Oxamyl is a highly toxic carbamate molecule with toxicological risk from contamination, used as an insecticide, nematicide, and acaricide on many field crops, vegetables, fruits, and ornamentals. Suspected poisoned animals and baits were collected between January 2018 and August 2021 from Lombardy and Emilia-Romagna regions and analyzed at the chemical toxicology laboratory of the Experimental Zooprophylactic Institute of Lombardy and Emilia-Romagna, located in Brescia. The analyses were carried out by an ion trap GC-MS system in 2467 suspected samples and showed the presence of oxamyl in 67 of these. In this study, we analyzed 47 (out of 67) positive baits: the provinces in which more cases have been recorded are Mantua, Ferrara, and Cremona, which overall had 72% of positivity. The nature of the analyzed samples was mostly corn (55.3%), followed by bird carcasses (19.1%), apples (14.8%), meatballs (2.1%), bread (2.1%), and other (8.5%). The use of oxamyl to produce poisoned baits is constantly increasing, proving that it must be considered as a public health risk for the possible consequences on target and non-target organisms, including humans. N-monomethyl carbamates, such as carbaryl, oxamyl, fenobucarb, and ethienocarb, are used as insecticides. N-dimethyl or ethyl methyl carbamates such as the natural alkaloid physostigmine, neostigmine, or rivastigmine that derive from physostigmine can also be used in medicine , the possession, use and abandonment of any food prepared in such a way as to cause intoxication or injury to the person who ingests it’ . In addition, ‘rodent-control and pest-control operations, carried out by specialized companies, shall be carried out in such a way as not to harm in any way people and other non-target animal species and shall be publicized by the companies themselves by means of notices displayed in the areas concerned […]’  .Previous studies ,9 have sThe high toxicity of carbamates results from their mechanism of action, which is based on the inhibition of AChE, resulting in prolonged overstimulation of the nervous system and skeletal muscles . TypicalWithin the carbamate family is oxamyl (methyl 2-(dimethylamino)-N-(methylcarbamoyloxy)-2-oxoethanimidothioate) , which oIt is used as an insecticide, nematicide, and acaricide on many crops, vegetables, fruits, and ornamentals such as apples, bananas, carrots, celery, citrus, cotton, cucumbers, eggplants, garlic, ginger, melon, onions, peanuts, pears, peppers, peppermint, pineapples, bananas, potatoes, pumpkins, soybeans, spearmint, squash, sweet potatoes, tobacco, tomatoes, and watermelons . It can Bacillus thuringesis, it improves the growth response of eggplant and reduces the development of the nematode Meloidogyne incognita [In addition, when added with horse manure, sesame cake, or ncognita .It has a high solubility in water (280 g/L at 25 °C), a very low soil absorption coefficient (Koc = 25), where it is degraded by hydrolysis into its non-toxic metabolite, methyl N-hydroxy-N′, N′-dimethyl-thioxamimidate . The forResults of previous studies show that its presence in soils induces changes in the abundance of microorganisms that degrade it and in the diversity of the soil bacterial community . FurtherThe formulations currently on the market in Italy can be consulted in the database of plant protection products, which the Ministry of Health makes public and keeps up to date: generally marketed under the name Vydate, oxamyl is available in granular form or as a soluble concentrate .Carbamate pesticides are used intensively as pesticides in agriculture due to their broad spectrum of action  and are,The onset of symptoms following exposure to carbamates depends on the dose, route of exposure, and type of carbamate involved ,23.Acute oxamyl exposure leads to a cholinergic seizure, with more rapid effects following ingestion and inhalation than dermal exposure. Signs and symptoms may include increased salivation, lacrimation, bradycardia, sweating, spontaneous defecation and urination, miosis, blurred vision, tremors, muscle twitching, mental confusion, convulsions, and coma. Gastrointestinal symptoms include abdominal pain, diarrhea, nausea, and vomiting. Dyspnoea and pulmonary edema may also occur .It is a highly toxic compound, so much so that the EFSA’s recommended risk classification places it within Class IB: highly hazardous . It is, The main reasons for deliberate poisoning are intolerance towards wild and domestic animals , the ecoWhen a bait is found, the competent health authority draws up a report of suspected poisoning, giving all the anamnestic information needed to direct the search and identification of the toxic substance contained. Then, the bait and the relevant report form are sent to the Experimental Zooprophylactic Institutes competent for the territory, as reference laboratories for carrying out the inspection of the baits and the toxicological analyses necessary to confirm the diagnostic suspicions .The aim of this work is to examine the presence of oxamyl in poisoned baits, organic liquids, and animal carcasses sent by the various territorial offices of the Experimental Zooprophylactic Institute of Lombardy and Emilia-Romagna to the toxicology chemistry department in Brescia, in the period between January 2018 and August 2021.2 and 14.4 million inhabitants [Lombardy  and Emilia-Romagna , which are two regions located in northern Italy, with a total area of 46,308 kmLombardy borders Switzerland to the north, and Trentino-Alto Adige and Veneto to the east;Piedmont to the west and Emilia-Romagna to the south. Emilia-Romagna is bordered to the north by Lombardy and Veneto, to the west by Lombardy and Piedmont, to the south by Liguria, Tuscany, Marche, and the Republic of San Marino, and to the east by the Adriatic Sea.The study area can be divided into:Overall, three zones can be distinguished: mountains, hills, and plains, as well as large natural wetlands  located in the eastern part of Emilia-Romagna .Almost half of the territory (48%) is characterized by the presence of a large plain, the Pianura Padana, a flat region of fluvial origin, with fertile conditions for cultivation and intensive livestock activities .Lombardy, with more than 50,000 farms and 8000 food companies, is at the top of the Italian agricultural sector, and whose activities cover 69% of the national territory with traditional products and quality agri-food productions .In addition, in Emilia-Romagna, agricultural land covers about 68% of the regional surface and includes areas with high agricultural yields in the lowland part, followed by forest and semi-natural areas (25%), artificial surfaces (5%), water bodies (1%), and wetlands (<1%) .Wild animals are also relevant in the pre-Alpine area and in the wooded and shrubland area of the hills and high plains .In this context, the presence of pesticides in the environment could be related to production activities.The determination of carbamate oxamyl was performed through a semiquantitative GC-MS method, as described in the work of Chiari et al.  and BertA typical 30 g of sample was weighed, added to 30 g of dry sodium sulfate, and extracted with acetone RPE for analysis (100 mL) by shaking for 1 h in a blender. After filtration on paper, 2 mL of filtrate was collected and the solvent was stripped out under a gentle nitrogen flow at 40 °C. The residue was reconstituted with 2 mL of acetone Chromasolv for pesticide’s residue analysis and injected (1 microliter) into an ion trap GC-MS system . The chromatographic separation was carried out by a capillary column DB 5MS  using helium as gas carrier. The detection was performed by the ion-trap mass analyzer: full mass spectrum of the target compound was acquired, providing the best identification.The limit of instrumental detectability was 25 pg (S/N = 3). The limit of quantification of the method was 0.1 mg/kg (S/N = 5).Acetone RPE (purity ≥ 99.8%) was purchased from Carlo Erba Reagents . Acetone Chromasolv for Pesticides Residue Analysis was purchased from Sigma-Aldrich . Oxamyl standard (purity 99.5%) was purchased from LGC Dr. Ehrenstorfer .During the period under review, the toxicology laboratory received 2488 samples for oxamyl. Of these, 2467 samples were analyzed and oxamyl was detected over the LOQ in 67 (2.7%). The semiquantitative results range from 0.3 mg/kg to 70,888 mg/kg, with an average value of 2531 mg/kg.Two-thirds of the positive samples (47) were baits, from seven of the twenty provinces in the whole study area. Twenty-one samples (0.8%) could not be tested because they were unsuitable or insufficient.The 47 positives found in the baits are distributed over time as follows: seven in 2018, eleven in 2019 and 2020, and eighteen in January–August 2021 .In terms of the nature of the samples analyzed, the absolute number and percentage of each is given below: maize , poultry carcasses , apples , bread , meatballs , other  .The study area covered the whole of Lombardy and Emilia-Romagna, but only in some of these provinces were positives reported in the baits tested .In particular, the provinces of Mantua, Ferrara, and Cremona together recorded 72% of positivities, with the number of annual reports increasing steadily since 2018 .Mantua: thirteen samples of maize and one of gastrointestinal content;Ferrara: six samples of apples, two samples of maize, three of gastrointestinal contents, and one chicken carcass;Cremona: six samples of maize, one sample of bread, and one meatball;Brescia: six chicken carcasses and one maize sample;Milan: four samples of maize;Parma: two poultry carcasses;Forlì: one apple sample.Finally, examining the nature of the matrices found to be positive in each of the provinces, the following results were obtained:Baits containing maize were the most frequently reported in Lombardy, especially in the provinces of Mantua, Cremona, and Milan .The effects of oxamyl exposure have been studied in terrestrial and aquatic organisms, following both acute and chronic exposures.The molecule is highly toxic when administered as a single oral dose: Kennedy  calculatWhen administered by intraperitoneal injection, it was found to be highly toxic to rats, mice, and guinea pigs. It is also mildly irritating to the eyes. With regard to contact reactions, oxamyl produced mild skin irritation and the toxicity of skin absorption in rats (LD50 is >1200 mg/kg) and rabbits (740 mg/kg) was relatively high, suggesting limited absorption. Oxamyl is highly toxic by inhalation with the 1-h LC50 value in rats being 0.17 mg/L  and 0.12 mg/L .Again, Anguiano et al.  demonstrSturnus vulgaris) [Similar results were obtained in starlings (ulgaris)  and honeulgaris) .With regard to chronic toxicity, studies in rats, mice, and rabbits suggest that oxamyl has no carcinogenic, teratogenic, or embryotoxic effects. However, in one- and three-generation breeding studies, litter size was seen to be slightly lower in rats fed oxamyl at 100 or 150 ppm than in those fed 50 ppm. No evidence of a teratogenic response was observed in the rat, although a reduction in the rate of weight gain was observed in pregnant rats fed oxamyl at 100 ppm or more .In the case of suspected poisoning, when the local IZS receives a sample, it carries out an inspection of the material received in order to target the search for certain categories of molecules. The suspicion of the presence of oxamyl arises from the fact that, although it is a colorless substance, in commercial products, dyes are added to give the bait a characteristic color; for example, the commercial product Vydate has tartrazine added, which gives it a green color, the same color as the artificially contaminated bait. Therefore, the color becomes a danger indicator.Myocastor coypus), a small, alien mammal that causes a great deal of damage to both crops and the ecosystem [The decision to use apples as bait is probably due to one of the animal species for which the bait is intended, namely the nutria  was coined recently, the concept related to it has long been recognized both nationally and globally. The only way forward is the realization that we live within a system of which people, animals, plants, and generally, the environment in which we are all immersed are a part. So, there are not only individuals and communities, but there is also not only the human species to be preserved: the health of the planet and all its inhabitants must be given equal dignity if we are to create a sustainable, resilient, and enduring ecosystem. We are all elements of one system, in which the health of each human, animal, or environmental element is closely interdependent on that of the others.The data compiled in this study provide a picture of oxamyl poisoning in Lombardy and Emilia-Romagna. To date, numerous toxicity studies on various animal species are available in the literature, but data on the presence of oxamyl throughout the country are scarce. Our analysis confirms that, in spite of numerous restrictions and bans on the use of toxic pesticides, they are still widely used in the preparation of poisoned baits and that insecticides are one of the most commonly used categories in this respect.In addition, there has been a steady increase in the number of cases reported during the time period covered by this work. This is all the more worrying given that only the first eight months of 2021 were taken into account.This demonstrates the need to strengthen monitoring and control activities by health authorities, which would otherwise lead to an underestimation of the actual cases, and to inform about the risks to human and animal health.In this regard, and as our study shows, the type of bait, together with the particular coloring, must give priority to the search for oxamyl.


