The infants in the control group were given routine nursing, while those in the observation group received family participatory nursing. In the observation group, the length of stay and duration of oxygen therapy were shorter, the weight gain speed was higher, and the incidence rate of nosocomial infection was lower than those in control groups (P < 0.05). The time of reaching full gastrointestinal feeding was earlier, the daily milk intake and exclusive breastfeeding rate were higher, and the feeding intolerance rate was lower in observation group than those in control group (P < 0.05). Observation group exhibited significantly higher body weight, Z-value of body weight, NBNA score at 7 d after hospitalization, 15-month mental development index (MDI), and psychomotor development index (PDI) scores than control group (P < 0.05). In addition, the families' somatization, depression and anxiety factor scores, and total score of SCL-90 in the observation group were lower than those in the control group, while each dimension score and total score of MPOC-20 were higher than those in control groups (P < 0.05). Family participatory nursing is of great significance in ameliorating the clinical outcomes of premature infants in the NICU, which can shorten the length of stay, improve feeding conditions, facilitate infant growth and development, and enhance their families' psychological status and nursing satisfaction.The purpose of this study was to explore the effects of family participatory nursing on the clinical outcomes of premature infants in the neonatal intensive care unit (NICU) and their families' psychological status. Total of 150 premature infants were admitted to the NICU of our hospital from December 2019 to December 2021, and their families were selected by convenience sampling method and divided into control group ( Due to low body weight and immature development of organs, premature infants are prone to serious health problems, so long-term monitoring in the neonatal intensive care unit (NICU) is often required , 2. The n = 78, from December 2019 to December 2020) and observation group  according to the admission time. Inclusion criteria were as follows: (1) infants with a birth weight of <2500 g and gestational age of 28–37 weeks, (2) those with a duration from birth to NICU admission for <12 hr., (3) those whose families could provide care and spend >8 hr./day with them, and (4) those whose families gave informed consent and voluntarily participated. Exclusion criteria were as follows: (1) infants receiving invasive respiratory support, (2) those complicated with congenital, hereditary, or metabolic diseases, (3) those who needed to undergo surgical treatment, and (4) those whose families had cognitive dysfunction or communication barriers. This study complied with the declaration of Helsinki. The general clinical data had no statistically significant difference between the two groups (P > 0.05)  .(1) In the control group, the infants were given routine nursing. The nurses undertook all relevant nursing tasks for the children, including basic vital sign monitoring, comfortable position adjustment, breathing management, basic nursing of the umbilicus and buttocks, touch and hearing stimulus, reasonable feeding, infection prevention, strengthening of environmental management, routine health education for families, and introduction of the infants' conditions. The infants' families could visit through the video system until the infants were discharged. The infants were followed up in the hospital every 3 months after discharge. (2) In the observation group, the infants received family participatory nursing. (1) Preparation stage: the nursing intervention team was set up, the nursing intervention system was developed, and the families received training. (a) The nursing intervention team was set up, consisting of 2 attending physicians, 1 head nurse, 2 supervisor nurses, 4 nurses, 1 prolactinist, and 1 psychological counselor. The members had different responsibilities determined according to the nature of work. Specifically, the attending physicians were responsible for disease assessment, assessment of intervention effect, answering other medical-related questions, and feasibility assessment of nursing implementation. The head nurses were mainly responsible for managing, supervising, and adjusting the nursing implementation plan. The nurses took charge of the implementation of the nursing plan. (b) The nursing intervention system was developed according to the “NICU Visiting System of Toronto Children's Center in Canada,” as follows: The nursing was given from 9:00 AM to 17:30 PM. The families' involvement should be the same ones with good health, normal cognition, basic nursing ability, and good compliance. During the nursing period, the families should be able to cooperate with the nursing intervention team, possess good awareness of cleaning and disinfection, and implement nursing under the guidance of nurses rather than acting without permission. (c) Family training: the nurses and prosthodontist provided training for the families on the admission instructions, nursing system, cleaning and disinfection, basic neonatal nursing, sensory perception intervention , breastfeeding, basic vital sign  monitoring, pain manifestation and assessment, use of commonly-used instruments in the NICU, emergency handling, etc. The training could be given in the form of Microsoft Office PowerPoint (PPT) presentation, picture demonstration, one-to-one guidance, and mobile Apps. After the training, the families received assessment regarding the above training content, and they could enter the neonatal intensive care unit (NICU) for nursing after passing the assessment. (2) Implementation stage: the family participatory nursing could be started when the infants' signs became stable and no ventilator was needed, as follows: (a) the families were responsible for all the life nursing and nonmedical nursing. The father/mother of the infant entered the NICU with the nurse, and then completed bathing, diaper changing, skin care, oral care, and breastfeeding (self-provided breast milk if the father entered) under the guidance and supervision of the nurse. The families were instructed to record the general signs of the infants every day and communicate with the nurse about the infants' conditions and the daily diagnosis and treatment plan during the nursing period. (b) The families were instructed to make physical contact with the infants every day at 10:00, 12:00, 14:00, and 16:00, touch the infants' head, face, chest, abdomen, limbs, hands, feet, and back in turn in a quiet and comfortable environment, with the force from weak to strong, and rub the large muscle groups. (c) The infants' sleep cycle was introduced to the families, so as to help infants go through complete sleep cycles as far as possible and improve their sleep quality. (d) The infants' items were disinfected regularly to avoid infection, and the room temperature of the ward was kept at 28–32°C and the humidity at 60–70%. (e) Psychological support was provided for the families, and they were encouraged to actively ask questions. The questions should be patiently answered to make the families free from worry and anxiety. The families with an adverse psychological status could seek help from a psychologist. (3) Follow-up stage: the nursing ended after the infant was discharged, and the infants were followed up in the hospital every 3 months after discharge.α coefficient of this scale was 0.825 in this study. Moreover, the intellectual and psychomotor development of infants at the age of 15 months was assessed using the Bayley scales of infant development (BSID). The scale developed by Nancy Bayley in 1933, which was introduced into China in 1992, includes the mental development index (MDI) and psychomotor development index (PDI), and MDI or PDI <80 points indicates developmental retardation. The Cronbach's α coefficient of this scale was 0.816 in this study. (4) Families' psychological status: the families' psychological status was assessed using the symptom checklist-90 (SCL-90) at discharge. Developed by derogatis in 1973, the scale was introduced into China in the 1980s, covering 90 items of 9 factors , using a 5-level scoring method. The higher the total score, the poorer the mental health of the infants' families. The Cronbach's α coefficient of this scale was 0.805 in this study. (5) Nursing satisfaction: the nursing satisfaction of infants' families was assessed using the measure of processes of care 20-item (MPOC-20) at discharge. The scale was translated into Chinese by Lin et al., covering 20 items in 5 dimensions  and using a 7-level scoring method. A higher total score corresponds to higher nursing satisfaction. The Cronbach's α coefficient of this scale was 0.821 in this study. The scales used in the text are internationally recognized and have a high degree of reliability. They are now widely used.(1) Hospitalization conditions: the length of stay, duration of oxygen therapy, weight growth speed, and incidence rate of nosocomial infection were recorded. Weight growth speed = (body weight at discharge - body weight at admission)/length of stay. (2) Feeding conditions: the time of reaching full gastrointestinal feeding ), daily milk intake, exclusive breastfeeding rate at discharge, and feeding intolerance  rate were recorded. (3) Clinical outcomes: the body weight of infants at discharge was recorded, and its Z-value was calculated: /standard deviation of weight at corrected gestational age. At 7th day after hospitalization, the neurobehavioral function of infants was assessed using the neonatal behavioral neurological assessment (NBNA) . NBNA waχ ± s) and compared between two groups by independent-samples t-test. Enumeration data were expressed as [n(%)], and when subjected to χ2 test or Fisher's exact probability test, P < 0.05 was considered statistically significant.Statistical product and service solutions (SPSS) 20.0 software  was used for data analysis. Normally-distributed data were expressed as (‾P < 0.05)  .P < 0.05)  .P < 0.05)  .P < 0.05)  .P < 0.05)  .NICU admission after birth is an important measure to improve the growth and development of premature infants. Previously , it was Different from the traditional nurse-centered nursing mode, family participatory nursing promotes family participation to a new level on the basis of ability training for families. Tan et al.  introducIn terms of the clinical outcomes of infants, observation group had higher body weight at discharge, Z-value of body weight, NBNA score at 7th day after hospitalization, and 15-month MDI and PDI scores than control group, suggesting that family participatory nursing can greatly enhance the neuromotor and intellectual development of premature infants in the NICU. Premature infants often suffer from immature neurobehavioral development and have higher risks of neuromotor dysfunction and mental retardation after discharge . In famiThe families of premature infants often have unhealthy emotions such as anxiety and depression due to mother-baby separation and worry about the future development of premature infants. As reported , the advIn conclusion, family participatory nursing is of great significance in ameliorating the clinical outcomes of premature infants in the NICU, which can shorten the length of stay, improve feeding conditions, facilitate infant growth and development, and enhance their families' psychological status and nursing satisfaction.


