Ginkgo biloba (maidenhair tree), which have been reported to exert anti-inflammatory and antioxidant properties and repair a variety of active cellular damage, have been applied therapeutically for centuries. In this study, we established a rat SCI model to investigate the effects of Ginkgo biloba leaves on decompression at different stages of SCI. The application of Ginkgo biloba leaves during the decompression of SCI at different time points, the neurological recovery of SCI, and the underlying molecular mechanism were explored. The findings provide reliable experimental data that reveal the mechanism of GBI (Ginkgo biloba injection) in the clinical treatment of SCI.Spinal cord injury (SCI) is a severe trauma of the central nervous system characterized by high disability and high mortality. Clinical progress has been achieved in understanding the pathological mechanism of SCI and its early treatment, but the results are unsatisfactory. In China, increasing attention has been paid to the role of traditional Chinese medicine in the treatment of SCI. In particular, extracts from the leaves of  In recent years, the incidence of spinal cord injury (SCI) has been increasing. Progress has been made in understanding the pathological mechanism of SCI and its early treatment, but the results have been unsatisfactory , 2. At pGinkgo biloba  reportedly exert anti-inflammatory, antioxidant, and neuroprotective properties and can repair a variety of active cellular damage. These extracts have been used therapeutically for centuries [Ginkgo biloba extract Tanakan effectively eliminated free radicals generated during lumbar ischemia and reperfusion in rabbits and reduced reperfusion injury [Ginkgo biloba extract improved neuronal cell damage after spinal cord ischemia and reperfusion via the mitochondrial pathway [Ginkgo biloba extract Ginkgolide B against acute SCI in rats, which may be related to the JAK/STAT signaling pathway [Ginkgo biloba extracts on neuronal apoptosis and their neuroprotective effects after SCI [Ginkgo biloba extracts inhibit spinal cord inflammation after secondary injury while simultaneously exerting neuroprotection after early decompression has not yet been reported.The development of traditional Chinese medicine has led to increasing attention being focused on its application in the treatment of SCI in China. The extracts from the leaves of enturies  in the centuries –10. The n injury . Meanwhi pathway . Song et pathway . Currentfter SCI , 15. HowGinkgo biloba leaves can alleviate inflammatory reaction after secondary SCI and protect functional cells, such as neurons and oligodendrocytes, thereby promoting the repair of SCI. This study aims to provide a reference for the application of traditional Chinese medicine in clinical SCI.Based on the above theory, we hypothesized that Sprague-Dawley rats weighing 200–210 g (6-7 weeks of age) were purchased from Liaoning Changsheng Bio. Co., Ltd. with approval from the ethics committee and divided into two groups . All animal experiments were performed based on the Guidelines for Animal Care and Use of the Model Animal Research Institute at Wuhan Myhalic Biotechnology Co., Ltd. The Institutional Review Board confirms that the scheme of this project was properly designed, the number of animals required was limited to the minimum, the investigators were qualified to carry out the proposed project, and all animals were handled with sufficient care and protection.Ginkgo biloba injection (GBI), simple decompression 48 h after SCI without drug intervention, and simple decompression 48 h after SCI with GBI. All rats were sacrificed 3 and 60 days after injury via administration of an overdose of sodium pentobarbital.Sprague-Dawley rats subjected to SCI modeling were anesthetized intraperitoneally using 1% pentobarbital sodium at 30–40 mg/kg. An acute SCI model of continuous static compression was established in rats by cyclization . ThroughGinkgo biloba  was dissolved in 0.5% sodium carboxymethyl cellulose solution. GBI was performed via daily intraperitoneal injection (4 mg/kg of body weight) for two weeks after SCI. Meanwhile, control and nondrug-treated rats received the same volumes of 0.5% carboxymethyl cellulose solution at the same times.Neurological assessments were performed daily after SCI. The open-field test assessed the rats' locomotion, weight support, and coordination, and the results were evaluated using the Basso, Beattie, and Bresnahan (BBB) scale (range from 1 to 21), as previously described . A score of 0 represented no spontaneous movement, whereas a score of 21 represented complete mobility. The BBB curve was constructed using data from selected time points.After sacrifice, half of the spinal cord tissues were obtained and stored at −80°C, and the remaining half of the samples were fixed in 4% paraformaldehyde. The nerve myelin sheath, morphology, and number of neurons were observed by Luxol fast blue (LFB) (servicebio.cn) and Nissl staining. The number of nerve fibers was examined by NF-200 staining  and astrocytes and glial scars were assessed by glial fibrillary acidic protein (GFAP) staining . All images were acquired using a microscope  and the Leica Application Suite image system was used to collect and analyze relevant parts of the sample.μl of protease K (20 μg/ml) for 20 min. After two washes with phosphate-buffered saline for 3 min each, the samples were incubated with a TUNEL apoptosis detection kit  according to the manufacturer's instruction. The sections were sealed, dried, and observed using an optical microscope .Paraffin-embedded tissue specimens were sliced and underwent conventional dewaxing and hydration. The sections were placed into 3% hydrogen peroxide methanol for 15 min, and each sample was incubated with 100 Flow cytometry  was performed to detect the infiltration of inflammatory cells in spinal cord tissues, including neutrophils  and peripheral mononuclear macrophages . Immunohistochemical staining of F4/80 was carried out to assess the infiltration of macrophages . All samples were obtained three days after SCI.μL of lysate per 20 mg of tissue. The tissue was then homogenized until complete lysis was achieved. The lysed sample was centrifuged at 12,000 ×g for 1 minute at 4°C, and the supernatant was quantified and stored at −80°C. The expression of the following factors was detected: Gro-α , a chemotactic activity for neutrophils; monocyte chemoattractant protein 1 ; macrophage inflammatory protein 2 , MIP1 , and MIP1b , produced by activated monocytes and neutrophils; tumor necrosis factor-α ; interleukin 1b , IL6 , inducible nitric oxide synthase , and cyclooxygenase-2 . Secondary antibody incubation was performed with horseradish peroxidase-conjugated goat anti-rabbit IgG . GAPDH  serves as an internal reference.Dorsal white matter strips were isolated and stored at −80°C for the detection of inflammatory proteins and cleaved caspase-3 , Bax  and Bcl-2  in the spinal cord. The spinal cord tissue was cut into small pieces, and radioimmunoprecipitation assay buffer containing was protease and phosphatase inhibitors added at a ratio of 150–250 P < 0.001.All data were expressed as the mean ± SD. Data analysis was performed using GraphPad 7.0 software. Repeated-measures analysis of one-way analysis of variance was performed to compare the inflammatory infiltrate and proteins among groups. Differences were considered statistically significant at P < 0.0001; All rats initially had a BBB score of 21, and rats subjected to SCI showed a BBB score of 0–2 on the first day, which reflected total hindlimb paralysis. At 1 and 3 days after injury, the movement of the injured rats remained severely impaired, but decompression treatment at 8 h and 48 h after SCI resulted in significant improvements in mobility. Compared to decompression only, injured rats subjected to GBI (4 mg/kg/d) after 8 h after SCI decompression showed greater locomotor activity that continued throughout the experimental period  , and GBI treatment promotes Bcl-2 expression. . However, after GBI treatment, the expression of the above inflammatory factors decreased significantly, which further confirmed that GBI treatment could inhibit the inflammatory response of spiral core tissue.After SCI, a large number of activated glial cells and macrophages infiltrate the injured site. This results in the secretion of inflammatory proteins, which in turn aggravates tissue damage. To further study the activation of microglia after SCI, CD11b and CD45, which are markers of activated glial cells, were detected by flow cytometry 3 days after rats were subjected to SCI . The resGinkgo biloba is a product of lipid peroxidation that exerts potential protective function in spinal cord myelin, glial, and neuronal membranes and other cellular elements. Studies have pointed out that the Ginkgo biloba extract EGb761 has protective effects against brain death-induced kidney injury, and this effect may be related to SAPK and JAK-STAT signaling [Ginkgo biloba can inhibit the inflammation of the spinal cord after secondary injury with simultaneous neuroprotection after early decompression has not been reported.SCI is a severe trauma of the central nervous system that affects a large proportion of young adults. It has become a global medical and social problem that results in burden such as disability, high cost of treatment, and low survival . Ginkgo ignaling . ClinicaMyelin integrity is an important factor in maintaining the physiological function of the central nervous system. The preservation and regeneration of myelin integrity after SCI plays a key role in the recovery of spinal cord function , 19. Acuα, COX-2, CCLs, and other neurotoxic factors, leading to neuronal death and aggravating nervous system damage. We examined the expression of ten inflammation-related proteins to verify the combined therapeutic effect of GBI and decompression in rats with SCI. Western blot revealed that the combination of decompression and GBI significantly reduced the expression of IL-6, TNF-α, CCL1-3, and COX-2, suggesting the effectiveness of combined GBI and decompression therapy against inflammatory factors in vivo. The observations from Nissl and TUNEL staining showed consistent results.The myelin sheath is damaged during SCI, while a large number of neuronal cells undergo apoptosis. Microglia activation and macrophage infiltration also occur in injured tissues, resulting in the exacerbation of SCI , 21. In Although GBI has been reported to have neuroprotection and antioxidant activity in different in vitro and in vivo models, it has not been combined with decompression for postspinal injury treatment. Our study validated the effectiveness of combined GBI and decompression therapy in vivo, aiming to provide a reference for the clinical treatment of SCI.Ginkgo biloba, which further reduced inflammatory response after pots-SCI decompression, protected functional cells such as neurons and oligodendrocytes, and inhibited apoptosis to promote SCI repair. As a traditional Chinese medical ingredient, Ginkgo biloba can be combined with surgical decompression treatment in clinical practice and contribute to the clinical treatment of SCI.Based on previous studies that elucidated the molecular mechanism of secondary injury SCI, we found that early surgical decompression significantly ameliorated SCI in rats. Our study employed the classic clinical drug