Memorials are increasingly used to encourage people to reflect on the past and work through both individual and collective wounds. While much has been written on the history, architectural forms and controversies surrounding memorials, surprisingly little has been done to explore how visitors experience and appropriate them. This paper aims to analyze how different material aspects of memorial design help to create engaging experiences for visitors. It outlines a matrix of ten interconnected dimensions for comparison: (1) use of the vertical and horizontal axis, (2) figurative and abstract representation, (3) spatial immersion and separation, (4) mobility, (5) multisensory qualities, (6) reflective surfaces, (7) names, (8) place of burial, (9) accommodating ritual, and (10) location and surroundings. With this outline, the paper hopes to provide social scientists and practitioners  with a set of key points for reflection on existing and future memorials and possibilities for enhancing visitor engagement with them. As such, they work to affirm and define a community, through creating and strengthening its memory for events in certain ways  has a lot of movement. And now I’m seeing all the names written here [touches names]. I’m seeing ‘m56’. I don’t know what that means. I’m seeing a rainbow down there. That’s very pretty. [pause] And how the water all goes into that pit. Hmmm. It is kind of ominous in a way. Just like you can’t see the bottom [gestures downward again with hands] and it is kind of like the water just slowly goes over the edge. I think it is cool. I like it, but it has something [pause], to me it reflects some of the darkness that happened in addition to being a nice place. It reminds me of like the gravity of it all. It also reminds me of in my hometown there are some man-made lakes and they have, I don’t know what you call it, it is a drain basically, and it is called the waterhole. When I was young and the lakes were full the water would flow into it. It was always terrifying [laughs]. It has like little ropes that make like don’t swim or take your boat past this. It was like terrifying. What if someone somehow slipped down there, it would be awful. You’d see somebody in a little tin boat just outside the buoys and think oh my god.”.The participant transitions from a description of the sensory features of the memorial to an interpretation of what they stand for . Ecological psychology would describe this as a movement from direct to indirect perception of the environment, from sensory to symbolic qualities . This trThe inscription of names on memorials radically changes their character, as it provides a connecting link to concrete individuals that have died. In traditional memorials, names were reserved for only significant individuals that can stand in for a nation or cause. For example, at VF, only the names of the Spanish dictator Francisco Franco and the founder of Spanish fascism José Antonio Primo de Ribera are present together with their bodies see . Franco. The morA Strong Clear Vision.). The memorial presents the names chronologically according to the day of their death, which created some controversy, especially in that it presents the progression of the war in terms of the number and scale of individual dead.In contrast to VF, counter-memorials aim to avoid creating hierarchies around names, preferring to dwell on individual losses without attaching them to any higher cause. Thus, they aim to create differentiation from a mass subject. The practice of listing the names of the individual dead began as a local memorial practice in the US after the Civil War, in Australia after the Boer War in 1903 and in England after WWI . It has The 9/11 memorial, by contrast, organizes names of the dead around the location of death , group affiliation  and ‘meaningful adjacency,’ where individuals sharing a personal, work or family relationship were placed close together . Located“I find myself looking at the names to see if perhaps there was someone I knew. It’s interesting [pause]. I don’t even know why that would be an actual desire [pause]. Maybe it is a desire to feel more connected to them. I’ve seen names that look familiar but no one that I knew. And also when my friends visit they look to see if ‘oh that person has my first name,’ ‘oh that person has my last name’. I think people are just trying to find some connection. It’s somewhat unconscious.”Finally, at the newly opened Holocaust Names Memorial, the 130,000 individuals from the Netherlands that died in the concentration camps are listed alphabetically, together with their birthday and age at which they died. This allows visitors to search for particular names more easily and see family connections—for example, Anne Frank and her family. Similar to what we found at 9/11, one visitor we spoke to searched for his own name at the site, and on finding it, he did historical research on who the person was and their life trajectory, discovering some parallels to his own family migration history. This is again a strategy of building a personal connection to the unique individual represented by a name. It contrasts sharply with MMJE, where visitors struggled to find a point of personal connection with the abstract site, with few indicators as to what it stands for.Rituals can be seen as routine behavior with a high symbolic load . This sy“Key components of memorial spaces relate specifically to the ritual use of space in memorial practices. There is a need for those who have incurred a loss to search for meaning, to grieve, to contemplate and reflect on the person or event, and, at times, to use the memorial as a place to catalyze change. Many successful memorials contain these ingredients through the symbolic and functional use of siting, materials, and spatial organization”.However, memorials accommodate rituals and commemorative events (such as on the anniversaries of the tragic events) in different ways, and with greater and lesser ease . MemoriaIn fact, the ongoing discussions about the appropriateness and meaning of MMJE are part of what continues to animate it as a site of memory—as different perspectives are continually woven into the meaning people give to the place . In our Anyone who has been in the presence of a corpse knows they have a special power over the human psyche. Anthropologists have carefully outlined the myriad of different rituals for handling bodies of the dead and their link to cosmological beliefs . For exaWhile the material tangibility of corpses can facilitate the grieving process by helping relatives to acknowledge death, preventing them to be “left in a state of persistent and tormenting ambiguity, torn between hope and resignation” , sometiHowever, it is also the case that necropower , exercisWhen a memorial is constructed at the site of the traumatic events, it is already affectively charged with the site’s history. The 9/11 memorial is designed to mark and contain the absence of the twin tours and the people who died there. The museum similarly focuses on mangled material objects from the site of destruction, reminding the visitor of the force of violence. There is also a tree highlighted on-site that survived the destruction and thus provides symbolism of resilience and hope. At the Hiroshima Peace Park (where an atomic bomb was dropped) they adopt a similar strategy, including the retention of the ruin of the Genbaku Dome. When new memorials are planned, there is often a discussion around their placement at the site of trauma or in another location. Chile’s Museum of Memory and Human Rights  was criticized for its placement outside the city center and with no connection to the repression (while many sites that were could have been used). A similar critique was made of MMJE, against the counter-argument that it should be placed in the middle of the capital where people cannot help but be confronted with it.The surroundings of a site are equally important. VF was built near the town of El Escorial, where the Spanish royalty is buried, conferring the city’s symbolic value onto the site through its proximity. Moreover, by placing it on top of the Sierra, it creates a visual spectacle seen from miles around; in a literal and symbolic sense, Franco’s legacy is above the kings and queens. Similarly, VVM, 9/11 and MMJE are all central nodes in an urban network of memorial sites: VVM directly to the Washington Monument and Lincoln Memorial; 9/11 to smaller memorials located throughout NYC and New Jersey ; and MMJE to memorials commemorating other groups targeted by the Nazis, such as the Memorial to the Homosexuals Persecuted by the National Socialist Regime, and Sinti and Roma Memorial. One of the critiques of MMJE was in fact that it separates victims into groups as the Nazis had done. The National Memorial for Peace and Justice  goes a sWe can also consider the surroundings more directly as the physical space around the memorial site. Our participants at 9/11 frequently commented on the transition from the busy streets of NYC’s financial district into the peaceful park-like space of the memorial site. Being at the center of a major international city can also create tensions: both 9/11 and MMJE have become must-see tourist attractions, which encourage selfies for social media over reflection on the site’s meaning . FinallySince ancient times, memorials have played a crucial role in practices of mourning and memory. While they contribute to the commemoration of a collective loss, they also enable individuals to make meaning of events and to find ways of personally connecting with the collective past. In conducting our research, we were fascinated by the power of architecture to set up fields of mnemonic experience for our participants. This is not to say that the way people experience memorial sites is determined by their material design or their intended meaning. As we have shown, people interpret and engage with memorials in manifold ways through different personal associations and memories that are aroused while exploring the site. Drawing on Gibson’s  notion oWhile the dimensions included in the matrix contain aspects that can be found in both traditional and counter-memorials, the results empirically support Stevens’ and Franck’s observation that counter-memorials “have given visitors more and more choices for how to engage with them, framing them as actors, not just viewers” . For exHowever, as noted above, we should be cautious in drawing general conclusions about the effects of memorials’ design. As Watkins et al. remind us, “an effective design is not style-specific [but] context-specific” . Experi