Stenocereus queretaroensis (S. queretaroensis). The present study evaluates the toxicity of these AgNPs through cytotoxicity and mutagenicity tests in vitro, as well as genotoxicity tests, including the evaluation of acute oral, dermal, and inhalation toxicity, along with dermal and ocular irritation, in vivo, according to guidelines of The Organization for Economic Co-operation and Development (OECD). We evaluated cell cytotoxicity in L929 cells, and the half-maximal inhibitory concentration was 134.76 µg/mL. AgNPs did not cause genotoxic or mutagenic effects. Furthermore, in vivo oral, dermal, and acute inhalation toxicity results did not show any adverse effects or mortality in the test animals, and after the dermal and ocular irritation assessments, the in vivo models did not exhibit irritation or corrosion. Therefore, the results show that these previously synthesized S. queretaroensis AgNPs do not represent a risk at the tested concentrations; however, little is known about the effects that AgNPs induce on physiological systems or the possible risk following long-term exposure.Silver nanoparticles (AgNPs) synthesized with plants are widely used in different industries, such as the medical, industrial, and food industries; however, their hazards and risks remain unclear. Here, we aimed to evaluate the toxicological effects of AgNPs in both in vitro and in vivo models. Previously, we developed and characterized green synthesized AgNPs based on  The synthesis of nanoparticles is considered an emerging improvement and development tool in various industrial, food, medical, diagnostics, electronics, and aeronautical applications. Notably, the interest in and production of silver nanoparticles have exponentially grown due to their possible applications in the medical, cosmetic, and food industries due to their physicochemical properties ,2.There are many ways to synthesize AgNPs through different chemical, physical, and biological methods. Physical and chemical methods are expensive and not environmentally friendly, as their production requires toxic chemicals and releases toxic waste products to the environment. The green synthesis of AgNPs stands out as an environmentally friendly method, as it generates nanoparticles using plant extracts, with a lesser impact in the environment; therefore, it is an emerging approach to produce cheaper and less harmful nanoparticles. Moreover, plants are a good resource for reducing agents to synthesized AgNPs because of their alkaloids, saponins, tannins, phenols, and terpenoids contents ,3,4,5. G3 . However, to set up the regulation of AgNPs-based products, it is imperative to consider the type of product, purpose, and the exposure type it will have on humans and the environment; the FDA recommends detecting, quantifying, and characterizing these nanoparticles in products to regulate each of them, since nanomaterials are susceptible to batch-to-batch variation in their physicochemical properties [Despite the AgNPs current use and applications, there is also a gap in silver nanoparticle regulations. Regulatory institutions such as the European Chemicals Agency (ECHA), European Food Safety Authority (EFSA), European Medicines Agency (EMA), U.S. Food and Drug Administration (FDA), and the U.S. Environmental Protection Agency (EPA) are still working on defining the regulatory status of nanomaterials . The peroperties ,15. In toperties ,17. S. queretaroensis; this was the first report of a green synthesis of AgNPs based on this plant extract, which has a high betalain content that confers both antioxidant and anti-inflammatory properties. Furthermore, S. queretaroensis peel contains ascorbic acid (vitamin C) and reducing sugars that have an important role in silver reduction for the green synthesis of nanoparticles. The produced S. queretaroensis based AgNPs were characterized by ultraviolet visible spectroscopy (UV-Vis), and dynamic light scattering (DLS) analysis determined a size distribution between 20 and 600 nm, with an average particle size of 98.96 nm. We also assessed their antimicrobial activity, highlighting that the results were promising against fungi, Gram-negative and Gram-positive bacteria, and a methicillin-resistant strain of Staphylococcus aureus, which makes them an attractive option to be used in antimicrobial products; however, in order to further use these AgNPs as part of a product formulation, their safety needs to be assessed [In addition, there are a few gaps in the AgNPs research. First, the properties of AgNPs are directly related to the synthesis method used, as well as the particle size ; the sizassessed . S. queretaroensis green synthesized AgNPs at the different biological levels  in both in vitro and in vivo systems. Particularly, we evaluated their cytotoxic effects using in vitro models to determine their toxic potential at a cellular level. Furthermore, we evaluated their toxic effects in animal models through different exposure pathways such as oral, dermal, and inhalation. We evaluated the toxic capacity of these S. queretaroensis AgNPs through several assays, including cell cytotoxicity, oral and dermal acute toxicity, dermal and ocular irritation, mutagenicity, genotoxicity, and inhalation toxicity, according to the OECD guidelines.In this work, we aimed to perform extensive analysis on the toxicological effects of these Stenocereus queretaroensis was acquired at a market in Guadalajara City. The bacterial strains TA 100 and TA 98 were obtained from Molecular Toxicology Inc. . The L929 cell line was acquired from the American Type Culture Collection (ATCC), Virginia, USA. The animals used were Wistar rats, BALB/c mice, and New Zealand rabbits acquired from Bioterio Morelos . Triton X-100 rea-gents, 3--2,5-diphenyltetrazolium bromide (MTT), histidine, biotin, and Vogel-Bonner (VB) salts were acquired from Sigma-Aldrich . Dimethylsulfoxide (DMSO), sodium chloride (NaCl), sodium hydroxide (NaOH), and glucose reagents were acquired from KARAL , while the reagents used for the cytotoxicity tests were acquired from Thermo Fisher Scientific . Giemsa Kit reagents and immersion oil were purchased from HYCEL . Agar reagents, sodium azide, 2-nitrofluorene, silver nitrate, and sodium pentobarbital were obtained from BD Bioxon , Affymetrix , and Fermont , respectively.The fruit of S. queretaroensis peel extract. AgNPs were synthesized by adding the aqueous S. queretaroensis peel extract to a 2mM silver nitrate (AgNO3) solution at a volume ratio of 1:20. The pH was adjusted to 8 using 1M NaOH with an Orion 3 Star potentiometer Thermo Scientific) . The mixture was stirred for 30 min at a temperature of 90 °C using a Cimarec stirring hot plate Thermo Scientific , allowed to cool, and subsequently left to and stand for 24 h at room temperature to end the reaction. Then, the AgNPs solution was centrifuged at 15,000× g rpm for 20 min at 4 °C in a Sorvall Legend XTR centrifuge , and the supernatant was discarded. Finally, the pellet was washed with distilled water five times and once in 90% ethanol to obtain pure S. queretaroensis-mediated AgNPs [We had previously produced AgNPs with an aqueous Cell cytotoxicity was assessed with the L929 mouse fibroblast cell line, obtained from ATCC. L929 cells were cultured with Dulbecco’s Modified Eagle Medium (DMEM), 10% Fetal bovine serum (FBS), and 1% penicillin-streptomycin.After reaching a 95% confluence in a T75 culture flask Falcon, Thermo Scientific , cells were detached and seeded in 96-well plates Corning  of 10,000 cells/well. The culture media were changed 24 h after seeding, and the AgNPs extract was added to the culture media to reach the specific concentrations of 10, 20, 30, 50, 60, 80, and 500 µg/mL to test the cytotoxicity at different doses. Simultaneously, positive cytotoxicity controls were performed using TritonX-100 at different concentrations . All tests were performed in triplicate.2 for 4 h. Later, the MTT solution was discarded, and plates were left to dry at room temperature overnight. Finally, 100 µL of dimethyl sulfoxide (DMSO) was added to each well and the wells were incubated at room temperature under continuous agitation for an hour. Plates were read using a multi-well reader spectrometer model X-mark BIORAD  at 570 nm. IC50 was calculated using the Statgraphics XVI.I software.The cytotoxicity of AgNPs was determined using the MTT 3--2,5-diphenyltetrazolium bromide) method. Next, 24 h after treatment exposure, cells were washed twice with 1X Dulbecco’s phosphate-buffered saline (DPBS), a 0.5 mg/mL MTT solution was added to cell cultures, and cultures were incubated at 37 °C 5% COv/v). Cultures were incubated for 48 h at 37 °C, and two positive controls were used: Sodium azide (5 µg/plate), and 2-nitrofluorene (10 µg/plate) for TA100 and TA98, respectively.The mutagenicity of the AgNPs was assessed through the bacterial reverse mutation, or Ames, test under the OECD 471 guideline . A pre-iAll the animals were handled according to the guidelines and regulations promulgated by the Federal Government of Mexico NOM-062-ZOO-1999 . Animalsp ≤ 0.05.Two weeks before the experiment, 12 male Wistar rats (±180–200 g) were housed in cages under a twelve-hour light/dark cycle at 22 ± 3 °C and 60% humidity; they were fed with a standard diet, and their weight was recorded. Rats were randomly distributed into two groups (n = 6). AgNPs were orally administered daily (at 20 mg/mL for 60 days) to evaluate the genotoxic potential following the in vivo micronucleus test according to the 474 OECD guideline and the methods of Hayashi et al. (2016) ,22. Bloo−1 of AgNPs with a stainless steel cannula. After administration, we observed the mice closely every 30 min for the first 4 h, after which we periodically monitored the mice every 24 h for 14 days. At the end of the test, the animals were weighed and sacrificed to analyze their organs  and to search for signs of toxicity or macroscopic changes.The acute oral toxicity was evaluated according to the 425 OECD guidelines . Five BA−1) to the shaved area using a micropipette. Next, the treated area was covered with a gauze dressing. The animals were observed at 30 min, 2 h, and 6 h, and then monitored once daily for 14 days after treatment. We weighed the rats on days 1, 7, and 14. Once the test period was completed, the rats were sacrificed with sodium pentobarbital (150 mg/kg−1), and a necropsy was performed to evaluate the state of the organs and their physical characteristics . All experiments were conducted in duplicate.The acute dermal toxicity was evaluated in female Wistar rats (±250 g) following the OECD 402 guidelines . First, Dermal irritation was assessed on three New Zealand albino rabbits according to the OECD 404 guidelines . The rabEye irritation was evaluated according to the OECD 405 guidelines in three albino New Zealand rabbits . We instThe acute inhalation toxicity of AgNPs was evaluated according to the OECD 403 guideline . Six rat50) of both the AgNPs and the Triton X100 positive control; the IC50 values were 134.76 µg/mL and 85.59 µg/mL, respectively. Additionally, we analyzed the cell morphological changes in cultures after exposure to AgNPs  that is, those colonies that