Uterus biopsies were taken before, during, and after ischemia, with or without reperfusion, and were subsequently stored under cold (4ᵒC) or warm (37ᵒC) conditions. TUNEL cell death assay was done at various time points along the combined in vivo/ex vivo experimental timeline. We found that Gap27 protected against storage-related cell death under cold but not warm conditions when the uterus had experienced in situ ischemia/reperfusion. For in situ brief ischemia without reperfusion, Gap27 reduction of cell death was delayed and significantly less, suggesting that protection critically depends on processes initiated when the organ was still in the donor. Thus, the inclusion of the connexin channel inhibitor Gap27 during cold storage protects the uterus against cell death, and the degree of protection depends on the history of exposure to warm ischemia. Gap27 protection may be indicated for uteri from deceased donors, in which ischemia is likely because life-saving organs have retrieval priority.Uterus transplantation is an experimental infertility treatment for women with uterine factor infertility. During donor uterus retrieval and subsequent storage, ischemia and other stressors are likely to occur, resulting in the delayed restoration of organ function and increased graft rejection. The uterus expresses connexin-based hemichannels, the opening of which can promote ischemic cell death, as well as gap junctions that may expand cell death by bystander signaling. We investigated if connexin channel inhibition with connexin channel inhibitor Gap27 could protect the uterus against cell death during the storage period. The study involved 9 female patients undergoing gender-change surgery. Before uterus removal, it was exposed to  Uterus transplantation is an experimental infertility treatment option for women with a dysfunctional or absent uterus, also known as absolute uterine factor infertility. After extensive research on several animal species , Brännstin situ while still in the donor, influenced the uterus outcome after storage.Here, we investigated whether connexin channel inhibition with Gap27 during the storage period between donor uterus retrieval and its implantation in the recipient patient, could improve the uterus condition in terms of cell death scoring. We tested both cold (4°C) and warm (37°C) static storage conditions and also checked whether warm I/R applied to the uterus Tissue samples were taken from the uteri of healthy transgender patients of Western European descent undergoing a laparoscopic hysterectomy and ovariectomy. Transgender patients in the age range of 18–27 years were included. Patients with complex medical history like diabetes, heart and lung disease or thrombo-embolic disease were excluded from the study; no organs were procured from prisoners. In total, 9 uterus donors were recruited in the study, 4 in Experiment 1 (recruitment from December 2014 to January 2016) and 5 in Experiment 2 (recruitment from July 2017 until April 2018). The donors were females, in transition; their mean age was 21.6 years old (median age 22), 7 were still studying and two were working, one patient was from Holland, and eight from Belgium. The recruitment took place during outpatient clinic visit, transgender patients planning a surgery were asked to participate in this study after a thorough explanation. The recruitment was done in the Ghent University Hospital and the research was done in the Department of Basic and Applied Medical Sciences, Ghent University.®.Before the surgery, the patients were hormonally treated for approximately a year with NebidoAll material from patients was collected after their written informed consent before surgery according to a protocol that was approved by the ethical committee of the Ghent University Hospital. All the experiments were carried out in accordance with the protocol approved by the ethical committee of the Ghent University Hospital.in situ warm ischemia, plastic surgeons performed the bilateral mastectomy. Upon finishing the mastectomy, the uterine vessel clips were removed. The last biopsy (UB5) was taken 10 min after reperfusion; thus, warm I/R involved 3 h of ischemia followed by 10 min of reperfusion. All biopsies during surgery were hysteroscopically guided, taken with a Spirotome Cervicore device  and had approximately 1.6 mm in diameter. The uterine biopsies were taken from the fundus of the uterus, along an inside to outside direction (endometrium to myometrium) over the thickness of the wall. Uterus biopsies were taken in transgender patients (N = 4) during laparoscopic hysterectomy and ovariectomy combined with mastectomy. The hysterectomy was performed to near completion except for the ligation and transection of the uterine arteries and veins. Each of the four patients had five uterine biopsies taken during the surgery; the first uterine biopsy (UB1), was then taken before bilateral clipping of the uterine arteries and veins, the second (UB2), 10 min after clipping, corresponding to 10 min of warm ischemia. The third biopsy (UB3) was taken after 1 h of warm ischemia and the fourth (UB4) just before removing the vessel clips, corresponding to 3 h of warm ischemia. During these 3 h of ® perfusion solution  to wash the organ from the blood , it was flushed three times (500 ml) with cooled Custodiollood see . Custodiolutions , 24, 25 ex situ) with a Biopsy Punch . Ex-situ biopsies were kept in small containers filled up with Custodiol® solution with or without Gap27 peptide either in the fridge (4°C) or warm incubator (37°C), to mimic cold or warm storage conditions. After storage, samples were prepared as described under Sample preparation and TUNEL staining.Uterus biopsies (3 biopsies per patient per storage condition) were collected immediately after that see ex situ in situ exposure to 20 min warm ischemia  were only collected after organ retrieval with a Biopsy Punch  and were kept in perfusion solution with or without Gap27 peptide and cold-stored at 4°C for 0 h, 1 h, 5 h, 12 h or 24 h. After storage, biopsy samples were prepared as described under Sample preparation and TUNEL staining.We investigated cell death during cold storage of uterus biopsies after ischemia  induced in situ terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL), using In Situ Cell Death Detection Kit . Samples were incubated with the TUNEL reaction mixture for 1 h at 37ᵒC in the dark. Samples treated with DNase I  for 10 min were used as a positive control and not exposed to TdT enzyme, as a negative control. Next, samples were washed with PBS and stained with 1 μg/ml DAPI (Life Technologies Europe) in PBS and were mounted with Vectashield mounting medium . Three random slides per replicate and three images across the sample were acquired with a Nikon TE300 epifluorescence microscope equipped with a 10× objective , and a Nikon DS-Ri1 camera . The number of TUNEL-positive cells was expressed relative to the total number of nuclei stained with DAPI. The calculations were performed using FIJI ImageJ after the application of a threshold corresponding to the upper level of the background signal.At the end of the storage period, biopsies were fixed in 4% formaldehyde  at 4°C overnight. After washout, samples were embedded in paraffin and cut into 5 μm sections. On the day of the staining, slides with tissue samples were deparaffinized and permeabilized for 8 min in 0.1% TritonX-100  dissolved in PBS . Apoptosis was detected by 50 of 20–30 μM [50 for hemichannel inhibition is ~161 μM , was used in this study to block connexin gap junctions and hemichannels during storage of uterus biopsies. Inhibition of gap junctions by Gap27 is characterized by an IC20–30 μM  and we unt of 2;  giving ~nt of 2; . Gap27 wData are expressed as mean ± SEM, with ‘n’ denoting the number of replicates; ‘N’ indicates the number of patients. The number of patients from which samples were taken is specified per experiment. Statistical analysis was performed using GraphPad Prism 7 software . In Experiment 1, we used one-way ANOVA with Sidak’s multiple comparisons. For Experiment 2, we used a two-way ANOVA with Sidak’s multiple comparisons. The level of statistical significance level was set at p < 0.05.in situ and ex situ part, an overview of which is presented in in situ part of Experiment 1, we investigated cell death as estimated from TUNEL scores in uterus biopsies taken at various time points during 3 h of in situ warm ischemia (clipping of uterine arteries and veins in the patient) followed by 10 min of reperfusion (release of clips) (The study design was divided into two parts: an f clips) . Based of clips) . Surprisf clips) . As a rein situ warm ischemia and 10 min of reperfusion, the uterus was removed, biopsies were taken and subsequently kept under either cold (4°C) or warm (37°C) storage conditions in Custodiol® solution. We used 3 biopsy samples from each of the 3 donor patients included in this study per storage condition. As observed in We next tested cell death after 5 h of cold or warm storage. To that purpose, after 3 h of in situ warm ischemia period was reduced to 20 min. Given the fact that reperfusion did not provoke significant cell death in Experiment 1, we further decided not to apply a reperfusion phase for Experiment 2. Because of the more limited time available during surgery, we only took uterus biopsies after removal of the uterus from the body, and its flushing with Custodiol® solution. We then exposed the samples to various periods of cold storage (4°C) up to 24 h, in control, or with Gap27 added to the solution , reflecting the shorter in situ warm ischemia period (20 min vs. 3 h) and the absence of in situ reperfusion. Gap27 did not protect as inferred from the TUNEL scores in the first 12 h period, but it significantly reduced TUNEL positivity after 24 h of storage by ~35%   .We evaluated how initial warm ischemia, and subsequent different storage conditions, influenced the viability of the human uterus. We found that Gap27 protects the uterus against cell death, measured by TUNEL staining, during cold storage while it was ineffective against warm storage. Interestingly, protection by Gap27 was stronger and quicker when the uterus had previously experienced warm I/R (3 h/10 min) compared to 20 min ischemia without reperfusion, in which case protection was only detectable after 24 h cold storage.During