Pneumocystis jirovecii (Pj) pneumonia (PCP). However, differentiating PCP and colonization remains difficult. This study aimed to establish the performances of the commercialized qPCR MycoGENIE® Pj kit (Ademtech) to distinguish PCP and Pj colonization. Patients with a positive Pj qPCR on bronchoalveolar lavage (BAL) or upper respiratory tract (URT) samples were prospectively included between May 2019 and December 2020 at Bordeaux University Hospital. They were classified in “PCP” or “Pj colonization” groups based on the revised EORTC/MSGERC criteria. The two groups’ results were compared; ROC curves were produced to determine the best thresholds. Excluding the low number of HIV-positive subjects, there were 100 PCP  and 70 Pj colonization . Pj loads were significantly higher in PCP compared to Pj colonization group (p ≤ 0.01). The best cut-offs for PCP diagnosis were 31.45 Cq/8275 copies/mL for BAL and 32.33 Cq/8130 copies/mL for URT . Fungal load quantification using MycoGENIE® Pj qPCR helps discriminating PCP from colonization, high fungal loads being indicative of probable PCP. Low load results should be interpreted with caution, in accordance with clinical and radiological signs.Quantitative PCR (qPCR) is highly sensitive to diagnose  Pneumocystis jirovecii (Pj) is an opportunistic fungus responsible for severe pneumonia in patients with various immunosuppressive conditions affecting lymphocytes-mediated immunity [Pneumocystis pneumonia (PCP) has long been advanced HIV infection with a CD4 T lymphocyte count lower than 200 cells/mm3, but its incidence has decreased since the introduction of chemoprophylaxis and highly active antiretroviral therapy. PCP now mainly happens in non-HIV immunocompromised patients, such as patients with solid or hematopoietic malignancies and patients with long-lasting immunosuppressive treatments for a solid organ transplant or a hyper-inflammatory disease [Pj in vitro and the lack of sensitivity of microscopic examination of respiratory samples in this context [Pj quantitative PCR (qPCR) has grown to be an indispensable tool for PCP diagnosis given its high sensitivity [Pj DNA (hundreds of genome copies/mL) in respiratory samples, revealing PCP disease as well as Pj colonization [immunity ,2,3. The disease ,2,4. The disease . However context . Pj quansitivity . Hence, sitivity . It allonization .Pj qPCR, in numerous populations considered as immunocompetent throughout the world (ranging from 15% to 70%) and with a particularly high prevalence among children [Pj colonization as a major reservoir of infections , and as a comorbidity factor of certain diseases, especially chronic obstructive pulmonary disease (COPD) [Indeed, asymptomatic colonization is being increasingly identified using children ,11,12. Te (COPD) ,14.Pj pulmonary colonization is also encountered in immunosuppressed subjects, sometimes complicating PCP diagnosis in case of positive qPCR. Therefore, fungal load quantification or semi-quantification has been proposed to help physicians in discriminating active PCP from Pj colonization with mitigate results [However,  results . However® Pneumocystis jirovecii Real-Time PCR kit  which includes a quantification range, in its ability to distinguish PCP from colonization. The Pj qPCR quantification results have thus been compared between the two groups (PCP or colonization patient groups) established on the basis of the 2021 revised EORTC/MSGERC criteria for PCP [Pj colonization using this qPCR kit, and to evaluate the test performances with these cut-off values.In this context, this study aimed to evaluate the commercialized MycoGENIE for PCP . Mainly,® Pneumocystis jirovecii qPCR, performed on a respiratory sample between May 2019 and December 2020 at Bordeaux University Hospital. Exclusion criteria were patients under 18 years old and a preventive or curative treatment for PCP more than one day before the date of the concerned respiratory sampling. Due to the well-known significant difference between the fungal load of HIV-positive and HIV-negative patients, we decided to split them for two distinct analyses [All prospectively included patients had a positive MycoGENIEanalyses . A distranalyses ,15. URT analyses . All included patients were informed about the anonymous use of their medical chart data for clinical research purposes. The samples were collected from research subjects, during the course of routine care management at the discretion of the clinician, and patient information was anonymized prior to analysis. Therefore, this protocol did not require approval from an ethics committee and was exempt from formal informed consent. It complies with the ethical and legal requirements of French law (15 April 2019) and the Declaration of Helsinki.Pj colonization group according to the revised EORTC/MSGERC criteria [The patients were classified in PCP or criteria , as follPj colonization” group. Importantly and in concordance with EORTC/MSGERC criteria, the fungal load was never taken into account for this classification.In addition to these two subgroups defined by EORTC/MSGERC criteria, a “putative PCP” group was added in this study, in order to take into account the immunosuppressed patients without corticosteroids  who did2 < 80 mmHg)), presence of typical radiological features on chest radiograph or computed tomography scan , use of anti-Pneumocystis treatment after sampling, immunosuppressive treatment , and clinical evolution and outcome (death or not) in the 6 weeks after diagnosis. Biological data including total lymphocyte and CD4 T cells blood counts were collected within a maximum of 7 days before and after the respiratory sample for Pj PCR was performed.We collected retrospectively the clinical histories of all the patients. The collected data included the following: age, sex, underlying diseases , HIV status), pertinent clinical symptoms at diagnosis  in a 1:1 (v:v) ratio. After a centrifugation step , each sample was submitted to microscopic examination to detect fungal cysts and/or trophozoites using Musto and May-Grünwald-Giemsa staining [Unlike BAL, URT samples were first homogenized for 30 min at 37 °C with Dithiothreitol  before performing DNA extraction. DNA extraction and amplification were carried out using the automated ELITe InGenius® system . DNA extraction was performed using ELITe InGenius® SP 200 kit  and Pj DNA amplification using MycoGENIE® Pneumocystis jirovecii , according to the manufacturer instructions. MycoGENIE® Pneumocystis jirovecii assay is a real-time PCR kit targeting the P. jirovecii mitochondrial large sub-unit rRNA gene (mtLSU) that includes a quantification standard range to estimate the fungal load of each sample in genome copy number/mL and an internal inhibition control. Of note, this kit is CE-IVD marked. Results of fungal loads were expressed as quantification cycles  and genome copies/mL.A volume of 250 µL pre-treated pellet was added to 250 µL of Cobas2 tests were used when appropriate. Alternatively, the Fisher’s test was used for a low number of patients. Receiver operator characteristic (ROC) curves were used to define the best cut-off values for BAL and URT samples. All the statistical analyses were performed using the software GraphPad Prism . p < 0.05 was considered statistically significant.Descriptive analyses have been performed to summarize the studied patient population. Continuous variables were presented as medians with interquartile range . Qualitative variables were expressed as absolute numbers and percentages (%). The Mann–Whitney, Kruskal–Wallis (and subsequent Dunn’s test in case of significant difference), and the χ® Pneumocystis jirovecii qPCR on a respiratory sample. Among them, 66 were excluded because they were under 18 years old (n = 23) or had received an anti-Pj treatment before sampling (n = 43). On the 187 remaining patients, there were 170 HIV-negative patients and 17 HIV-positive patients. Given the limited number of HIV-positive patients, the added value of this Pj quantification for PCP diagnosis was evaluated on the HIV-negative patient population. Among them, 66 had positive BAL samples and 104 had positive URT samples . Concerning the BAL, thirty-two (48%) were from patients classified in the PCP group  while among URT samples, sixty-eight (65%) were from patients classified in the PCP group   .Pj colonization group for URT. Regarding clinical symptoms, hypoxemia was significantly more frequent in the PCP group for both BAL and URT , as was fever  and dyspnea for URT (p < 0.0001). As expected, the group of patients with PCP received significantly more curative anti-Pj treatment after the positive qPCR result compared to the colonized patients (p < 0.0001 for both sample types), reflecting the physicians’ decision. Of note, about 25% of patients classified in the Pj colonization group received an anti-Pj curative treatment. The characteristics of the included patients according to their subgroup of PCP  are detailed in Epidemiological, clinical, radiological, and biological data are summarized in Pj colonized group , p < 0.01  compared to the p < 0.01 . As expele types .Pj colonization, ROC curves were produced to display the fungal load ability of binary (PCP versus Pj colonization) classifiers for each sample type (BAL and URT). The area under the ROC curves for Cq were 0.678 for BAL and 0.792 for URT  and 32.33 for URT . The putative PCP group having been added to the original EORTC/MSGERC classification by us  on both sample types, yet they were associated with highly poor sensitivities (27.3% for BAL and 8.8% for URT). On the opposite, a perfect sensitivity was associated with thresholds as low as only hundreds of genome copies/mL that were not helpful for PCP diagnosis (2% specificity)  . The besPj load remains of interest to determine PCP mostly when fungal load is high (as in case of millions of genome copies). However, in case of moderate or low loads, excluding a PCP is not possible and requires a global evaluation with the clinical/radiological context. A system of two thresholds has been proposed in previous works on Pj qPCR: one low threshold under which Pj colonization would be highly probable and a high threshold over which a PCP is greatly probable, and a gray-zone in-between that requires an interpretation with the global clinical and radiological context of the patient [Pj loads is in concordance with previous reports of qPCR evaluations [From these results, quantifying pulmonary  patient ,16,17, bluations ,16,18 anFirstly, a disconnection between the fungal load and PCP severity has been reported in humans and in rodent models. Indeed, the pulmonary symptoms would be related to an overstated and inadequate immune host response rather than to the fungus itself ,19,20,21® Pneumocystis jirovecii) has been described [Pj load in the lungs, and can consequently reduce the efficiency of such quantifications to predict PCP diagnosis [Secondly, an inter-strain variability of the copy number of the genes targeted by the qPCR  are currently presented as a continuum rather than two distinct states; mination .Pj colonization status. Use of immunofluorescent assay could have improved the performances of the microscopic examination given its superior sensitivity to conventional staining [Of note, microscopic examination after staining was only positive in 6% of PCP cases, confirming the major role of qPCR in PCP diagnosis despite some interpretation difficulties related to the staining ,8; howevPj colonization groups, and both leading to interpretation cut-off values. Finally, β-D glucan blood concentration was not available in this study. While this indirect biomarker can help differentiating Pj colonization and PCP, and has been associated with results of qPCR quantification to diagnose PCP [3 is required for all other types of immunosuppression [3 [This study has some limitations. Firstly, we selected patients and samples according to a stringent cut-off based on prolonged PCP treatment/prophylaxis (more than one day before the sampling date) to assess efficiently the PCR performances. Given the low number of HIV-positive patients excluding a significant statistical analysis, this population was not analyzed. Although BAL is consensually considered as the reference sample for PCP diagnosis , the numnose PCP , it is nnose PCP . The usenose PCP ,16,17. Hpression . Howeverssion [3 , leadingssion [3  and was ssion [3 ,16,25, ePj treatment before the qPCR were also excluded from the study since their fungal load could have been modified under treatment.Children (23 in our cohort) were excluded from these analyses to avoid the cases of primo-infections that could be associated with different fungal loads in comparison to the mechanism of reinfections that is now largely accepted for PCP pathophysiology in adults . PatientFinally, our population of HIV-negative patients exhibited characteristics similar to populations previously reported: median age about 65 years old; various immunosuppressive conditions including mainly transplanted patients; a large population of solid and hematological cancers and inflammatory diseases; about 70% of patients receiving corticosteroids ,28. ® Pneumocystis jirovecii qPCR helps to discriminate PCP from Pj colonization in our experience, with Cq < 31.45 (or loads > 8275 genome copies/mL) for BAL and Cq < 32.33 (or loads > 8130 genome copies/mL) for URT samples being indicative of PCP. However, as observed in previous works on qPCR, low fungal load results, being possibly associated with either PCP or colonization, should be interpreted with caution, in accordance with clinical and radiological signs.To conclude, quantification of fungal loads using MycoGENIE