spontaneously repair the mutation and are used as test control, after treatment . Based op = 0.8037) in peripheral blood MNPCE in the test group that received AgNPs orally for 60 days in comparison to the negative control that received distilled water (DW). MNPCE indicated a clastogenic effect (p ≤ 0.05) in the test group vs. the control group (DW).We assessed the genotoxic potential of these AgNPs through the in vivo micronucleus test. We did not find any significant increase .After assessing the acute dermal toxicity, we monitored all groups carefully and did not observe any changes after the first 30 min, 2 h, and 6 h in the area exposed to the AgNPs. During the rest of the monitoring, we did not observe changes in the skin, coat, eyes, mucous membranes, behavior patterns, or the respiratory and circulatory systems. After the 14-day observation period, there was no mortality, and the animals were weighed on days 0, 7, and 14 . On day We performed dermal irritation tests for these AgNPs in New Zealand male albino rabbits. We did not observe any severe dermal response . The skiWe evaluated the degree of eye irritation according to the OECD 405 guidelines by scoring the cornea, iris, and conjunctiva lesions at specific intervals after treatment. Irritation was graded on a scale from 0 to 4 . The classification of irritation scores from 24 h is listed in 50 is greater than 5 mg/L.The acute inhalation toxicity of the AgNPs was evaluated according to the OECD 403 guideline; the mean aerodynamic mass was determined to be 1.33 microns, with a geometric standard deviation (σg) of 2.17. This test did not produce any mortality 14 days after inhalation exposure, and no severe clinical signs were observed after the 14-day evaluation period . At the S. queretaroensis, and these showed a high antimicrobial potential; however, in order to further explore their application, it is necessary to assess their toxicologic activity in biologic systems. In this work, we evaluated the toxicological effects of S. queretaroensis AgNPs using in vitro and in vivo models.Lately, there has been an increase in the usage of AgNPs in consumer and scientific applications, such as food packaging, antimicrobial agents, textile and pharmaceutical production, etc. However, there is still a lack of information on their nano-toxic potential. Previously, we had developed AgNPs based on an Additionally, we performed an Ames mutagenicity assay, the gold-standard method for determining the mutagenic potential of chemical substances such as plants or products for initial testing . We testWe also evaluated the genotoxicity of these AgNPs and determined their cytogenetic damage potential by quantifying the formation of micronuclei in vivo, which is the result of chromosomal damage in the erythroblasts of the test individual. Our results show no significant difference between the peripheral blood MNPCE of the test and the control groups (1.62 ± 1.32 test group against 1.44 ± 0.81 control). Similarly, Kim et al. (2008) evaluated genotoxicity after a 28-day oral exposure  . Kim et −1), we did not observe any changes in physiological and conduct patterns reflecting any signs of toxicity, and there was no significant difference between initial and final body weights; this suggests that the half-lethal dose (LD50) is greater than 2000 mg/kg; as for acute dermal toxicity, we did not find any signs of physiologically adverse effects nor significant difference in body weights; in fact, both experiments were successfully carried out without the deaths of any individuals. Alwan et al. (2021) reported the safety of the oral administration of biogenic AgNPs; their results did not show any difference in body weights or the health state of mice, and there were no changes in biochemical blood and histopathological parameters [We separately assessed the acute toxicity of biogenic silver nanoparticles through oral and dermal exposure according to the OECD 425 and 402 guidelines. These evaluations are important to determine the biological risks of substances such as biogenic AgNPs. After 14 days of oral exposure to AgNPs . After continuous inhalation exposure to AgNPs, there were not any relevant changes or statistical differences compared to the control groups . HoweverS. queretaroensis peel extract, as well as skin and ocular irritation following the OECD test guidelines. We observed a dose-dependent manner of cytotoxicity for AgNPs (IC50 134.76 µg/mL). The green synthesized S. queretaroensis did not exhibit mutagenic damage in the Ames test in a concentration up to 0.03 µg/plate. Acute toxicity through oral and dermal exposure of AgNPs to in vivo models was not detected during the 14-day trial period at 2000 mg/kg doses. There was no sign of adverse reactions after ocular and dermal irritation assessments. The testing of green S.queretaroensis-based AgNPs did not show any adverse effects from its application in in vitro and in vivo models, which would allow these nanoparticles to be used as part of an antimicrobial or therapeutic product; however, these evaluations were short-term, and the effects of AgNPs on full biological systems under long-term exposure have not yet been addressed. It is necessary to further evaluate the interactions between AgNPs and complex biological systems in a time-dose dependent manner.In conclusion, we evaluated the cytotoxicity, genotoxicity, and mutagenic potential, and also performed acute oral and dermal toxicity tests, to determine the toxic effects of green synthesized AgNPs based on


Neoadjuvant immunochemotherapy has attracted much attention as a treatment for locally advanced non-small-cell lung cancer. However, there is scarce evidence of the safety and efficacy of camrelizumab as neoadjuvant in lung cancer. Here, we present three patients who were diagnosed with IIIA squamous non-small-cell lung cancer from September to December in 2020 and received two cycles of neoadjuvant camrelizumab plus nab-paclitaxel and nedaplatin, followed by surgical resection. All three patients had a reduction in the tumor size on CT image and not delayed planned surgery. We did not observe grade 3 or 4 adverse events. Two of the three patients achieved a major pathological response (MPR), including one complete tumor regression of the primary lung tumor. Multiplex fluorescent immunohistochemistry revealed that CD8+ T cells, FoxP3+ regulatory T cells, and PD-L1 expression on immune cells in the surgical specimen were much higher than in the pretreatment biopsy sample in patients with MPR. This was not observed in the patient without MPR. Camrelizumab plus chemotherapy could potentially be a neoadjuvant regimen for resectable IIIA squamous non-small-cell lung cancer, with a high MPR proportion, and did not compromise surgical procedure. Our findings should be validated in a future randomized clinical trial. Neoadjuvant therapy is one of the many approaches to locally advanced non-small cell lung cancer (NSCLC) . HoweverThis single-group study was developed by the author’s medical center. Three patients who were diagnosed with stage IIIA squamous lung cancer between September and December in 2020 were evaluated to undergo lobectomy surgery. Patients received the following drugs intravenously: camrelizumab (200 mg) on day 1; nab-paclitaxel (100 mg/m²) on days 1, 8, and 15; and nedaplatin (80 mg/m²) on day 1 of every 21 days for 4–6 cycles. Surgery was performed after the first two treatment cycles with an interval of 3–6 weeks . All theAll three patients received the two planned cycles and underwent complete tumor resection. The clinical characteristics of these patients are presented in All three patients were operated by video thoracoscopy approach and had an R0 surgical resection. The durations of the surgery were 90, 115, and 130 min, respectively. No significant hemorrhaging occurred during surgery. The length of postoperative hospital stay was 3, 4, and 5 days, respectively, without stay in the intensive care unit (ICU). No postoperative complications, such as pneumothorax, hemoptysis, chylothorax, and pneumonia, occurred.Two of the three patients (patient A and patient B) achieved MPR, including one complete tumor regression of the primary lung tumor but had residual lymph-node metastases (patient A). One patient (patient C) had an incomplete pathological response to the neoadjuvant treatment. Reduction in the tumor size was noted in all patients’ CT images after two cycles of treatment Figure 1Hematoxylin–eosin staining (HE staining) revealed that residual non-viable tumor and lymph nodes of patients with MPR composed of extensive necrosis and a large amount of inflammatory cell infiltration, foamy histiocytes, and multinucleated giant cells can be seen locally. These features were not evident in areas distant from the tumor bed, which was consistent with immunological response, but this response was barely noticeable in the patient without MPR Figure 2In our study, we observed that neoadjuvant administration of two cycles of camrelizumab plus nab-paclitaxel and nedaplatin in patients with stage IIIA squamous NSCLC was not associated with additional adverse events than in previous studies –11. Thervs. 2.2%). The study also showed that, compared with chemotherapy, the improvement of nivolumab plus chemotherapy on pCR was consistent in key subgroups, including disease stage , PD-L1 tumor proportion score , and tumor mutational burden (low [22.4% vs. 1.9%]); high [30.8% vs. 2.7%])  . In term. 2.7%]) . The het. 2.7%]) , 11. The. 2.7%]) , 11. We Compared to adjuvant therapy, neoadjuvant immunotherapy may improve efficacy by reducing metastasis or recurrence in early-stage NSCLC . PD-1 blIn conclusion, all three patients in this study had a radiographic response, and two of them reached a major pathological response. There were no new safety signals identified. Neoadjuvant camrelizumab plus chemotherapy could potentially be a therapeutic option for patients with stage IIIA squamous NSCLC, which requires confirmation in future prospective multicenter randomized studies.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was reviewed and approved by Review Board of Tianjin Medical University General Hospital. The patients/participants provided their written informed consent to participate in this study.XL, CX, ML, HL, and JC wrote the manuscript. XL, CX, ML, JL, MD, HZ, SX, SW, DW, ZS, and GC took care of the patients, collected, and analyzed the data. JC and HL supervised the research. All authors contributed to the article and approved the submitted version.This study was supported by grants from the National Natural Science Foundation of China , Natural Science Foundation of Tianjin , Shihezi University Oasis Scholars Research Startup Project (LX202002), Special Support Program for the High Tech Leader and Team of Tianjin (TJTZJH-GCCCXCYTD-2-6), Tianjin Municipal Education Commission Natural Science Foundation (2019KJ202), and The Fundamental Research Funds for the Central Universities (3332018180). The funding sources had no role in study design, data collection, and analysis; the decision to publish; or the preparation of the manuscript.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.