ischemia, due to restricted or reduced blood flow, the tissue is deprived of oxygen and blood nutrients, causing a gradual loss of cellular homeodynamics. At reperfusion, these changes induce inflammation and oxidative stress that can lead to an organ’s loss of function . The detin situ warm ischemia and the apparent absence of reperfusion injury following restored circulation in the patient. Adachi et al., 2016 and Kisu et al., 2017 used a similar experimental set-up in non-human primates to investigate the allowable exposure time to warm ischemia induced by clamping the uterine vessels for up to 8 h followed by 3 h of reperfusion [We observed low sensitivity of the uterus to 3 h erfusion , 34. In erfusion , 34. Bräerfusion , 36. Ourin situ warm I/R experiment mimics what the uterus would experience during other types of procedures where the uterine artery is temporarily blocked. For example, transient blocking of the uterine perfusion by clipping uterine arteries is used to prevent excessive blood loss during laparoscopic myomectomy [Moreover, the omectomy . Studiesomectomy , 39. Howin situ warm I/R compared to brief warm ischemia. This indicates that warm ischemia initially triggered a cellular stress response which evolves to cell death during subsequent storage. When the organ was retrieved within 20 min, cell death significantly increased only after 12 h of cold storage, which confirms the observations made by Wranning et al. who found an allowable cold storage time of at least 6 h for human uterine smooth muscle tissues in protective solution [Apoptotic cell death progressed faster after warm than after cold storage. Moreover, cold storage injury was two-fold higher when the organ was previously exposed to the long solution . Some stsolution  or even solution . Recentlsolution . HoweverIt has been confirmed that gap junctions play a role in the pathogenesis of ischemia, cold storage, and reperfusion injury in mice , 43. Thein situ warm ischemia, indicating that the peptide protects against cell death processes that were initiated when the uterus was still in the patient rather than by storage-induced processes. Cell death processes initiated during long, in situ warm ischemia, continue during the warm storage. In this case, cell death probably overwhelms the protective potential of Gap27, possibly because other, non-connexin signaling pathways are activated. Collectively, this indicates that the peptide does not primarily target intrinsic storage-linked processes but rather events initiated in the preceding period of uterus retrieval from the donor.Here, Gap27 strongly protects the uterus against cell death during cold storage when surgery involved pre-exposure to long warm I/R. Thus, Gap27 protection seems to depend on the preceding vs. necrotic cell death because the readout was based exclusively on TUNEL staining. Caspase-3 staining was tested, but statistical variability was large, precluding the extraction of accurate data. Further studies evaluating additional readouts are necessary, including uterus morphology, oxidative stress markers and cellular, biochemical or proteomic markers of distinct cell death modes such as necrosis, apoptosis, necroptosis, ferroptosis, autophagic cell death, cellular energy catastrophe and others.There are several limitations to our study. First, the experimental warm ischemia only represents the first phase during organ retrieval but lacks a second warm ischemia phase that would occur during re-anastomosis upon organ implantation. Second, we did not evaluate the endometrium separately since before surgery, patients went through long hormonal treatment and the endometrial layer was thin and non-active. Third, the number of available patients was low and statistical power suboptimal, making it possible that the results obtained do not necessarily apply to a larger patient cohort, therefore, the results should be interpreted with caution. Lastly, we could not distinguish between apoptotic in situ I/R appears to be well tolerated by the human uterus. However, it triggers significant cell death that becomes apparent during storage. Inclusion of Gap27 during cold storage could be useful to protect the uterus obtained from deceased donors where marked warm ischemia is likely because priority retrieval of life-saving organs. This study is a promising lead to the discovery of new measures for uterus protection during the long period between organ retrieval and re-implantation, which may promote implantation success.In conclusion, the setting of our I/R experiment is unique because it is performed inside the human body. It mimics conditions where the uterus would be removed from a deceased donor after experiencing long warm ischemia because of its delayed retrieval after removal of life-saving organs. Long warm


Although found in lower quantities, ARG-containing phage particles were more prevalent among the Antarctic than the Mediterranean seawater samples and Antarctic bacterial communities were confirmed as their source. In the Mediterranean area, ARG-containing phages from anthropogenic fecal pollution might allow ARG transmission through the food chain. ARGs were detected in phage particles isolated from fish , the most abundant being β-lactamases. Some of these particles were infectious in cultures of the fecal bacteria Escherichia coli. By serving as ARG reservoirs in marine environments, including those with low human activity, such as the Antarctic, phages could contribute to ARG transmission between bacterial communities.Anthropogenic activities are a key factor in the development of antibiotic resistance in bacteria, a growing problem worldwide. Nevertheless, antibiotics and resistances were being generated by bacterial communities long before their discovery by humankind, and might occur in areas without human influence. Bacteriophages are known to play a relevant role in the dissemination of antibiotic resistance genes (ARGs) in aquatic environments. In this study, five ARGs ( Antibiotic resistance is one of the biggest threats to public health this century. The growing environmental presence of antibiotics and their derivative metabolites is mainly due to anthropogenic activities, such as medicine, agriculture, and industry. Antibiotics have been overused both in clinical settings and the livestock industry, including for prophylaxis, although their use as growth supplements has been prohibited in Europe since 2006 (EC Regulation No. 1831/2003) [Depending on their transport and persistence in soil or water, antibiotics can spread to surface water and groundwater, exerting a selective pressure on aquatic bacteria and the microbiota of aquatic animals, and leading to the emergence of resistant bacteria and the dissemination of antibiotic resistance genes (ARGs) ,5.A high demand for fish products has led to an increase in aquaculture in recent years . HoweverMicroorganisms have been producing natural antibiotics for billions of years to gain a selective advantage over competitors , and the31 phage particles have been estimated) and can be isolated from any habitat where bacteria exist [Bacteriophages are the most abundant entities in the world  Mediterranean coastal water, subjected to a high anthropogenic impact and (ii) Antarctic seawater, where the anthropogenic impact is low. Considering the negligible human influence in the Antarctic area, we also examined if phages induced from the bacterial fraction of marine communities could be confirmed as the source of the ARG-containing phage particles. Additionally, the impact of ARG-containing phage particles in Mediterranean seawater on the food chain was studied by analyzing their presence in marine fish, shellfish, and farmed fish.n = 10 samples), Mediterranean fish (n = 10), Atlantic fish (n = 10), frozen fish (n = 10), and farmed fish (n = 10) were obtained in local supermarkets during 2018–2019. All the samples were collected in sterile containers, kept at 4 °C and analyzed within 24 h.Twenty seawater samples were collected from eleven different sampling sites (Med-1 to Med-11) off the Barcelona coast in February–October 2019 a. Ten se® filter holders , followed by filtration through 3 μm mixed cellulose ester membranes (MCE) (Merck), and filtration through 0.2 μm Isopore™ polycarbonate membranes. Phage particles from the final filtrate were recovered by chemical flocculation [2-0.2M ascorbic acid buffer to a final volume of 5 mL.Five liters (L) of Antarctic seawater samples were filtered by 5 μm Swinnexculation . An ironFor Mediterranean seawater samples, 100 mL was filtered through 0.22 μm low protein binding polyethersulfone (PES) membranes. Viral particles in filtered samples were then concentrated with PEG8000 following previously reported protocols ,26.® Ultra Centrifugal Filters to volumes of 0.5 mL. The concentrated phage suspensions of both origins were used for the phage DNA extraction.Lastly, the suspensions of phages of both origins were dialyzed and concentrated with Amicon® Ultra 15 mL centrifugal filters as described above. The suspensions were stored at −20 °C until processed for phage DNA extraction.Antarctic seawater samples were processed within 48 h after collection for phage induction from the bacterial communities. One liter of each sample was filtered by 0.2 μm Isopore™ polycarbonate membranes (Merck) to retain the marine bacterial communities. In order to enrich the bacterial communities, filters were incubated in 7 mL of Difco™ Marine Broth at 18 °C for 48–72 h without shaking. Phages were induced from the possible lysogens present in these bacterial communities by treatment with mitomycin C (0.5 µg/mL) and incubation overnight at 20 °C in the dark without shaking . After iv/v)) to rule out the possible presence of vesicles containing DNA, vigorously vortexed for 5 min and centrifuged at 16,000× g for 5 min. The aqueous phase was incubated with DNase I (100 units/mL) to eliminate any free DNA in the samples outside the phage particles. DNase was inactivated by heating for 5 min at 75 °C.As in previous studies ,24, filtThese suspensions were then used to extract DNA from the phage capsids. Before breaking the capsids, aliquots were taken to confirm DNA removal by the DNase treatment and complete inactivation of DNase by heat treatment.v/v) and chloroform treatment. DNA was precipitated using 100% ethanol and 3 M sodium acetate and resuspended in 50 µL of ultrapure sterile water. DNA was quantified using a Nanodrop ND-1000 spectrophotometer.Viral capsids were broken using proteinase K (0.5 µg/mL) in 250 μL of proteinase K buffer and then incubated for 1 h at 55 °C . The enc® Environmental Master Mix 2.0 . The reaction contained 9 μL of the sample DNA or standards with known DNA concentration. The results were analyzed with the Applied Biosystems StepOne™ Instrument program.Quantitative real-time PCR (qPCR) with TaqMan hydrolysis probes was