Polianthes tuberosa) is an ornamental flowering crop of the Amaryllidaceae family. Tuberose mild mosaic virus (TuMMV) and tuberose mild mottle virus (TuMMoV), members of the genus Potyvirus, are ubiquitously distributed in most tuberose growing countries worldwide with low biological incidence. Here, we report the first coding-complete genomic RNA of TuMMV and TuMMoV obtained through high-throughput sequencing (HTS) and further, the presence of both the viruses were confirmed using virus-specific primers in RT-PCR assays. Excluding the poly (A) tail, the coding-complete genomic RNA of TuMMV and TuMMoV was 9485 and 9462 nucleotides (nts) in length, respectively, and contained a single large open reading frame (ORF). Polyprotein encoded by both the viral genomes contained nine putative cleavage sites. BLASTn analysis of TuMMV and TuMMoV genomes showed 72.40–76.80% and 67.95–77% nucleotide sequence similarities, respectively, with the existing potyviral sequences. Phylogenetic analysis based on genome sequences showed that TuMMV and TuMMoV clustered in a distinct clade to other potyviruses. Further studies are required to understand the mechanism of symptom development, distribution, genetic variability, and their possible threat to tuberose production in India.Tuberose ( Polianthes tuberosa, also known as tuberose, is an important perennial herb within Amaryllidaceae family. In the country’s tropical regions, it is frequently grown as an ornamental blooming plant for the development of long-lasting flower spikes. It is also known as “Rajanigandha” or “Nishigandha” in popular parlance. It is believed to have originated in Mexico. In India, it is popularly grown in Andhra Pradesh, Assam, Gujarat, Karnataka, Punjab, Rajasthan, Tamil Nadu, West Bengal, and parts of Uttar Pradesh. It is rich in fragrance constituents as well as secondary metabolites, including polyphenols, nonpolar volatiles, and benzenoid derivatives. Furthermore, the flower and bulb extracts exhibit anti-inflammatory, antispasmodic, diuretic, and emetic effects [ effects .Potyviridae contains about 200 plant virus species that are now allocated to the twelve genera Arepavirus, Bevemovirus, Brambyvirus, Bymovirus, Celavirus, Ipomovirus, Macluravirus, Poacevirus, Roymovirus, Rymovirus, Tritimovirus, and Potyvirus .Potyviridae members can be monopartite or bipartite (flexuous filamentous particles 650–950 nm long and 11–20 nm wide), single-stranded positive-sense RNA genomes with sizes ranging from 8.2 kb to 11.3 kb, with an average size of 9.7 kb. Genomic RNA is translated into polyproteins that must be proteolytically processed to provide ten mature and one fusion protein required for replication and motility: P1 (modulator of replication and translation), helper component proteinase HC-Pro (aphid transmission and silencing suppression), P3 (movement virus and replication), P3N-PIPO (cell-to-cell movement), 6K1 (formation of replication vesicles), cytoplasmic inclusion, CI (replication and helicase involved in virus movement), 6K2 (formation of replication vesicles), genome-linked protein VPg , NIa-Pro (polyprotein processing), NIb (RNA-dependent RNA polymerase), and CP  [Some members have a narrow host range, whilst others can infect a wide range of hosts . SeveralThe rapid development of high-throughput sequencing (HTS) technology in recent years has not only enabled the investigation of diseases with unknown etiology but also facilitated the identification and characterization of known/novel viruses ,7,8,9,10Tuberose is an economically important, vegetatively propagated flower crop grown worldwide . In 2021RNA of pooled symptomatic leaf samples was sequenced using the Illumina HiSeq 2000 platform to identify viruses that may be associated with the symptoms. In the two libraries, the Illumina sequencing data generated was approximately 90 million 75 bp paired-end reads and after trimming, 90,380,608 reads (average length 75 bp) were acquired . A totalPotyvirus [In present study, the obtained TuMMV and TuMMoV genomes contained 9485 and 9462 nt, respectively excluding the poly(A) tail. TuMMV and TuMMoV genomic RNA contained a large ORF, encoding for a polyprotein of 3070 aa and 3081 aa, which began with an ATG codon at 138 and 164 nt and ended with UAG and UAA termination codon at 9350 and 9490 nt, respectively. The characteristic domains, conserved cleavage sites, and motifs specific to the genus otyvirus ,22,23 we1-2A6–7 motif at positions 2604–2859 nts and 2625–2888 nts, respectively. This motif resembled the highly conserved G1-2A6–7 motif found in other members of the family Potyviridae [Furthermore, a PIPO ORF resulting from viral RNA polymerase slippage in the TuMMV and TuMMoV P3 cistrons was predicted downstream of the Gyviridae . Like ot256H-(X)8-D-(X)33-34-S298 in P1; 349K-I-T-C352 (aphid transmission), 494C-D-N-Q-L-D499 (symptomatology), and 607P-T-K609 (aphid transmission) in HC-Pro; 1455V-A-T-N-I-I-E-N-G-V-T-L1466  and 1506G-R-V-G-R1510 in CI; 1237G-(X)2-G-X-G-K-S1244, 2466F-T-A-A-P2470, 2480C-V-D-D2483, 2584G-N-N-S-G-Q-P-S-T-V-V-D-N-S-L-M-V2600 (RNA-dependent polymerase activity), 26288GDD2630 (RNA-dependent polymerase activity), and 2670W-F-M-S2673 in NIb; 2817DAG2819 (aphid transmission) and 3023QMKAAA3028 in CP. Similarly, in TuMMoV, the predicted motifs in mature proteins are 206H-(X)8-D-(X)33-34-S250 in P1; 350K-I-T-C353 (aphid transmission), 495C-D-N-Q-L-D500 (symptomatology) in HC-Pro; 1456V-A-T-N-I-I-E-N-G-V-T-L1467  and 1507G-R-V-G-R1511 in CI; 2466F-T-A-A-P2470, 2480C-V-D-D2483, 2584G-N-N-S-G-Q-P-S-T-V-V-D-N-S-L-M-V2600 (RNA-dependent polymerase activity), 2628GDD2630 (RNA-dependent polymerase activity), and 2670W-F-M-S2673 in NIb; 2817DAG2819 (aphid transmission) and 3033QMKAAA3038 in CP [Furthermore, potyvirus-specific conserved motifs were discovered in proteins encoded by the genomes of both the viruses. The predicted motifs in TuMMV cleavage products are 38 in CP . Few of In Blast analysis, coding-complete genomic RNA sequence of TuMMV shared 72.40–76.80% sequence identities with complete genomes of other potyviruses like pokeweed mosaic virus KU133475), plum pox virus (OK562680), turnip mosaic virus (AB093615), leek yellow stripe virus (AJ307057), Japanese yam mosaic virus (KJ789139), Moroccan watermelon mosaic virus (MN688647), sunflower chlorotic mottle virus (GU181200), and sweet potato feathery mottle virus (MH763680) while that of TuMMoV shared 67.95–77.00% sequence identities with other viruses like Pennisetum mosaic virus (JX070146), turnip mosaic virus (AB093615), leek yellow stripe virus (AJ307057), jasmine virus T (KX398054), and achyranthes bidentata mosaic virus (MT648692). Pairwise sequence identities (%) of the coding-complete genomic RNA sequence of TuMMV (ON116187) shared nucleotide (nt) identities 48.10–59.0% and amino acid (aa) identities 41.30–61.80%, and TuMMoV (and ON219793) shared nt identities 47.40–59.0% and aa identities 42.0–62.30% with some other closely related potyvirus isolates reported globally . Accordi3475, pluTo analyze the evolutionary relationship between TuMMV, TuMMoV, and 44 other potyviruses, a phylogenetic tree based on their complete genomes was constructed. The results showed that these potyviruses can be divided into two distinct groups, subgroup I and subgroup II. Further, subgroup I divided into three distinct groups, subgroup Ia, subgroup Ib, and subgroup Ic. TuMMV and TuMMoV genomes that shared 65.20% nt identity clustered together in a single subgroup Ia, and both the viruses were showed genetic relatedness with papaya ring spot virus and Moroccan watermelon mosaic virus .RT-PCR assay using primers mentioned in To better understand the genetic variability of the TuMMV isolates, a phylogenetic tree was constructed based on the partial CP coding sequences of TuMMV and TuMMoV from different geographical locations. In the phylogenetic tree, the TuMMV isolate of this study fell in sister clade to the TuMMV Taiwanian isolates while the TuMMoV isolate obtained in the current study formed a sister clade to the TuMMoV isolates of China A.Phylogenetic analysis based on the genomic RNA sequence  and partSymptoms of viral disease were observed in the experimental field of tuberose cv. Mexican-single, Hyderabad-single, Rajni-phule, and Sikkim-selection during January 2021 at the Horticulture Research Center, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, India. Leaf samples exhibiting typical symptoms of viral diseases such as mosaic, puckering, and stunting were collected and stored at −80 °C till further analysis along with asymptomatic samples. Sap obtained from symptomatic leaf samples were examined under transmission electron microscope (TEM) after staining with 2% aqueous uranyl acetate (UA) by following the leaf dip method .Leaf samples of Mexican-single and Hyderabad-single cultivar were subjected to total RNA extraction using GeneJET RNA purification kit  in accordance with the user instructions manual. The quality and quantity of total RNA were assessed using Qubit™ fluorometer  and TapeStation . From the isolated pool of total RNA, ribosomal RNA was depleted using the Ribo-Zero rRNA Removal Kit . Sequencing libraries were prepared from the ribodepleted RNA pool using truseq-stranded-total RNA library preparation kit  by following the manufacturer’s instructions. Bioanalyzer  and TapeStation were used to verify the quality of the prepared libraries and the libraries were sequenced using Illumina HiSeq 2000 platform at NxGenBio Life Sciences  to produce 2 × 75 bp paired-end reads.https://www.biobam.com/omicsbox, accessed on 23 February 2022). The outcomes showed in a report have a number of assembled reads and total used reads. Open reading frames (ORFs) encoded by the putative viral genomes were determined by NCBI ORF Finder . Conserved domains in the virus proteins were predicted using NCBI Conserved Domain-Search tool , and the genome organization of the identified viruses was constructed using the Bioedit 7.2 program [Raw reads obtained were imported into CLC Genomic workbench (20.0.4) and quality trimmed using the trimming tool to remove ambiguous and adaptor sequences. Trimmed reads were assembled using the de novo assembly tool in CLC Genomic workbench (20.0.4) to obtain longer contigs. Obtained contigs were subjected to BlastX analysis against non-redundant (NR) database in the workbench. The alignment against reference viral genomes in the database were made using OmicsBox 2.1 ( program .Genome sequences of identified viruses along with the available genome sequences of known potyviruses were retrieved and subjected to multiple sequence alignment using the CLUSTALW tool in MEGA-X . PhylogeTotal RNA isolation was performed from four tuberose cvs. Mexican-single, Hyderabad-single, Rajni-phule, and Sikkim-selection as described in Virus diseases limit tuberose production worldwide. With increased global trade, the possibility of introduction of tuberose viruses into newer areas grows by the day. As a result, rapid and reliable tuberose virus detection techniques are required to halt this inadvertent introduction and ensure virus-free tuberose production. We used HTS in this study and determined the first coding-complete genome sequences of TuMMV and TuMMoV. The genome sequences would be useful in providing a clearer picture of the virus evolution and in inferring biological properties of the viruses, which ultimately would help in devising management strategies. However, biological studies are required to understand the mode of transmission and effects of single/mixed viral infection on tuberose plants.