Adrenal cortex produces glucocorticoids, mineralocorticoids and adrenal androgens which are essential for life, supporting balance, immune response and sexual maturation. Adrenocortical tumors and hyperplasias are a heterogenous group of adrenal disorders and they can be either sporadic or familial. Adrenocortical cancer is a rare and aggressive malignancy, and it is associated with poor prognosis. With the advance of next-generation sequencing technologies and improvement of genomic data analysis over the past decade, various genetic defects, either from germline or somatic origin, have been unraveled, improving diagnosis and treatment of numerous genetic disorders, including adrenocortical diseases. This review gives an overview of disorders associated with the adrenal cortex, the genetic factors of these disorders and their molecular implications. Adrenal glands are the major regulators of body homeostasis and endocrine stress response . They arThe adrenal glands are comprised of two distinct parts, the cortex and the medulla. The medulla found in the center of the adrenal gland is composed of chromaffin cells and it is dependent on tissue interactions with the adrenal cortex . The corAdrenal cortex cellular function is finely regulated by complex mechanism that involve paracrine and endocrine responses. Dysregulation of signaling pathways in the adrenal cortex is associated with the development of adrenal tumors, some are benign and most rarely, malignant tumors .PRKACA mutation and compared to GNAS and CTNNB1 mutant CPAs. NGS analysis revealed differences between PRKACA mutant and GNAS and CTNNB1 mutant CPAs, such as increased cortisol production in PRKACA mutant CPAs . The results indicated a very low-level mosaicism (less than 0.5%) in the germline DNA, while all adrenocortical cells tested from 11 different nodules harbored the disease-causing variant. This finding has implication in patient prognosis and, family risk prediction (The advance of new technologies in the field of genetics made possible to determine variations and structures at a genone-wide level . Next geant CPAs . This stediction . In thisvia LDL-receptor mediated endocytosis (via the scavenger receptor class B type 1 (SR-B1), or produced de novo from the acetyl coenzyme A (CoA)  particles. Briefly, LDL particles are taken up by adrenal cells ocytosis , 16. The A (CoA) . Cellula A (CoA) . In the  A (CoA) .The secretion of glucocorticoids is regulated by the HPA axis. Their synthesis is stimulated by ACTH, which is released into the bloodstream by the anterior pituitary as part of a 241-amino acid precursor, POMC. In turn, ACTH production is regulated by corticotropin-releasing hormone (CRH), which is released by the neuroendocrine neurons in the paraventricular nucleus of the hypothalamus. Secretion of CRH is dependent on circadian rhythm, as well as various stressors  acting on the hypothalamus. The HPA axis is a negative feedback system, in which cortisol acts as a direct inhibitor of the synthesis of both ACTH and CRH.CYP11B2), while they also stimulate aldosterone production and glomerulosa cell proliferation   major histocompatibility complex locus  deficiency  is rare, and severely damaging variants in CYP17A1 result in absent cortisol as well as androgens, causing puberty failure and sexual infantilism  and HSD3B2, respectively. HSD3B1 is expressed in the placental and peripheral tissues , while HSD3B2 is expressed exclusively in the adrenals and gonads  overproduction. DHEA is converted to testosterone by extra-adrenal 3βHSD1, and patients present in infancy with underdeveloped 46,XY genitalia and – rarely – 46,XX virilization . LCAH is caused by defects in the steroidogenic acute regulatory protein (StAR) and is characterized by deficiency of all steroid hormones. StAR regulates the transfer of cholesterol from the outer to the inner mitochondrial membrane, a vital step in the initiation of steroidogenesis. As a result, cholesterol cannot be mobilized. Adrenal lipid droplets subsequently accumulate and are seen on the autopsy, thus the name of the disorder. In both 46,XY and 46,XX patients, it presents with female external genitalia and an adrenal crisis in the neonatal period .Regarding the current treatment for CAH, there is no consensus yet, therefore, it still remains a challenge. It usually includes glucocorticoid and mineralocorticoid replacement therapy .Adrenocortical tumors (ACTs) can be sporadic or familial, unilateral or bilateral, and non-secreting or secreting. The latter secretes various adrenal steroid hormones; the exact hormone varies depending on the tumor type. Unilateral ACTs are common, and approximately 10% of the general population appears to have an adrenal cortical lesion . They arCushing’s syndrome (CS) has an estimated incidence of 39-79 per million people per year in various populations, with a female-to-male ratio of 3:1 –56. DataCS is characterized by cortisol overproduction. The cause of 80% of endogenous CS cases is over-secretion of ACTH by a pituitary corticotroph adenoma or – less frequently – by a neuroendocrine tumor –63. In rMC2R, a G protein-coupled receptor (GPCR). This triggers an increase in cAMP levels, which binds to the PKA regulatory subunits causing the release from the catalytic subunits. The catalytic subunits, then translocate to the nucleus, where they phosphorylate, and thus activate, transcription factors that promote cortisol synthesis , autonomous macronodular adrenal hyperplasia (AMAH), bilateral macronodular adrenal hyperplasia (BMAH), and ‘huge’ or ‘giant’ macronodular adrenal disease. Another term, ACTH-independent massive bilateral adrenal disease (AIMBAD), has also been used in the past, but in later studies the secretion of cortisol appeared to be regulated by corticotropin and thus, this term is not used anymore .MC2R leading to activation of cAMP/PKA pathway and thus excessive cortisol secretion , the macronodules exhibit ectopic or excessive expression of G-protein coupled receptors, including luteinizing hormone/choriogonadotropin (LH/hCG) responsible for Cushing syndrome during pregnancy and after menopause , glucose-87%, theecretion . So far,ecretion .via a variety of mechanisms have been reported in patients with PBMAH. For example, variants in PDE11A, that encodes phosphodiesterase type 11A, have a prevalence of 24-28%, whereas inactivating germline variants in PDE8B, encoding phosphodiesterase type 8B, and PRKACA copy number gains, have also been encountered  resulted in autonomous cortisol secretion via constitutive activation of the cAMP/PKA pathway , multiple endocrine neoplasia type 1 (MEN1) and hereditary leiomyomatosis (FH)  , 87–89. sis (FH) .ARMC5) have been found to implicate in the pathogenesis of PBMAH, with their prevalence estimated between 21%-26%  and HDAC9 (that encodes a histone deacetylase)  . In addi studies .A meaningful update of the 2022 WHO classification of adrenocortical tumors was recently summarized by Mete and co-workers early this year. As a result of the advance on next generation sequencing studies, it is possible to recognize that PBMAH is caused by germline variants in one out of many susceptible genes, with a second hit in the somatic cells. These findings strongly suggest a neoplastic instead of hyperplastic condition. To avoid a misnomer for the disease, the 2022 WHO classification changed the nomenclature of primary bilateral macronodular adrenal hyperplasia to bilateral macronodular adrenocortical disease .PPNAD is most commonly diagnosed in children and young adults and is a rare cause of ACTH-independent hypercortisolism. It is most commonly part of Carney complex (CNC), an autosomal dominant tumor predisposition syndrome. CNC presents with various endocrine tumors including pituitary adenomas, thyroid benign tumors, and testicular Sertoli cell-calcified tumors, as well as non-endocrine tumors, most commonly pigmented skin lesions, skin and cardiac myxomas , 103. PPhttps://PRKAR1A.nichd.nih.gov/hmdb/mutations.html). The majority of them are located in exons 2,3,5,7, and 8 while about 20% are located in intronic sequences and affect splicing . PRKAR1A variants are found in 37% of patients with the sporadic form of the disease and in more than 70% in the typical familial forms, with almost 100% penetrance , 106. PRsplicing , 106. Mosplicing . In almosplicing –109. Larsplicing , 111. Ocsplicing , 112.PRKACB gene  , 114. ThACB gene . MoreoveACB gene .PDE11A and PDE8B have emerged as putative causes of PPNAD. The loci harboring these genes had the most significant associations in a genome-wide association study performed on individuals lacking genetic defects in GNAS or PRKAR1A, while the locus harboring PDE11A showed the largest loss-of-heterozygosity in tumor samples  was detected in a 2 year old female patient diagnosed with PPNAD; the variant was inherited from the patient’s father. This variant was subsequently shown to lead to decreased PDE8B activity in vitro , haploinsufficiency of PDE11A has been implicated in ACA and ACC , were reported in a patient with CNC that presented with myxomas, acromegaly, and abnormal skin pigmentation. The patient was found to have increased cAMP-induced kinase activity in lymphocytes, resembling what is seen in CNC patients with PRKAR1A variants. Moreover, increased Cβ levels were found in several cell types as well as in breast myxoma cells  were found in two (11%) patients with PPNAD in a previous study, with one of these patients also harboring a PRKAR1A variant. These variants were encountered in larger adrenocortical adenomas that arose within the context of PPNAD, and were absent from the surrounding hyperplastic adrenocortical tissue -β-catenin pathway has also been suggested to play a role in micronodular BAH. Somatic variants in the β -catenin gene  and the most common variant has been reported to be p.Leu206Arg  were overexpressed, while in GNAS-mutant tumors, there was increased expression of extracellular matrix receptor interaction and focal adhesion pathways   .FH, <1%) and rarely, carcinomas (<1%)  –137.FH, <1%) and aldosterone-producing ACC (<1%)  is the most frequent secondary form of hypertension, accounting for approximately 10-20% of patients referred with resistant hypertension and 5% of patients in primary care , 139. PACC (<1%) . The vasCC (<1%) .FH (type I-type IV) have been described so far and they are inherited in an autosomal dominant manner  is the formation of a chimeric gene, resulting from an unequal fusion of the regulatory regions of CYP11B1, which encodes 11β-hydroxylase, and CYP11B2, that encodes aldosterone synthase. Both enzymes are responsible for the last steps of cortisol and aldosterone synthesis, respectively (CYP11B1/CYP11B2) should be considered for patients who are diagnosed with PA and have a family history of the disease, onset of PA before the age of 20 years, or family history of stroke at a young age , which is encoded by KCNJ5. Genetic defects in this gene cause a lack of ion selectivity and increased sodium influx, which results in cell depolarization triggering calcium entry into the cells. This signals an increase in CYP11B2 expression and increase in aldosterone production. The severity of hyperaldosteronism has been shown to be related to the type of KCNJ5 variant in some patients  have also been described in patients with PA; these variants occur exclusively de novo. These patients present with a severe early-onset form of hyperaldosteronism associated with a complex neurological disorder, with the phenotype also including seizures and neurological abnormalities (PASNA) ( (PASNA) .PDE2A, PDE3B and ARMC5 have also been reported in patients with PA   being responsible for 36% of cases  , 159–161of cases . Variant cohorts –166 (~70 cohorts . Variantfrequent .APC). Binding of the Wnt ligand to its receptor causes β-catenin to dissociate from the axin complex and translocate to the nucleus where it induces the expression of the transcription factors T cell factor (TCF) and lymphocyte enhancer factor (LEF)  and LH/hCG receptors  . Somaticadic APA , 171; sieceptors . Based oeceptors . In rare as well , 174. Ho as well , 175–177ACCs are rare tumors derived from the adrenal cortex. They affect both adults and children with an annual incidence of 0.7-2.0 cases per million per year , 179. ThTP53, the underlying genetic cause of LFS, have been identified in 70% of cases, while de novo variants have been shown to have a prevalence of 7-20%   . Loss ofhood ACC . BWS is hood ACC . In addihood ACC –201. Thehood ACC .MEN1 is inherited in an autosomal dominant manner and is caused by germline heterozygous variants in the MEN1 gene on chromosome 11q13. Its main manifestations are hyperparathyroidism (95%), entero-pancreatic neuroendocrine tumors (50%) and pituitary adenomas (40%). Associated adrenal lesions, mostly ACA and hyperplasias, are present in 20-55% of MEN1 cases . Patients have a significantly increased risk of cancer, especially colorectal and endometrial cancer, and thus screening for Lynch syndrome is recommended in all patients that are diagnosed with colorectal cancer  –213. Lynl cancer , 214. Thl cancer .ACC has been reported in patients with neurofibromatosis type 1, familial adenomatous polyposis, and Werner syndrome , 216–223In general, the discovery of genetic syndromes that confer an increased risk for ACC has yielded important clues into the molecular basis of ACC development. For example, the association between FAP and adrenal tumors provided the basis for the insights into the role of β-catenin signaling in adrenal tumors, while the link between ACC and BWS combined with gene expression profiling suggested the IGF-1 receptor as a target for ACC therapy. The latter hypothesis has now been tested in clinical trials .Patients with unilateral adrenal adenomas that are hormonally active receive treatment with unilateral adrenalectomy . IndividThere are at least 15 clinical trials currently recruiting patients diagnosed with ACC. Location includes United States, Europe and China. Over 20 studies, all over the world, are recruiting patients diagnosed with hyperaldosteronism, comprising either observational or interventional studies. There are also about 20 studies currently recruiting patients with CS and many other studies are now recruiting for other adrenal cortex-related diseases. Information about current and past trials, as well as institution and collaborators, can be found at clinicaltrials.gov, a resource provided by the United States National Library of Medicine.Here, we summarized the main genetic and molecular aspects of adrenocortical diseases, which are, in some cases, difficult to diagnose. The rapid progress of next generation sequencing (NGS) techniques has opened new horizons to examine and diagnose adrenocortical diseases with unbiased mechanisms. For example, exome sequencing may be used in patients with a clinical phenotype but no identified variant in one of the known causative genes, in order to perform an unbiased scan and potentially identify causative variants in genes previously not associated with the disorders. This not only yields a diagnosis, but also provides new clues into disease pathophysiology. Despite the progress, diagnosis based on genetic screening is still limited to either large centers and/or patients with financial access to these analyses. Therefore, heath care providers still face limitations in offering access to precise medicine to all patients. Considering that a great number of adrenocortical diseases is due to genetic onset, we believe that increasing access to NGS will greatly improve early and precise diagnosis of adrenocortical disorders. Consequently, more accurate treatments will be delivered to individuals that harbor genetic alterations leading to disorders of the adrenal cortex.GP: Writing - original draft; AM: Writing - review & editing; FF: Conceptualization; Supervision; Writing - review & editing. All authors contributed to the article and approved the submitted version.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.