performed using the StepOne Real Time PCR System in a 20 μL reaction mixture with the TaqManblaTEM, blaCTX-M-1 group, blaCTX-M-9 group, blaOXA-48 and blaVIM), two quinolone resistance genes (qnrA and qnrS), mecA, a gene commonly found in Staphylococcus that confers resistance to methicillin [sul1, which confers resistance to sulfonamides and is frequently found in environmental and clinical bacterial populations [tetW, which confers resistance to tetracycline and is commonly found and used in aquaculture environments [armA, which encodes aminoglycoside resistance and is widely distributed in Enterobacteriaceae [blaTEM, blaCTX-M-1 group, blaCTX-M-9 group, tetW and sul1), and the fish samples were analyzed for the eleven ARGs.A total of 11 ARGs with a range of mechanisms and clinical relevance were evaluated. Five genes conferred resistance to β-lactam antibiotics  of known concentration were used to generate the standard curves. All samples were run in triplicate (including the standards and negative controls). The number of gene copies (GC) was defined as the mean of the triplicate data obtained. To quantify the ARGs, we considered the results obtained within the threshold cycle and within the limit of quantification. This was determined by the last valid threshold cycle for each ARG assay , which were used to analyze the bacterial indicators. Somatic coliphages were analyzed as indicated below.Microbial indicators were evaluated in fish samples and Mediterranean seawater. To isolate bacteria and somatic phages from fish and shellfish samples, 20 g of each sample was homogenized for 2 min in 60 mL of phage buffer with the Stomacher homogenizer . Stomacher bags with filters  were used to enhance the separation of solid waste from the liquid fraction containing the microorganisms. For Mediterranean seawater, 100 mL of each sample was filtered through 0.45 µm mixed cellulose ester (MCE) EZ-PakEscherichia coli and E. coli resistant to Amp were determined on Chromocult® Coliform Agar or Chromocult® Coliform Agar with Amp (100 μg/mL), respectively. Incubation was first performed for 2 h at 37 °C to accommodate potentially damaged microorganisms and then overnight at 44 °C. Presumed E. coli colonies were confirmed in MacConkey agar. Total Enterococcus were enumerated by membrane filtration, according to the ISO standard method [Enterococcus agar (BD). Suspected Enterococcus colonies were confirmed by growing them in bile esculin agar for 3 h at 44 °C.Total aerobic microorganisms resistant to ampicillin (AmpR) were evaluated on Tryptone Soy Agar (TSA) with Amp (100 μg/mL) at 37 °C. Total d method . Filtersg and the supernatant was filtered through 0.22 μm low protein binding PES membranes (Merck). Seawater samples (100 mL) were filtered through 0.22 μm low protein binding PES membranes (Merck) and concentrated with Amicon® Ultra 15 mL centrifugal filters (Merck). The filtrates were analyzed in duplicate for the presence of somatic coliphages according to the ISO standard method [E. coli strain WG5 (ATCC 700078) as the bacterial host, and incubated at 37 °C for 18 h.Somatic coliphages were evaluated as fecal viral indicators in the samples . The homd method , which uEach sample was analyzed for all the indicators in duplicate.E. coli WG5 host strain was evaluated. This strain, used for the somatic coliphage count, was selected for its sensitivity to phage infection [The ability of phage particles in the fish and shellfish samples to infect and propagate in enrichment cultures of the nfection  and becanfection .E. coli WG5 at the exponential phase (OD600 0.3) in 8 mL of Luria-Bertrani broth (LB) and incubated overnight at 37 °C with shaking. After incubation, phages were purified by filtration through 0.22 μm low protein binding PES membranes and treated with chloroform and DNase, as indicated above.The propagation cultures were prepared with 1 mL of each phage suspension after the DNAse treatment and 1 mL of The infectivity of the phage particles carrying ARGs was evaluated using qPCR by comparing ARG abundance in packaged DNA before (direct quantification from the sample) and after phage propagation in the enrichment cultures. If the GC number of the ARGs in the phage particles increased after propagation, it was considered that the phage particles in this sample were able to infect and propagate in the host strain. Otherwise, if ARG levels did not vary or decreased, the particles in this sample were considered incapable of propagating.blaTEM was the most prevalent ARG in both sites, found in 100% of Antarctic and 75% of Mediterranean samples . The second most prevalent ARG in the Mediterranean was blaCTX-M-1 (45%) but much less than in the Antarctic (80%) (ARGs were detected in the phage DNA fraction isolated from Mediterranean and Antarctic seawater . For the samples a. sul1 wic (80%) a.10 units lower than in the Mediterranean samples  in the Antarctic samples was 3–5 log samples b. blaTEMblaTEM were produced in 100% of the lysates, and particles carrying blaCTX-M-1 group were produced in 75%  were excluded from the ARG prevalence study.E. coli cultures, with 10–100% of samples testing positive  only after propagation . After the enrichment step, five ARGs were detected in an increased number of samples of Mediterranean fish, three of them  observed in a farmed fish sample. In contrast, despite being one of the most prevalent ARGs, blaCTX-M-1, was the least abundant, detected at less than 5 log10 GC/25 g in most of the samples tested. The second most abundant ARG was blaCTX-M-9, particularly in Mediterranean and frozen fish, with average values close to 6 log10 GC/25 g. A similar level of abundance was found for the prevalent gene tetW, with the highest value of 6 log10 GC/25 g obtained in frozen fish samples, 90% of which were positive for this ARG. The sul1 gene was detected in both Atlantic and farmed fish at average values higher than 5 log10 GC/25 g.The most prevalent gene, f sample  and the Comparing the abundance of all ARGs in the phage fraction of each type of fish reveals higher values in shellfish, Mediterranean, and Atlantic fish than in farmed and frozen fish, with some exceptions .Antibiotic resistance is as ancient as some of the earliest bacterial communities, but little is known about the presence and persistence of antibiotics in the environment before they were harnessed for the benefit of humanity . Seawatesul1, in agreement with previous reports of β-lactamase genes  in remote polar areas [sul1, which expresses resistance to synthetic or semi-synthetic antibiotics such as sulphonamides. A recent study detected resistance against these antibiotics in bacteria recovered from freshwater samples collected close to Antarctic research stations [sul1 is not necessarily an indicator of human activity, as it is usually present in environmental bacteria from soils and waters, even in the Antarctic [sul1 is associated with an integron, which could explain its wide dissemination even in the absence of selective pressure by antibiotics [The most prevalent ARGs in Antarctic seawater were β-lactamase genes and ar areas . There wstations , which wntarctic . Furtheribiotics .Despite the samples analyzed from Antarctica were not close to human bases, the growing human activity in the Antarctic area, including scientific expeditions, military stations, and tourism , could bTEMbla) and tetracyclines. Although a clear association has not yet been established, these findings shed light on the selective pressure generated by humans in natural environments, where bacteria can take advantage of phages to spread their genetic content in difficult conditions.The greater abundance of ARGs in the Mediterranean samples can be explained by a high antibiotic selective pressure derived from human activity in the Mediterranean coast and the impact of human fecal pollution. These results are in line with the relative abundance of ARGs recently reported by Calero-Cáceres et al.,  in the vThe ARGs detected in the phage DNA fraction isolated from lysogens of Antarctic bacteria allow us to suggest that these bacterial communities are the source of the ARGs in the phage particles. The particles would be formed after the packaging of bacterial DNA (including ARGs) by prophages inserted in the bacterial chromosome. Several studies reveal that temperate phages are widely distributed in Antarctic bacterial communities and prioritize lysogeny when bacterial concentration is low .Our results therefore confirm the existence of a diversity of ARGs in an area as pristine as the Antarctic, and that bacteriophages in marine environments represent a potential vehicle for the horizontal transmission of ARGs between bacterial communities. The role of phages as HGT elements in marine environments has already been demonstrated, as they can disseminate key genes involved in oxygenic photosynthesis in cyanobacteria, which allows various bacterial communities to adapt to surface waters . The demblaTEM [On the other hand, the high abundance of phage-associated ARGs found in Mediterranean seawater may have important consequences for fish and shellfish, as well as consumers of fishery products. According to our results, fish microbiota showed a high percentage of resistance to β-lactam antibiotics, which represents both a direct health risk to consumers as well as a general health hazard if this resistance is transferred to human microbiota. These results are in accordance with a recent study in which ARGs were detected in wild fish, the most prevalent being blaTEM .tetW in our samples may reflect the overuse of tetracyclines in aquaculture and their spread in diverse environments [sul1 has been reported as one of the most prevalent ARGs in phage DNA of environmental and food samples [The most commonly used antibiotics in aquaculture are fluoroquinolones, sulfonamides, and tetracyclines . The preronments . Althoug samples ,23,24, i samples .E. coli and 185 CFU/100 mL of intestinal enterococci in a sample indicate sufficient quality for consumption [European food safety regulations establish that foodstuffs should not contain microorganisms in quantities representing a risk for human health , The samsumption . HoweverThis study confirms the presence of ARG-containing phage particles in aquatic environments subjected to a low or high anthropogenic influence, demonstrating that seawater is a reservoir of ARGs and that phages could play an important role in their transmission. In addition, human activity has a clear impact on the evolution of antibiotic resistance, the strongly anthropogenic Mediterranean area showing a higher abundance of ARGs than the relatively pristine Antarctic seawater. This high abundance of ARG-containing phage particles may have an important impact on fish and shellfish microbiota, contributing to the dissemination of those ARGs into microbial communities relevant to human health.


