using an FIB on a single‐crystal gold flake grown on a silicon substrate using a wet chemical synthesis method. As structural deformities may affect SPP propagation in a lossy manner, the FIB working current was set to a low value (7 pA) to achieve effective results. To realize different milling depths in the waveguide and antenna, each was patterned separately using the bitmap function of the FIB . The structured flake was transferred upside down and aligned at the desired location.The device was fabricated by depositing a 300 mm insulating layer of silicon dioxide on a silicon substrate using an electron beam evaporation system (ULVAC VT1‐10CE). The insulating layer restricted any contact between the electrode and silicon layer. Therefore, the photocurrent contributed by silicon was not detected by the source meter (B2901A). Graphene was transferred onto the sample using the wet transfer method, followed by oxygen plasma etching51 usi3)3 solution (33 wt.%) at room temperature (23–26°C) for ≈12 h. Consequently, the copper foil was fully etched away, leaving a photoresist/graphene stack. The stack was cleaned for 1 h using deionized (DI) water to remove etchant residue. To enhance surface energy, the target substrate was treated with UV‐ozone before the transfer, providing a location adjustable transfer process without damaging the graphene. The stack with the substrate was left in an oven at room temperature to complete dehydration, then immersed in acetone to remove the photoresist and subsequently washed in isopropanol (IPA).A monolayer graphene sheet grown on a copper foil by the chemical vapor deposition (CVD) method was transferred using the wet chemical technique. Graphene was protected by spin coating a thin layer of photoresist (PMMA‐A4). The copper foil was removed by placing the stack in a Fe(NOFew applications require the upside‐down transfer of nanostructures. In the proposed device, this technique plays a crucial role in creating a light source at the nanoscale and precludes direct illumination of the graphene sheet. First, the waveguide was patterned on a single crystalline gold flake, then 2 µL of photoresist was applied on the target flake with a pipette and left to dry on a hot plate for 2 min at 150°C. The resist/gold stack was peeled from the native substrate with tweezers, aided by a few drops of DI water on the edge. The stack was placed on a thin PDMS stamp and immersed in acetone for 2 h to reveal the gold flakes. The PDMS, along with the flake, was mounted onto a micromanipulator and aligned with the device using a microscope. The device and stamp stack were heated to 120°C to provide better contact energy between the flakes and device. Finally, the PDMS stamp was slowly peeled off from the device, leaving the patterned flake at the desired location. For clear schematics, please refer to Figure The authors declare no conflict of interest.C.‐H. W., C.‐J. K., and M.‐W. Y. performed sample fabrication, simulation, and optical characterization. J.‐H. Y., P.‐Y. W., C.‐B. H., T.‐C. L., J.‐S. H., S. I. and K.‐P. C. helped analyze the experimental data. C.‐H. W., M.‐W. Y. and K.‐P.C. wrote the manuscript. All authors discussed the results and commented on the manuscript.Supporting InformationClick here for additional data file.


The resulting composite is a solid film exhibiting magnetic properties. XRD, FTIR, Raman, TEM, and HRTEM methods were used to study all the obtained materials. It was shown that nickel nanoparticles on the surface of the reduced graphene oxide had an average diameter of 27 nm and were gradually distributed on the surface of reduced graphene oxide sheets. The data obtained allowed us to conduct a reconnaissance discussion of the mechanism of composite fabrication in supercritical isopropanol.Nanocomposites based on ferromagnetic nickel nanoparticles and graphene-related materials are actively used in various practical applications such as catalysis, sensors, sorption, etc. Therefore, maintaining their dispersity and homogeneity during deposition onto the reduced graphene oxide substrate surface is of crucial importance to provide the required product characteristics. This paper demonstrates a new, reproducible method for preparing a tailored composite based on nickel nanoparticles on the reduced graphene oxide surface using supercritical isopropanol treatment. It has been shown that when a graphene oxide film with previously incorporated Ni Reduced graphene oxide (RGO) is a nanomaterial obtained by the chemical or thermal reduction of graphene oxide, which is increasingly attracting the attention of scientists due to its unique properties and application prospects. The term “reduced graphene oxide” was defined in ISO/TS80004-13:2017 ,6,7. At Metal nanoparticles on the RGO surface are capable of catalyzing a range of organic reactions ,14,15,162O3, Ni(OH)2, or NiOOH, which creates additional difficulties during synthesis and can decrease catalytic properties , w, w2+, an−1 originates from the breathing modes of aroma rings activated by defects. This band is forbidden in graphite, and it becomes active in the presence of any defect in the carbon structure. The G band at 1570–1590 cm−1 is due to the E2g phonon appearance at the Brillouin zone center. 