Flavonoids are types of natural substances with phenolic structures isolated from a variety of plants. Flavonoids have antioxidant, anti-inflammatory, anticancer, and antiviral activities. Although most of the research or applications of flavonoids are focused on human diseases, flavonoids also show potential applicability against porcine virus infection. This review focuses on the recent progress in antiviral mechanisms of potential flavonoids against the most common porcine viruses. The mechanism discussed in this paper may provide a theoretical basis for drug screening and application of natural flavonoid compounds and flavonoid-containing herbs to control porcine virus infection and guide the research and development of pig feed additives. Swine pathogens, including viruses, bacteria, parasites, mycoplasma, etc., seriously endanger the development of the pig industry. Swine viruses, such as transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), Influenza A virus (IAV), African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus (PCV), and pseudorabies virus (PRV), cause severe diseases through single infection and/or coinfection ,2,3,4,5.Although vaccination is the primary strategy to prevent some virus infections, vaccination is not feasible or practical in many cases because of virus mutation, diverse virus subtypes, and poor cross-protection effect of vaccines. Furthermore, the antiviral activity of chemical drugs currently used in the clinic is narrow, usually accompanied by cytotoxicity, biological toxicity to organisms, and high cost.etc.), and mineral medicine. Evidence proved that many herbal medicines have antiviral and anti-inflammatory effects because they contain phytochemicals, such as flavonoids [Traditional Chinese medicine (TCM) is widely used in China and Chinese communities outside China, which can effectively relieve the disease from severe to moderate or mild, improve the cure rate, reduce the mortality rate, and promote the recovery of patients ,13,14,15avonoids ,11,12,13To date, more than 6000 flavonoid compounds have been identified. Flavonoids have three benzene rings, in which A ring and B ring are connected by a three-carbon heterocyclic pyran ring (C ring) to form a basic C6–C3–C6 carbon skeleton ,17. BasePrevious studies demonstrated that flavonoids could directly inhibit virus infection via several mechanisms, including interfering with and blocking the processes of attachment, entry, replication, and release ,20. MoreAlphacoronavirus genus of the family Coronaviridae [TGEV is an enteropathogenic coronavirus that belongs to the aviridae ,62. TGEVaviridae ,62,63. Taviridae ,62. Theraviridae ,62,63. Iaviridae ,64, whic6 to 103.9). Moreover, intracellular reactive oxygen species (ROS), which are considered to be factors inducing inflammatory responses, were almost entirely inhibited by (+)-Catechin with a concentration of 80 μM [(+)-Catechin flavonoids, abundantly found in green tea, are bioflavonoids with great potential for anti-oxidative effects. Evidence showed that (+)-Catechin flavonoids inhibit cancer, cardiovascular disease, and virus infection . Liang eof 80 μM . These rIn addition, receptor tyrosine kinases (RTKs) play essential roles in cell proliferation , and it Alphacoronavirus genus of the family Coronaviridae, another top ten swine pathogen, which poses a significant threat to the swine industry in the US and worldwide [Porcine epidemic diarrhea virus (PEDV) also belongs to the orldwide ,62,66. Torldwide ,62,66. Torldwide ,67, the pro) through binding with Cys144, Asn141, and His162 residues of PEDV 3CLpro [pro [Quercetin, a phytochemical abundant in fruits and vegetables , has a promising therapeutic prospect. Quercetin has many properties such as antiviral, antioxidative, anti-inflammatory, and anticancer . Li et aV 3CLpro . MoreoveV 3CLpro . Additiopro [pro ,33. ThesHypericum monogynum and Crataegus pinnatifida. Hyperoside is a flavonoid glycoside with multiple pharmacological effects [Hyperoside can be extracted from various plants, such as  effects . Recentl effects . On the  effects . These rEnterococcus genus, Lactobacillus genus, and Enterobacteriaceae family, in the intestine but decreasing the number of Clostridium coccoides in the caecum [Puerarin, a medicinal and edible isoflavonoid compound naturally existing in the pueraria, was validated as a medical component with immense therapeutic activities in various health disorders . As repoe caecum . Moreovee caecum ,60. TherOrthomyxoviridae family and includes four genera, Alphainfluenzavirus, Betainfluenzavirus, Gammainfluenzavirus, and Deltainfluenzavirus, corresponding to influenza virus A (IAV), B (IBV), C (ICV), and D (IDV), respectively [HA and NA genes of epidemic strains may affect the vaccine’s efficacy [The influenza virus belongs to the ectively . IAV hasectively ,73,74. Iectively ,76. The ectively ,77. Furtefficacy ,77.Glycyrrhiza inflata showed inhibitory effects on NA from various IAV strains [Chalcones are α, β-unsaturated ketones which widely exist in plants and have been used in clinical treatment for a long history as bioprecursors of flavonoids. Chalcones presented various pharmacological activities, such as antimicrobial, antiviral, anti-inflammatory, immunosuppressive, etc. . The pre strains . Among t strains . Further strains . These rIn addition, epigallocatechin gallate, also called epigallocatechin-3-gallate (EGCG), mainly extracted from green tea, is a polyphenolic flavonoid with the catalysis of epigallocatechin and gallic acid. It was identified that EGCG possesses varieties of biological activities, including antioxidant, anti-inflammatory, and antimicrobial properties ,87. Xu eAsfarviridae family, is a causative agent of African swine fever (ASF). ASF is a highly contagious viral disease of domestic pigs and wild boars, which causes a devastating infectious disease with acute onset, rapid progression, and up to 100% mortality [African swine fever virus (ASFV), only one member of the ortality ,94. WOAHortality . Unfortu50) of 8.4 μM [Myricetin  is a natural flavonol compound in numerous plants, including oranges, grapes, herbs, and teas, etc. . It has f 8.4 μM . The inhf 8.4 μM . Moreovef 8.4 μM . These rApium genus. Genkwanin is the O-methyl derivative of apigenin. These flavonoids have various biological activities, such as the induction of autophagy and apoptosis and the suppression of cellular proliferation and inflammatory reactions [Apigenin, an abundant fruit and vegetable flavone, belongs to the eactions ,98,99. Heactions . The reseactions . Based oeactions . As expeeactions . Furthereactions . These reactions ,100,101,Genistein and kaempferol are the major flavonoids of sophora fruit extracts. Genistein, a phytoestrogen extracted from soybeans, has multimodal action against diseases, including cell cycle arrest, autophagy and apoptosis induction, metastasis inhibition, antioxidant, and anti-inflammatory . ArabyanArteriviridae family, containing a single positive-stranded RNA of about 15 kb in length [Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped virus belonging to the n length ,103. PRRn length ,103. Then length ,103.Ge et al. found that EGCG, a polyphenolic compound from green tea, exerts multiple roles in suppressing PRRSV infection in a time and dose-dependent manner . First, Ruansit et al. reported that quercetin modulated the immune responses by enhancing type I and II interferon and type I interferon-regulated genes (IRGs) and decreasing pro- and anti-inflammatory cytokine expressions against highly pathogenic (HP)-PRRSV challenge in vivo and in vitro . In addimyxovirus resistance 1, interferon regulatory factor 3 (IRF3), IRF7, 2′-5′-oligoadenylate synthetase 1, stimulator of interferon genes (STING), osteopontin (OPN), IFNα, IFNβ, IFNγ, and TNFα [IRF3, IRF7, STING, OPN, IFNα, IFNβ, and IFNγ genes significantly increased, and the levels of TNFα and TGFβ were reduced in the HP-PRRSV-infected group after being stimulated with rutin [Rutin is a quercetin glycoside in several medicinal and eatable plants, which can effectively improve metabolic function due to its effective antioxidation and anti-inflammatory properties . Rutin cand TNFα . Howeverth rutin . These rPsoralea corylifolia, which was used in TCM for several centuries [Isobavachalcone (IBC) is a flavonoid extracted from the plant enturies ,105,106.enturies . HoweverHumulus lupulus L., which has broad biological activities, including anti-inflammatory, inhibition of cholesterol accumulation, and inhibition of cell proliferation [nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HMOX1), glutamate-cysteine ligase catalytic subunit (GCLM), and NAD(P)H quinone oxidoreductase 1 (NQO1) [Xanthohumol (Xn) is a prenylated flavonoid isolated from hops feration . Recentlferation . Moreove1 (NQO1) ,55. Afte1 (NQO1) ,55. In a1 (NQO1) ,55. The Soy isoflavones (ISF), including genistein and daidzein isoflavones, have many potential pharmacological activities. For example, ISF suppresses arterial stiffness, regulating intestinal flora and mimicking estrogen via its special structure to affect the physiological state of animals ,108. In Varicelloviru genus, the Alphaherpesvirinae subfamily, and the Herpesviridae family [Porcine pseudorabies virus (PRV) belongs to the e family . It has e family ,112. In e family ,114,115.e family . CurrentIE180 and the expression of latency-associated transcripts, thus alleviating the pathological changes in these organs [Several groups reported that flavonoids, including quercetin, kaempferol, EGCG, dihydromyricetin (DMY), and luteolin, have anti-PRV activities via various mechanisms ,116,117.e organs . In addie organs . Huan ete organs . Furthere organs . Moreovee organs . Ampelopsis grossedentata (A. grossedentata) and Nekemias grossedentata (N. grossedentata) [Dihydromyricetin (DMY) is a flavonoid extracted from several plants, such as dentata) . DMY hasdentata) . DMY candentata) . Sun et dentata) .Verbascum lychnitis, Carex fraseriana, and other herbs, exhibiting antioxidant, anti-inflammatory, apoptosis-inducing, chemopreventive, and antiviral activities [nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) [Luteolin is a natural dietary flavonoid found in tivities ,120,121.tivities . Furthertivities . In addi (COX-2) . TherefoPolygonum hydropiper L., Garcinia species, and Licorice, also exhibit anti-PRV activity due to the abundant antiviral ingredients in these herbs [Polygonum hydropiper L. (FEA) [iNOS and COX-2, and cytokines in PRV-infected cells [Meanwhile, widely used herbs in TCM, such as se herbs ,122,123.L. (FEA) ,124. Fured cells . Moreoveed cells . These rGarcinia parvifolia leaf using ethyl acetate (45 L Ea), ethanol (45 L Et), and hexane (45 L H) solvents, respectively [Additionally, Adnan et al. extracted bioactive compounds from ectively . As a reectively . In contectively . Furtherectively . These rCircovirus in the family Circoviridae [Porcine circovirus 2 (PCV2) is a non-enveloped DNA virus with a diameter of about 20 nm, belonging to the genus oviridae . PCV2 isoviridae ,6. In adoviridae ,6. To daoviridae . Althougoviridae , the viroviridae ,128. Spatholobus suberectus Dunn (S. suberectus) is a widely used herb in traditional Chinese medicine, which can improve blood circulation and be anti-platelet, anti-inflammatory, antibacterial, neuroprotective, and anticancer [S. suberectus Dunn (TFSD) extracted from S. suberectus [ticancer . Chen etberectus . They foberectus . In contberectus . Furtherberectus . These rKd = 98.03 ± 4.76 μM, blocking the binding of virions with heparan sulfate, a cellular receptor on the surface of host cells [EGCG also exhibits anti-PCV2 activity . Li et ast cells . Furtherst cells . These rst cells . TherefoAlthough flavonoids have exhibited effective inhibition of virus infection, some limitations must be overcome. Garcinia parvifolia leaf using ethyl acetate (45 L Ea), ethanol (45 L Et), and hexane (45 L H) solvents are distinct and showed diverse antiviral activities on PRV infection [First, most flavonoids are extracted from plants containing various bioactive ingredients in the extract. However, although advanced technologies, such as microwaves, ultrasound, pressurized liquids, supercritical fluids, and electric fields, are used to extract flavonoids, different bioactive components could be obtained by disparate extraction techniques or the same techniques under other conditions ,130. Fornfection . TherefoSecond, the effective concentration of each flavonoid is critical for its antiviral activities. Some natural flavonoids may be metabolized into another bioactive ingredient in vivo, the fundamental antiviral components. However, characteristics of natural flavonoids such as poor water solubility, high instability, and low oral bioavailability limit their application. Therefore, new delivery strategies, such as nanospheres, nano-capsules, micro and nano-emulsions, micelles, solid lipid nanoparticles, and capsules , are proNotably, whether flavonoids have cytopathic effects in vitro or adverse effects in vivo is controversial. Some groups reported that when flavonoids were used in non-cytotoxic concentrations in vitro or in vivo, flavonoids could inhibit virus infection in a dose-dependent manner, thus improving the survival rate of cells or animals or providing preventive protection against virus attack ,37,53,59To date, more than 30 kinds of swine viruses, including 20 emerging or re-emerging swine viruses, have been confirmed to infect pigs and cause severe diseases, seriously affecting the swine industry and the world economy. Unfortunately, there are no effective drugs and treatments for most of these viruses, and some existing prevention programs, including vaccines, have certain limitations or low effectiveness. This paper reviewed the antiviral activities of flavonoids against porcine viruses in vivo and/or in vitro. Generally, flavonoids inhibit porcine virus infection by inhibiting viral attachment, entry, replication and translation, assembly, and/or release . MeanwhiSome flavonoids play antiviral roles in multiple stages of the virus infection and can be used to prevent and treat the diseases. Flavonoids, such as EGCG, kaempferol, rutin, IBC, and quercetin, exhibit antiviral activities against numerous porcine viruses, indicating that these flavonoids have broad-spectrum antiviral activities. Multiple targeted flavonoids mainly modulate cellular inflammatory, oxidation, and immune responses. In contrast, some are single target agents, such as hyperoside and chalcones, which are only effective in a single step of virus infection and only act on a single viral protein. Notably, the same flavonoid may inhibit different viruses in different ways. For example, quercetin inhibits PRRSV infection by immunomodulatory, anti-inflammatory, and antioxidant activity but inhibits PEDV infection by direct interaction with PEDV protein. However, most published data on flavonoid antiviral activities are based on in vitro studies. Therefore, future research should focus on field applications of flavonoids and their derivatives against porcine viruses, including efficacy, safety, dosage, and application stages. Moreover, the combination of flavonoids extracted from herbs and berries showed synergistic antiviral effects, further confirming the effectiveness of flavonoids containing-TCM in the antiviral activities against swine viruses. Therefore, oral administration of purified active ingredients of flavonoids or dietary supplementation of the plant containing flavonoids is promising for preventing and treating virus infection in the swine industry.


Since May 2022, an outbreak of monkeypox has been ongoing in non-endemic countries. We report four cases in Italy in young adult men reporting condomless sexual intercourse. The patients are in good clinical condition with no need for specific antiviral drugs. Biological samples from seminal fluid were positive for monkeypox viral DNA. For many other viruses found in semen there is no evidence of sexual transmission. The possibility of sexual transmission of monkeypox virus needs to be investigated. Orthopoxvirus genus of the Poxviridae family. Two genetic MPXV clades have been characterised: West African and Central African. Outside of Africa, the first cases of monkeypox were reported in 2003 in the United States (US) [Human monkeypox virus (MPXV) is a double-stranded DNA virus of the tes (US)  when an All four monkeypox patients were young adult men who have sex with men (MSM). Two of them (Patients 1 and 3) had an HIV infection and received effective antiretroviral therapy (ART); the other two (Patients 2 and 4) were on antiretroviral pre-exposure prophylaxis (PreP).All patients travelled in the first 2 weeks of May 2022: three patients participated in a mass gathering event in Gran Canary island (GCI) and one travelled for sex work. During the travel, they had condomless sexual intercourse with different male partners.All four patients had a history of sexually transmitted infections (STIs) .All four monkeypox patients were in good clinical conditions and were admitted to two different hospitals in central Italy, in combined droplet and contact isolation measures plus filter face piece-2 (FFP2) for clinical care management.The first case was a male in his 30s who referred to our centre after accessing an emergency department where an MPXV infection was suspected. During travel made in mid-May, he had been treated with oral ciprofloxacin and acyclovir and one single dose of benzylpenicillin for the appearance of skin lesions. At admission, multiple asynchronous deep-seated and well-circumscribed lesions with central umbilication were present on the genital area, with inguinal lymphadenopathy; a single lesion was present on the anterior and posterior thorax and on the left calf.Patient 2 was a male in his 30s, taking daily-PreP. He was admitted for fever and asthenia starting mid-May. Three days later, perianal lesions appeared as raised, itchy papules secreting serous, with concomitant painful inguinal lymphadenopathy. Multiple anal lesions appeared over the next 3 days, followed by few single lesions with different timing on the skin of the back, legs and the sole of one foot.Patient 3 was a male in his 30s. He was admitted for a 2-day history of fever, followed by the appearance of clustered itchy papular lesions in the anal region and single lesions on head, thorax, legs, arms, hand and penis. He reported smallpox vaccination during childhood, 30 years earlier.Patient 4 was a male in his 30s, taking event-driven PreP. After a 2-day history of myalgia, vesicular-papular genital lesions appeared, followed by further skin lesions that appeared 6 days later in the suprapubic area and chest.In all patients, skin lesions progressed with an asynchronous evolution .Samples obtained from skin, genital and anal lesions, serum, plasma, seminal fluid, faeces, and nasopharynx were all positive for MPXV DNA in real-time PCR . The patViral DNA was extracted by Qiamp Viral RNA mini kit (Qiagen), and two real-time PCRs were used to assess the presence of MPXV DNA. Real-Star Orthopoxvirus PCR Kit (Altona Diagnostics GmbH) was used as screening PCR. This method recognises a region common to all Orthopoxviruses without distinction of species. The second PCR (G2R_G assay) published by Li et al. targets the tumour necrosis factor (TNF) receptor gene and was used as confirmatory PCR . The RotWe performed Sanger sequencing of the amplicon obtained from a third end point PCR (in-house method), targeting the complete gene for the viral haemagglutinin (HA) to identify which of the two known MPXV clades, the virus in the patient samples belonged to. We obtained the HA gene sequence only for the first three patients. The sequence of the fourth patient is in progress.For the first case, next generation sequencing was performed on an Ion Torrent GSS5 platform (ThermoFisher), using a metagenomic approach starting from 100 ng extracted DNA. The library was prepared using the Ion Xpress Plus Fragment Library Kit (Thermofisher). After quality filter, the reads were de-novo assembled using SPAdes , to receWe performed phylogenetic analyses using INMI sequences (HA and full genome)  and sequThe clinical description of, to our best knowledge, the first four monkeypox cases detected in Italy warrants closer investigation for several reasons. Firstly, it has been suggested that the MPXV in the currently ongoing outbreak is transmitted from human to human , even thSexual behaviour of the cases in the present series and the initial appearance of lesions mostly in the anal and genital areas all suggest that close contact during sexual intercourse was important for virus transmission. The seminal fluid obtained from three monkeypox patients at the time closest (5–7 days) to symptoms onset, was found to be positive for MPXV DNA in all four patients, with a Cq range from 27 to 30. The correlation between Cq value and infectious viral load in MPXV infections is not yet known, but the range of Cq values we measured in semen makes viral isolation unlikely. However, detection of viral DNA in the three seminal fluid samples we analysed, excludes the hypothesis of biological sample contamination. Although these findings cannot be considered definitive evidence of infectivity, they demonstrate viral shedding whose efficiency in terms of transmission cannot be ruled out. It has been observed that it is likely that MPXV can be transmitted through substances of human origin ,12 and iWe need to consider that many other viruses causing viraemia can be found in semen ,14 with The characteristics of these four patients reflect those described in other European countries in the current outbreak. The phylogenetic characteristics of the virus could support the hypothesis of a recent introduction of the West African clade of the MPXV into the community in non-endemic countries. Moreover, the characteristics of the population involved, as well as reported exposure to multiple, condomless sexual contacts, suggest that human-to-human transmission through close physical contact in sexual networks plays a key role in the current outbreak. Further studies are needed to assess presence, persistence and contagiousness of MPXV in different body fluids.