Pollen is a major cause of allergic respiratory diseases. In Qatar, data on the presence and prevalence of allergenic airborne types of pollen is quite limited.The study aimed to determine and correlate the most frequently implicated airborne pollen detected by aerobiological monitoring samplers in respiratory allergy symptoms. An aerobiological survey was started on May 8, 2017. Airborne pollen was collected using two Hirst type seven-day recorder volumetric traps. Skin prick test in patients attending allergy clinics in Doha using commercial extracts was conducted.P = 0.016) and allergic rhinitis .Twenty-five pollen types representing the native, as well as the introduced plants, with a relatively low daily mean concentration were observed from May 2017 to May 2019. The highest pollen concentrations were reached by Amaranthaceae (58.9%), followed by Poaceae (21.7%). SPT revealed a comparatively higher degree of sensitization to pollen. Among 940 patients, 204 were sensitized to pollen  with 135 (66.2%) and 114 (55.8%) to Amaranthaceae and Poaceae, respectively. Some patients had polysensitization. There was a statistically significant association between Amaranthaceae, and asthma (r = 0.169, This is the first study to monitor airborne pollen in the state of Qatar. The main pollen detected were Amaranthaceae and Poaceae. Pollen may represent a possible exacerbating factor in adult patients with allergic diseases such as asthma and allergic rhinitis. The prevalence of asthma and allergic rhinitis is high and increasing in western countries . HoweverAllergic diseases related to pollen are called pollinosis and include rhinitis, conjunctivitis, and asthma. During the last few decades, pollen has received increasing attention due to its strong allergenic potential, with severe impacts on human health . There iAirborne pollen monitoring has become a standard practice in many countries . During To date, a few studies have been conducted in Qatar in order to determine and characterize sensitization to most common airborne pollen types and inhalant allergens. One-thousand one-hundred and six patients with clinical signs of allergy, attending a clinic at a tertiary hospital in the capital city of Doha, were evaluated , 15. 51.The undertaken study was conducted to estimate the prevalence of common pollen sensitization and allergic respiratory diseases among adult patients attending allergy clinics in a tertiary hospital in the state of Qatar; a country with hot desert climate, and to investigate the association with an aerobiological airborne pollen monitoring study carried out during the studied period.2 and has a coastline of 900 km in length. It lies between latitude 24° 27′ and 26° 10′ and longitude 50° 45′ and 51° 40′, connected to Saudi Arabia in the south, and bordered by a semi-enclosed sea, the Persian Gulf, characterized by hyper-salinity [Acacia spp., Prosopis juliflora, Ziziphus nummularia, and Lycium shawii [Qatar is a peninsula that occupies an area of more than 11,000 kmsalinity . Geograpsalinity . Doha issalinity . The maim shawii .3). The traps have a pump with a mechanism to suck a constant flow of 10 L per minutes and a regular check of the flow by an air flow controller is done weekly when the drum is changed. A recording tape, coated with a special type of silicone fluid as an adhesive substance, was collected weekly. Preparation, mounting, and counting of microscopic slides were done according to the standard protocol proposed by the Spanish Aerobiology Network; the REA [3 is the sum of the daily mean pollen concentration over the given period [Airborne pollen monitoring was carried out using two Hirst type seven-day recording volumetric spore traps (VPPS 2000 Lanzoni), from 8 May 2017 to 7 May 2019, representing two subsequent years. Two traps were placed on the roof of Building 310, Hamad Medical City, Doha (east Qatar) and Al Khor Hospital, Al Khor (north Qatar), at 15 m above ground level. Both traps were fixed on the corner of the rooftop of the selected buildings, 1 m above the roof. There were no adjacent higher buildings or air conditioners to cause airflow obstruction. The selection of these two areas was based on the surrounding vegetation and the presence of no physical barriers. The monitoring sampler operates on the principle of impaction through suction, with a 2 mm clockwise movement on the drum each hour (airflow rate was 10 L/min), and thus, the concentration of airborne particles could be calculated hourly . All clinical investigations were conducted according to the principles expressed in the 1964 Helsinki declaration and its recent amendments. Written informed consent was obtained from all the participants in the study. Between May 2017 and May 2019, all patients referred for skin prick testing for inhaled allergens from the adult allergy and immunology clinics of the Hamad General Hospital were included.® Extracts™, Greer Laboratories, Inc or AllerMed, San Diego, USA), including tree, grass, and weed pollen were used. The corresponding allergens were from the Poaceae family: Bermuda grass (Cynodon dactylon), seven grasses mix (Kentucky Blue (Poa pratensis), Meadow Fescue (Festuca pratensis), Orchard (Dactylis glomerata), Ryegrass (Lolium spp.), Redtop (Agrostis gigantean), Sweet Vernal (Anthoxanthum odoratum), Timothy (Phleum pretense), Dandelion, Fabaceae: Mesquite tree (Prosopis sp.), Alfalfa, Amaranthaceae: Chenopodium album, Rough pigweed (Amaranthus retroflexus), Russian thistle , and Compositae: Mugwort (Artemisia sp.). Readings were performed by a physician 15 min after the allergens over the skin were pricked. After that, in order to evaluate the results, the diameter of the wheal was measured. A positive reaction was considered when the wheal was at least 3 mm larger than the induced concurrent negative control skin testing, and the positive control of histamine reaction was at least 3 mm in diameter or the allergen reaction was greater than or equal to 50% of the histamine reaction [Standardized allergen extracts of nine allergens from Allergopharma  with adding questions about the diagnosis made by the treating physician and frequency of exacerbation occurring over the previous 12 months of the year .P ≤ 0.05 was considered statistically significant.The analyses were performed, using data obtained from May 2017 to May 2019. The results were presented as means with a range for normally distributed data and frequency (number and percentage) of participants as appropriate. Correlations between symptoms and airborne pollen concentrations were demonstrated using the Spearman correlation test. Data were analyzed using the Statistical Package for Social Sciences software  for Windows, Version 21.0. A Prosopis , Ziziphus, Dandelion (one of the species included in Asteraceae family) as well as introduced plants such as Cupressaceae, Casuarina, Conocarpus and Alfalfa (one of the species included in Fabaceae family). Detailed percentages of the annual contribution are provided in Table During the study period (May 2017–May 2019), low pollen concentrations were recorded in both sampling sites due to the low-density cover vegetation typical of arid area. An APIn of 1977 was detected in the atmosphere of Qatar, from which 1359 corresponded to Al Khor and 618 to Doha. The pollen belonged to 25 different taxa. In both years, pollen from weed plants had the principal contribution to the airborne pollen spectrum , followed by trees , and grass . The most common taxa detected, which made up the total amount of pollen recorded in the atmosphere of Qatar, proceeded from native plants, such as Amaranthaceae, Poaceae, 3 recorded at Al Khor station . Poaceae, on the other hand, showed their highest pollen concentration between the beginning of February to the end of March. Higher pollen concentration was obtained at both stations during the year (2018–2019), with a total of 39 pollens. Poaceae showed one peak value of eight pollen grain/m3 during March 2018 in Al Khor  with a mean (range) age of 30 (20–61) years, agreed to participate in this study. Qatari nationals comprised 43.7% of the study population, and the remaining were mainly nationals from other Arab , Asian , and other countries . Three-hundred and eighty-three (40.7%) tested negative to all allergens.The skin prick test reaction was positive in 557 patients, with a total of 839 89.2%) inhaled allergens. Among these patients, 382 patients (68.6%) had polysensitization; 204 (21.7%) had positive sensitization to pollen, 324 (34.5%) to dust mite, 48 (5.1%) to fungal spores, and 263 (28%) to animal proteins . Among the patients with sensitization to pollen, 120 were males (59.1%), and 84 were females (40.9%), with a mean (range) age of 37 (31–40) years , followed by the Poaceae (55.8%). The detailed frequency and percentages of positive skin reactions for all studied pollen allergens are summarized in Table Chenopodium album 67 (32.8%). The relative proportions of patients positive to specific pollen allergens are shown in Fig. The frequencies of sensitization were Bermuda grass 102 (50%), Russian thistle 100 (49%), Rough pigweed 93 (45.6%), and The symptoms questionnaire was performed for all the examined patients with positive SPT to pollen allergens to stratify the cohort into the disease type . Each of the 204 patients had positive symptoms suggesting at least one allergic disease. A comparison was made between the percentage of patients with allergic diseases and those with positive sensation and showed that 38 (18.6%) patients had asthma, 104 (51%) allergic rhinitis, and 12 (5.9%) atopic dermatitis. Moreover, 50 (24.5%) had more than one disease. Detailed percentages are presented in Fig. P = 0.016) and allergic rhinitis  were found. Moreover, positive correlation coefficients, but not reaching statistical significance, were noted in all of the common diseases associated with individual allergens  were the most common airborne pollen types detected in the aerobiological study carried out in Qatar. This finding is in accordance with the data obtained from other studies carried out worldwide, which rank Poaceae and Amaranthaceae among the major aeroallergens causing allergy symptoms, especially in North America and Europe , 4.The annual pattern of variation in pollen concentration was very similar at both stations in the 2 years, with maximum daily mean concentrations recorded during August for the Amaranthaceae pollen (2017–2018) and March for the pollen of Poaceae (2018–2019). A similar pattern was found in other countries in the Middle East, such as Kuwait , 11 and