SCA1, SCA2, and SCA3 are the most common forms of SCAs among the polyglutamine disorders, which include Huntington’s Disease (HD). We investigated the relationship between leukocyte telomere length (LTL) and the phenotype of SCA1, SCA2, and SCA3, comparing them with HD. The results showed that LTL was significantly reduced in SCA1 and SCA3 patients, while LTL was significantly longer in SCA2 patients. A significant negative relationship between LTL and age was observed in SCA1 but not in SCA2 subjects. LTL of SCA3 patients depend on both patient’s age and disease duration. The number of CAG repeats did not affect LTL in the three SCAs. Since LTL is considered an indirect marker of an inflammatory response and oxidative damage, our data suggest that in SCA1 inflammation is present already at an early stage of disease similar to in HD, while in SCA3 inflammation and impaired antioxidative processes are associated with disease progression. Interestingly, in SCA2, contrary to SCA1 and SCA3, the length of leukocyte telomeres does not reduce with age. We have observed that SCAs and HD show a differing behavior in LTL for each subtype, which could constitute relevant biomarkers if confirmed in larger cohorts and longitudinal studies. The spinocerebellar ataxias (SCAs) due to CAG expansion are the most common of the polyQ diseases along with Huntington’s Disease. The ataxias are a group of neurodegenerative disorders, which are clinically and genetically heterogeneous. So far, more than 40 genes and loci have been identified . Seven SClinically, SCA1, SCA2, and SCA3 share a set of features including cerebellar dysfunctions, which encompass gait ataxia, intention tremor, and eye-movement abnormalities .From a neuropathological point of view, degeneration of Purkinje cells and cerebellar circuits are considered the predominant pathogenic mechanism for SCAs, underlying the characteristic ataxic symptoms . HoweverThe cause of the susceptibility of neurons in specific brain regions to the toxic effects of mutant proteins is still unclear, but it is becoming evident that the response of cells surrounding the neurons, such as glial cells, including microglia and astrocytes may play a role in SCA pathophysiology. This has been shown at least for SCA1 and SCA3. In SCA1, analysis of human autopsy material indicated a significant glial pathology , and, reHuman telomeres consist of repeated TTAGGG nucleotide sequences present at the ends of chromosomes, where they play a protective role against DNA damage. During physiological DNA replication, telomeres progressively shorten with each cell division, due to the inability of the DNA polymerase to replicate the 3′-end of the DNA strand. Telomerase, a ribonucleoprotein complex, counteracts such shortening of the telomeres ,16, but,Due to tissue availability, the measurement of leukocyte telomere length (LTL) is also widely used in studies on aging and neurodegeneration, on the basis that telomere lengths from different tissues of the same individual are statistically correlated ,25.A number of epidemiological and population studies provided evidence that leukocyte telomere shortening is associated with aging ,19,20,25Among the CAG repeat disorders, LTL analysis has previously only been performed in HD, indicating that telomere shortening could be a useful biomarker for disease conversion in pre-manifest subjects . SimilarA cohort of 107 ataxic patients, molecularly diagnosed as SCA1 (n = 52), SCA2 (n = 26), and SCA3 (n = 29), collected at the Ataxia Centre, National Hospital for Neurology and Neurosurgery UCL/UCLH, London, UK , and at the Laboratory of Neurogenetics of the Institute of Translational Pharmacology of the CNR, Rome, Italy, was examined. In The control subjects are blood donors recruited at the Department of Experimental Medicine and Surgery of the University of Tor Vergata, Rome. A sex- and age-matched control group was selected for each patient group, with two controls for each patient, with the exception of the SCA3 group, where two controls were selected for 86% of patients.Written informed consent was obtained from all participants. DNA was collected for research purposes, according to the principles expressed in the Declaration of Helsinki.Genomic DNA was extracted from patient peripheral blood leukocytes using a FlexiGene DNA kit  in accordance with the instructions of the manufacturer.Molecular diagnosis of the three SCA subtypes was performed according to Orr et al.  for SCA1Leukocyte telomere length was measured by monoplex real-time PCR quantitative analysis (monoplex qPCR) on a 7300 real-time PCR instrument . This method allows the determination of the number of copies of telomeric repeats (T) compared to a single-copy gene (S) used as a quantitative control (T/S ratio) . The telp < 0.05. The relationship between the T/S ratio and age at blood sampling was evaluated by regression analysis. When necessary, LTL was adjusted for age by multiple regression. A comparison of the regression lines slopes was carried out by a t-test. ROC curve analysis was performed through the easy ROC web interface. The optimal cut-point was identified according to the Youden Index method.Statistical analyses were performed using Statistix software . Parametric (ANOVA) and nonparametric  tests were used to compare the distribution of quantitative variables between patients and controls and the distribution of the mean T/S ratio across age and repeat number classes. The level of significance was set at The demographic, genetic, and clinical features of the SCA1, SCA2, and SCA3 cohorts are summarized in p < 0.00001). The same highly significant correlation was found in 26 SCA2 patients, when CAG repeats and age at onset were analyzed using linear regression (p < 0.00001). For the 29 SCA3 subjects, the linear regression was highly significant (p = 0.002).The SCA1 group cohort consisted of 52 patients and 5 pre-symptomatic subjects. The linear-regression analysis between the number of CAG repeats in the expanded allele and age at onset was highly significant (p < 0.00001) (p = 0.03) and from SCA1 manifest patients (p = 0.04)  compared to controls  . In the .04) see .p = 0.20, SCA1 patients: p = 0.79).No difference in mean LTL was observed between males and females in the two groups . A significant relationship was observed between LTL and age in the SCA1 patient group as well . The two regression lines show similar slopes (p = 0.88), but different elevations (p < 0.0001) indicate that from the age of about 20 years LTL of SCA1 patients and controls shows a similar shortening rate per year (0.0035 T/S vs. 0.0037 T/S), and SCA1 LTL values are lower than those of controls at any age . The same result was observed between age-adjusted LTL and disease duration, which was calculated as the difference between age at blood sampling and the reported age of onset (p = 0.4).No relationship was observed between LTL values and CAG repeat number, after adjusting for age by means of multiple-regression analysis, (We then assessed the accuracy of LTL as a biomarker of SCA1 disease through Receiver Operating Characteristic (ROC) curve analysis. In discriminating symptomatic SCA1 patients from controls, LTL displayed extremely high accuracy [AUROC(AUC) = 0.916, see p < 0.00001), with longer telomeres in SCA2 patients  and 52 controls  .No difference was observed in mean LTL between males and females in the two groups , but, in SCA2 patients, the relationship between LTL and age was not statistically significant   .To investigate further, the relationship between age at blood sampling and LTL in SCA2 patients, we analyzed the LTL values for different age categories of SCA2 patients, compared to controls . Again, p = 0.23) and (p = 0.88), respectively. These results suggest that telomeres maintain a constant length throughout the duration of the disease.No relationship was observed between LTL values and CAG number and disease duration, after adjusting for age by means of multiple-regression analysis (We assessed the accuracy of LTL as a biomarker of SCA2 disease through Receiver Operating Characteristic (ROC) curve analysis. In discriminating symptomatic SCA2 patients from controls, setting the cut-off point of LTL at 0.99 [AUROC (AUC) = 0.792, see p = 0.003), with lower levels in SCA3 subjects  and SCA3 patients  .There was no difference in LTL mean between males and females in the two groups . A similar negative relationship of LTL with age at blood draw was observed for the SCA3 patient group . The two regression lines show similar slopes (p = 0.64) but different elevations (p = 0.007), indicating that from the age of about 20 years LTL of SCA3 patients and controls shows a similar shortening rate per year (both 0.003 T/S), and that SCA3 LTL values are lower than that of controls at any age . The effect of disease duration on LTL was examined in SCA3 patients. A significant negative linear relationship between LTL and duration  was observed (p = 0.02), suggesting that leukocyte telomeres shorten as the disease progresses, independent of age.No relationship was observed between LTL values and CAG number after adjusting for age by means of multiple-regression analysis  curve analysis. In discriminating symptomatic SCA3 patients from controls, setting the cut-off point of LTL at 0.91 [AUROC(AUC) = 0.718, see There is wide evidence that leukocyte telomere shortening is a common hallmark of conditions associated with increased systemic oxidative stress and chronic inflammation . PreviouInterestingly, despite these diseases being caused by mutations in three different genes, they all belong to the group of polyQ conditions ,42,43, aSCA1. Analysis of LTL in SCA1 patients showed significantly reduced values compared to controls. The analysis of a small number of pre-symptomatic SCA1 subjects suggests that the telomeric erosion could start at this stage. After clinical onset, telomere length depends on age but not on disease duration. The picture of LTL in SCA1 appears similar to what has been observed in HD [ed in HD ,46, in wed in HD  than in The reduced LTL in SCA1 patients could suggest a state of peripheral inflammation secondary to neuroinflammation, as observed in other neurodegenerative diseases such as AD or HD . InitialROC curve analysis distinguishes SCA1 patients and controls in a sensitive and specific way (AUC 0.916), making LTL a possible biomarker useful for clinical trials to help stratification of SCA1 patients. In order to verify this as a potential biomarker for progression of the disease, a larger sample of patients should be recruited in a longitudinal study. The latter will be relevant for understanding the annual decrement in patients.SCA2. The most relevant result of the LTL analysis in SCA2 is the observation of a significantly higher LTL in patients than in controls. This difference is more evident in those age groups in which LTL begins to decrease in controls, while LTL of SCA2 patients remains more stable. Furthermore, the LTL value seems independent from the CAG number and disease duration. The observation of longer telomeres in neurodegenerative disorders and in some forms of familial cancer is quite rare. Some familial cancers, caused by germline mutations in the TERT gene, are referred to as “long telomere syndromes”. Furthermore, somatic mutations of the TERT promoter are observed in 70% of solid tumors, indicating that telomerase abundance is critical for cancer initiation and progression [gression ,51. Amongression . Amyotrogression ,54,55. Sgression , the resgression , may progression . Some stgression . In our SCA3. A different picture emerges from the analysis of LTL in SCA3. The LTL values of SCA3 patients were significantly reduced compared to controls, similarly to SCA1, but to a lesser extent. The telomere length of SCA3 patients was also found to depend on both patients’ age and disease duration. This suggests a possible different mechanism from SCA1, in which LTL is not dependent on either patient age or disease duration.Involvement of the inflammatory system and dysregulation of cytokine expression has been reported in SCA3 ,58,59,60The LTL trends and their relationship in SCA1, SCA2 and SCA3 are reported in We previously published the results of LTL in HD ,46, and In HD, SCA1, and SCA3, LTL was significantly shorter compared to controls. There was a variability of LTL among these three conditions, in which HD was significantly shorter than SCA1 and SCA3 .In SCA1 and HD, we have a similar behavior in which the decrease in LTL could be a biomarker of the disease prodromal stage in SCA1 , such asConversely to HD and SCA1, SCA3 LTL was significantly negatively correlated with age and disease duration. A larger sample size and a longitudinal study could confirm SCA3 LTL as a promising biomarker of disease progression . StatistLTL is an indirect biomarker of inflammatory response and oxidative damage. This study provides support for the literature data elucidating the relevance of these mechanisms early in the disease process in SCA1 and HD ,62, whilIn SCA2, the persistence of long telomeres in leukocytes is an intriguing observation that needs to be investigated to verify whether this is due to the persistence of telomerase, as observed in ALS patients , or to t