2+ composite before and after treatment in the SCI. All obtained spectra have characteristic D and G bands, which for the original graphene oxide correspond to 1344 cm−1 and 1583 cm−1, respectively  and increases with the mean distance rise between two defects . The ID/IG value for RGO is higher than that for GO, indicating a higher degree of in-plane defect and edge defect in the RGO due to the SCI reduction process.As a fast, simple, and nondestructive tool, Raman spectroscopy provides valuable information on the structural properties of carbon-based materials . The D bectively a. The sipitation b. In thepitation c, obtain defects . The higIn these tests, it does not matter that all samples were thin films. When interpreting the optical properties and Raman spectra of graphene-based materials, characteristics such as thickness, optical constants, band gaps, and the reflectance of both the dielectric substrate and the material under study should be taken into consideration. Due to the heterogeneity of the flake size and the distribution of oxygen-containing groups or vacancies, individual flakes of both GO and RGO are highly inhomogeneous, and, consequently, rather different values for the peak positions and widths in Raman spectra can be found in the literature . It shou0 composite , the formation of C-C bonds occurs in the reduced graphene oxide. In this way, new defects can be formed, for example, five-membered rings  formate and the deoxygenation of oxygen-containing functional groups of graphene oxide. Also, when carrying out the process in the SCI medium, the surface defects of graphene oxide are healed, which opens possibilities to obtain a reduced graphene oxide material with larger lateral flakes. The surface of reduced graphene oxide sheets acts as a matrix for nickel nanoparticles and prevents their further growth and agglomeration. The reduction of graphene oxide in supercritical isopropanol media allows the reaction to be carried out at a relatively low temperature (280 °C) with 100% conversion. In addition, when using supercritical fluids, there is no formation of toxic by-products that can be obtained when using, for example, sodium borohydride. Thus, reduction in supercritical isopropyl alcohol is an inexpensive, scalable, and environmentally friendly technology for producing composites based on nickel nanoparticles and reduced graphene oxide. The resulting composite material with potentially good conductive and magnetic properties can be used as a catalyst for hydrogenation reactions of unsaturated hydrocarbons and also as a sorbent of organic toxic substances in filtration systems.As a result of this study, a new facile method for obtaining small Ni


Ovis aries papillomavirus 3 (OaPV3) is the main cSCC viral determinant and oncogenic properties of viral E6 and E7 proteins were preliminarily investigated. However, E6 and E7 role and mechanisms resulting in cSCC have not been fully clarified, mainly due to the lack specific immunological tools, such as antibodies for in situ detection of ovine papillomavirus. This paper reports the development of specific serological tools for the investigation of OaPV3 pathogenicity, and their preliminary use to screen 4 ovine cSSC formalin-fixed paraffin embedded tissues. Relevance of immunological tools to investigation of viral biological properties and diagnosis are also discussed.Cutaneous squamous cell carcinoma (cSCC) is a malignant lesion characterized by proliferation and transformation of keratinocytes in the epidermis and infiltrating derma. cSCC is reported in domestic and wild animal species, worldwide. The occurrence and development of cSCC rely on synergic multifactorial conditions, most importantly sunlight exposure and Papillomavirus (PV) infection. In sheep, the development of such lesions represents a threat both to animal welfare and milk production.  Cutaneous squamous cell carcinoma (cSCC) is the second most common non-melanoma skin cancer and is reported in humans and several other animal species  non-enveloped viruses with a circular dsDNA genome of about 8 kbp in size. PVs cause or contribute to the development of lesions in both cutaneous and mucosal tissues, and were detected in most vertebrate species including humans, mammals, birds, reptiles, and fishes  were identified in cutaneous lesions and/or in cSCC of different host species and are considered risk factors in cSCC development  was identified in cSCCs in UV-exposed skin regions of sheep  as GST-tagged recombinant proteins. Briefly, E6 and L1 genes were amplified from a plasmid vector containing the whole OaPV3 genome (pUC19/OaPV3)  cells, and clones containing the recombinant vector (pGEX-2T/OaPV3_E6 and pGEX-2T/OaPV3_L1) were selected for ampicillin resistance. The two recombinant plasmids were purified with the PureLink™ Quick Plasmid Miniprep Kit (Invitrogen). Cloning was confirmed by automated Sanger sequencing. In order to express recombinant proteins, E. coli BL21-CodonPlus® (DE3)-RIPL cells (Stratagene) were alternatively transformed with the pGEX-2T/OaPV3-E6 or pGEX-2T/OaPV3-L1, and recombinant colonies were selected for ampicillin resistance. In order to optimize expression and to improve solubility in aqueous phase, several induction conditions and were tested. Shortly, IPTG was added to E. coli cultures at the final concentration of 0.1 mM, 0.2 mM, or 1 mM. Induction was also alternatively conducted at 37 °C, 30 °C, 25 °C, or room temperature for 4 h collecting bacterial cultures aliquots at different time-points (T0 and every hour). Moreover, O/N induction was attempted at 4 °C.PCRs were performed by using the Platinum™ Pfx DNA Polymerase (Invitrogen) and the following cycling conditions: 94 °C for 2 min initial denaturation, 2 cycles of denaturation, annealing and extension at 94 °C for 15 s, 50 °C for 30 s, 68 °C for 30 s, and 30 cycles set up at 94 °C-55 °C-68 °C for 15 s, 30 s, and 30 s respectively; a final extension was conducted at 68 °C for 10 min. PCR products were visualized on agarose gel and purified with MinElute® PCR Purification Kit (Qiagen). E6 and L1 genes were double - digested with EcoRI and BamHI. After electrophoresis, bands were gel purified with the QIAquick® Gel Extraction Kit (Qiagen). After digestion and purification, amplicons were cloned into pGEX-2T  digested with the same enzymes, by using the Rapid DNA Dephos & Ligation Kit (Roche). Ligation products were used to transform One Shot TOP10 Chemically Competent E. coli cultures were spun at 4000 x g and pellets were stored at -80 °C for further analysis.At every time point, 1 ml aliquots of Moreover, in the attempt of obtaining a hydrosoluble protein -RIPL transformed with the original pGEX-2T vector without insert were used as negative controls.E. coli pellets were resuspended in Laemmli buffer, denatured at 95 °C for 5 min, and resolved on 10% polyacrylamide gels. In order to evaluate