Salsola, Halopeplis, Arthrocnemum, or Halocnemum, with Salsola being one of the principal causes of pollinosis in arid lands [A possible explanation for this observation is that in the studied area, the weather is characterized by two periods; the cold season (October to March), when the temperature is generally under 25 °C and the warm season  , thus faid lands . Moreoveid lands , with diBased on the result of the skin prick tests carried out on patients with allergic diseases, the Amaranthaceae species remained a dominant cause of respiratory allergy. Due to their seasonality, and because these plants are wind-pollinated, their pollen are among the crucial causes of summer allergies in the Mediterranean countries , as wellThe grasses are the second abundant airborne pollen in the atmosphere of Qatar. Worldwide, at least 40% of allergic patients are sensitized to grass pollen allergens . In EuroIn Qatar, Bermuda grass is common and found mostly in the residential, cultivated areas, and along roadsides. It is often grown as lawns but grows abundantly on its own. Our data showed that 114 (55.8%) patients had sensitization to grass pollen. Bermuda grass  and the seven grass mix  were the most common among the sensitizing agents. Similar results were found in other countries such as Kuwait , and AusIn this study, the extracts of other outdoor pollen types, with limited contribution to the total airborne pollen concentrations, showed a positive SPT among the allergenic patients. For example, Mesquite trees are wind-pollinated and endemic species in Qatar. The air monitoring samplers recorded lower daily mean concentrations without any specific peak during the 2 years of the study; however, the SPT revealed 37 (18.1%) positive results. These data may not indicate that this pollen type is not allergenic as reported in other Asian countries , 39 and P < 0.05) between the Amaranthaceae extract SPT results and the symptoms of asthma and allergic rhinitis. Similar results were reported in countries such as Spain [The result of our study showed that there is a highly significant association . Based on differences recorded over several years of aerobiological studies in different countries, long-term samplings are recommended to better establish daily and annual patterns for the different pollen types in a locality , 32. HowFinally, our findings further support the conclusion that this study is an important step to establish a more comprehensive aerobiology network throughout the Arab Gulf region.Aerobiological monitoring can be an essential tool for identifying the atmospheric pollen concentration across the variable seasons, improving the quality of the urban environment and protecting pollen-allergy sufferers by using prevention measures and allergen specific immunotherapy. The data presented is limited in time; however, pollen can be one of the main sensitizing allergens among patients with allergic rhinitis and asthma in Qatar, along with other known indoor allergens and may contribute to the pathogenesis of allergic respiratory diseases. There are no previous studies in the same region to make any comparison between environmental factors, such as social practices, increasing mobilization, and introduction of new potentially allergen-producing plant species in urban areas for greening, the causation of allergic diseases and our results. The result of this study is mostly consistent with the result of other studies in other parts of the world, with some variations due to differences in environmental conditions and can be a foundation for new research areas in the desert climate.


Recent phylogenomic analyses based on the maternally inherited plastid organelle have enlightened evolutionary relationships between the subfamilies of Orchidaceae and most of the tribes. However, uncertainty remains within several subtribes and genera for which phylogenetic relationships have not ever been tested in a phylogenomic context. To address these knowledge-gaps, we here provide the most extensively sampled analysis of the orchid family to date, based on 78 plastid coding genes representing 264 species, 117 genera, 18 tribes and 28 subtribes. Divergence times are also provided as inferred from strict and relaxed molecular clocks and birth–death tree models. Our taxon sampling includes 51 newly sequenced plastid genomes produced by a genome skimming approach. We focus our sampling efforts on previously unplaced clades within tribes Cymbidieae and Epidendreae. Our results confirmed phylogenetic relationships in Orchidaceae as recovered in previous studies, most of which were recovered with maximum support (209 of the 262 tree branches). We provide for the first time a clear phylogenetic placement for Codonorchideae within subfamily Orchidoideae, and Podochilieae and Collabieae within subfamily Epidendroideae. We also identify relationships that have been persistently problematic across multiple studies, regardless of the different details of sampling and genomic datasets used for phylogenetic reconstructions. Our study provides an expanded, robust temporal phylogenomic framework of the Orchidaceae that paves the way for biogeographical and macroevolutionary studies. The family has a striking morphological and ecological diversity and evolved complicated interactions with fungi, animals and other plants5 and a diverse array of sexual systems8. Numerous efforts have been made to understand the natural history, evolution and phylogenetic relationships within the family13. To date, there are seven nuclear genome sequences available, i.e., Apostasia shenzhenica14, Dendrobium catenatum15, D. officinale16, Gastrodia elata17, Phalaenopsis equestris18, a Phalaenopsis hybrid cultivar19, P. aphrodite20, Vanilla planifolia21, 221 complete plastid genomes and 2,678 sequence read archives for Orchidaceae in NCBI (accessed 22 August 2020) .Orchidaceae, with 23, but extensive uncertainties remain regarding the phylogenetic placement of several subtribes24. This knowledge-gap stems from a dearth of both taxonomic and genomic sampling efforts that would be required to comprehensively cover all major orchid clades (subtribes/groups of genera). Givnish et al.2 published the first well-supported analysis of Orchidaceae based on plastid phylogenomics. They performed a maximum likelihood (ML) analysis of 75 genes from the plastid genome of 39 orchid species, covering 22 subtribes, 18 tribes and five subfamilies. This robust but taxonomically under-sampled study corroborated relationships of the subfamilies and tribes, observed in previous studies13.Phylogenomic approaches have been implemented to infer relationships between major orchids clades in deep and recent time27 because of reduced taxon and data sampling28. This is particularly true for Cymbidieae and Pleurothallidinae, the two most species-rich groups in which generic relationships are largely the product of rapid diversification29 that is difficult to resolve using only a few loci30. Cymbidieae comprise 10 subtribes, ~ 145 genera and nearly 3800 species1, 90% of which occur in the Neotropics29. Four of these subtribes are among the most species-rich in the Andean and Chocoan region . Pleurothallidinae include ~ 5500 exclusively Neotropical species in 47 genera. Pleurothallid orchids are one of the most prominent components of the cloud forest flora in the northern and central Andes and Central America32, also being very well represented in the humid forests of eastern Brazil.Previous orchid studies have failed to resolve relationships in rapidly diversifying clades33. This group comprises four mostly terrestrial tribes, 25 subtribes and over 3,600 species. The subfamily occurs on all continents except the Antarctic. Previous efforts to disentangle the phylogenetic relationships in the subfamily have mostly relied on a small set of nuclear and plastid markers34, and more recently on extensive plastid coding sequence data2.Another group in which phylogenetic relationships are unresolved is Orchidoideae37, showing independent transitions to terrestrial, rupicolous and epiphytic habit. Moreover, they have evolved a diverse array of pollination systems40, rewarding species offering scent, oil and nectar, and even food- and sexual deceptive species42. However, the absence of a solid phylogenetic framework has precluded the study of how such systems evolved and the diversification dynamics of Cymbidieae, Pleurothallidinae and Orchidoideae more broadly.The wide geographical range of these groups in the tropics and temperate regions and their striking vegetative and reproductive morphological variability make them ideal model clades for disentangling the contribution of abiotic and biotic drivers of orchid diversification across biomes. Occurring from alpine ecosystems to grasslands, they have conquered virtually all ecosystems available in any elevational gradient43. High-throughput sequencing and modern comparative methods have enabled the production of massive molecular datasets to reconstruct evolutionary histories and thus provide unrivalled knowledge on plant phylogenetics44. Here, we present the most densely sampled plastid analysis of Orchidaceae, including data from 51 newly sequenced plastid genomes,. We apply two general approaches: (a) maximum likelihood phylogenetic analysis conducted on 78 plastid coding regions to inform relationships; (b) Bayesian inference in combination with strict and relaxed molecular clocks and a birth–death model applied to a subset of the plastid coding regions to produce a temporal framework of the orchid family. Our study expands the current generic representation for the Orchidaceae and clarifies previously unresolved phylogenetic relations within the Cymbidieae, Pleurothallidinae and Orchidoideae. The results reported here provide a robust framework for the orchid family and new insights into relationships at both deep and shallow phylogenetic levels.Phylogenetic analyses are crucial to understanding the drivers of diversification in orchids, including the mode and tempo of morphological evolutionDendrobium: 3; Cymbidium: 1; Fig. The maximum likelihood (ML) tree derived from the 78 plastid genes is provided in Fig. Absolute times of divergence under strict and relaxed clocks for Orchidaceae, subfamilies and most tribes are provided in Table ycf1 as the most informative locus, which attained the highest values at a reference time (phylogenetic depth) of 0.51. On average, plastid loci attained their highest PI value at a reference time of 0.85 (SD = 0.16). In contrast, the highest PI values of the 10 most informative loci