Leptospira spp. infection in cows from the Caatinga biome, Brazil. Samples of the blood, urinary tract  and reproductive tract  were collected from 42 slaughtered cows. Diagnostic tests included were the microscopic agglutination test (MAT), polymerase chain reaction (PCR) and bacterial isolation. Anti-Leptospira spp. antibodies were found in 27 (64.3%) of the animals analyzed using MAT at a 1:50 dilution (cut-off 50), while 31 (73.8%) animals had at least one organ/fluid where the presence of Leptospira spp. DNA was identified, and 29 animals (69%) were positive at bacteriological culture. The highest sensitivity values for MAT were obtained at the cut-off point of 50. In conclusion, even under hot and dry climate conditions, it is possible that Leptospira spp. can spread through alternative routes such as venereal transmission; moreover, a cut-off of 50 is recommended for the serological diagnosis of cattle from the Caatinga biome.Bovine leptospirosis causes economic losses and raises public health concerns. It is possible that there are peculiarities in the epidemiology of leptospirosis in regions with a semiarid climate, such as the Caatinga biome in Brazil, where the climate is hot and dry, and the etiological agent require alternative routes of transmission. This study aimed to close knowledge gaps to the diagnosis and epidemiology of  Leptospira species, has several hosts, and exposure ensues through direct contact with infected animals or indirectly via water and soil contaminated with urine [Leptospira spp. This occurs despite the hot and dry climate conditions of this region, which are adverse to the survival of this bacterium in the environment. Leptospirosis is widespread globally and is among the diseases that impair livestock production rates. It raises public health concerns because of its zoonotic impact and that, even after more than a century of research, the vaccines available do not confer lasting immunity or cross-protection between serovars . The ageth urine ,3. Transth urine . The losth urine . In cattth urine . Studiesth urine ,10,11,12th urine ,14 preseLeptospira spp. serovars.Among the methods for diagnosing leptospirosis, there are indirect methods that investigate the immune response of the host and direct methods that target the detection of leptospires or their DNA. The microscopic agglutination test (MAT) has been recommended as a serological test by the World Organization for Animal Health (OIE) because of its capacity to demonstrate the most likely infecting serogroup . MoreoveLeptospira spp. as well as short seroconversion periods [Given that there are differences in incidence related to climatic conditions  and to i periods ,22. Furt periods ,12. ThusTamandua tetradactyla), preá (Cavia aperea), mocó (Kerodon rupestris) and cachorro-do-mato (Cerdocyon thous), offers unique epidemiological conditions that can influence the occurrence of infectious diseases such as leptospirosis. Therefore, this study aimed to evaluate the serological, molecular and microbiological methods applied to the diagnosis of leptospirosis in cows from the Caatinga biome in Northeastern Brazil.Low rainfall and high temperatures characterize the semiarid region of Brazil, and when associated with the peculiarities of the existing vegetation, the Caatinga, a biome exclusive to Northeastern Brazil with abundant wild fauna—such as tamanduá-mirim  in the state of Paraíba, Northeastern Brazil. The animals originated from areas located in the Caatinga biome and were slaughtered within a maximum period of two days. This biome is an exclusively Brazilian biome and has a semiarid climate characterized by long periods of water scarcity and stunted vegetation. The climate is hot and dry, with a rainy season in summer/autumn and rainfall concentrated between March and April. However, precipitation may occur at any time between January and May. Droughts can last for more than a year, resulting in a negative water balance plus high solar radiation ,24. The The minimum sample size was determined using the following formula for proportion analysis :n=p0×q0×n = minimum sample sizeα/2 = 1.96 Z1−β = 1.64 ZP0 = 33% (reference proportion for PCR positivity) [itivity) P1 = 61.40%  [ in PCR) q0 = 1−p0q1 = 1−p1According to these parameters, a minimum of 37 animals were needed to assess true prevalence within a confidence interval of 95%; however, 42 cattle were used. They were all healthy female, aged greater than or equal to 24 months, cross-bred and had no history of vaccination against leptospirosis. According to the data taken from animal movement forms held by the State Veterinary Service of Paraíba, these female cattle came from rural farms located in the semiarid region, from municipalities belonging to two federative units: Paraíba  and Pernambuco .The blood samples were collected from jugular vein using 8 mL labeled sterile tubes containing a coagulation activator just prior to slaughter of the animals. After collection, the tubes were sent to the laboratory, where they were centrifuged at 1512 g for 10 min, and the serum samples were stored in microtubes at −20 °C.Leptospira spp. The tissues were immediately fragmented by using autoclaved surgical materials and sterile slides for each tissue. After that, the fragments were immediately transferred to a specific room in the slaughterhouse, where there was a Bunsen burner, and then they were deposited onto autoclaved Petri dishes while avoiding contact between the fragments. The material was fragmented into smaller portions of ≈ 2 gm (in duplicate), placed into DNA and RNA-free microtubes and stored at −20 °C for molecular detection and bacteriological isolation. Sterile swabs were used to collect vaginal fluid directly from the cervical–vaginal region, and urine was taken by cystocentesis during evisceration, using 5 mL sterile syringes. Both materials were also stored in duplicates in DNA- and RNA-free microtubes with 0.5 mL of phosphate-buffered saline [The bladder, kidney, ovary, uterine tube, uterus and placenta (placentome region) were collected for the direct detection of d saline .Leptospira spp. antibodies using a collection of 24 serovars belonging to 17 different pathogenic serogroups of five species provided by the Laboratory of Veterinary Bacteriology of the Fluminense Federal University, Niterói, Rio de Janeiro, Brazil, originating from the Pasteur Institute, France. The Leptospira species and serovars were L. interrogans serovars Copenhageni, Canicola, Autumnalis, Wolffi, Hardjoprajitno, Icterohaemorrhagiae, Pomona, Kennewicki, Hebdomadis, Pyrogenes, Bratislava and Australis; L. santarosai seorovars Guaricura, Shermani and Canalzoni; L. borgpetersenii serovars Javanica, Tarassovi, Ballum, Mini and Castellonis; L. kirschneri serovars Grippotyphosa and Cynopteri; L. noguchi serovars Panama and Lousiana [The microscopic agglutination test (MAT) was used to detect anti-Lousiana . The serLousiana .Immediately after the collection, three drops of urine, the swab with vaginal fluid and approximately 2 gm of each tissue were inoculated into tubes containing 5 mL of liquid EMJH medium  with amphotericin B (0.05 mg/mL), 5-fluorouracil (0.1 mg/mL), fosfomycin (0.4 mg/mL), trimethoprim (0.2 mg/mL) and sulfamethoxazole (0.4 mg/mL) . After 2LipL32, specific to pathogenic leptospires and, therefore, of public health importance, was amplified with LipL32-45F (5′-AAG CAT TAC CGC TTG TGG TG-3′) and LipL32-286R (5′-GAA CTC CCA TTT CAG CGA TT-3′) primers [Leptospira interrogans serovar Copenhageni, Fiocruz L1-130 (ATCC BAA-1198) was used as positive control, and ultrapure water was used as a negative control.The Dneasy Blood and Tissue Kit  was used to extract the DNA from the urine, vaginal fluid and tissues, as well as vaginal fluid and urine EMJH cultures that exhibited the growth of leptospires under dark-field microscopy examination, according to the manufacturer’s recommendations. The gene  primers  followin primers . PrimersLipL32-45F and LipL32-286R primers [http://www.ncbi.nlm.nih.gov, accessed on 19 November 2022) using the BLAST tool . SeaView4 software [http://tree.bio.ed.ac.uk/software/figtree/, accessed on 26 November 2022), as viewed through the FigTree v1.4.3 . The phylogenetic reconstruction included Leptospira sequences for comparison. primers  were use primers  were use primers , and thep ≤ 0.05). The sensitivity and specificity of MAT at the cut-off points of 50, 100, 200 and 400 were calculated using DAG_Stat software [The proportions of positive animals and samples were compared by using the chi-squared test with Yates’ continuity correction or Fisher’s exact test using the BioEstat 5.3 software  with a 5software , deemingLeptospira spp. antibodies at the cut-off point of 50, and Sejroe, Tarassovi, Australis, Ballum, Djasiman and Hebdomadis were the reactive serogroups. At the cut-off of 100, 16 animals  were seroreactive for Sejroe, Tarassovi and Hebdomadis serogroups, and at titer of 200, 10 animals  reacted for the serogroups of Sejroe, Tarassovi and Hebdomadis, and eight animals  were seroreactive for Sejroe, Tarassovi and Hebdomadis at the titer of 400. Overall, the most frequent serogroups were Sejroe (55.6%) and Tarassovi (22.2%), with titers ranging from 50 to 1600  presented with anti- to 1600 , considep < 0.05) between urine and kidney, urine and uterus, uterus and uterine tube, and the placenta differed from all of the biological materials . Among the 309 samples, PCR detected leptospires DNA in 90 (29.1%). The most frequent PCR positive samples were the placenta (13 samples: 86.7%), uterus (17 samples: 40.5%) and kidneys (14 samples: 33.3%). There were statistically significant differences  from the kidney, uterus and ovary, and the placenta differed from all of the biological materials , and the biological materials with the highest frequencies were uterus (eight samples: 19.1%) and placenta (two samples: 13.3%).In microbiological culture, the pathogen was visualized in at least one sample in 29 animals , and 73 cultures (23.6%) out of 309 were found to be positive, highlighting the placenta (10 samples: 66.7%), uterus (13 samples: 31%), ovary (11 samples: 26.2%) and kidneys (11 samples: 26.2%). Urine differed statistically  and two from cultures (kidney and ovary) from different animals. These samples showed a 99% similarity with tersenii .Leptospira spp. infection, with high sensitivity (100.00%) and specificity (84.6%) deeming microbiological culture results as gold standards, especially for detecting leptospiral DNA from the uterine tube, with 100% sensitivity and specificity , suggesting that there are different sources of infection, although Sejroe (55.6%) and Tarassovi (22.2%) were more prevalent. In other investigations conducted in semiarid regions, Sejroe (58.17%) was the most prevalent serogroup, followed by Icterohaemorrhagiae (17.32%) and Australis (4.58%) [Leptospira suggests that contact with other animals exists, but there is the possibility that cattle without signs and symptoms of Leptospira spp. infection may carry and spread other strains within the species (adaptive process) [The high frequency of