solubility of rGST-OaPV3 E6 and L1 E. coli pellets were first resuspended in PBS and lysed by three cycles of liquid nitrogen freezing and thawing at 42 °C. Supernatants and cellular debris were subsequently separated by centrifugation and analysed by SDS-PAGE.Production of recombinant proteins was assessed by SDS-PAGE, as previously described  and briefly sonicated on ice. MgCl2 was then added to 5 mM final concentration and lysates were incubated for 1 h at room temperature. Then, urea was added to the final concentration of 3.5 M and lysates were incubated for an additional hour at room temperature prior to clarification by centrifugation. Both supernatants and pellets were analysed by SDS-PAGE.In order to tentatively promote solubilization and improve affinity chromatography purification, pellets were resuspended in 10% Triton X-100 in PBS. Also, solubilization of recombinant proteins from inclusion bodies was attempted by following Bishop and collaborators  and images were acquired by means of a GelDoc EZ® system (Bio-Rad).E. coli expressing the recombinant proteins were resolved by SDS-PAGE. Gels were stained as described above and bands corresponding to recombinant GST-OaPV3 E6 and L1 were manually excised from the gels. After three washing steps, bands were homogenized in 400 µL sterile PBS by using a TissueLyser II (Qiagen) and 0.5 mm glass beads. Briefly, gel slices were subjected to 10 cycles of homogenization , freezing for 20 min at -20 °C, and thawing. Homogenates were clarified by centrifugation and used for immunization.The total lysates of Two male Wistar rats, weighing 250 ± 30 g, were obtained from Charles River . Animals were kept at standard housing conditions , 12 h light/dark cycle) and supplied with standard laboratory chow . Rats were intraperitoneally injected respectively with rGST-OaPV3 E6 and rGST-OaPV3 L1. Immunisation was repeated two times at 15 days intervals. Before injection, homogenates were diluted 1:1 with Freund’s complete (first inoculum) or incomplete (second and third inocula) adjuvant. One ml volume was used for each immunisation.0 (before first immunisation), T1 (15 days after first inoculum), T2 (15 days after the second inoculum), and T3 (15 days after the third inoculum). Sera were obtained from all blood samples by centrifugation. All procedures were conducted based on the Guide for the Care and Use of Laboratory Animals and were approved by the local Animal and Use Committee .Blood samples were collected at TE. coli respectively expressing rGST-OaPV3 E6 or rGST-OaPV3 L1. Briefly, total lysates were resolved by SDS-PAGE on 10% gels, and proteins were subsequently blotted onto nitrocellulose membrane (1 h at 250 mA). Membranes were blocked with 5% skim milk in PBS-0.05% Tween (PBS-T) for 1 h. Rat sera collected at T0, T1, T2, and T3 were used at final dilutions of 1:100, 1:500, and 1:1000 in 2% skim milk PBS-T. Incubation was carried in a Mini-PROTEAN II Multiscreen Apparatus (Bio-Rad) for 1 h. After three washing steps, membranes were incubated for 1 h with an anti-rat IgG-HRP secondary antibody (Southern Biotech) diluted 1:50.000 in 2% skim milk PBS-T. After five washes, membranes were developed with the Chemiluminescent Peroxidase Substrate (Sigma-Aldrich) and images were acquired with a VersaDoc MP 4000 Imaging System (Bio-Rad).Sera collected from rats immunized with rGST-OaPV3 E6 and rGST-OaPV3 L1 were tested by western immunoblotting against total lysates obtained from 3) α-rGST-OaPV3 E6 or α-rGST-OaPV3-L1 sera diluted 1:100 in ImmPRESS Reagent. Slides were washed and incubated with an anti-rat secondary antibody conjugated with HRP (Southern Biotech) diluted 1:500 in ImmPRESS Reagent for 30 min at RT. After washing, the slides were incubated with DAB Peroxidase Substrate  until the desired stain intensity developed, lightly counterstained with hematoxylin, and finally mounted with Eukitt Mounting Medium (BiOptica). Images were captured by means of a Nikon Eclipse 80i microscope equipped with a Nikon DS-Fi1 camera (Nikon Instruments Inc.).Rat α-rGST-OaPV3 E6 and α-rGST-OaPV3-L1 sera were tested against 4 ovine formalin-fixed, paraffin-embed (FFPE) cSCCs by immunohistochemistry, as previously described .E. coli under different induction conditions, as demonstrated by the appearance of bands of about 43 kDa and 83 kDa in E. coli lysates  after induction at the same conditions .E. coli cultures incubation at 37 °C. Optimal induction time was estimated at 3 h for rGST-OaPV3 E6 and at 4 h for rGST-OaPV3 L1.Finally, the optimal protein production was achieved at 0.1 mM IPTG and E. coli expressing alternatively rGST-OaPV3 E6 or rGST-OaPV3 L1 , while in the case of rGST-OaPV3 L1 IgGs were detectable since T2 (30 days after the first inoculum and 15 days after booster).Since the combination of different induction conditions and protein extraction protocols failed to generate hydrosoluble forms of both rGST-OaPV3 E6 and rGST-OaPV3 L1, bands corresponding to recombinant proteins were excised from polyacrylamide gels, homogenised and used to inoculate two rats. Immunizations and sample collection were scheduled as shown in Fig.  L1 Fig. b. Specif L1 Fig. b. rGST-O0 were used as negative controls and were not able to recognize rGST-OaPV3 E6 nor rGST-OaPV3 L1 specific bands.Pre-immune sera collected at TBased on western immunoblotting results, hyperimmune sera were tested by immunohistochemistry against FFPE samples from 4 ovine cSCCs Fig. .All OaPV3-positive cSCC tissue samples positively reacted with both α-rGST-OaPV3 L1  is the most common type of skin cancer in sheep and is mainly localized in the sheep head, back, groins, udder, and perineal regions , thus covering both the The use of E6 as an infection marker can overcome the difficulties linked to the low number of PVs DNA in the tumour Doorbar . Sera prThis study reports the development of tools suitable for the study of biologic features of ovine papillomaviruses, suitable to confirm their role in cSCCs carcinogenesis and representing a promising tool for early diagnosis and prognosis of ovine SCC.