occurred at an average reference time of 0.63 (SD = 0.11) and 0.80 (SD = 0.17) for per-site and net PI calculations.Phylogenetic informativeness plots are provided on Fig. 28. Our study sampled 264 species from all subfamilies, representing 18 tribes (out of 22), 28 subtribes (out of 46) and 74 genera  and Pleurothallidinae (Epidendreae) are presented for the first time. Our 78-coding sequence plastid ML analysis led to similar results as reported by Givnish et al.2, Niu et al.13 and Li et al.28 but with an overall clear increase in support: 22% of branches with LBS < 85 in Givnish et al.2 and 21% in Li et al.28vs 11.5% in this study. This is particularly evident in relationships inferred within Orchidoideae, Cymbidieae, Epidendreae and Collabieae. For the latter, high support for the previously unresolved relationship of Podochilieae + Collabieae28 was attained for the first time.Previous phylogenomic studies of the orchid family included up to 74 species representing 18 tribes, 19 subtribes and 66 genera47. Nonetheless, our ML tree also identifies intricate relationships that have been consistently recovered as unsupported in several studies. These include poorly supported basal branches in Epidendroideae representing Sobralieae, Nervilieae and Tiphoreae48, Arundina + remainder of Arethuseae28, and the position of Eulophiinae in the Cymbidieae49  might hamper the phylogenetic placement of clades with robust support.The absolute age estimates derived from our strict and relaxed molecular clocks and five of the most informative plastid loci are in line with previous nuclear-plastid multi-locus and phylogenomic plastid-only chronograms30. Relationships among subtribes have recently been estimated using the plastid genes psaB, rbcL, matK and ycf1 combined with the low-copy nuclear gene Xdh26. Here, Cymbidiinae was sister to the remainder of Cymbidieae. Poorly supported and incongruent relationships were found among Catasetinae, Eulophiinae and Eriopsidinae, however, when compared with the topologies obtained by Whitten et al.30, Freudenstein & Chase48 and Pérez-Escobar et al.7.Multiple studies have inferred evolutionary relationships in Cymbidieae from morphological and molecular characters2 included 8 of 11 subtribes of Cymbidieae, but some inter-subtribal relationships were unresolved: Stanhopeinae (20 genera), Maxillariinae (12 genera), Zygopetalinae (36 genera), Oncidiinae (65 genera) and Eulophiinae (13 genera). A clade formed by Stanhopeinae and Maxillariinae had poor support (LBP = 62) and their relationship to Zygopetaliinae also had low support (LBP = 72). The relationship between Eulophiinae and a clade of Stanhopeinae, Maxillariinae, Zygopetalinae and Oncidiinae also had poor support (LBP = 42). One of the outcomes of our expanded sampling (nine subtribes) is the improvement of support in Cymbidieae, more specifically for branches of some groups involved in rapid diversifications that historically have been problematic to resolve30. In particular, Maxillariinae + Stanhopeinae and Catasetinae + Cyrtopodiinae are now both strongly supported (LBP = 100). In addition, our results also support the placement of Dipodium (Dipodiinae) as sister to the rest of Cymbidieae, a relationship which was previously recovered from a few loci26. However, our plastid phylogenomic framework is still incomplete due to absence of representatives of Eriopsidiinae and Coleopsidinae.The most complete taxonomic sampling conducted to date under a plastid phylogenomic framework56. The most extensively sampled analysis of Zygopetalinae inferred from plastid markers (matK-ycf1)30 included 60 species and 27 genera, but relationships between most genera attained only low support. Our expanded molecular, but taxonomically reduced, matrix (i.e. 20 genera and 21 species) produced greater support for the backbone relationships in the subtribe, including the radiation of the Huntleya clade . Nonetheless, relationships between the Huntleya grade (i.e. Huntleya clade + Cryptarrhena) and the remainder of Zygopetalinae still remains unresolved.One other novelty of our study is the inference of relationships in Zygopetalinae, a subtribe in which relationships have previously been poorly understoodCheiradenia and Hoehneella with moderate to strong support , with Hoehneella being related to the Huntleya clade (i.e. Huntleya and Chaubardia). Our ML tree supports both assumptions, placing Cheiradenia as sister to Paradisanthus with maximum support and Hoehneella as sister to Chaubardia in a moderately supported clade  clustered with Acacallis and not with Otostylis and Paradisanthus, and therefore we confirm that Koellensteina in the strict sense is related to Acacallis. In addition, Otostylis is recovered as sister to Warrea and not to Paradisanthus as previously suggested by Williams et al.56 based on a weakly supported placement. Our results also highlight the extensive and independent terrestrial and epiphytic habit transitions occurring in this clade, as most sister genera show different habit types.Our phylogenetic analyses further place for the first time in the orchid tree of life rt Figs. , S1. Che45. The rapid radiation of Pleurothallidinae occurring in the last ~ 20 Myrs29 is associated with the evolution of a diverse suite of pollination systems ranging from food deception59 to pseudocopulation60 linked to dipterans62 and a complex array of reproductive and vegetative morphologies32. Understanding of relationships in the subtribe has relied mostly on relatively small number of markers65, which have informed with some confidence the phylogenetic placement and monophyly of genera in Pleurothallidinae, yet basal branches in these trees have often lacked good support.One of the most spectacular Neotropical plant diversifications is perhaps that of the Pleurothallidinae, for it involves the evolution of ~ 5000 species that have conquered virtually all biogeographical regions in the American tropicsmatK markers64. A synthesis of the phylogenetic relationships in the subtribe based on such studies was conducted by Karremans66. Here, a cladogram depicting the commonest topologies of relationships between genera was provided and nine clades were defined (termed “affinities” by the author) but without considering the magnitude of the support for these . Our plastid phylogenomic analysis recovered well-supported relationships in Pleurothallidinae that are mostly in line with previously published studies63. However, these previous trees based on a handful of DNA nuclear and plastid markers yielded poor resolution and low support for backbone branches as well as infrageneric relationships. In contrast, our plastid phylogenomic inferences recovered high support along the backbone, thus recovering novel placements. Some of these noteworthy well-supported relationships are the position of Acianthera as sister to Myoxanthus and Dresslerella as sister to Barbosella + Restrepia . In contrast, our analysis robustly places Dreslerella as sister to Restrepia and Barbosella, a result that does not support the monophyly of the Restrepia affinity.29. The remarkable relationship recovered here for Acianthera + Myoxanthus could yield more clues about the biogeographic history and evolution of the subtribe because Brazil harbours a high species diversity of Acianthera and some of the early divergent clades in Myoxanthus (particularly the species close to M. lonchophyllus), whereas Myoxanthus is notably absent in the Antilles. In addition, other early divergent clades such as Octomeria and Barbosella are more diverse in Brazil. These clades share the lack of stem annulus as a morphological symplesiomorphy, a character that later appears in more diverse groups such as Masdevallia + Dracula, Lepanthes, and Pleurothallis + Stelis69. Members of these clades probably diversified after a migration to the mountainous areas of the northern Andes ca 16 ± 5 Ma and together account for almost 80% of the species in the subtribe29. The modern range extends mostly along the Andean and Central American mountain ranges. Here, another noteworthy relationship is that the less diverse Specklinia clade  was recovered as sister to the most species-rich clades of the subtribe . In previous phylogenetic analyses Specklinia clade was recovered as sister to Pleurothallis29.Although estimates of the ancestral distribution of the Pleurothallidinae are still uncertain, most of the early divergent Pleurothallidinae and their sister groups are found in the Antilles or BrazilLepanthes affinity  were largely weakly supported, demonstrating the need for increased taxon sampling, principally in Lepanthopsis and Tubella32. In particular, the early diversification of the Lepanthes affinity (> 1500 spp.), inferred to have occurred around 8 Ma, has been linked to colonisation of newly formed environments in the Andean Cordillera, accelerated mountain uplift and the evoluton of specific pollination systems (pseudocopulation and food mimicry60).Likewise, relationships between early divergent members in the Teagueia , which resembles Platystele73. Karremans74 had suggested a close relationship between Teagueia and Scaphosepalum, but our results place Teagueia as sister to Platystele with high support, thus corroborating the long-standing hypotheses of their sister relationship based on the similarity of their reproductive structures73.Another novel placement concerns 35 in which Diurideae is sister to Cranichideae and Codonorchideae to Orchideae. Our findings differ from Givnish et al.2 and Salazar et al.34, in which Diurideae/Cranichideae are sister to Codonorchideae, with Orchideae sister to all these . Similarly, our analyses provide a well-supported result for Orchidoideae. Although taxon sampling was sufficient to resolve the relationships between the major clades in the family, sampling of unrepresented genera and representatives of Eriopsidiinae, Goodyerinae, and Coleopsidinae would further enhance our understanding of phylogenetic relationships.This study presents a well-resolved, more densely sampled and strongly supported analysis of Orchidaceae and their absolute times of divergence than all previous similar studies. For deep branches and recent diversifications in Cymbidieae and Epidendreae, support is improved, yet several recalcitrant branches that historically have been challenging to resolve were also recovered as poorly supported  were conducted at Rapid Genomics LLC , BGI Genomics  and Genewiz GmbH . Pair-end reads of 150 bp were obtained for fragments with insert size of 300–600 bp. Overhangs were blunt ended using T4 DNA polymerase, Klenow