seroreactivity found (64.3%) indicates that, even under adverse climatic conditions, jroe 58.1% was the (4.58%) . Surveysjroe 58.1% was the (4.58%) ,36,37; 7 (4.58%) ,39; 30.3 (4.58%) ; 47.01%  (4.58%) ,42,43,44 (4.58%) ,46. The Mus musculus). Asymptomatic mice harbor bacterium in their kidneys, which makes them an important source of infection for humans or animals [The contrast between unfavorable climatic conditions  and a significant proportion of seroreactive animals, especially regarding the Sejroe serogroup, provides evidence for intraspecies transmission, which is less dependent on environmental factors because cattle are maintenance hosts ,48. Tara animals . The conp < 0.05) that demonstrated that leptospires accumulate preferentially in the placenta, uterus and kidneys when compared to the urine. This can be explained by the fact that the size, morphology  and translational motility of the agent facilitate access to these organs, allowing it to escape the immune system due to the physical barrier that prevents contact between antibody and antigen molecules. The broad presence of leptospires in the reproductive tract reinforces that this is a site of extra renal bacterial colonization in cattle, which can act as adapted hosts, as has already been elucidated in other studies [PCR data can generate difficulties in interpreting the interrelation of results, as some factors, such as the amount of bacterial DNA in the sample and its quality, may not be favorable ,52. Lept studies ,54.When analyzed from an epidemiological perspective, it is clear that, especially in the Caatinga biome, where climatic conditions are adverse for bacterial survival in the environment, extra renal colonization by leptospires is a strong indication that, in this region, venereal transmission and vertical transmission of leptospires are alternative routes in contrast to transmission through the urine, in which the external environment is part of the epidemiological cycle. The PCR results confirmed that, for animals maintained under field conditions, as well as urine, vaginal fluid could be valuable for identifying carriers both in slaughterhouses and farms.Leptospira spp. was isolated from 29 animals (69%). By comparing the positivity rates between the different materials, there were statistically significant differences (p < 0.05) that demonstrated the tropism of the microorganism for the placenta, uterus, kidney and ovary, while the frequency of occurrence in the urine was lower, as explained earlier. The results from the cultures revealed that working on microbiological isolation, using tissue from the placenta, uterus, kidney and ovary can increase the chances of success. However, considering that access to tissue from the uterus, kidney and ovary is difficult, the collection of such material is appropriate in postmortem cases. Leptospires-specific DNA was identified in 19 cultures, among which there was one kidney sample and one ovary sample from different animals. The isolation and characterization of autochthonous strains are important with regard to understanding epidemiology and refining diagnostic tools. Moreover, this introduces the possibility of discovering new species and/or variants. If these circulating strains can be incorporated into vaccines, greater protection for animals in the region can be provided [provided .L. borgpetersenii, which belongs to the pathogenic clade and, according to virulence, to subgroup 2, along with the species L. santarosai, L. mayottensis, L. weilii and L. alexanderi [L. borgpetersenii causes early embryonic loss and estrus repetition, resulting from uterine inflammation and damage caused by embryo invasion [The sequenced DNA from four samples demonstrated 99% similarity with exanderi . L. borginvasion . The ideinvasion ,60,61,62Leptospira spp., except in microclimates, which would have been unlikely in the present study, considering that the animals were living on several different farms.Out of the 31 positive animals on PCR, 15 showed positivity in both the reproductive and urinary tracts, 13 were positive only in the reproductive tract and three only in the urinary tract. Animals with urogenital tract involvement potentiate the diffusion of the agent, especially in the rainy season of a semiarid region, due to the possibility of simultaneous transmission via the urinary, venereal and congenital/transplacental routes. The occurrence of a bovine genital leptospirosis syndrome has been proposed , and it Leptospira spp. infection and showed high sensitivity (100%) and specificity (84.6%). This reinforces the importance of molecular methods, and many factors contribute to this accuracy, e.g., the extraction kit, thermostable DNA polymerase, laboratory equipment and operating procedure. Reports of leptospiral DNA detection in animals negative upon MAT are common, and if the animal presents with a high antibody titer, the outcome of PCR tends to be negative. The high sensitivity of PCR often eliminates the need for isolation as a confirmatory result, and it is suitable for emergency situations, given that it is fast and enables early diagnosis based on blood analysis during leptospiremia [Under the conditions of the Caatinga biome, molecular detection proved to be reliable for diagnosing pathogenic spiremia .Leptospira spp. infection. Similar results were also found by our research group in sheep from Brazilian semiarid regions [The findings of this research reinforce the hypothesis that, although very useful for herd diagnosis, serology may be an insufficient tool to identify carriers individually, thus making it necessary to detect the presence of the agent to identify and safely treat the carriers . Therefo regions ,12.Leptospira spp. infection are imperfect, and the appropriate combination of methods and specimens for each stage of infection improves diagnostic accuracy. The principle behind MAT is simple, but it requires maintaining a panel of live leptospires, representing a biological risk and restricting its practice to specialized laboratories [It is worth mentioning that the diagnostic tests for ratories . In PCR,ratories ; in addiratories . Microbiratories ; in addiratories .Our data show that a cut-off point of 50 may be more suitable for cattle from the Caatinga biome, and, in this scenario, high sensitivity values are essential to reduce the number of false-negative results upon MAT. In addition, given the results of this survey, cut-off points below 50 should also be considered as they are relevant in other species, such as sheep and swine. This is very important from epidemiological and infection control points of view as it avoids the maintenance of infected animals in the herd since subclinical infections are very common .It is important to emphasize that although the recommendation by the World Organization for Animal Health (OIE) of the use of cut-off point of 100 for the serological diagnosis of leptospirosis in cattle , this stLeptospira spp. infection in cattle from the Caatinga biome of Brazil. The results indicate that, even under the adverse environmental conditions of the Brazilian Caatinga biome, leptospires may survive and propagate through alternative routes of transmission, such as sexual pathways, and the high proportion of PCR-positive cows in the genital tract highlights the possible role of females in venereal transmission. Moreover, a cut-off of 50 should be considered for the serological diagnosis of cattle from the Caatinga biome. The study also shows that cows are commonly exposed to leptospires in the Caatinga biome, and this constitutes a One Health-based concern, demonstrating the importance of broad studies where large numbers of humans and animals coexist when investigating zoonotic infections and when planning and implementing control measures for cattle-associated leptospirosis.This study provides important information relating to the diagnosis and epidemiology of


Results: Significant differences in chronic respiratory pathology are observed between patients with and without obesity. The inflammatory markers CPR, ferritin, NLR, and PLR are elevated during the evaluated period, while changes in leukocyte populations are present on day 1 (eosinophils) and day 3 (lymphocytes). Finally, a persistent elevation of D-dimer level is observed, presenting significant differences on day 7 between patients with and without obesity. Obesity had a positive correlation with admission to the critical patient unit, invasive mechanical ventilation, and length of hospital stay. Conclusion: Patients with obesity hospitalized for COVID-19 present marked elevations of inflammatory and hemostasis parameters, with a correlation between obesity, changes in laboratory biomarkers, and the risk of adverse clinical outcomes also observed.Background and aims: It is reported that patients with obesity are more frequently hospitalized for COVID-19, and evidence exists that obesity is a risk factor, regardless of other comorbidities. The objective of this study was to evaluate the association of obesity with changes in laboratory biomarkers in hospitalized Chilean patients. Materials and methods: A total of 202 hospitalized patients (71 with obesity and 131 without obesity) with a diagnosis of COVID-19 were included in the study. Demographic, clinical, and laboratory  data were obtained. We performed a statistical analysis, assuming significance with a value of  There cases , althougre cases ,12,13. Ire cases . This haglobally . This isglobally ,16,17. Aglobally . Moreoveglobally ,19. In tally [2) . For thi2) and 131 did not present obesity, with a diagnosis of COVID-19, and who were treated at the Hospital Dr. Hernán Henríquez Aravena, Temuco, Chile, were included. Exclusion criteria considered children under 18 years of age, pregnant women, and low-weight patients. All the patients were diagnosed according to established criteria (WHO interim guidance) by a reverse transcriptase real-time polymerase chain reaction (RT-PCR) assay of nasal and pharyngeal swab specimens, between March 2020 and April 2021. The severity of COVID-19 was defined according to the WHO Clinical Progression Scale [Through a retrospective study, a total of 202 hospitalized patients (>18 years old) of whom 71 had obesity  and was carried out in accordance with the ethical requirements established in the Declaration of Helsinki.