To the Editor: Idiopathic pulmonary fibrosis (IPF) is the most common manifestation of interstitial lung disease, with a median survival of 3–5 years after diagnosis. IPF is characterized by progressive fibrosis with the development of fibroblastic foci in the interstitium. Despite the short median survival, there is a striking variance in the clinical course of IPF, with some patients characterized as stable progressors (SP) and some as rapid progressors (RP) based on their decline in lung function and loss of forced vital capacity (FVC) (ty (FVC) . While thttps://doi.org/10.1172/JCI165976DS1). We identified 5 SP and 4 RP at the time of diagnosis based on loss of at least 10% FVC within the first year as well as 3 patients with terminal-stage IPF delineated for transplant (TP) and 3 individuals with age-matched normal lung tissues (NC) (clear margins from excision for pulmonary hamartoma). We investigated the presence of key transcriptional signatures using spatial transcriptomics  were sufficient to discriminate between SP and RP . SP sampfibrosis , was fouLRRC38, LUM, COA5, and ITM2A, were mainly under the control of TBX3 (http://www.ipfcellatlas.com/)  , and TBXas.com/) .HLA-DQ haplotypes were expressed in the SP and RP groups, suggesting differential immune responses in the two groups. Furthermore, MMP7 was highly expressed in the RP group compared with that in the SP and TP groups (Given the increased transcriptional activity found in the vasculature of the RP group, we investigated whether there could be transcriptional crosstalk between the vascular bed and the fibroblastic foci. In the fibroblastic foci , distincP groups , D and IP groups . While oIn conclusion, this study suggests a key role of the vascular bed in the progression of IPF, warranting further investigations. This study has limitations owing to its preliminary nature; use of a large control group with minimal lung disease would allow for statistically robust multivariate analysis in future larger studies. Furthermore, these results show similarities with publicly available single-cell transcriptional data sets, and they also highlight key differences. These could be attributed either to technical aspects of transcriptional profiling or selection of specific IPF subphenotypes. Future studies with a controlled patient cohort will be needed to discern the true variability between gene profiling by single-cell RNA-Seq and spatial transcriptomics. Nevertheless, these data highlight distinct pathways of crosstalk between fibroblasts and the vascular bed, which could prove central to the pathophysiology of the disease. Targeting such pathways and modulating the interaction between the vasculature and fibroblasts may usher the development of future therapeutics to limit the progression of IPF.


Three-dimensional (3D) printing is a versatile manufacturing method widely used in various industries due to its design flexibility, rapid production, and mechanical strength. Polyurethane (PU) is a biopolymer frequently employed in 3D printing applications, but its susceptibility to UV degradation limits its durability. To address this issue, various additives, including graphene, have been explored to enhance PU properties. Graphene, a two-dimensional carbon material, possesses remarkable mechanical and electrical properties, but challenges arise in its dispersion within the polymer matrix. Surface modification techniques, like polydopamine (PDA) coating, have been introduced to improve graphene’s compatibility with polymers. This study presents a method of 3D printing PU scaffolds coated with PDA and graphene for enhanced UV stability. The scaffolds were characterized through X-ray diffraction, Fourier-transform infrared spectroscopy, mechanical testing, scanning electron microscopy, and UV durability tests. Results showed successful PDA coating, graphene deposition, and improved mechanical properties. The PDA–graphene-modified scaffolds exhibited greater UV resistance over time, attributed to synergistic effects between PDA and graphene. These findings highlight the potential of combining PDA and graphene to enhance the stability and mechanical performance of 3D-printed PU scaffolds. Three-dimensional 3D) printing is often used for industrial applications due to its flexibility and capability to provide ample mechanical strength and support after fabrication . FurtherD printinTo overcome the abovementioned problem, various additives have been introduced into PU to improve its performance . Among tGraphene is a single-layered, two-dimensional material formed by a network of carbon atoms . It has Polydopamine (PDA) has a similar chemical structure to melanin, which can act as a UV absorber to absorb UV rays and eliminate free radicals, thereby preventing the photodegradation of polymers . A recenIn this study, we 3D-printed a robust composite material with excellent UV stability using PU with PDA/graphene coatings . FirstlyPhotocurable water-based PU resin  was first heated and dehydrated to 60% of its original weight. Then, 30% 2-hydroxylethyl methacrylate  and 1.5% 2,4,6-trimethylbenzoyl-diphenyl-phosphineoxide  were mixed evenly, then added to the cooled PU and stirred. The above processes were carried out in a dark environment at room temperature. For the fabrication of scaffolds, 60 mL of the PU was loaded into the photocurable printing machine  and fabricated according to our pre-designed Solidworks model. The design of the scaffold used in this study was 5.0 mm× 5.0 mm× 6.5 mm with 1 mm pores and a thickness of 0.25 mm. The scaffolds were fabricated layer-by-layer (50 µm per layer) with a 15 s UV exposure per layer. For surface modifications, the scaffolds were placed in different concentrations  of 3-hydroxytyramine hydrochloride  with 25 mM tris buffer (pH 8.5) and stirred overnight. Then, the scaffolds were rinsed and dried in an oven. For those groups containing graphene, 0.5 mg/mL of graphene was added to the dopamine HCl solution.