In this paper, a method to control the lime reaction by different slaking conditions is proposed to reduce the occurrence of cracks in newly repaired earthen city walls. The effects and mechanisms of the slaking time , lime content , and moisture content  on the cracking and mechanical properties of lime soil were analyzed by the test results of surface cracks, triaxial compression, particle gradation, pH value, X-ray diffraction and scanning electron microscope. The results show that proper slaking of lime soil specimens can reduce surface cracks and improve mechanical properties. After 12 h of appropriate slaking, the crack rate of the lime soil with 20% content decreased by 97.13%, the cohesion increased by 20.27%, and the internal friction angle decreased by 11.27%. However, the mechanical properties decreased when the slaking time was too long. After 72 h of slaking, the cohesion of 20% lime soil decreased by 8.21% and the internal friction angle increased by 2.82%. Further analysis shows that the appropriate slaking conditions can regulate the reaction rate and alkali environment, control the lime produced cementitious substances, improve the particle gradation and further reduce the occurrence of surface cracks. These results provide a basis for the restoration technology of newly repaired earthen city walls. Lime and soil are mixed to form lime soil, which has the characteristics of high strength, good integrity, economic material and convenient construction ,2. As a The stable reaction of the lime soil has been widely investigated. As a chemical-physical improvement method, lime can significantly improve the mechanical properties of soil through pozzolanic reactions, carbonation reactions, cation exchange and crystallization ,22,23,242 has better dispersion and the higher activity when the digestion temperature was 84 °C, the mass ratio of water to lime was 5:1 and unslacking was performed. Wei et al. [2 prismatic crystal changes to plate crystal, which improves the plasticity of putty. Rosell et al. [In order to stabilize the reaction of lime, improve the performance of lime soil and optimize the construction technology, scholars across the world have carried out a lot of research on the “lime slaking” of lime soil. Gu et al.  found thi et al.  concludei et al.  determini et al. ,35,36,37l et al.  concludel et al. . In factl et al. ,41. Due l et al. ,43,44. ITo explore the influence of slaking on the surface cracks and mechanical properties of the mended lime soil of the Kaifeng city wall, lime soil was prepared according to the mix proportions of the mended lime soil. Combined with the engineering construction technology, tests were designed considering the slaking time, lime content and moisture content. The influence of these factors on surface cracks, mechanical properties, particle gradation, pH value and microstructure was studied and the reasonable composition and slaking time of lime soil materials for wall repair in the Kaifeng area were determined.3. The physical property indexes are shown in The soil used in the test was obtained from the peeling of a soil site in Kaifeng City. The soil is silty clay, with unfavorable characteristics, such as loose structure, low strength, strong capillarity and poor water stability. The tests also show that the natural moisture content is 13.20%, the plastic limit is 21.03%, the liquid limit is 32%, the plasticity index is 16.6, the optimum moisture content is 14.32% and the maximum dry density is 1.68 g/cmThe effects and mechanism of the slaking time, lime content, and moisture content on the cracking and mechanical properties of lime soil were analyzed by the test results of surface cracks, triaxial compression, particle gradation, pH value, X-ray diffraction and scanning electron microscope.A series of tests were carried out to explore the deterioration mechanisms of surface cracks and mechanical properties of lime soil with slaking time, lime content and moisture content. The tests include surface cracks, triaxial compression, particle gradation, pH value, X-ray diffraction and scanning electron microscope. According to the relevant literature ,46,47, tExperiments on the slaking time, lime content and moisture content of specimens were conducted according to the Standard for Geotechnical Engineering testing method (GB/T50123-2019) . The dryTo observe the change in particle gradation of lime soil during slaking, 600 g of dried specimens (divided into three parts) were taken every 12 h for sieve analysis. The fine screen apertures were 2 mm, 1 mm, 0.5 mm, 0.25 mm, 0.1 mm and 0.075 mm, respectively. The numerical error was controlled within ±1 g.The pH5S  puncture pen pH meter is used for pH detection. The pH5S puncture electrode is inserted into 7.00 pH, 4.00 pH and 10.01 pH solutions in turn for 3-point calibration, then inserted into three different positions of lime soil for pH detection, and the pH value error shall be controlled within ±0.05. The results are taken as the average value. To observe the pH change in lime soil during slaking, the pH is measured every 1 h for the first 24 h and every 4 h for the next 48 h.The slaked lime soil specimens must be photographed at regular intervals in the natural environment. Each image is processed by Photoshop, and the crack ratio  is calcuTsz-10  strain-controlled triaxial apparatus was used in the triaxial compression experiments. Considering the actual confining pressure conditions, the low confining pressures were set to 50 kPa, 100 kPa and 150 kPa. The shear rate was 0.8%/min. The lime soil was slaked for 0 h, 12 h, 24 h, 48 h, and 72 h to produce specimens of 39.1 mm in diameter and 80 mm in height. The specimens were put into the triaxial apparatus and subjected to unconsolidated undrained tests at different low confining pressures.The 2 g specimens with different slaking times were dried and ground to powder. The phase composition of the specimens was tested by Bruker D8 Advance’s X-ray powder diffractometer . The instrument parameters were as follows: ceramic X-ray tube, Cu target, power 2.2 kw. The angle scanning range (2θ) was 20~70°, the scanning speed was 5°/min, and the step interval was 0.02°.After the triaxial compression test, 2 g specimens were taken into a drying oven and dried at 105 °C for 12 h. The specimens were then put into a coating machine, and platinum was sprayed on the surface of the specimens. The micro morphology of the specimen fracture was observed with an FEI QUANTA 250  environmental field emission scanning electron microscope. The instrument parameters were as follows: secondary electron image, high vacuum mode, test voltage of 1 kV and test current of 10 mA.2 reacts with CO2 to form CaCO3 crystals, which form a cohesive polymer through bonding. The attraction between particles increases, resulting in flocculation and coagulation [gulation ,51, whicFrom the changes in particle size and composition of lime soil under different slaking time, it can be divided into two aspects. On the one hand, the mass percentage of lime soil with particle sizes less than 2 mm, 1 mm, 0.5 mm and 0.25 mm gradually decreases with the increase in slaking time. On the other hand, the mass percentage of lime soil with particle sizes less than 0.1 mm and 0.074 mm increases after 12 h of slaking. When lime soil continues to be slaked, the percentage of mass with particle size less than 0.1 mm and 0.074 mm decreases. Flocculation and coagulation between particles increase during lime soil slaking, gradually increasing the percentage of mass with particle sizes greater than 0.25 mm and 2 mm. Although the addition of lime changes the composition of the particles, the lime destroys the connection between soil particles during the slaking process, producing more small particles with particle sizes less than 0.1 mm and 0.074 mm. Compared with 0 h of slaking, after 12 h of slaking, the mass percentages of particles with particle sizes less than 0.1 mm increases by 3.65%, 2.94% and 15.05% and the mass percentage of particles with particle sizes less than 0.075mm decreases by 2.72%, 1.48%, and 14.04%, respectively. At this time, the particle grading curves of the soil specimens evidently tend to be stable, indicating that the reaction rate of lime slows down and the particle influence on size composition of soil specimens slows down.− is produced in the slaking reaction.The relationship between the pH values of the specimens with different lime contents and slaking times is shown in 2+ and 2(OH)−, and the pH of the mixture increases. CO2 in the air participates in the reaction and consumes OH−, resulting in a decrease in the pH value of the mixture [During slaking, the clay lime mixture begins to react upon the addition of water. The hydrated lime is decomposed into Ca mixture . In pracThe surface cracks and crack ratios of specimens with different slaking times, lime contents and moisture contents under natural conditions are shown in The surface cracks of the lime soil specimens are also closely related to the content of lime and water. The occurrence of surface cracks in lime soil specimens is directly proportional to the content of lime and inversely proportional to the moisture content. Compared with 10% lime content (0 h), the crack ratios are increased by 11.82% and 123.81% for the specimens (0 h) with 15% and 20% content after 6 h of natural curing, respectively. Compared with 14% moisture content (0 h), the crack ratios are decreased by 77.89% and 80.93% for the specimens (0 h) with 18% and 22% content after 2 h of natural curing, respectively. In the process of natural curing, the increase in lime content and the decrease in moisture content will accelerate the chemical reaction rate of the specimens, produce more surface cracks and reduce their durability.2 generated by the reaction of quicklime with water in lime soil reacts with CO2 in the air to generate white CaCO3 material. If the reaction continues after the lime soil hardens, it will expand due to the continuous reaction of the lime, resulting in uplift and cracking [Combined with the results of particle gradation and the pH value of lime soil under different slaking times, lime contents and moisture contents, the reasons for the significant increase in surface cracks over time in unslaked lime soil specimens in natural environment can be attributed to the following: (1) the cracks are caused by chemical reaction of lime soil, and the increase in slaking time, lime content and moisture content will accelerate the chemical reaction rate of the specimen. During the experiment, the surface cracks of the unslaked lime soil specimens increase with time in the natural environment. At the same time, white substance precipitation and water loss are observed on many different parts of the specimen surface. It is considered that Ca(OH)cracking . (2) TheThe principal stress difference axial strain curves of lime soil with different lime contents with different slaking times and moisture contents are shown in The figures show that the larger the lime content, the higher the confining pressure and the smaller the moisture content, the greater the peak value of the stress–strain curve and the more obvious the softening characteristics. On the contrary, the less lime content, the lower the confining pressure and the greater the moisture content, the lower the peak value of the stress–strain curve and the more obvious the strain hardening characteristics. When the lime content is 20% and the moisture content is 14%, the stress–strain curves under low confining pressures show strain softening characteristics. The particle size of quicklime powder selected in the experiment is 150–200 mesh, which can be used as “fine aggregate” in the silty clay to fill, bond and solidify soil particles and