Piper mollicomum Kunth (PM), in its reproduction period, as well as register inflorescence visitors, microclimate and phenological information. The essential oils (EOs) obtained from the different fresh organs by hydrodistillation were identified and quantified by Gas Chromatography/Mass Spectrometry (GC/MS) and by GC coupled to a Flame Ionization Detector (GC/FID), respectively. The cercentage content of some volatiles present in reproductive organs, such as limonene, 1,8-cineole, linalool and eupatoriochromene, increased during the maturation period of the inflorescences, and decreased during the fruiting period, suggesting a defense/attraction activities. Furtermore, a biosynthetic dichotomy between 1,8-cineole (leaves) and linalool (reproductive organs) was recorded. A high frequency of bee visits was registered weekly, and some correlations showed a positive relationship between this variable and terpenes. Microclimate has an impact on this species’ phenological cycles and insect visiting behavior. All correlations between volatiles, insects, phenology and microclimate allowed us to present important data about the complex information network in PM. These results are extremely relevant for the understanding of the mechanisms of chemical–ecological plant–insect interactions in Piperaceae, a basal angiosperm.The aim of this study was to monitor the volatile chemical composition from leaves and reproductive organs of  Piper mollicomum Kunth (Piperaceae) is a heliophilous or cyophilous species, which means it can grow both in high luminosity environments and in humid and shady places. It has membranous, tomentose leaves and spicule inflorescences [escences ,2. This escences ,2,3. Thiescences ,3,4,5.P. mollicomum are widely recognized, and include increased gastrointestinal motility [The bio-pharmacological effects of motility , antifunmotility ,8, antibmotility , larvicimotility  and antimotility . Some etmotility ,13,14,15P. mollicomum are used as mystical resources to achieve spiritual contact with deities in rituals of African-based religions. Amaci is one of these important rites [In addition to its medicinal applications, specimens of nt rites .Carollia perspicillata  and C. castanea  [Piper L. specimens may efficiently disseminate their seeds in their habitat with the aid of these animals. [Piper and animals [This species also contributes to several biotic interactions. For example, its infructescences have already been described as the main food resource for two species of frugivores bats, k, 1890) ,20. Becaanimals. ,22. In t animals .Some authors point out that the reproductive organs are anatomical structures characterized especially as mutualistic in their ecological relationships ,24. HowePiper species and Eois caterpillars  [P. mollicomum, that are continuously defoliated by herbivores, emit EOs attractive to predatory wasps who feed on their herbivorous rivals, promoting negative pressure on them. These damaged organs released terpenes that were detected in very low percentages in intact leaves. This mechanism infers that some constituents may benefit the plant by attracting opportunistic insects, such as parasitic predator wasps, which attack their natural enemies [Despite the great entomological biodiversity in neotropical forests, many plant specimens are preyed by specific groups of insects ,43. This., 1818) ,50,51,52 enemies .Essential oils are complex mixtures of volatile and lipophilic substances, biosynthesized in various plant organs within intracellular compartments. These volatiles are composed of a wide variety of complex compounds, however, the main constituents in these mixtures are terpenes, organic substances derived from isopentyl diphosphate (IPP) or 3,3-dimethylallyl diphosphate (DPP) units ,54,55,56Several studies correlate the participation of substances that can be found in EOs in some important ecological relationships ,64,65,66Piper species and in-sects. It is important to note the harmonious relationships between these two groups, particularly pollination, a subject that has received regular attention in recent years [Not only disharmonious relationships predominate between nt years ,71,72,73Piper gaudichaudianum Kunth at different seasons and periods of the year. This study made it possible to propose a new chronotype, in addition to other important findings, such as a new micromolecular index that allows inferences about the reduction–oxidation of mixtures (Ramos and Moreira Index) [Ramos et al. (2021) analyzed the chemical phenotypic plasticity of EOs from a Index) .P. mollicomum that showed a high percentage of linalool, eupatoriochromene and E-nerolidol in the reproductive structures [Piper throughout its reproductive period.Furthermore, some studies were carried out by our group with ructures ,3. Howevructures . The corructures ,78,79,80v/w). Stages 1, 2, 3 and 4 presented the lowest percentages , and stage 5, the highest   . The volα-terpineol (11.41%), and the constituent with the most prominent relative percentage in the different stages of the reproductive organ was 1,8-cineole (26.07% at 58.77%). In December, the constituents that stood out in the foliar EOs were the non-oxygenated monoterpenes α-pinene (11.30%) and β-pinene (6.49%), the oxygenated monoterpene linalool (11.28%) as well as the non-oxygenated sesquiterpene β-elemene (8.62%). On the other hand, in the different stages of the reproductive organ, the main identified constituent was 1,8-cineole (22.96% to 32.01%), in addition to α-pinene (3.50% to 14.7%) and linalool (0.00% to 10.46%). In the last month of this research (January) 1,8-cineole was the constituent that presented the highest relative percentages in the EOs, not only in the leaves (8.62%), but also in the different stages of the reproductive organ (13.50% at 49.32). It is important to highlight that the constituent desmethylencecalin (eupatoriochromene) showed a significant difference (p < 0.05) in the relative percentage of leaves (0.00% to 2.56%) and stages of reproductive organs (0.12% to 32.92%) from September to January.In the initial months of flowering (September and October), the oxygenated monoterpene linalool was the chemical constituent that registered the highest relative percentages, both in leaves and in most stages of the reproductive organ . Also noteworthy is the non-oxygenated monoterpene limonene, which showed high percentages in most EOs in this period, including in the reproductive organs (0.01% to 20.07%). In November, the major constituent in the leaves was identified as the oxygenated monoterpene r2) between EOs’ constituents vs. seasonality. For this analysis, we highlighted all the constituents of the EOs that were identified at least once in the periods and stages of the analyzed reproductive organs. The results show negative correlations for the constituents eupatoriochromene , α-pinene , and myrcene . This information infers that there are changes in the content of EOs from the first stage of floral development to the beginning of fruiting, a fact that leads to a significant decrease in these compounds, while the development of reproductive structures occurs. As result of this Spearman’s correlation, we proposed to investigate the trend on the ontogenic pattern of the percentage contents of the main constituents of the EOs along each stage of the reproductive organ  and 3 (anthesis), and decreases in the period of fructification (stages 4 and 5). The graphs  was the floral visitor that registered the highest frequency (n = 3042). Insect 6 (Hymenoptera—Colletidae sp.), another bee, was the second hymenopteran with the highest number of visits (n = 820). The other bees, insects 5  and 4 , recorded frequencies of 238 and 67, respectively. Diptera had significantly different visit rates among them. While insect 3 (Diptera—Syrphidae sp. 2) was the third insect with the highest number of visits (n = 164), insect 1 (Diptera—Syrphidae sp. 1) was observed only once (n = 1). The data of the weekly sums referring to all frequencies of visits are described in In order to evaluate correlations between the chemical constituents of the EOs and the frequency of visits by potential pollinators, weekly observations were carried out. Data collection indicates that bees and flies were the main groups of insects observed in all inspections . In totaE-β-ocimene; in addition to the acyclic oxygenated monoterpene linalool. The results also indicate a strong positive correlation between sesquiterpenes E-caryophyllene and aromadendrene, with the visits of the insects to inflorescences. The graph also shows that the constituents that can attract insects were biosynthesized mainly in the first months of observation, September and October, when the first stages of inflorescence sprouting began. Concurrently, the sesquiterpenes E-nerolidol and β-selinene, in addition to the monoterpenes E-β-ocimene, Z-β-pinene, α-pinene, 1,8-cineole, as well as eupatoriochromene, did not show a significant positive correlation with respect to the constancy of visits. Additionally, Z-β-ocimene, in addition to the acyclic oxygenated monoterpene linalool, and the sesquiterpenes E-caryophyllene and aromadendrene. The constituents E-nerolidol, β-selinene, α-pinene, 1,8-cineole, E-β-ocimene, limonene and eupatoriochromene did not show significant correlations with the variables in the analysis.Canonical correspondence analysis was carried out to assess a potential correlation between insect visits and volatile components . This anP. mollicomum, as confirmed in the two canonical correspondence analyses. These data denote the value of terpenes in this important ecological function. Furthermore, these patterns enable us to deduce the possibility that the compounds that increase insects’ attraction to P. mollicomum inflorescences may have a synergistic effect. It is known that the volatile constituents of leaves, and not only those of inflorescences or mature flowers, influence the attraction of insects [These results show that some major constituents present in the EOs may influence the frequency of visits by potential pollinators to stage 3 (anthesis) of  insects . These cP. mollicomum, we performed Spearman’s correlation analysis to confirm the relationships between visitors and volatile constituents vs. abiotic microclimate factors  , inflorescences temperature  and leaf temperature  for insects 2 , 3 (Syrphidae sp. 2), 5  and 6 (Colletidae sp.), respectively. A weak positive correlation was jointly demonstrated for light intensity and frequency of visits by insects 4  and 5 ; . This information allows us to infer that insects are more attracted to plants in hot periods. This fact is probably due to the intense volatilization of the constituents that possibly attract insects.In addition to canonical correspondence analyses on the insect–plant