−1 and range of 500–4000 cm−1 was used to analyze the molecular structures of the PU. To improve the signal-to-noise ratio, 32 scans were performed for each spectrum to ensure the reliability of the data, which was performed thrice, with the averages recorded. Compression tests were used to evaluate the mechanical properties of our composite materials. Compression tests were performed using an electronic universal testing machine  that is able to provide constant, consistent compressive strength and rates. The samples were first fixated onto the holder of the testing machine, and a compressive rate of 1 mm/s was applied to the samples. The compressive force and deformation rates were recorded simultaneously and shown as a stress–strain curve emphasizing the Young’s modulus and fracture properties. Each group was tested in sextuplicate. Field emission scanning electron microscopy  was used to observe the composite materials’ surface morphology and elemental analysis. The samples were first fixated onto a self-designed platform, and carbon glue was applied homogenously to the surfaces of the samples. The samples were then left to dry to remove excess water or solvent, and a fine layer of metal coating was then deposited onto the surfaces to improve conductivity further. FE-SEM at an accelerating voltage of 3 kV was used to observe surface morphology.X-ray diffraction  analysis was used to analyze the crystalline structure and characteristics of the scaffolds. A 12 mm diameter by 2 mm height scaffold was fabricated for this study. Prior to XRD analysis, the scaffolds were first fixated onto the platform to ensure the accuracy and repeatability of the results. Copper Kα radiation (Cu Kα radiation) (λ = 1.5406 Å) was used as the source with a pre-determined operating voltage and current as per our previous protocols. The diffraction signals for this test were conducted in the range of 2θ angles (10–60°), and the corresponding diffraction intensities were then recorded and analyzed to determine the crystalline structure, orientation, and characteristics. Fourier transform infrared spectroscopy  at a resolution of 4 cm2. The samples were placed into the in-built chamber and exposed to UV light for different durations  at 30 °C. FTIR, mechanical properties, and microscopic imaging were conducted at each timepoint to evaluate its durability and stability.To investigate the UV durability of composites, we performed accelerated durability tests in an in-built chamber  equipped with a UV irradiation wavelength of 365 nm and 9 W/mp-value of <0.05 was considered significant.One-way analysis of variance (ANOVA) was used to analyze differences between groups. Significant deviations were assessed using Scheffe’s multiple comparison test. A Photo images of the 3D-printed PU scaffolds are shown in −1, 1714 cm−1, and 1534 cm−1 corresponded to the N–H, C=O, and N–H bands of PDA, respectively. The bands at 1142 cm−1 and 748 cm−1 corresponded to the C–O–C and –OH bands of PU, respectively [−1 and 1282 cm−1 ranges, indicating that PDA was successfully coated onto PU [The XRD results of PU and PDA-modified scaffolds are shown in ectively . Further onto PU . Taken t onto PU . InteresThe mechanical properties of the scaffolds were evaluated, and the stress–strain curves were as shown in −1 could be attributed to the –OH groups on graphene. Furthermore, 1539 cm−1 also corresponded to the C=C and C-O-C bonds of graphene, thus further confirming the presence of graphene on PU surfaces [Photo images of the scaffolds after PDA–graphene coating are shown in surfaces . The strsurfaces . It has surfaces . Furthersurfaces . In addisurfaces . In addisurfaces .The UV absorption capabilities of the scaffolds were evaluated at the wavelength range of 300–1000 nm, as shown in The mechanical properties of the various scaffolds were evaluated after 1, 2, and 3 weeks of UV exposure, and the results are shown in In this study, we successfully developed a method for enhancing the UV stability and mechanical properties of 3D-printed PU scaffolds by coating them with PDA and graphene. The PDA coating, achieved through surface modification, enabled effective bonding between PU and graphene, facilitating the deposition of graphene nanosheets onto the scaffold surfaces. The resulting composite exhibited significantly improved compressive strength and Young’s modulus compared to pure PU scaffolds. Furthermore, the PDA–graphene-modified scaffolds demonstrated enhanced UV durability, with their mechanical properties remaining stable even after prolonged UV exposure. This phenomenon was attributed to PDA’s UV-absorbing properties and graphene’s mechanical reinforcement capabilities. This study’s results underscore the potential synergistic effects between PDA and graphene in enhancing the performance of 3D-printed materials. The introduced coating method offers a cost-effective and environmentally friendly approach to improving polymer composites’ stability and mechanical strength. As 3D printing technology continues to evolve, these findings have implications for developing durable and high-performance materials for various applications, including automotive, aerospace, and biomedical engineering. Further research could delve into optimizing the PDA–graphene coating process and exploring its applicability in other polymer systems, paving the way for innovative advancements in material design and engineering.