® R Max coagulation analyzer . Measurements of inflammatory biomarkers were performed using the cobas 8000 series modular analyzer (Roche Diagnostics), by immunoturbidimetric assay (PCR) and electrochemiluminescence (ferritin).For the group of subjects included in the study, epidemiological, demographic, and clinical data were obtained from the medical history of each patient. Demographic variables, signs, and symptoms were recorded. In addition, laboratory biomarker results were obtained from the laboratory information system (LIS), at days 1, 3, 7, and 15 of hospitalization. All the samples for day 1 were collected within the first 24 h of hospital admission. The results were obtained by complete and differential blood count of leukocyte population through hematology analyzer MINDRAY CAL 6000 , obtaining the neutrophil–lymphocyte ratio (NLR) and platelet–lymphocyte ratio (PLR). Hemostasis tests  and immunoturbidimetric (D-dimer) assays, using a STAp < 0.05) between the population with and without obesity that presented some comorbidity or clinical manifestation, whilst Student’s t-test was used to evaluate differences (p < 0.05) over time  of the variations of laboratory biomarkers. The relationship between obesity and laboratory parameters was evaluated over time  using Pearson’s correlation, and categorical principal component analysis (CATPCA) was used to determine a pattern over time  of the patients with obesity with respect to the evaluated laboratory parameters. Statistical analyses were performed in the IBM SPSS Statistics for Windows program .Chi-square was used to determine significant differences (p = 0.001).A comparison was made between the population with or without obesity in terms of comorbidities and clinical manifestations . On evalp = 0.041) and dyspnea , whereas for clinical manifestations such as anosmia and dysgeusia there is a greater predominance in patients with obesity . In addition, no significant differences were observed regarding symptoms before hospitalization between the groups (5.43 + 3.8 days in patients with obesity vs. 4.73 + 5.1 days in patients without obesity (p = 0.162).In clinical manifestations, patients without obesity represent a segment with a greater number of cases reported for coughing . On the other hand, no significant differences were observed in PT and APTT between the groups; however, a sustained elevation of D-dimer was evidenced. On day 7, patients with obesity had higher D-dimer values (7.7 μg/mL) than those without obesity (2.3 μg/mL).p = 0.038) and patients with obesity; likewise, a significant direct correlation is found between patients with obesity for the neutrophil parameter . By day 3, there is a statistically significant direct correlation between patients with obesity and WBC . On day 7, a significant direct correlation persists between the patients with obesity and WBC , as well as a significant direct correlation with NLR  and neutrophils . On the other hand, there is a statistically significant inverse correlation with patients with obesity for the MCHC parameter at this time point . By day 15, no significant direct or inverse correlations between the laboratory tests and the patients (with and without obesity) are observed.Pearson’s statistical analysis was used to determine significant correlations between the laboratory parameters and obesity in patients , highligSince obesity constitutes a recognized risk factor for severity and mortality in individuals infected with SARS-CoV-2, in this retrospective study, we discuss the association of obesity with changes in laboratory biomarkers in Chilean patients hospitalized for COVID-19.p = 0.001). The results show that patients without obesity have a high frequency of type 2 diabetes mellitus and cardiovascular disease. These results could be explained, at least in part, by the characteristics of the population included in the study, with an important component of the Native American population (Mapuche people) and high multidimensional poverty. Various studies show that the Mapuche population presents a greater susceptibility to diabetes and cardiometabolic risk factors, especially in those who migrate from rural to urban areas. A study shows that conditions such as urbanization and sedentary behavior influence insulin resistance to a greater extent in Chilean Mapuches than in Chileans of European descent [It has been reported that patients with obesity are more frequently hospitalized for SARS-CoV-2 . Additio descent . In addi descent .p = 0.041) and dyspnea , while for symptoms such as anosmia and dysgeusia there was a greater incidence in patients with obesity . To our knowledge, such differences in clinical manifestations within the general population, with or without obesity, have not been described before [When we studied the clinical manifestations, patients without obesity represented the segment with the highest number of cases reported for coughing  and neutrophils   . This asp = 0.043). Mao et al. [In this context, Lu and Wang  describeo et al.  associatp = 0.011). In addition, obesity was observed to be a risk factor for intensive care unit (ICU) admission  and invasive mechanical ventilation (IMV) .A meta-analysis considering 12,591 patients showed that obesity was associated with an increased need for ICU intervention and invasive mechanical ventilation [When evaluating the association of obesity with clinical results, no significant differences were observed regarding death; however, significant differences were observed in admission to the critical patient unit, invasive mechanical ventilation, and length of hospital stay  and day 3 (lymphocytes). Finally, a persistent elevation of D-dimer was observed, presenting significant differences on day 7 between patients with and without obesity. Obesity was associated with admission to the critical patient unit, invasive mechanical ventilation, and length of hospital stay. This study contributes to the knowledge of risk stratification on the prognosis of COVID-19 hospitalized patients with obesity.


Tissue accidents provide numerous pathways for pathogens to invade and flourish, causing additional harm to the host tissue while impeding its natural healing and regeneration. Essential oils (EOs) exhibit rapid and effective antimicrobial properties without promoting bacterial resistance. Clove oils (CEO) demonstrate robust antimicrobial activity against different pathogens. Chitosan (CS) is a natural, partially deacetylated polyamine widely recognized for its vast antimicrobial capacity. In this study, we present the synthesis of four membrane formulations utilizing CS, polyvinyl alcohol (PVA), and glycerol (Gly) incorporated with CEO and nanobioglass (n-BGs) for applications in subdermal tissue regeneration. Our analysis of the membranes’ thermal stability and chemical composition provided strong evidence for successfully blending polymers with the entrapment of the essential oil. The incorporation of the CEO in the composite was evidenced by the increase in the intensity of the band of C-O-C in the FTIR; furthermore, the increase in diffraction peaks, as well as the broadening, provide evidence that the introduction of CEO perturbed the crystal structure. The morphological examination conducted using scanning electron microscopy (SEM) revealed that the incorporation of CEO resulted in smooth surfaces, in contrast to the porous morphologies observed with the n-BGs. A histological examination of the implanted membranes demonstrated their biocompatibility and biodegradability, particularly after a 60-day implantation period. The degradation process of more extensive membranes involved connective tissue composed of type III collagen fibers, blood vessels, and inflammatory cells, which supported the reabsorption of the composite membranes, evidencing the material’s biocompatibility. Regenerative medicine seeks to restore tissues by incorporating cells or growth factors that facilitate recovery . It is aTo repair and replace injured tissues, tissue transplants from the same patient (autografts) or other individuals  are used. However, these treatments have problems associated with their application . In the Typically, scaffolds are designed from synthetic and natural polymers, which offer various advantages and disadvantages during use. Biopolymers have the advantage of being biocompatible and biodegradable, as well as stimulating interaction with the cellular biological environment without generating aggressive responses. However, they have poor mechanical and barrier properties . On the On the other hand, another strategy used to improve the bioactivity of scaffolds is to generate porous three-dimensional nanocomposites by incorporating nanomaterials in the polymer mixtures , which f2, Na2O, CaO, P2O5, and others. However, the most used is 45S5, because its composition does not allow for the production of necrotic tissue or pus [Among the most interesting bioactive materials are nanobioglasses (n-BG), which stimulate a calcium and phosphate layer and advance angiogenesis on the surface of the scaffolds without producing an adverse immune reaction ,13,14,15e or pus ,17. The e or pus ,19,20. Oe or pus . On the other hand, significant benefits have also been reported in the design of nanocomposite scaffolds incorporating essential oils, such as a higher rate of degradation, greater porosity and biocompatibility, and lower inflammatory response ,23. EsseSyzygium aromaticum L.  is widespread in Madagascar, Sri Lanka, Indonesia, and China. It is a plant that produces an essential oil with antioxidant, insecticidal, antibacterial, and antifungal properties, and is classified as safe for consumption (GRAS) by the Food and Drug Administration (FDA), facilitating its application in the cosmetic, food, and perfumery fields [y fields . Accordiy fields . Among ty fields , analgesy fields , anesthey fields , anticany fields ,31,32, ay fields  that proy fields .Despite all the advantages of CS-PVA composites that have previously been reported, including CS/PVA/TTEO tea tree essential oil, which accelerates and stimulates subdermal tissue regeneration after 90 days of implantation without necrotic tissue , the app6 Da), a deacetylation degree of >80%, and a viscosity of 20–300 cP (in 1% acetic acid), according to the provider, while the polyvinyl alcohol showed 90% hydrolysis and contained 95,000 g/mol.All the reagents used in this study were obtained from Sigma, Aldrich. The chitosan (CS) used had a low molecular weight (1 × 102:40%-CaO:6%-P2O5) have been reported elsewhere [The synthesis and characterization of n-BG with a ternary system (54%-SiOlsewhere .Syzygium aromaticum L.) was obtained from Marnys . Essential oil chemical characterization was performed using gas chromatography mass-coupled  with a Cu anode and a wavelength of Kα1 (0.154 nm). The diffraction 2θ angles were between 5–50°, with a scan speed of 2.63 s, a scan rate of 2 degrees/min, and a step size of 0.01°.