enhance the soil strength. The increase in lime content and the decrease in moisture content can enhance the bonding effect and improve the strength of the samples. However, due to the high lime content and the small moisture content, uneven particle gradation, brittle failure easily occurs, resulting in the softening of the stress–strain curve.The relationship between the cohesion and internal friction angle of the specimens with different slaking times, lime contents and moisture contents is shown in With the increase in lime content and the decrease in moisture content, the cohesion and internal friction angle of the specimens increase. Compared with 10% lime, the cohesion of the specimens with 15% and 20% lime after 0 h slaking increased by 95.23%, and 188.42%, and the internal friction angle decreased by 0.21% and 1.97%, respectively; Compared with the moisture content of 22%, the cohesion of the specimens with moisture contents of 14% and 18% increased by 68.24% and 24.60% and the internal friction angle decreased by 47.50% and 13.08%, respectively. Although the shear index of the specimens with high lime content and low moisture content is good, the specimens exhibited many cracks and decreased in durability. Comparing 2 increased first and then decreased, and the diffraction peak of calcite increased. During slaking, some active CaO in lime soil is hydrated to produce Ca(OH)2. The reaction formula is as follows (1). The increase in Ca(OH)2 content during lime soil slaking increases the pH value. In an environment with a high pH value, the particle size composition, surface crack and mechanical properties of lime soil are improved in the slaking process. The lime soil continues to slake, and Ca(OH)2 reacts with CO2 in the air to form CaCO3. The reaction formula is as shown in (2). This reaction will consume part of the OH− generated by the reaction of CaO with water. Therefore, the content of Ca(OH)2 decreased and the content of CaCO3 increased continuously. As the slaking time of lime soil increases and the pH value decreases, the particle gradation becomes worse and the mechanical properties decrease. Lime provides lime soil with a strong alkaline environment that hydrates the clay minerals silicates and aluminates [2 and highly polymerized amorphous silica gel are the source of strength growth of hardened bodies [uminates  under thd bodies . The cem2 and CaCO3 crystals generated by various reactions, such as hydration and carbonization of lime, “grow” in the pores of soil particles, showing an irregular flake crystal growth structure, forming a bridge structure between the soil particles, promoting the bonding of the dispersed soil particles into a whole, and effectively improving the mechanical properties of lime soil.The specimens were magnified 2000 times by a scanning electron microscope to observe the particle morphology and pore characteristics of the lime soil specimens . The 20%2 substance produced by the reaction of lime and water endows lime soil with extremely high strong alkalinity. Lime with the particle size of 150–200 mesh acts as the “fine aggregate” in silty clay and plays a bonding role in the slaking process of lime soil, which makes some lime and soil particles form a cohesive body, increasing the particles with particle sizes greater than 2 mm and reducing the particles with particle sizes less than 0.074 mm. The microstructure of lime soil before and after slaking shows that the reaction process of lime soil has the ability to “destroy” silty clay particles on the one hand, and the generation of cementitious materials on the other hand. Therefore, the slaking process of lime soil involves the generation of cemented large particles and the reproduction of small particles. This helps to explain why the cohesion of lime soil has increased after slaking (12 h), and the internal friction angle has decreased. However, the number of large and small particles bonded in the lime soil slaked for a long time tends to be stable, the cementitious material of the specimen is limited, and the bonding performance decreases, resulting in a decrease in the cohesion of the specimen and an increase in the internal friction angle.Based on the principles of slaking reactions and carbonization, tests on lime soil with different slaking conditions confirmed that the slaking time can improve the surface cracks and mechanical properties of the specimens. The main conclusions are summarized as follows.With the increase in slaking time, the crack ratio of the specimen decreases, the cohesion first increases and then decreases, and the internal friction angle first decreases and then increases. The particle curve of lime soil shows an overall downward trend; the pH value first increases and then decreases. Proper slaking time (12 h) of lime soil can accelerate the reaction rate of lime in a high alkali environment, improve particle gradation, further reduce surface cracks and improve mechanical properties. However, it is worth noting that the long-term slaking process has a negative impact on the shear index of lime soil.The less lime and more water, the less the specimen crack ratio. The more lime and less water, the stronger the mechanical properties of lime soil. The more lime and water, the more the number of particles with particle size less than 2 mm, and the fewer particles of sizes less than 0.074 mm, the greater the peak value. In order to avoid the surface diseases caused by the chemical reaction of lime soil and improve the durability, it is suggested that the specimen should have a high moisture content and be slaked for 12 h in practical engineering.2, CaCO3, and other cementitious substances produced by reasonably slaking lime soil can improve the crushing of clay particles in a high pH environment, form aggregates, fill the pores between the specimen particles and effectively improve the mechanical properties.After the lime soil is slaked for 12 h, the particles less than 0.075 mm increase and the pH value increases to 13. Under a high pH environment, the unslaked lime causes the crushing of clay particles, and the resulting fine cracks explain the cracks on the outer surface of lime soil specimens. Ca(OH)Proper slaking of lime soil for 12 h and a high moisture content can accelerate the reaction speed of lime, reduce the damage to specimen particles in a high pH environment, improve particle gradation, further reduce surface cracks and improve mechanical properties. By controlling the slaking time and moisture content of lime soil, the durability and surface performance of the repaired city wall can be improved, which is of great significance to the restoration of Kaifeng city wall.In addition, the particle size, carbonization condition, and carbonization time of lime will affect the cracking and mechanical properties of the material, which need to be further studied.


Minimally invasive gynecological surgery is rapidly evolving in the Caribbean. Hysteroscopic myomectomy is the procedure of choice for the removal of submucosal uterine leiomyomas. In Trinidad and Tobago, advancements in minimally invasive surgery have allowed submucosal myomectomies to be performed hysteroscopically with results that are on par with international standards of care. This report highlights a successful hysteroscopic myomectomy performed for the largest submucosal uterine leiomyoma documented in the Caribbean. Uterine leiomyomas are benign tumors of the myometrium that are present in up to 80% of women in the reproductive age group in the Caribbean . It is tThe mainstay of fertility-sparing surgical treatment for leiomyomata is uterine myomectomy . TraditiTrinidad and Tobago, a twin-island developing state in the Caribbean, have been at the forefront of minimally invasive gynecological surgical techniques in the region and has been keeping up with international advancements in gynecological surgery. In this report, we highlight the case of a successful submucosal myomectomy, and, to the best of our knowledge after an extensive literature review, the removal of the largest submucosal leiomyoma done by hysteroscopy in the Caribbean.A 39-year-old nulliparous female presented to the Gynaecology clinic with menorrhagia, dysmenorrhea, and symptomatic anemia for one year. She denied experiencing weight loss, fever, and urinary or gastrointestinal symptoms. She had a history of symptomatic uterine leiomyoma that was treated with an abdominal myomectomy five years earlier. She subsequently had menorrhagia and dysmenorrhea after three years. An ultrasound at this time revealed a submucosal fibroid measuring 6.1 cm in diameter. Before her visit to our clinic, the patient used different medical treatment options such as goserelin (3.6 mg subcutaneously once per month for six months), medroxyprogesterone acetate, and tranexamic acid with minimal improvement over one year. Her medical history was otherwise unremarkable.On clinical examination, a 10-week-sized firm uterus was palpated in the pelvis. Speculum examination demonstrated a healthy-appearing cervix. Blood investigations revealed microcytic anemia  and normal renal and liver function tests. Pelvic ultrasonography revealed an enlarged uterus with a 6.9 cm × 3.6 cm solitary mass within the endometrial cavity  fluid management system. A panoramic inspection of the uterine cavity was first performed to assess the intracavitary topography, and a solitary submucosal G0 leiomyoma  was observed on the right lateral wall of the uterine cavity which was consistent with preoperative imaging  was inserted into the endocervical canal under direct visualization. The leiomyoma was resected down to its base under direct visualization, and the morcellated tissue fragments were suctioned through the device into a collecting pouch for postoperative histopathological analysis  Table  10]. La. La10]. TM hysteroscopic morcellator) and hysteroscopic resectoscopes [Several techniques have been developed to effectively achieve complete hysteroscopic resection of G0 and G1 leiomyomas. The two major modalities available to facilitate leiomyoma removal include the hysteroscopic tissue removal system (TruCleartoscopes . Severaltoscopes ,12. In ctoscopes . In sometoscopes . NotwithTM hysteroscopic morcellator and Thermedx FluidSmartTM fluid management system allowed for the seamless disintegration, removal, and collection of the myoma specimen for later histopathological analysis. The total operative time in our case was 145 minutes compared to the international average of 105 minutes [This operation was completed by a one-step hysteroscopic procedure that achieved complete excision of the large G0 6.9 cm × 3.6 cm leiomyoma. To our knowledge, after an extensive literature review, this is the largest submucosal leiomyoma removed by hysteroscopic myomectomy in the Caribbean. The largest submucosal leiomyoma managed by hysteroscopic myomectomy in the Caribbean measured less than 4 cm in diameter . The lea minutes . One facTM fluid management system [Additionally, an intrauterine pressure between 70 mmHg and 100 mmHg facilitated using distending medium is required for adequate visualization of the uterine cavity . As notet system . Accordit system . In our The field of minimally invasive gynecology is rapidly advancing in the Caribbean with rising popularity among both gynecologists and patients. Complex hysteroscopic interventional procedures should be performed by trained surgeons to maintain proper quality control and minimize adverse procedural outcomes. Hysteroscopic resection of submucosal leiomyomas improves fertility, menorrhagia, symptomatic anemia, and reduces early pregnancy complications. This case highlights the largest submucosal leiomyoma removed by single-session hysteroscopic myomectomy in the Caribbean as a day case.