We demonstrate the use of a novel, integrated THz system to obtain time-domain signals for spectroscopy in the 0.1–1.4 THz range. The system employs THz generation in a photomixing antenna excited by a broadband amplified spontaneous emission (ASE) light source and THz detection with a photoconductive antenna by coherent cross-correlation sampling. We benchmark the performance of our system against a state-of-the-art femtosecond-based THz time-domain spectroscopy system in terms of mapping and imaging of the sheet conductivity of large-area graphene grown by chemical vapor deposition (CVD) and transferred to a PET polymer substrate. We propose to integrate the algorithm for the extraction of the sheet conductivity with the data acquisition, thereby enabling true in-line monitoring capability of the system for integration in graphene production facilities. Since graphene was first made available by mechanical exfoliation of graphite , this twTerahertz time-domain spectroscopy (THz-TDS) has been demonstrated to be a versatile technique for the non-destructive retrieval of the electrical properties of graphene ,10,11,12THz-TDS was first introduced in 1989 through the utilization of photoconductive antennas (PCAs) and femtosecond (fs) lasers . In a tyInstead of generating THz radiation using pulsed lasers, it was proposed in 1999 to excite PCAs with a temporally incoherent light source and measure the cross-correlation between the emitted incoherent radiation and the incoherent pump light ,19. SimiThe main components of a typical CCS setup using PCAs is shown schematically in Both emitter and detector operate by the standard principles of THz-frequency photomixing . AssuminIn the detector, as in the emitter, the incoherent light generates free carriers inducing photoconductance quations  and 2)  σd(t),(relationJCC(t)∝∫−Upon insertion of a sample between the emitter and the detector, the field is modified. The interaction is described in the frequency domain by a transfer function The transfer function is set up by analyzing how plane waves of light propagate through the sample in a transmission experiment as displayed in n internal reflections, i.e.,i and j and d and refractive index The total transmitted field When a thin film of a conducting material such as graphene is present on the interface between medium 1 and 2, the relevant transmission and reflection coefficients are described by the Tinkham equations  given bys, a, and f refer to substrate, air, and film, respectively, using either deterministic or stochastic fitting algorithms such as basin hopping or differential evolution [To extract the conductivity experimentally, a sample measurement volution ,30.Two transmission mode experiments on the same sample of graphene at normal angle of incidence have been executed. The experimental setup is shown in Key metrics for the used devices as operated are summarized in x by calculating the energy contained in each trace proportional to Additionally, the spot sizes were determined through knife-edge scans in ambient air, shown in A is a normalization constant, x is the spatial position, and w is the beam width.The spot sizes are found to be 1.3 mm for the TDS device and 5.6 mm for the CCS device, where the spot size is defined as twice the width extracted from fitting the beam intensity profile with a Gaussian function of the formThe graphene sample is grown by chemical vapor deposition (CVD) on copper foil and then transferred to a transparent PET substrate with standard wet transfer techniques . During e sample . The 2D graphene ,34.For better extraction of the graphene conductivity parameters, the thickness and dielectric permittivity of the substrate have been characterized experimentally with the TDS device and determined to be 208 quencies . The reaquencies , and we The Debye model describes the relaxation processes and displays the same qualitative behavior of the dielectric function. Hence, it is frequently used to model the dielectric properties of polymers in this range ,38.A 9 cm × 9 cm sheet of graphene was raster-scanned in ambient air in a serpentine pattern with a stepsize of 3 mm, corresponding to 961 single-pixel measurements. For each pixel, the Drude conductivity was extracted by fitting with Equation  using thSince the TDS system has a small spot size compared to the pixel dimensions, >99.99% of the THz radiation is contained within each pixel, assuming a Gaussian beam profile. For the CCS device with a much larger spot size, only 35.6% of the radiation is contained, leading the extracted conductivities to be a weighted mean of the pixel in question and its neighbors. To provide a reasonable basis for comparison, the pixel values measured using the TDS device are therefore convolved with a Gaussian filter with a width equal to the beam width of the CCS device.The spatial maps of the measured conductivities using both devices are shown in Since the transmission decreases when the DC conductivity increases, the peak-to-peak value is correlated with the DC conductivity. In Equation  is reducThe CCS device is capable of measuring the DC conductivity of graphene with a standard deviation on the error distribution of We believe that correctly compensating for the drift can remove the bias. This is currently being explored.In the previous section, we demonstrated how the DC conductivity can be determined with higher precision from the peak-to-peak value of the trace. In Extracting We have demonstrated that our novel THz-CCS device is capable of measuring the electrical properties of graphene. The measurements have been compared to what is achievable with a state-of-the-art commercial THz-TDS device, resulting in a standard deviation on the relative errors of