−1 range using an FT-IR-8400 instrument  with a spectral resolution of 4 cm−1 and 32 scans.On the other hand, attenuated total reflectance–Fourier transform infrared spectroscopy (ATR-FTIR) was used to determine functional groups in the 500–4000 cmg), melting temperature (Tm), and crystallization temperature (Tc) were calculated from the midpoint, endothermic, and exothermic peaks, respectively. The data were processed using TA Instruments Universal Analysis Software 2000 version 4.5A.For thermogravimetric analysis (TGA), we used a NETZSCH TG 209 F1 Libra instrument . The samples were heated from 25–800 °C at 10 °C/min . The thermal transition of the pieces was studied under differential scanning calorimetry (DSC)  using nitrogen, with 25–250 °C heating at 10 °C/min. The glass transition temperature  in the secondary electron mode at 20 kV.The preliminary biocompatibility of the four composite formulations was assessed through the subcutaneous implantation method in a biomodel , as previously reported ,35.For the macroscopic examination and trichotomy, the samples were preserved in plastic containers after formalin and alcohol dehydration. Then, a xylol diaphanization and infiltration with paraffin using the Auto-technicon Tissue Processor™ from Leica Microsystem  process was utilized. The Thermo Scientific™ Histoplast Paraffin™ kit, provided by Fisher Scientific in Waltham, MA, USA, facilitated the cutting process of the samples for histology studies.Paraffin blocks with the immobilized tissues were cut at a 5 µm thickness with a Leica microtome instrument. The histology studies used the hematoxylin–eosin (HE) and Masson’s trichotomy (MT) staining techniques. A Leica DFC 295 camera and Leica DM750 optical microscope were used for the image analysis with the Lecia Microsystems 4.12.0 software .The Biomedical Experimentation Animal Ethics Committee (CEAS) at the Universidad del Valle provided ethical approval for the study, which adhered to the guidelines established by the “Animal Research: Reporting of In Vivo Experiments” (ARRIVE) guidelines . Our previous work reported the synthesis and characterization of the n-BG 45S5 type .trans-β-Caryophellene (4.0%) as the main components. Several authors have reported that essential oil compositions are usually influenced by age, soil composition, plant organs, edaphic conditions, genetic, extraction, and condition methods [S. aromaticum L. contains approximately ~20% EO, classified by the Food and Drug Administration (FDA) as safe (GRAS) and used in several cosmetic and food goods applications. The main component of CEO has been identified as eugenol (at least 50%), but eugenyl acetate, β-caryophyllene, and α-humulene account for the remaining 10–40%, with different biological activities reported for each [The GC-MS chemical composition of the CEO using the NIST and Wiley databases was based on the molecular weight and retention index. The chemical composition is summarized in  methods . S. aromfor each ,37,38. Cfor each . It is afor each ,40. Alsofor each ,40. The for each . There ifor each .−1 related to the -OH groups present in both PVA and CS [2 at 2940 cm−1 was observed. In addition, the tension band of the carbonyl group (C=O) at 1659 cm−1 was related to the amido bonds I and II present in the CS [−1, as well as the flexural band of the C=O group at 1100 cm−1, were elucidated. Minor changes were observed concerning the formulations, especially in formulations F3 and F4, which contained CEO in their formulations. In this sense, variations in the intensity of the C=C band located at 1560 cm−1 are probably due to the presence of eugenol, the main component of CEO [A and CS . Likewisn the CS ,43, and t of CEO ,45. In aThe different diffraction peaks of the CS/PVA/CEO/n-BG/GLY composites are shown in TGA allows the analyst to provide information about the formulation according to the material’s thermal properties. For this purpose, the thermal degradation by weight caused by increasing temperature was evaluated. On the other hand, introducing glycerol and CEO to the F3 formulation (22.5%CS/70%PVA/5.0%CEO/2.5%GLY) was observed to decrease the thermal stability. In contrast to the F2 formulation (22.5%CS/70%PVA/5.0%n-BG/2.5%GLY), the addition of only Gly and n-BGs increased its stability compared to the control formulation, F1 (27.5%CS/70%PVA/2.5%GLY). This observation was because the introduction of n-BGs caused interactions between the hydroxyl groups found in the PVA, CS, and n-BGS components ; furtherm1) and PVA (Tm2), as well as the crystallization temperature (Tcc) attributed to n-BGs and the glass transition temperature (Tg) for each of the formulations, can be observed in The different thermograms showing the melting temperature of chitosan  , once euWhen performing histological analyses, it was observed that, 60 days after implantation, the implanted material had a high rate of resorption and replacement by connective tissue, in addition to the presence of adipocytes. Using Gomori trichrome (GT) staining, the presence of type III collagen in the implantation area was evident, as was the absence of a fibrous capsule A,B. FiguIn the subdermal implantation model , the areIn formulation F2, the decrease in the percentage of chitosan and the incorporation of n-BG affected the material resorption process. For this reason, after 60 days, large fragments of the composites in the process of degradation and resorption could be seen. Despite fragments of the material being observed, the healing process was carried out without a fibrous capsule in the implantation area. Like F1, this area appeared to be integrated with the rest of the tissue through type I collagen fibers from the muscle tissue A.In the implantation area , fragmenFormulation F3 differs from F2 in that the n-BG component was replaced by 5% CEO. An important histological finding was the presence of a fibrous capsule with parallel fibers surrounding the implantation area A. Using In As with the results of the previous implantation, after 60 days, a fibrous capsule delimiting the implantation area was observed, along with some fragments of the implanted material. The F4 formulations studied herein had CS, PVA, and Glycerol as standard components. CS is considered a biocompatible material with poor mechanical properties  but exceIn the studied formulations, the concentrations of PVA and glycerol always remained fixed; these are materials used as plasticizers that, in addition to improving the mechanical properties of the CS, are biocompatible . HoweverWith the other formulations, it was observed that by decreasing the percentage of chitosan, the degradation and reabsorption process of the material became slower, which explains the presence of remnants of the material in the results observed for formulations F2, F3, and F4. The presence of remaining material after 60 days was also an indication of the stability of the composites. When using these composites as scaffolds, the degradation and resorption process must be carried out progressively. At the same time, wound healing occurs since it aims to stimulate tissue formation to recover tissue architecture, which is achieved through controlled degradation .Histological results also change when new components are incorporated. In this way, integrating n-BG into F2 and F5 stimulated the formation of connective tissue with a different appearance than that observed in formulations F1 and F3. In general, n-BG have been widely used in scaffolds for tissue regeneration due to their bioactivity, which allows for the release of calcium and phosphorus . This coIn Clove essential oils have been used as analgesics and antimicrobials in different applications ,70. It hAltogether, the histological results of the skin implantations show that the different formulations allowed the healing process to be carried out adequately. F1 is the formulation that presented the highest percentage of CS (27.5%), which induced a faster inflammatory response. It generated an almost complete degradation of the material in the absence of a fibrous capsule and with tissue recovery, as observed in The in vivo reabsorption of chitosan depends on several factors, such as molecular weight and the preparation method. Still, it is accepted that the inflammatory response is a mild and transient reaction to a foreign body. Some view chitosan as an inert biomaterial that induces no more than a gentle, brief foreign-body reaction . By reducing chitosan and incorporating n-BG in F2, the inflammatory response changes, and a more significant presence of inflammatory infiltrate with less reabsorption of the material can be observed, but the fibrous capsule is still absent. Despite fragments of the material persisting, the absence of the capsule can be explained by the presence of the bioactive n-BG. This material is recognized for accelerating tissue healing by stimulating the more rapid formation of granulation tissue and a deposit. It also promotes accelerated collagen formation and angiogenesis .In F3, the CS percentage of F2 was preserved, but the CEO replaced the n-BG. An interesting finding was the presence of a fibrous capsule that appeared to transform into scar connective tissue occupying the implantation area as the material was degraded and reabsorbed . In addiOn the other hand, formulation F4 is the only one that contains CEO and n-BG. The implantation results show a faster resorption process than that of F3. Still, both present some features in common, such as the formation of highly organized connective tissue in the implantation area that seems to be formed at the expense of the fibrous capsule. It is thick and contains a large number of blood vessels. This early formation of an organized connective tissue can be explained by the effect of the release of ions from the n-BG with the capacity to positively affect the migration of epithelial cells, angiogenesis, and proliferation of fibroblasts .In addition, as expected, CEO-containing membranes show the best signs of connective tissue regeneration and, therefore, a normal healing process. The above is probably because the addition of CEO through its main component (eugenol) promotes tissue healing through a foreign body mechanism. Several studies have shown that eugenol promotes protective action on liver cirrhosis by preventing liver cancer characterized by abnormal cell proliferation and decreasing oxidative stress, which could be the protective mechanism against liver cirrhosis . On the −1 region corresponding to C=C bonds of the aromatic ring, mainly due to the contribution of eugenol present in the essential oil. Furthermore, we observed that the inclusion of n-BGs promoted increases in the thermal properties and influenced the heat-resistant properties of CEO, which was particularly evident in formulations F3 and F4. The morphological examination conducted using scanning electron microscopy (SEM) revealed that the incorporation of CEO resulted in smooth surfaces, in contrast to the porous morphologies observed with n-BGs. Subdermal implantation of the CS/PVA/CEO/GLY/n-BG nanocomposites was assessed in a biomodel for 60 days, revealing high biocompatibility in this tissue. The excellent healing, hair recovery, and tissue remodeling without any normal immune response upon material degradation and resorption, along with the raising of type III collagen fibers, blood vessels, and inflammatory cells, demonstrated the material’s biocompatibility. We believe that these results are promising for a safe and eco-friendly approach to regenerative medicine.In this study, we synthesized four membranes comprising CS/PVA/CEO/GLY/n-BG composites, which exhibited enhanced thermal stability compared to their components. CEO and n-BGs were confirmed through various characterization methods such as FTIR, XRD, TGA, and DSC. In FTIR analysis, we observed a sharp increase in the bands in the 1200–700 cm