interaction in  factors . The anar2 = −0.667, p < 0.05; r2 = −0.622, p < 0.05) and rainfall index  with the frequency of insect visits 2 , 3 (Syrphidae sp. 2) and 6 (Colletidae sp.), respectively. These findings support earlier research that indicated that increase in humidity impairs bee visits to flowers of different plant types.In contrast, our findings also describe a moderate negative correlation between relative air humidity , aromadendrene , camphor , α-terpinene , tolualdehyde , and δ-cadinene  showed positive correlations with the environmental variable wind speed (m/s). The data also describe that both the temperature of the inflorescences in stage 3 and the temperature of the leaves (°C) can negatively influence the percentage of β-pinene . According to our results, rain and relative humidity have a strong influence, directly proportional, on the chemical constitution, mainly α-pinene , β-selinene , myrcene , δ-cadinene , tolualdehyde , γ-terpinene , benzyl benzoate  and eupatoriochromene .It is known that the meteorological microclimate variables may influence the attraction of pollinators, however, in addition to this important information, our purpose was to assess whether these factors also influence the biosynthesis of chemical constituents identified in the EOs of P. mollicomum . Additionally, there is a strong positive correlation between rainfall (mm) and leaf sprouting . This correlation indicates that the greater the rainfall, the more pubescent leaves initiate the sprouting process. Moreover, data showed that the leaf sprouting phenophase is negatively correlated with the microclimate light intensity . This trend indicates that higher luminosity inhibits the sprouting process. However, this correlation needs to be further evaluated in detail, as the opposite is expected [P. mollicomum, which would support these results. These issues can be addressed by experiments using carefully regulated in vitro culture.We also performed Spearman’s correlations between abiotic factors of microclimate vs. pattern of phenological events . Data shexpected . We specP. mollicomum. Four of the five were registered between September and November 2020. The last stage was only found in the last two months of the analysis (December 2020 and January 2021). The results show that at the beginning of flowering, there was mainly biosynthesis of the volatile constituents linalool and limonene. In this period, the plants still had their immature inflorescences, with the absence of enough pollen grains to favor the pollination process (and consequent reproduction). With that, the fundamental issue to keep the specimens viable for propagation is the defense of these relevant and important organs. Some studies show that plant species exude toxic volatiles, against herbivores, most markedly in the early stages of flowering, mainly to maintain the accessibility of their buds and, as a result, ensure successful reproduction [Lavandula angustifolia Mill., Lamiaceae), tobacco  and mint , showed that genes that significantly contribute to protection from different reproductive organs were mainly stimulated during the early stages of pubescent tissue development. Another study related to the defensive behavior of Japanese pepper  corroborates these findings [During the observation period of this research, we examined and described five stages of ontogenic development of the reproductive organ of oduction ,83. Zhenfindings .Myzus persicae Sulzer., 1776). This article explains that limonene also protects plants against herbivory by attracting ladybirds , natural enemies of aphids [Glycine max (L.) Merrill). Coleoptera of the species Coccinella septempunctata Linnaeus., 1758, were attracted to plants that contained considerable amounts of limonene and that were extensively damaged by aphids  [Phytoseiulus persimilis Athias, 1957, and Neoseiulus californicus McGregor, 1954, protecting specimens of Lamiaceae (Mentha spicata L.) that emit volatiles against herbivory caused by insects [Citrus sinensis L., Rutaceae) and, consequently, induce an attack by herbivores that directs a negative trait to these plants [Musca domestica L. can reduce limonene toxicity by catalytic activity, converting this monoterpene into less toxic compounds [The initial stages of development of reproductive organs tend to be stressful for many plant species, as in these stages, plants need to reallocate their energy reserves, both for the growth of their flowers or inflorescences, and for the development of defensive strategies. There is an intense investment of plant metabolism in the biosynthesis of compounds of primary and secondary metabolism . The stuf aphids . These rf aphids ,85. Thes., 1917) . In addi insects . Other s insects ,88. Howee plants . Howevere plants . For exaompounds .P. mollicomum was the monoterpene linalool. Previous studies have already suggested that this constituent would be able to help attract insects [Colletes). Approximately 30 years later, some scientists reinforced this hypothesis by demonstrating that, in addition to linalool, the cyclic monoterpene 1,8-cineole, could also aid in the pollination process by attracting bees [Lippia alba Mill. Brown., when exposed to high temperatures, volatilize a higher content of linalool [P. mollicomum specimens [As mentioned, another biosynthesized compound in the early stages of flowering of  insects . Accordiing bees . These dlinalool . Glinwoolinalool . Recentlpecimens . Linaloopecimens , explainpecimens ,95,96,97Arabidopsis thaliana (L.) Heynh (Brassicaceae), showed promising evidence. Besides, recent findings showed that linalool may not have a direct role in plant defense, but an indirect importance in this applicability by the fragmentation of metabolites derived from its oxidation. The work by Boachon et al. (2015) described that one of the enzymes that plays a fundamental role in the metabolism of linalool, CYP76C1, may be involved in its oxidation process in A. thaliana flowers. This CYP catalyzes a metabolic cascade for the formation of soluble oxidizing derivatives, such as 8-carboxy-linalool, in addition to volatile cyclic derivatives, such as alcohols and aldehydes [A. thaliana flowers altered the behavior of adverse visitors. The results of these tests showed that thysanopterans, in addition to other herbivorous insects, have a greater predilection for plants with a high content of linalool than for those with derivatives of this monoterpene, demonstrating the repulsive role of linalool oxidation products [It has been known for years that the enzymes of the linalool–synthase complex facilitate the production of linalool throughout the ontogeny of flowers ,100,101.ldehydes . This stldehydes . In the ldehydes . These aproducts . It is aproducts ,106,107.Mature inflorescences were noticeably more prevalent from November 2020 to January 2021 so that the biosynthesis of components that aid in luring prospective pollinators would be more feasible and support the reproduction process ,106,107.Ageratin adenophora (Spreng.) R.M.King and H.Rob (Asteraceae) throughout its life cycle, high contents of hydroxylated derivatives of desmethoxyencalin  were shown to influence the ontogenicity of floral organs in the early stages [A. adenophora leaves identified high percentages of chromene derivatives in pubescent leaves and low percentages in senescent leaves [E-nerolidol, β-elemene and D germacrene in senescent leaves of P. mollicomum, which allows us to infer about the protective role of these constituents in leaf structures. As the eupatoriochromene content is generally higher in immature leaves, this compound may assist both in the development process and in leaf protection [P. mollicum leaves exhibit this intriguing dichotomy in the time  and space (various compartments) of chromene/sesquiterpene biosynthetic activity. Besides, eupatoriochromene had already been identified as the main constituent present in the EOs of dry leaves and inflorescences of other Piper species [The important data presented in the bar graphs  show they stages . Anothert leaves . The relt leaves . These dotection ,118,119. species ,121,122.E-nerolidol, 1,8-cineole, linalool, limonene and α-pinene. As their respective percentages in the EOs of the reproductive parts decreased proportionally to the ontogenic development of the inflorescences, our inference is that these compounds act in a synergistic effect to assist in the process of protecting these organs against herbivores, which may cause, for example, damage to the palatability of these structures. It is known that fruits and infructescences can be preyed on by animals, which leads to seed dispersal. Toxic compounds present in these reproductive organs are important in the early stages of development, as they help to protect them [E-nerolidol, 1,8-cineole, linalool, limonene and α-pinene in P. mollicomum: development and protection.Returning to the discussion started earlier and, as a reinforcement of the idea, our dataset makes us reflect on the presence, besides eupatoriochromene, of other constituents present in EOs, such as ect them ,123. Howect them ,123. It Otolemur crassicaudatus Geoffroy, 1812) and kangaroo species, such as Macropus rufogriseus Desmarest, 1817, causing damage to the digestive process of these marsupials [Phascolarctos cinereus Goldfuss, 1817), manage to reverse the toxicity of 1,8-cineole, and ingest a significant content of leaves that contain a high content of this monoterpene in their vegetative structures, such as species of the genus Eucalyptus L. [P. mollicomum. Our hypothesis is that 1,8-cineole is related to the repellency process of herbivores because there is a gradual increase in the early stages of inflorescence development and a significant decrease in the fruiting stage.It must also be considered that infructescences and fruits accumulate a greater content of primary metabolites, therefore, a drainage effect must also be considered . Some strsupials ,126,127.rsupials ,129,130.yptus L. ,131. AccTherefore, from the foregoing, it appears that the constituents in the EOs of P. mollicomum may present activity in attracting potential pollinating insects ; attracting natural enemies of herbivores (limonene); participating in the ontogenic development of inflorescences, and possibly of leaves (eupatoriochromene); and acting in the defense of vegetative structures .The results of this manuscript demonstrated by canonical correspondence and Spearman’s correlation, that linalool may play a major role in attracting potential pollinators, while 1,8-cineole is important in herbivore repellency. This evidence helps to support the hypothesis that in the reproductive period there is a pattern of conversion of chemical resources in the different organs, that is, 1,8-cineole in the leaves and linalool in the reproductive organs . This isβ-pinene. Previous studies mention the action of temperature and radiation on the β-pinene content [β-pinene isomers [α-pinene content and found that this monoterpene undergoes EOs significant deterioration by photooxidation when exposed to high temperatures [Vitis vinifera L. (Vitaceae), showed that when there was an increase in UVB radiation, there was also a significant increase in the percentage of oxygenated cyclic monoterpenes such as 1,8-cineole [Ocimum basilicum L. (Lamiaceae), it was evidenced that, when the 1,8-cineole content decreased, the amount of linalool increased significantly [However, our research was also designed to understand another question: is the biosynthesis of these constituents influenced by abiotic microclimate factors? Our results related to the Spearman’s analysis showed a negative correlation between inflorescence temperature vs. percentage content of cyclic monoterpene  content . Our res isomers ,134,135.