Background and Objectives: Post-COVID-19 syndrome is commonly used to describe signs and symptoms that continue or develop after acute COVID-19 for more than 12 weeks. The study aimed to evaluate a treatment strategy in patients with adhesive capsulitis (phase 1) developed in post-COVID-19 syndrome. Materials and Methods: The method used was an interventional pilot study in which 16 vaccinated patients presenting with the clinical and ultrasound features of adhesive capsulitis (phase 1) developed during post-COVID-19 syndrome were treated with infiltrative hydrodistension therapy under ultrasound guidance associated with early rehabilitation treatment. Results: Sixteen patients with post-COVID-19 syndrome treated with ultrasound-guided infiltration and early rehabilitation treatment showed an important improvement in active joint ROM after 10 weeks, especially in shoulder elevation and abduction movements. The VAS mean score before the treatment was 6.9 ± 1.66. After 10 weeks of treatment, the VAS score was 1 ± 0.63. Conclusions: The study demonstrated that the management of adhesive capsulitis (phase 1) developed in post-COVID-19 syndrome, as conducted by physiotherapists in a primary care setting using hydrodistension and a rehabilitation protocol, represented an effective treatment strategy. In the In strong similarity to COVID-19, the inflammatory cascade implicated in abnormal tissue repair and fibrosis of the shoulder AC is supported by similar cytokines and growth factors, particularly IL-1, IL-6, and TNF-α . MoreoveAdhesive capsulitis is prevalent in approximately 2% of the general population. The causes that could lead to this pathological condition can be idiopathic or traumatic  . As regaClinical examination and instrumental imaging are both used in diagnosing adhesive capsulitis.Ultrasonographic signs of adhesive capsulitis consist of a thickening of the inferior recess of the glenohumeral joint capsule on a longitudinal sub-axillary scan, thickening of the coracohumeral ligament and soft tissue structures in the rotator cuff interval, subacromial-subdeltoid bursa with hypervascularization, and inflammation of the bicep tendon sheath ,15,16,17Hydrodilatation is an effective therapeutic intervention that leads to rapid joint improvement in patients with adhesive capsulitis. This technique consists of injecting a saline solution or a saline solution combined with corticosteroids and anesthetic, which relaxes the capsule because it increases hydrostatic pressure  and thus the volume capacity of the shoulder ,19,20,21Regarding rehabilitation, interventions performed by physiotherapists are commonly used and often recommended to treat adhesive capsulitis of the shoulder. Traditional physiotherapy consists of patient education, physical applications such as joint mobilization, and exercises ,27. As pIn this study, we reported 16 vaccinated patients who developed adhesive capsulitis (phase 1) during the post-COVID-19 period and were treated with infiltrative hydrodistension therapy associated with early rehabilitation treatment ,31. The Sixteen consecutive patients referred to the outpatient clinic for adhesive capsulitis (phase 1) in post-COVID-19 syndrome were clinically and ultrasonographically evaluated. Patients were screened based on age (between 18 and 65) and the presence of clinical and ultrasound signs of adhesive capsulitis developed within 4 months of COVID-19 infection. The exclusion criteria consisted of patients who developed adhesive capsulitis of the shoulder where they received the COVID-19 vaccination. Among the patients enrolled in the study, 10 were women and 6 were men. The mean age of the patients was 52 ± 9.53. Patients had no other concomitant pathologies such as diabetes or hyperthyroidism, and all had received double vaccination against SARS-CoV-2. Six patients had developed adhesive capsulitis on the left shoulder and ten received this on the right shoulder. Nine patients were manual laborers and seven were office workers. Adhesive capsulitis developed between 1.5 and 3 months after the COVID-19 diagnosis (mean time to onset was 2 months). In 7 of the patients, COVID-19 was asymptomatic, whereas in the remaining 9 patients, symptoms were mild. None of the patients were severely or critical ill. Patient demographics are shown in This interventional study was approved by the local ethics committee . The study was conducted in accordance with the ethical standards of the Declaration of Helsinki, and informed consent was obtained from all patients, including approval for photographic and/or video documentation.t-test; p < 0.05 was considered significant. The statistical analysis was performed using the statistical package for social sciences (SPSS), software version 15.0  by a statistical consultant from Rizzoli Orthopaedic Institute.All continuous variables are expressed in terms of mean ± standard deviation (SD) and range. Categorical variables are summarized in terms of absolute frequency and percentage. Correlations between the means of outcome measures pre-treatment and post-treatment were analyzed with a Active shoulder ROM, visual analogue scale for pain (VAS), the Shoulder Pain and Disability Index (SPADI) and the Disabilities of the Arm, Shoulder and Hand (DASH) questionnaire were used for clinical assessment. Data were recorded prior to the hydrodistension procedure and at 5 weeks and at 10 weeks post-treatment. The visual analogue scale (VAS) is a one-dimensional scale that evaluates the intensity of pain. The scale ranges from 0 to 10, with 0 being no pain and 10 being the greatest possible pain . The minUltrasound examinations of the shoulder were performed using Samsung HM70A with Plus Ultrasound System, equipped with a 5–14 MHz linear transducer with a musculoskeletal preset. All the patients were evaluated by experts of US-Diagnosis.(a)Coracohumeral ligament (CHL): for CHL assessment, patients were scanned in a sitting position, with the shoulder in a neutral position and the forearm extended. The scanning commenced in an axial oblique plane via positioning the transducer on the lateral border of the coracoid process, obtaining a longitudinal image of the CHL.(b)Rotator interval: the rotator interval is a triangular space in the anterosuperior rotator cuff bounded above by the anterior free edge of the supraspinatus tendon and below by the superior edge of the subscapularis tendon. The rotator interval was optimally visualized in the oblique plane with the patient’s fist held by their side.The following parameters were assessed:The criteria for the ultrasound diagnosis of adhesive capsulitis were a thickening of the inferior recess of the glenohumeral joint capsule, the performance of a longitudinal subaxillary scan , a thickPatients were treated with infiltrative hydrodistension therapy under ultrasound guidance. This technique consists of injecting a saline solution or a saline solution combined with corticosteroids and anesthetic which relaxes the capsule because it increases hydrostatic pressure , thus increasing the volume capacity of the shoulder ,19. An e-Commuting exercises: The patient bends the torso forward so that it is parallel to the floor and leans on a stool or table with the sound arm. They swing the affected limb back and forth for about 5 min. With the treated limb, the patient makes circles outwards with the palm facing out, and then circles inwards with the palm a.