Background: Suicide methods chosen by victims are particularly critical in suicide risk research. To differentiate suicide deaths, it is usual to categorize them as violent and nonviolent depending on the detrimental method chosen by the victims. Caustic ingestion, for example, is traditionally considered as a nonviolent suicide method. It results in severe consequences for the human body and it is associated with high levels of lethality. Methods: In this study, we retrospectively analyzed suicides that occurred between 1993 and 2021 in Milan  and that underwent autopsy. We compared a sample of 40 victims that ingested caustic substances with a sample of 460 victims of other chemical ingestion, and a sample of 3962 victims from violent suicide. Univariate analyses and univariate logistic regression models were performed. Suicides from caustic poisoning were significantly older, had a higher mean number of diseases and were more affected by psychiatric diseases compared to other chemical ingestion victims. By contrast, caustic suicides, compared to violent suicides, had a more balanced gender ratio, a higher mean number of diseases, were more affected by psychiatric diseases, had a higher rate of complex suicides , and had victims who died more frequently inside instead of outside. In logistic regression models, age was the only feature differentiating caustic from other chemical ingestion suicides while the features differentiating caustic from violent suicides were gender, mean number of diseases and suicide place. Conclusions: Suicides by caustic ingestion showed substantial differences compared to violent suicides, with a higher severe profile. However, some differences were reported comparing caustic ingestion to other chemical ingestion as well. Thus, we argue whether it is more appropriate to differentiate the suicidal ingestion of caustics from both violent and nonviolent suicide methods. Suicide is a leading public health concern causing the death of about 4000 people each year in Italy  and 700,Many efforts have been made in research to identify risk factors that drive people to suicide. Biology might play a key role determining a certain degree of familiarity for specific genes that predispose individuals to suicide ,4. TogetIngestion of corrosive agents with the intention to die by suicide is not rare : In Ital(1)We first wanted to investigate the differences in terms of socio-demographic, clinical and suicide-related features between caustic ingestion and other chemical ingestion. In research, caustic ingestion and chemical ingestion are commonly treated as the same suicide method, although the similarities and differences between these methods have not yet been explored in depth.(2)In the second analysis, we compared caustic ingestion victims to violent suicides to better understand the relationship between these two suicide methods. Similar to step (1), we analyzed socio-demographic, clinical and suicide-related features.Typically, the literature treats nonviolent methods as a homogeneous cluster that include, among others, suicide by substance poisoning such as pesticide ingestion, gas poisoning, caustic ingestion, and overdose ,12,22. HIn this study, a retrospective analysis was carried out comparing three groups of suicide cases. Group 1 included 40 cases that occurred due to ingestion of caustics. To classify caustics, we followed Zoja’s classification of caustics . See TabThe groups were compared in terms of sociodemographic features, such as gender, age, and ethnicity; to simplify, ethnicity was generally grouped in white Caucasian vs. other ethnicities. The groups were also compared in terms of clinical features, occurrence of both psychiatric and organic disorder(s); comorbidities; medication assumption; and in terms of psychotropic medication, such as antidepressants, anxiolytics, benzodiazepines, sedatives, and non-psychotropic medication, such as chemotherapeutics, cardiotonics, antiretrovirals, antihypertensives, anticonvulsants, and hypoglycemics. Moreover, the groups were compared in terms of suicide-related features, suicidal ideation and/or attempts, suicide place, simple vs. complex suicide, causes of death  and suicide place, the latter clustered in inside  and outside . Medico-legally speaking, suicidal ideation was defined as having verbally expressed to someone the intention to die by suicide; instead, previous suicide attempts were defined as having engaged in concrete acts of self-harm. Indeed, as it is usually shown in the literature, simple suicides and complex suicides have here been distinguished ,29,30: Fp ≤ 0.05.The IBM SPSS statistics 27 software  was used for statistical analyses. The groups were compared with regard to the socio-demographic, clinical, and suicide-related variables. We tested the normal distribution of the variables. Since all the continuous variables were not normally distributed, we used the Mann-Whitney U test only, while Chi-square test was applied for categorical variables. Univariate logistic regression models were used as well. We included in the models (Group 1 vs. Group 2 and Group 1 vs. Group 3) the variables that were found to be different among the groups in the univariate analyses. The analysis was conducted by setting statistical significance at The subjects involved in this study underwent a judicial autopsy at the Institute of Legal Medicine of Milan to identify the cause of death. Data collecting, sampling and subsequent forensic analysis were authorized by the public prosecutor. Therefore, data were acquired as part of a forensic judicial investigation and in accordance with Italian Police Mortuary Regulation. In accordance with Italian law, ethical approval is not required in these cases, however, the anonymity of the subjects must be guaranteed.n = 40) and of victims from chemical ingestion of other substances .The sample consisted of victims from caustic ingestion . However, Group 1 and Group 2 did not differ in terms of sex (χ2 (1) = 2.83, p = 0.09) and ethnicity (χ2 (1) = 0.11, p = 0.74). See The cases of Group 1 were older than Group 2 ones . Group 1 was significantly more affected by psychiatric disorders (χ2 (1) = 4.0, p = 0.05) than Group 2. No further significant differences have been found between the two groups. Details about the organic diseases are presented in As shown in 2 (1) = 0.04, p = 0.84), previous suicide attempt(s) (χ2 (1) = 0.23, p = 0.64), and the method of suicide  = 0.22, p = 0.64; Concerning suicide-related features, no significant difference was found between Group 1 and Group 2, referring to previous suicidal ideation (χp: 0.002) (Table 8). Group 1 had a slightly higher mean age compared to Group 2. Goodness of fit estimates are indicated in Explanatory variables showed no multicollinearity, all correlation values less than 0.5. Considering among covariants age, number of diseases, and presence of psychiatric disorder(s), the only variable significantly differentiating Group 1 from Group 2 in the univariate logistic regression model was age  and victims of violent suicide .We compared victims of caustic ingestion  = 11.06, p = 0.001). However, Group 1 and Group 3 did not differ in terms of age  and ethnicity (χ2 (1) = 0.54, p = 0.46). See The two groups differ in sex, with Group 3 predominantly composed of men compared to Group 1 . Moreover, Group 1 shown to be significantly more affected by psychiatric disorder(s) than Group 3 (χ2 (1) = 5.5, p < 0.02). Details about the organic diseases are presented in Statistically significant differences between Group 1 and Group 3 were found on the mean number of diseases , where G2 (1) = 5.27, p < 0.02). Furthermore, concerning the suicide place, the number of suicides inside were significantly higher in Group 1 victims rather than in Group 3 victims (χ2 (1) = 20.09, p < 0.001). See As shown in p < 0.001), mean number of diseases  and suicide place  , presence of complex suicide, and suicide place (inside vs. outside), the variables significantly differentiating Group 1 from Group 3 in the univariate logistic regression model were sex  . ORs shoThe aim of this study was to compare victims of suicide by caustic ingestion to victims of suicide by other chemical ingestion and to victims of violent suicide in order to evaluate the most appropriate categorization of caustic ingestion as a method of suicide. Caustic ingestion is traditionally classified as a nonviolent method of suicide , thus ouPrevious studies have shown the enhancement of suicide risk among the elderly ,33, whicSeveral studies have investigated the relationship between physical illness and suicide risk ,38,39. AAccording to regression analyses, we found no substantial differences between caustic and chemical suicides, the two groups appear not to significantly differ except for age. In fact, similarly to violent suicide ,16, our However, regression analyses of caustic vs. violent suicides showed more relevant differences. For instance, caustic ingestion victims had a more balanced gender ratio. Previous studies frequently associate the male gender with violent suicides and a high fatality of the suicide method ,14,41, bResults point out that suicides by caustics seem to differ from violent suicides, but might also show a more severe clinical picture: Our regression analysis highlights the higher mean number of diseases in suicides by caustics, corroborating the idea that these victims may be exposed to more consistent risk factors. Previous studies already showed that physical illness and functional impairment are relevant elements that increase the risk of suicide ,39,43. IThese considerations corroborate the idea that caustics ingestion as a means of suicide differ from other chemical ingestion nonviolent suicides. Therefore, we suggest that suicides by caustic ingestion should be categorized differently, and perhaps it would be more appropriate to consider them separately from both violent and nonviolent suicide victims following other chemical ingestion. In this study we aimed to search for a more defined and accurate categorization of suicide methods taking into consideration factors which have previously been underestimated, such as the choice of the different substances used to die by suicide. We propose that the vast differences in the effects on the human body from the ingestion of one toxic substance to another are a relevant factor in the subject’s choice of suicide method. The ingestion of caustic substances has one of the highest death rates ,24, and To the best of our knowledge this is the first study comparing suicide deaths that occurred due to caustic ingestion vs. other chemical ingestion vs. violent methods. Also considering the severity of the injuries caused by caustics, we conclude by proposing that caustic ingestion might be better addressed by differentiating it from both violent and nonviolent methods of suicide.This study has several limitations. We acknowledge that the number of victims of caustics poisoning analyzed was limited and this may have impacted our results. Future studies might consider a larger sample of victims of caustics ingestion to report more consistent findings. Moreover, all the data were collected post-mortem, therefore relevant clinical or anamnestic information may have been incomplete.