-cineole . In anotficantly . This faficantly .P. mollicomum. There are not many studies that list convergences between rainfall intensity and volatile constituents. However, some analyses have related this climatic variable with the production of sesquiterpenes in the EOs of several plant species [Rainfall intensity and relative air humidity also have a strong influence, directly proportional, on the chemical constitution of some monoterpenes and sesquiterpenes in the EOs of  species ,141,142.P. mollicomum. The results showed that the content of some volatiles, such as Z-β-ocimene, eupatorium-chromene, benzyl benzoate, α-terpinene, tolualdehyde and δ-cadinene, is positively influenced when the wind speed increases. There is some scarcity in the literature on the referred correlation, mainly, of the direct action of the wind on the content of the chemical constituents of the EOs of vegetative structures. However, a recent study carried out with two important tree species, Betula pendula Roth and Betula pubescens Ehrh. (Betulaceae), suggested that emission rates of monoterpenes and sesquiterpenes increased when the wind speed was measured, at relatively high rates in Betula pendula Roth [Our data also propose to discuss the role of the environmental variable wind speed in the biosynthesis and consequent emission of volatile constituents in ula Roth . More teTetragonisca angustula Latreille., 1811), 3 (Diptera—Syrphidae sp. 2), 5  and 6 (Hymenoptera—Colletidae sp.), in addition to a weak positive correlation for light intensity, and a frequency of visits by insects 4  and 5 . Rain and humidity can bring disadvantages to insect flight, meaning a decrease in visitation density [Apis, are active and extremely effective pollinators. Foraging typically begins in the morning when the lowest values of temperature, light intensity, and radiation are measured to be between 15.5 and 18.5 °C, 600 to 1700 lx, and 9 to 20 mW/cm2, respectively [Our additional objective was to evaluate the following question: do microclimatic environmental variables jointly interfere in the activity of visits by potential pollinators to inflorescences? Our results related to the Spearman’s correlation between microclimatic abiotic variables vs. frequency of visits by potential pollinators, showed a moderate positive correlation between air temperature, inflorescence temperature, leaf temperature, relative air humidity and rainfall index, by insects 2 , had much higher visitation rates than other potential pollinators, which is similar toother results [In addition to temperature, radiation and light intensity, another factor that influences the activities of insects that visit flowers is the altitude ,151,152. results ,155,156.T. angustula was the main floral visitor. This bee is considered a sociable forager, which uses a variety of chemical communication strategies with its partners, signaling them where to find rich food sources [T. angustula society [In the times under observation,  sources ,80. Give society ,80.Piper related to these variables.Concluding our analyses and correlations, we propose to relate whether microclimate meteorological variables influence the patterns of phenological events. The data showed a strong negative correlation between leaf fall and temperature; a strong positive correlation between rainfall and leaf sprouting; in addition to a strong negative correlation between light intensity and budding. Some published studies have shown that sudden drops in temperature can cause lesions and photoinhibition in the leaves of plant species, directly and indirectly, increasing the leaf abscission process, which corroborates our findings ,157,158.Piper mollicomum Kunth (PM) from this region. Licenses for the investigations were granted by SISBIO . The surroundings of the experimental plot consisted of native vegetation. Fertile specimens were previously identified by taxonomist Dr. George Azevedo de Queiroz at Botanical Garden of Rio de Janeiro (JBRJ), and the samples were deposited in the Herbarium of the University of the State of Rio de Janeiro (HRJ/UERJ)  (43°14′29.64″ W/22°58′9.80″ S), at an elevation that varies between 127 and 68 m between the studied specimens. The TNP is located in the South Zone of the City of Rio de Janeiro and has a tropical monsoon climate, according to the Köppen–Geiger classification . Six aduRJ/UERJ) .Digital Windmeter Anemometer —wind speed, temperature and relative air humidity; measured in the same period of observation of the frequency of visits by potential pollinators to mature inflorescences of P. mollicomum; Luxmeter —luminosity; performed once at each time of observation of the frequency of visits; and Infrared Laser Thermometer —for measuring the surface temperatures of leaves and reproductive organs; also collected once at each visit time. Therefore, the environmental variables measured were: (a) air temperature (°C), (b) inflorescence and infructescence temperature (°C), (c) leaf temperature (°C), (d) relative humidity (%), (e) wind speed (m/s), (f) rain (mm) and (g) light intensity (kJ/m2).Data on meteorological microclimate variables for the days of weekly collections were obtained with the aid of manual meters: The climatological data referring to the pluviometric indices were obtained from the Meteorological Station of Forte de Copacabana in the City of Rio de Janeiro, at the Brazilian Institute of Meteorology (INMET), in the weeks in which the observations took place. The sets of climatological variables were tabulated for linear correlation with other data: (a) frequency of insect visits to mature inflorescences; (b) chemical constituents identified in the EOs from leaves and from reproductive organs (five stages) and (c) phenological events.P. mollicomum phenology were carried out in the six individuals in the aforementioned period. The phenological events evaluated were:(a) leaf sprouting (emissions from vegetative buds); (b) leaf fall (reduction of leaves on stems or branches); (c) immature inflorescences or buds ; (d) mature inflorescences (anthesis flowers with the presence of pollen) and (e) immature infructescences (process ranging from the oxidation of mature inflorescences to the presence of infructescences with hardened pericarps). Due to the small size of the flowers and fruits, a manual lens (60X magnification) was used to observe the inflorescences and infructescences in the field, in addition to determining which flowers were in anthesis [The qualification and quantification of anthesis .Fournier Intensity (FI) methodology was used to quantify the phenophases mentioned above . The FI P. mollicomum were performed weekly, in the period and in locus described above, from 8:00 am to 5:00 p.m. . During this period, plant specimens contained significant amounts of mature inflorescences [Syrphidae sp. 1); Insect 2 (Hymenoptera—Tetragonisca angustula Latreille. 1811); Insect 3 (Diptera—Syrphidae sp. 2); Insect 4 ; Insect 5  and Insect 6 (Hymenoptera—Colletidae sp.).Observations of the frequency of visits by potential pollinators of escences ,165. Theescences ,165. Theescences . SpecimeP. mollicomum (5 g to 40 g) were collected monthly in the reproductive period, in the morning, between 9:00 and 10:00 a.m., for analysis involving ontogenic vs. constituents of the EOs. These materials were manually reduced with scissors and were submitted to the hydrodistillation method in a modified Clevenger-type apparatus. The reduced material was transferred to a 2 L round bottom flask, to which 700 mL of distilled water was added. The hydrodistillation process was carried out for 2 h. Upon completion, the pure EOs were separated from the aqueous phase, subjected to drying with anhydrous sodium sulfate  and stored in a closed amber vial, in a freezer at −20 °C, until the time of analysis [v/w) [P. mollicomum (Leaves (150 g) and five different stages of the reproductive organ of analysis ,55. Yielis [v/w) ,55. The llicomum  were desStage 1: Inflorescence in spike; presenting very immature development since its floral elements are protected by the bracts still in formation, arranged in inclined bands, with green color from the peduncle to the inflorescence.Stage 2: Inflorescence in spike; presenting an immature stage of development since its floral elements are still protected by bracts in formation, arranged in bands inclined to horizontal. The length of the peduncle is slightly longer and slightly changes its hue to yellowish-cream.Stage 3: Inflorescence in spike; presenting a more advanced stage of development from bottom to top since its floral elements are already more defined, where the triangular bracts  and open anthers with the release of pollen grains are morphologically visualized. The length of the peduncle is longer and the inflorescence changes its hue to intense yellow.Stage 4: Inflorescence in spike; presenting a more advanced stage of development from bottom to top since its floral elements are already more defined, where the triangular bracts  and open anthers with the release of pollen grains are morphologically visualized. The peduncle length is continuously longer, still green, while the inflorescence changes its tonality to yellow/brown interspersed bands, due to the presence of mature anthers.Stage 5: Inflorescence in spike; presenting initial stage of fruit in all its extension since one of the floral elements, the ovary, presents itself modified in its formation. At this stage, the triangular floral bracts are morphologically visualized, anthers already senescent, which may or may not keep their filaments adherent to the fruit, which is in development; arranged in horizontal bands, maintaining the length of the peduncle that still remains green, while the inflorescence changes its tonality to intense green.m/z) at 40—600 atomic mass units (u), with impact energy of 70 eV, operating in positive mode. A 1 μL sample of the EOs solution was injected, in splitless flow mode, and with the injector temperature at 270 °C [8-C28, Sigma-Aldrich, Brazil), performed in the same column and conditions used for the analysis by GC/FID. The identification of constituents was made by comparing the mass spectra obtained from data from the equipment’s library (NIST and Wiley https://webbook.nist.gov/chemistry/cas-ser/ (accessed on 1 January 2021)), and records in the literature [The EOs obtained by hydrodistillation were solubilized in dichloromethane  to obtain a final concentration of 1000 ppm. Subsequently, these diluted EOs were submitted to analysis by Gas Chromatography coupled to Mass Spectrometry (GC/MS) in an HP Agilent CG 6890—EM 5973N Equipment – Rio de Janeiro, to identify the constituents by their respective mass spectra. To determine the quantitative parameters of the EOs and calculation of the Retention Index (RI), the analyses were performed by Gas Chromatography Coupled to the Flame Ionization Detector (GC/FID), in an HP-Agilent 6890–Rio de Janeiro equipment. The GC/MS analyses were: HP-5MS capillary analytical column  and temperature ramp from 60 °C to 240 °C, with an increment of 3 °C/min, helium (~99.9999%) as carrier gas, at a constant flow rate of 1.0 mL/min; scanning range of masses . Spearman’s correlation test was processed to assess the relationships between abiotic factors, as well as volatile constituents vs. ontogeny vs. frequency of visitation of potential pollinators. The correlation coefficient of this inspection can be positive  or negative , assimilating quantitative values that suggest that the relationship between two or more variables is strong, moderate or weak: 0.00 to 0.19 (very weak); 0.20 to 0.39 (weak); 0.40 to 0.69 (moderate); 0.70 to 0.89 (strong) and 0.90 to 1.00 (very strong) . CanonicPiper mollicomum, a medicinal and ritualistic plant of fundamental value for the regeneration of deforested areas, such as Atlantic Forest. This is the first scientific work that studies the reproductive period of this Piper species. As a result, the information gathered is fundamentally important for comprehending the mechanisms of chemical–ecological plant–insect interactions in Piperaceae, a basal angiosperm.All correlations between volatiles vs. potential pollinators visits vs. ontogeny vs. microclimate vs. phenology allow us to convey important data regarding the complex information network in