-External rotation exercises: The patient is supine, with the arm close to the body, and the elbow flexed at 90°. The patient holds a stick with the healthy limb and places it on the palm of the affected limb, pushing on it so as to hyper-rotate the affected limb. The patient maintains this position for about 15–20 s and then returns to the starting position b.-Anterior elevation exercises: Standing upright, the patient holds a cane with both hands, raises their arms above their head as far as they can, holding the position for about 10 s and then returns to the starting position c.-Internal rotation exercises: The patient places the treated limb behind their back with the elbow bent. Using a towel and using the healthy limb, they bring the treated limb to the maximum internal rotation, maintaining this position for about 5 s d.-Retroposition exercises: Standing upright, the patient grasps a stick behind their back with both hands and brings their shoulders to the maximum retroposition, maintaining this position for 5 s e.-Abduction exercises: Standing next to a wall with the elbow flexed at 90°, the patient pushes their elbow and forearm against the wall f.We adopted a rehabilitation protocol with exercises aimed at recovering range of motion, avoiding manual loads for at least the first three months. Rehabilitation started immediately after the ultrasound-guided hydrodistension infiltrative treatment to improve the joint ROM of the affected glenohumeral joint right away ,29. RehaPatients were reviewed by two physiotherapist at 5 weeks (after 10 sessions) and at 10 weeks (after 20 sessions), and the data of the outcome measures were recorded.Sixteen patients with post-COVID-19 syndrome treated with ultrasound-guided infiltration associated with early rehabilitation treatment showed major improvement in ROM after 10 weeks, especially in shoulder elevation and abduction movements. In 16 patients with phase-1 adhesive capsulitis, before the treatment, a fairly reduced shoulder range of motion was seen with mean elevation values allowed to reach 55°, abduction allowed to reach 40°, internal rotation allowed to reach 30° with the arm abducted to 90°, and external rotation allowed to reach 40° with the arm abducted to 90°. After the infiltration treatment and the first 10 sessions of physiotherapy, there was an increase in the range of motion of the shoulder with elevation allowed to reach 100°, abduction allowed to reach 90°, internal rotation allowed to reach 50° with the arm abducted to 90°, and external rotation allowed to reach 70° with the arm abducted to 90°.Given the clinical improvement, it was decided, in agreement with the patients, not to carry out further infiltration but to continue with another 10 physiotherapy sessions, obtaining an almost complete recovery of range of motion at the end of the cycle . The data are reported in t-test showed significant improvements between pre-treatment and after 10 sessions, between pre-treatment and after 20 sessions, and between after 10 sessions and after 20 sessions for SPADI (p < 0.00001), between pre-treatment and after 10 sessions, between pre-treatment and after 20 sessions (p < 0.00001), between after 10 sessions and after 20 sessions (p < 0.00002) for DASH, and between pre-treatment and after 10 sessions, between pre-treatment and after 20 sessions (p < 0.00001), and between after 10 sessions and after 20 sessions (p < 0.02) for VAS. In addition, significant improvements were seen between all time points for range of motion in abduction and flexion (p < 0.00001), between pre-treatment and after 10 sessions, between pre-treatment and after 20 sessions (p < 0.00001) and between after 10 sessions and after 20 sessions (p < 0.005) in external rotation and between pre-treatment and after 10 sessions, (p < 0.00002), pre-treatment and after 20 sessions, and between after 10 sessions and after 20 sessions (p < 0.00001) in internal rotation. The data are reported in Pairwise comparisons with the p < 0.00001) and clinically significant changes at all time points from the baseline and between all time points, indicating a continued functional recovery of movement. The mean pain score went down significantly, from 6.9 (pre) to 1.5 (after 10 sessions) (p < 0.00001), showing a 78% reduction in pain after 10 sessions. This trend continued after 20 sessions to 1 (p < 0.00001), for an 86% reduction. These findings were confirmed in this study, in which we obtained a satisfying outcome from the treatment.Adhesive capsulitis in subjects who have contracted COVID-19 infection may be due to an exaggerated inflammatory response linked to an infection that also involves the musculoskeletal tissues. There was only one study in the literature that tried to describe the correlation between COVID-19 infection and the subsequent development of capsulitis ,6. In thThe main limitations of the study were the limited size of the study group, although it should be borne in mind that we enrolled highly selected patients who had recently suffered from COVID-19 and who had adhesive capsulitis of the shoulder with no other apparent causes, with the absence of a control group consisting of patients with adhesive capsulitis developed in post-COVID-19 syndrome not subjected to treatment.The results of this study demonstrated that ultrasound-guided hydrodistension with guided exercise provided by physiotherapists in primary care was clinically effective for patients with adhesive capsulitis (phase 1) developed in post-COVID-19 syndrome in terms of joint movement recovery and pain reduction. Although these findings did not provide new evidence on treatment efficacy, they were consistent with the previous findings of hydrodistension treatment associated with rehabilitation treatment. Future studies will be needed to compare these results given the small number of patients collected due to the recently discovered association between adhesive capsulitis and post-COVID-19 syndrome and the absence of a control group consisting of patients with adhesive capsulitis developed in post-COVID-19 syndrome not subjected to treatment.