The present study analyzed the characteristics of phase IV clinical trials in oncology using data from the ClinicalTrials.gov registry. The included trials were conducted between January 2013 and December 2022 and were examined for key characteristics, including outcome measures, interventions, sample sizes, and study design, different cancer types, and geographic regions. The analysis included 368 phase IV oncology studies. An amount of 50% of these studies examined both safety and efficacy, while 43.5% only reported efficacy outcome measures, and 6.5% only described safety outcome measures. Only 16.9% of studies were powered to detect adverse events with a frequency of 1 in 100. Targeted therapies accounted for the majority of included studies (53.5%), with breast (32.91%) and hematological cancers (25.82%) being the most frequently investigated malignancies. Most phase IV oncology studies lacked sufficient power to detect rare adverse events due to their small sample sizes and instead focused on effectiveness. To ensure that there is no gap in drug safety data collection and detection of rare adverse events due to limited phase IV clinical trials, there is a significant need for additional education and participation by both health care providers and patients in spontaneous reporting processes. Cancer is a complex and devastating illness that impacts millions of lives globally every year. Despite significant advances in cancer treatment, it remains the second leading cause of death worldwide . Phase IPost-marketing surveillance (PMS) is the practice where marketed medications are monitored for potential adverse events (AEs) post phase III clinical trials and may consist of active  or passive (spontaneous reporting) processes . PMS stuImportantly, roughly 20% of drugs received additional black box warnings during the post marketing phase, while 4% of the drugs were eventually withdrawn due to safety concerns . PMS stuClinicalTrials.gov is a database provided by the U.S. National Library of Medicine on behalf of the National Institutes of Health (NIH), which stores data concerning both privately and publicly funded clinical studies conducted around the world. It was first launched in February 2000 , and, stIn the present investigation, the general characteristics of registered phase IV oncology clinical trials in oncology were explored by analyzing studies on ClinicalTrials.gov that were initiated over the last decade (2013–2022). The analysis included a thorough investigation of the various types of interventions examined by the phase IV clinical trials, the indications, their outcome measures, funding, study design, and more. In this retrospective observational study, the characteristics of phase IV clinical trials in oncology that were registered with ClinicalTrials.gov with a start date during the timeframe of 1 January 2013, to 31 December 2022 were investigated. To be eligible for inclusion in this analysis, a registered trial must be interventional and evaluate an anti-cancer drug or device or combination of drugs or drug/device in a defined population of cancer patients. Drugs without an anti-tumor effect, such as anesthetics or supportive therapies , behavioral, diagnostic, prevention, or public health studies, were excluded. To facilitate the present analysis, a dataset of phase IV clinical studies from ClinicalTrials.gov, which was downloaded following the search and selection process described below and secured from the website on 31 March 2023, was obtained. Additionally, the authors of the present study designed a database to assist in the current analysis, analogically to previous investigations of ClinicalTrials.gov ,10,11. n = 372). Next, the remaining 872 studies were reviewed independently by two authors  to exclude all studies focusing on non-oncological pathologies  that were not excluded using the above filters and studies focused on broad cohorts with various indications (not specific to cancer patients). Moreover, if studies were not exclusive to or strictly focused on therapeutic agents (defined as drugs or devices) as a treatment for cancer or were non-pharmacologic studies (surgery or radiotherapy), they were excluded as per the a priori eligibility criteria (n = 504). After the eligibility review process, a total of 368 studies remained eligible and were included in this investigation. A list of the included studies with their identification number and title was added as An initial search of ClinicalTrials.gov for phase IV studies was conducted with a start date between 1 January 2013, to 31 December 2022, registered by 31 March 2023. The study selection process is presented in The ClinicalTrials.gov registry requires the sponsors or investigators of a clinical trial to report key trial data, which should include a set of data elements describing the study’s conditions, study design, sponsor, location, enrollment, eligibility criteria, and other protocol information. These data, which describe the general characteristics of the included studies, were extracted and analyzed. The said data included dates of the study, study design, outcome measures, cancer indication, age, sex, size of the cohorts, funding, location, and use of a data monitoring committee (DMC). The extracted characteristics of the clinical trials were later divided into more specific categories by a team of authors , with selected records checked for agreement by a third author (BMH). The outcome measures were divided into three categories: (1) safety, (2) efficacy, and (3) efficacy and safety, in accordance with the outcome measures recorded in the registry record. Due to limited data available, the authors of the present study did not attempt to distinguish between efficacy and effectiveness, thus using the term efficacy henceforth. The interventions were classified by type of anti-cancer therapeutics: (1), chemotherapy, which included alkylating agents, antimetabolites, antimicrotubule agents (mitotic inhibitors), topoisomerase inhibitors, and miscellaneous antineoplastics, (2) immunotherapy, which included cytokines, vaccine therapies, immunomodulatory drugs, and differentiating agents, (3) hormonal therapy, which consisted of antiestrogens, antiandrogens, aromatase inhibitors, luteinizing hormone-releasing hormone agonists, androgen biosynthesis inhibitors, androgens, corticosteroids, somatostatin analogs, and more, (4) targeted therapy, which included monoclonal antibodies, kinase inhibitors , proteasome inhibitors, mammalian target of rapamycin (MTORS), and more, 5) other anti-cancer therapy, (6) herbal/traditional medicine, (7) devices, and (8) other therapies. The indication for treatment  of cancers) was also extracted and analyzed according to system organ class. The included studies were also classified into sample size groups by total number of participants: (1) <300, (2) 300–599, (3) 600–2999, (4) ≥ 3000, and (5) missing. Same-size groups were determined based on the fact that, per binomial and Poisson distributions, enrollment must be greater than 300, 600, or 3000 to have a 95% chance of observing at least one AE with a probability of occurrence of 1%, 0.5%, or 0.1%, respectively  5,10].  other an.  other In cases where a data field was incomplete , we conducted a manual search  to attempt to retrieve the missing information. If the information was still unavailable on the website, we labeled the field as NA (not applicable) or missing. If a study presented an interventional model involving a single group and did not provide information on allocation and blinding, we made the assumption that allocation was non-randomized, and blinding was open. Nonetheless, if the single-arm studies were registered as randomized or blind, allocation or blinding was reported as “Missing/Uncertain/N.A” .p < 0.05.Results were analyzed using descriptive statistics, with categorical data reported as absolute number (n) and relative frequency (%) and continuous variables reported as the median and interquartile range (IQR). Differences in frequencies of a variable between studies grouped by outcome measures were compared using the Chi-square test. Statistical analyses were carried out with Prism 8  with statistical significance set at This study was conducted in compliance with the Declaration of Helsinki and within the terms of local legislation. No ethical committee approval was required for performing this retrospective analysis of publicly available clinical trial registry data. A total of 368 phase IV oncology studies started between January 2013 and December 2022 were included in the present analysis, 35.9% of which were currently reported as active as of 31 March 2023. The sample size of included studies ranged from 0 to 5000. The majority of included studies were small in size, with a median of 90 (IQR: 40–200), with 83.2% of studies consisting of <300 participants. Only one study (0.3%) was powered to detect AEs occurring at a rate of 0.1%, while only 21 (5.7%) and 32 (8.7%) studies were powered to detect AEs occurring at a rate of 0.5% and 1%, respectively. With respect to trial design, single-group assignment accounted for 50.0% of studies, while parallel assignment accounted for 47.0% of studies. All but 8.7% of included studies were open-label. The majority of studies were funded by industry (37.3%), followed closely by universities (or a similar institution) (34.5%) and hospitals (or a similar institution) (30.7%). Nearly half of all studies employed a Data Monitoring Committee . Over one-third of studies (39.4%) had started before submission and registration on ClincialTrials.gov. Exploring geographical distribution, a large majority of studies were conducted in Asia and Pacific regions (52.72%), which participated in more of the included studies than the next two largest regions, North America (17.9%) and Europe (17.1%) combined. Limited studies were performed in Africa (1.6%), Central and South America (3.3%), and the Middle East (1.4%). p = 0.178). No statistically significant differences were observed between safety-only vs. efficacy-only studies . Looking at the reported outcome measures, exactly half of the studies looked at both safety and efficacy, whilst 43.5% reported only efficacy outcome measures, and 6.5% reported only safety outcome measures. The majority of safety-only studies were small in size, with 75% having <300 participants, and, thus, they were not powerful enough to detect AEs occurring at a rate of 1 in 100 participants with 95% confidence. Safety studies were not found to frequently have larger sample sizes  compared to efficacy-only studies , chemotherapy (42.9%), hormonal therapy (17.6%), and immunotherapy (5.9%). Moreover, the characteristics of the study design regarding these interventions were also investigated. Unsurprisingly, the majority of the aforementioned anti-cancer therapies were open-labeled trials  . The intThe size of the sample used in a clinical trial is tightly related to the quality of safety surveillance of the investigated intervention . Phase IInterestingly, the lead sponsor of the analyzed studies varied significantly. The majority of the studies were sponsored by either an industry-related sponsor (37.2%), a hospital (or a similar institution) (30.7%), or a university (or a similar institution) (34.5%). The presence of DMC was also investigated in the present study. The results of the current study show that the DMC was reported in 49.4% of the clinical trials. However, it was absent in almost one-third of studies (29.1%) and not reported or missing in 21.4%. It is clear that taking advantage of DMC, even in open-label clinical trials, can help to monitor their safety, as well as assess their overall risks and benefits. Having a DMC in phase IV clinical trials provides an independent assessment of the safety, scientific validity, and integrity of the studies. If the DMC is used, it should be independent from the study sponsor. This independence enables the DMC to provide credible and unbiased recommendations to the sponsors. It ensures that sponsors can make study decisions without being influenced by their knowledge of interim study results. Alongside monitoring trial safety, the DMC also plays a crucial role in evaluating the trial’s ongoing validity and scientific quality. They ensure that clinical equipoise is maintained throughout the trial and monitor essential aspects, such as participant recruitment, protocol compliance, and data quality . The resThe present analysis demonstrates that the majority of the clinical trials were conducted in Asia and the Pacific region 52.7%). This was also demonstrated by other studies in the past 2.7%. Thi (3.3%). The present study has various limitations that need to be addressed. There is limited available data in the registry, and not all phase IV studies are registered. Hence, some ongoing clinical trials may not have been included in the present analysis. Furthermore, there is a high amount of missing/uncertain data; the sponsors/investigators should be further encouraged to fully complete all fields of the registration form. However, the authors of the present study made a manual screening of all the studied records; this helped address some missing data issues and improved the overall quality of the present report. Lastly, as mentioned earlier, almost 1/3 of studies started before being registered in ClinicalTrials.gov; this can create a bias in both the results of the said clinical trials, as well as the current analysis of study attributes. The present analysis demonstrated that targeted therapies (53.53%) account for the majority of all recent phase IV oncology studies, showing a rapid development of this field. Furthermore, the number of phase IV clinical trials has increased significantly over previous analyses. However, the clinical trials included in the present investigation were dominated by small sample sizes, with most of the studies enrolling less than 300 individuals (83.15%). Hence, most of the phase IV studies are not sufficiently powered to detect rare AEs and instead are focused on efficacy/effectiveness. Due to the limited number of ongoing safety studies, there is a significant requirement for additional education on and participation by both healthcare providers and patients in spontaneous reporting processes, thus ensuring that there is no gap in clinical safety data collection due to limited phase IV clinical trials. This would be especially true with respect to rare AEs, which may not have been identified in clinical development and which were only observed when the drug is utilized in a broader population in routine clinical practice.


Recently, the research on pig intestinal microbiota has become a hot topic in the field of animal husbandry. There are few articles describing the dynamic changes of porcine fecal microbiota and metabolites at different time points from birth to market.In the present study, 381 fecal samples were collected from 633 commercial pigs at 7 time points, including the 1st day, the 10th day, the 25th day, the 45th day, the 70th day, the 120th day, and the 180th day after the birth of swine, were used for microbiome analysis by Illumina MiSeq sequencing methods while 131 fecal samples from 3 time points, the 10th day, the 25th day, and 70th day after birth, were used for metabolome analysis by LC–MS methods.Lactobacillus increased significantly on day 120 compared with the previous 4 time points. From day 120 to day 180, the main porcine fecal microbes were Lactobacillus, Clostridium_sensu_stricto_1, Terrisporobacter and Streptococcus. Clostridium_sensu_stricto_1 and Terrisporobacter increased over time, while Lactobacillus, Escherichia-Shigella, Lachnoclostridium decreased with the time according to the heatmap, which showed the increase or decrease in microbial abundance over time. For the metabolome analysis, the PLS-DA plot could clearly distinguish porcine fecal metabolites on day 10, 25, and 70. The most different metabolic pathways of the 3 time points were Tryptophan metabolism, Sphingolipid signaling pathway, Protein digestion and absorption. Some metabolites increased significantly over time, such as Sucrose, L-Arginine, Indole, 2,3-Pyridinedicarboxylic acid and so on, while D-Maltose, L-2-Aminoadipic acid, 2,6-diaminohexanoic acid, L-Proline were opposite. The correlation between fecal metabolites and microbiota revealed that the microbes with an increasing trend were positively correlated with the metabolites affecting the tryptophan metabolic pathway from the overall trend, while the microbes with a decreasing trend were opposite. In addition, the microbes with an increasing trend were negatively correlated with the metabolites affecting the lysine pathway.For the microbiome analysis, the fecal microbial richness increased over time from day 1 to 180 and the β-diversity of fecal microbiota was separated significantly at different time points. Firmicutes were the main phyla from day 10 to 180, followed by Bacteroides. The abundance of In conclusion, this study elucidated the dynamic changes of porcine fecal microbiota and metabolites at different stages from birth to market, which may provide a reference for a comprehensive understanding of the intestinal health status of pigs at different growth stages. The gut microbiota of pigs has received a great deal of attention in recent years, as pigs are not only an important source of animal-derived food, but also an excellent biomedical model of human health . MicrobeCurrently, great progress has been made in understanding the swine gut microbiome. The porcine gut microbiome associated with different stages may be determined by differences in diet and gut physiology at different growth stages . Our preIn the present study, we investigated the changes of fecal microbiota and their metabolites at different growth stages of pigs, hoping to take some measures to regulate pig gut microbes to enhance pig gut health and improve pig growth performance in the near future. Fecal samples collected from 633 commercial pigs (Duroc × Landrace × Yorkshire crossbred pigs) were randomly selected, of which 381 samples  were subjected to microbiome analysis with Illumina MiSeq sequencing methods, while 131 samples  were subjected to metabolome analysis with LC–MS methods, which aimed to illustrate the following three key points: (i) the swine fecal microbiota from birth to market was characterized; (ii) the pan and core fecal microbiota and predicted metagenomic functions were investigated; and (iii) the fecal metabolite changes and their association with microbes were investigated.Here, the data of 381 microbial samples and 131 metabolic samples were analyzed. In terms of microbial data analysis, fecal samples from seven time points on day 1, 10, 25, 45, 70, 120, and day 180 with the number of 57, 70, 72, 24, 58, 50, and 50 were use, respectively. A total of 20,738,913 sequences were obtained and the average length of each sample was 416.17 ± 23.43. For metabolic data analysis, fecal samples from three time points on day 10, 25, and 70 with the number of 25, 60, and 46 were used, respectively. In pos ion mode, a total of 447 effective peaks were obtained, of which 312 metabolites in Library (such as HMDB and Lipid maps) and 151 metabolites in KEGG. As for neg ion mode, a total of 395 effective peaks were obtained, of which 309 metabolites in Library (such as HMDB and Lipidmaps) and 104 metabolites in KEGG.The α-diversity (Shannon and Chao index) and β-diversity of fecal microbiota of different time points from birth to market were shown in We further investigated the shifts of bacterial taxa from birth to market, including suckling, nursery and finishing stages. Principal coordinates analysis (PCoA) based on unweighted unifrac distance revealed the clustering of microbiota composition at the seven time points . The cloThe overall microbial composition at phylum  and genuLactobacillus increased significantly (p < 0.05) at the other 6 time points. On day 10, 25, 45, and 70, there was no significant difference in the relative abundance of Lactobacillus, but on day 120 there was a significant increase (p < 0.05) compared with these 4 time points. Curiously, the relative abundance of Lactobacillus on day 180 was reduced to the same level as day 10, 25, 45, and 70. Conversely, the relative abundance of Escherichia-shigella decreased significantly compared to day 1 at each time point starting from day 10. On the first day of birth, the fecal microbes of piglets were mainly Escherichia-shigella, Clostridium_sensu_stricto_1 and Fusobacterium. From day 10 to 45, the genus of fecal microbiota changed dramatically, but the abundance of Lactobacillus remained basically unchanged between the 3 time points. From day 70 to 180, the main fecal microbes were Lactobacillus, Clostridium_sensu_stricto_1 and Terrisporobacter. In addition, Streptococcus accounted for about 10% of the relative abundance of fecal microbes in pigs on day 120 and 180. Furthermore, the cladogram plot of LEfSe analysis was applied to find the distinct in microbial composition among different time points  were enriched in fecal microbiota. In order to specifically analyze the pattern of change in the abundance of a particular microbe with the growth time points of pigs, we screened for a particular microbe that had a gradient pattern over time  than day1, day 25 was significantly higher (p < 0.05) than day 10, day 120 was significantly higher (p < 0.05) than day 70 in genetic information processing. There was no significant difference in the 3 time points between day 25, 45, and 70, similar results were obtained on day 120 and 180, but they were significantly higher (p < 0.05) than the previous three time points. PICRUSt2 was used for potential metabolic function prediction of the swine microbiota in this study. On the general trend, genetic information processing increased with time, and the latter time was significantly different than the former time at KEGG level 1 among most time points . In detaLC–MS was applied to investigate the changes of porcine fecal metabolites on day 10, 25, and 70. The differences of metabolomics profiles over time were shown in p-values from the pathway enrichment analysis and impact values from the topology analysis. The metabolic pathways, including Tryptophan metabolism, Sphingolipid signaling pathway, Pyrimidine metabolism, Protein digestion and absorption, Glutamatergic synapse, Biosynthesis of plant secondary metabolites, Axon regeneration and ABC transporters pathway had the most significant differences. In order of the number of metabolites in the pathway that were enriched in the metabolic set, the differential metabolic pathways were biosynthesis of cofactors, ABC transporters, tryptophan metabolism, biosynthesis of plant secondary metabolites, protein digestion and absorption, pyrimidine metabolism and so on.Afterwards, we conducted differential metabolite pathway analysis affected at the 3 time points as pigs grew. As shown in According to the enriched metabolic pathway, we further identify the differential and pivotal metabolites affecting the pathway. As shown in Clostridium_sensu_stricto_1, Terrisporobacter, Clostridia_UCG_014, Ruminococcus_gauvreauii_group, Lachnospiraceae_XPB1014_group showed a positive correlation with 5-Hydroxyindoleacetic acid, 2-Formaminobenzoylacetate, 3-Indoleacetic Acid, 3-Methylindole, Serotonin, Indole, 4-(2-Aminophenyl)-2,4-dioxobutanoic acid, 2,3-Pyridinedicarboxylic acid, 4-(2-Amino-3-hydroxyphenyl)-2,4-dioxobutanoic acid and 5-(2’-Carboxyethyl)-4,6-Dihydroxypicolinate. While from the overall trend, the microbes with a decreasing trend were negatively correlated with the metabolites affecting the tryptophan metabolic pathway. Bacteroides, Lactobacillus, Escherichia-Shigella, Lachnoclostridium, Parabacteroides, Eubacterium_fissicatena_group, Fusobacterium, Streptococcus, Butyricimonas, Enterococcus showed a negative correlation with 5-Hydroxyindoleacetic acid, 2-Formaminobenzoylacetate, 3-Methylindole, Serotonin, Indole, 4-(2-Amino-3-hydroxyphenyl)-2,4-dioxobutanoic acid, 5-(2’-Carboxyethyl)-4,6-Dihydroxypicolinate. In addition, the above-mentioned microbes with an increasing trend were negatively (while the decreasing trend were positively) correlated with the metabolites  affecting the lysine pathway.In order to explore the functional correlation between changes of fecal microbiota and metabolites, Spearman correlation analysis was generated by calculating the Spearman correlation coefficient between microbial composition (genus level) affected by time points and metabolites. As shown in The importance of gut microbiota is widely recognized as they play a key role in animal health and their diversity provides beneficial functions for the host . AlthougAlpha diversity refers to the diversity within a specific area or ecosystem. Commonly used metrics include Chao, Shannon, Ace, and Simpson. In this study, we chose two indicators, Chao and Shannon, to describe the alpha diversity characteristics of pig fecal microbes. Our results indicated that as the pigs grew, the richness and diversity of the fecal microbiota increased significantly at most of the time. Beta diversity analysis explores the similarity or difference of community composition between different groups of samples by comparing the species diversity between different habitats or microbial communities. Our results showed that the swine gut microbiota had significant separation on day 1, 10, and 25, but not significant on day 45, 70, 120, and 180, suggesting that the fecal microbiota of pigs tended to mature and stabilize around day 45. Our results are basically consistent with those of Bacteroides, Escherichia, Clostridium, Lactobacillus, Fusobacterium, and Prevotella predominate in pre-weaning piglets, followed by Prevotella and Aneriacter transition to the predominant genera, Fusobacterium, Lactobacillus, and Astragalus were the comparative minor genera of postweaned piglets. Strikingly, In the dynamic distribution of 10 major bacterial genera across age, the abundance of Lactobacillus did not increase regularly with growth, which was consistent with our results at some time points.Our results showed that the abundance of Firmicutes in the fecal microbiota of pigs showed an overall trend of increase from birth to marketing, which were basically consistent with those of Changes in gut microbiota composition and abundance are accompanied by functional changes . PICRUStThe metabolome analysis methods can dynamically display the differences in fecal microbial metabolites in pigs at different growth stages. The results of our PLS-DA analysis showed that the metabolites of the porcine fecal microbiota were clearly segregated at different growth stages of pigs. In conclusion, this study combined microbiome and metabolome to illustrate the dynamics of pigs at different stages from birth to market. Raising a pig starts with maintaining gut health. Only when we fully understand the changes in the gut microbiota and their metabolites at different stages of pigs, can we then use human intervention to ensure the health of the porcine gut and promote the growth and production performance of pigs.All procedures were carried out in accordance with the relevant provisions of the university animal experiment, and the guidelines of the Zhejiang University  Institutional Animal Protection and Use Committee were strictly followed throughout the experiment.The breeding experiment was carried out in the Kaihua farm of Zhejiang Tianpeng Group Co., Ltd. The experimental animals were housed and managed as follows: The sows entered the farrowing room 7 day before delivery. During the lactation stage, the temperature of the piglets’ house was jointly controlled by fans, water curtains and heat lamps. Piglets were started on creep feed on day 10 and weaned on day 26. During the nursery and finishing stage, the pigsties were allocated reasonably, and the mangers and waterers were equipped according to the density of the pigs and the temperature and ventilation of the pigs’ house. The temperature was controlled by fans and water curtains to maintain a suitable temperature. After the end of each stage of the experiment, the stiff and weak pigs were eliminated. The feed compositions for different stages of pigs were shown in Stool samples from the litter pigs were obtained on day 1, 10, 25, 45, 70, 120, and 180 . In addition to the rectal swabs used on the 1st day, feces were collected from fresh feces excreted by animals at other time points. The collected samples were stored in −40°C refrigerator as soon as possible. A total of 381 samples from 7 time points were used for microbiome analysis and 131 samples from 3 time points, day 10, 25, and 70 , were used for metabolome analysis.® SPIN Kit  according to manufacturer’s instructions. The hypervariable region V3-V4 of the bacterial 16S rRNA gene were amplified with primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) . PCR amplification cycling conditions were as follows: initial denaturation at 95°C for 3 min, followed by 27 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°Cfor 45 s, and single extension at 72°C for 10 min, and end at 4°C. PCR products were then extracted, purified and quantified. Purified amplicons were pooled in equimolar amounts and paired-end sequenced on an Illumina MiSeq PE300 platform . The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: PRJNA918501). Bioinformatic analysis of the fecal microbiota was carried out using the Majorbio Cloud platform.p < 0.05). The metagenomic function was predicted by PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) based on OTU representative sequences.The methods and steps referred to the papers of other scholars . Total mTAAT-3′)  by an ABThe methods and steps referred to the papers of other scholars . The metp < 0.05 and defined as trends at p < 0.10.Spearman correlation analysis was generated by calculating the Spearman correlation coefficient between microbial composition (genus level) affected by time points and metabolites. The differences were defined as statistical significance at The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was approved by the Zhejiang University  Institutional Animal Protection and Use Committee. The study was conducted in accordance with the local legislation and institutional requirements.MJ and HH conceived and designed the experiment. HH, MY, and WL performed the experiment and collected the samples. HH performed the microbiome and metabolome analysis, as well as wrote the manuscript. MJ, ZL, and YW revised the manuscript. All authors reviewed and approved the final manuscript.This research was supported by the fund from Science and Technology Projects of Zhejiang , Li Ka Shing Foundation, China Agriculture Research System (CARS-35), National Center of Technology Innovation for Pigs, National Natural Science Foundation of China (U21A20249).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.