fragment and T4 polynucleotide kinase. Subsequently, a base 'A' was added to the 3′ end of the phosphorylated blunt DNA fragments. DNA fragments were ligated to adapters, which have a thymine (T) overhang. Ligation products were gel-purified by electrophoresis to remove all unbound adapters or split adapters that were ligated together. Ligation products were then selectively enriched and amplified by PCR. For each sample, between one and 10 million paired-end reads were generated.77 in order to eliminate sequencing artefacts, improve uniformity in the read length (> 40 bp) and ensure quality (Phred score> 20) for further analysis. Filtered sequences were processed with BBNorm78 to normalize coverage by down-sampling reads over high-depth areas of the genomes (maximum depth coverage 900 × and minimum depth 6x). This step creates a flat coverage distribution in order to improve read assembly. Subsequently, overlapping reads were merged into single reads using BBmerge79 in order to accelerate the assembly process. Overlapping of paired reads was evaluated with Flash80 to reduce redundancy. Merged reads were used to carry out the whole genome de novo assembly with SPAdes 81.Raw sequences were quality filtered using Trimmomaticde-novo approaches. The reference based approach was conducted on MIRA v. 482, a software that maps read data against a consensus sequence of a reference assembly (simple mapping). MIRA has been useful for assembling complicated genomes with many repetitive sequences85. MIRA produces BAM files as output, which were subsequently used to generate consensus sequences in SAMTOOLS86. We sourced 11 reference plastomes from the NCBI repository that represent related species, namely: Cattleya crispata, Goodyera fumata, Masdevallia picturata, M. coccinea, Oncidium sphacelatum and Sobralia callosa. The de-novo assembly approach relied on GetOrganelle87, using the recommended default settings for assemblies of green-plant plastid genomes.To produce contiguous, linear plastid genome sequences we relied on a refence-based and 88. Sequences were uploaded as fasta files, and running parameters were established as follow: BLAST protein search identity = 65%, BLAST rRNA, tRNA, DNA search identity = 85%, genetic code = bacterial/plant plastid, max intron length = 3,000, options = allow overlaps. Apostasia wallichii, Masdevallia picturata, Oncidium sphacelatum, Sobralia callosa and Goodyera fumata were set as the ‘Server Reference’ and Cattleya liliputana was set as the ‘Custom Reference’ for CDS and tRNA, rRNA, primer, other DNA or RNA specifications.Newly sequenced and datamined plastid genomes were annotated through the Chlorobox portal of the Max Planck Institute89 using MAFFT 790 and subsequently concatenated  of 3.0. Following Givnish et al.2, the two secondary calibration points were added to the root of the tree and MRCA of the Orchidaceae, using a normal distribution and mean values of 123.48 (SD = 2.0) and 90 (SD = 2.0). Because dating analyses conducted on dozens of gene alignments and hundreds of terminals are extremely computationally greedy, we estimated absolute ages on the five most phylogenetically informative genes (see below) and by constraining the tree topology to the ML tree derived from RAxML. For each clock model, we conducted two MCMC analyses with 250 million generations each with a sampling frequency of 10,000 generations. The convergence of the strict and relaxed molecular clocks parameters was confirmed on the software TRACER v1.6. (http://tree.bio.ed.ac.uk/software/tracer/). Maximum clade credibility trees were summarised from the MCMC trees in the program TreeAnnotator v.1.8. of the software BEAST. The individual gene alignments employed for ML and Bayesian phylogenetic infereces are freely available at 10.6084/m9.figshare.14068892A set of 78 plastid genes was used to reconstruct phylogenetic relationships in Orchidaceae. These were aligned94 using in the web application PhyDesign http://phydesign.townsend.yale.edu/). The input files were the consensus ML ultrametric tree converted with the function chronos of the R-package APE (http://ape-package.ird.fr/) using an smoothing rate of 1 and a relaxed clock model, and the partitioned concatenated gene alignments.To estimate the phylogenetic informativeness (PI) of plastid genes we calculated the per-site and net values for each assessed locus with the HyPhy substitution rates algorithm for DNA sequencesSupplementary caption 1.


PPARG) belongs to the nuclear receptor superfamily functioning as transcription factors to regulate cellular differentiation, development and metabolism. Moreover, it has been implicated in the regulation of lipid metabolism, as well as the maturation of monocytes/macrophages and the control of inflammatory reactions. The aim of this study was to evaluate the relationship between the Pro12Ala (rs1808212) PPARG gene polymorphism on immune molecular and cellular components in mothers and their offspring participating in the PREOBE study.Peroxisome proliferator activated receptor gamma  rs1808212 polymorphism, . Only CD19 levels in peripheral blood were significantly higher at delivery in pregnant women carrying the CC (Pro12Pro) genotype (p ≤ 0.001). Moreover, there were statistically significant differences in leukocytes and neutrophils maternal levels at 34 weeks of gestation, being lower in carriers of Pro12Ala genotype .Study findings revealed no effect of rs1808212 PPARG polymorphism may have an effect on some cell and immune parameters in pregnant women during pregnancy and at time of delivery. However, newborn innate immune system does not seems to be influenced by PPARG Pro12Ala polymorphism in cord blood.Results suggest that Pro12Ala  The immune system is our first line of defense against microbial infection. It is composed of leukocytes that secrete various immune molecules such as cytokines, chemokines, immunoglobulins, and complement proteins in order to eliminate pathogens as well as mediate cell-to-cell communication. A successful pregnancy requires a robust, dynamic and responsive immune system. Immune modulation and tolerance induction are required at the beginning of the pregnancy to allow implantation, but a successful pregnancy also depends on a responsive immune system that can protect both the mother and the fetus against environmental insults when necessary . Indeed,PPARs) family belong to the nuclear receptor superfamily that functions as transcription factors to regulate cellular differentiation, development and various metabolic processes , in general there was no effect on those levels by PPARG Pro12Ala SNP distribution (p > 0.05), except at 34 weeks of gestation where statistically significant differences were found in leukocytes and neutrophils levels, being lower in carriers of Pro12Ala genotype .About the effect of CC and the CG genotypes: results show significant changes in CD19 cells at delivery (p < 0.001) and marginally significant at 24 and 34 weeks of pregnancy. Conversely, CD4/CD8 ratio varied significantly at 24 and 34 weeks of pregnancy  between mothers with different genotypes, and also at delivery, although not reaching statistical significance in this last case. In these weeks of pregnancy (24 and 34) and at delivery, CD19 levels and CD4/CD8 ratio were increased in homozygous mothers compared against heterozygous mothers  counts and CD4/CD8 ratio between mothers carrying the CC, 8 (8.99%) CG and 1 (1.12%) GG; allele frequencies were: 93.85% C and 6.15% G, and no deviation from Hardy–Weinberg equilibrium was detected (p = 0.15). Due to low frequency of minor homozygotes in the newborns, subsequent statistical analyses were done comparing CG + GG vs. CC. No statistically significant variation (p > 0.05) has been found in cord blood levels of immune cells or cytokines between babies who carry CC major homozygous genotype and carriers of G (CG + GG)  PPARG polymorphism could be associated with immune cell and cytokine levels in mothers during gestation and in newborn cord blood. Our results suggest that during pregnancy, the majority of immune components are not altered in Ala12 PPARG carrier mothers with respect to Pro12Pro subjects. However, CD19 molecules seem to vary slightly at second and third trimester of pregnancy and at time of delivery, although not reaching statistically significant differences. B-lymphocyte antigen CD19 is a clinical biomarker linked to the lymphocyte B development, with relevance during early life. CD19 is a transmembrane protein which acts as an adaptor protein to recruit cytoplasmic signaling proteins to the membrane. Moreover, CD19 is critically involved in establishing intrinsic B cell signaling thresholds through modulating both B cell receptor dependent and independent signaling. It plays roles in the antigen-independent development, as well as the immunoglobulin-induced activation of B cells. CD19 is thus critical for the body to mount an optimal immune response [The main goal of the current study was to determine if rs1808212 response .PPARG Pro12Ala polymorphism during pregnancy, as there are statistically significant differences in mothers’ carriers of CC vs. carriers of CG genotypes in weeks 24 and 34, although not at 40 weeks. CD4 helper/inducer cells and CD8 cytotoxic/suppressor cells are T lymphocytes phenotypes, characterized by distinct surface markers and functions that mostly reside in lymph nodes but also circulate in the blood. CD4/CD8 ratios between 1.5 and 2.5 are generally considered normal; however, a wide heterogeneity exists because sex, age, ethnicity, genetics, exposures, and infections may all impact the ratio. A low or inverted CD4/CD8 ratio is an immune risk phenotype and is associated with altered immune function, immune senescence, and chronic inflammation [CC and GC genotypes at the different times of the study overlap, so the differences may not be biologically significant.Results also suggest that CD4/CD8 ratio may be influenced by ammation . Thus, tPPARG is expressed in blood cells and induced during macrophage differentiation [PPARG is expressed in B lymphocytes [Nuclear receptor PPARG has a major role in transcriptional regulation of genes involved in various physiologic processes of clinical significance, such as developmental, metabolic and anti-inflammatory processes. PPARG plays a pivotal role in adipogenesis, fatty acid uptake, lipid storage and systemic energy homeostasis, as well as in the control of inflammation . PPARG intiation . It enhantiation , which antiation . PPARG iphocytes  and T lyphocytes .CG compared to those carrying CC genotype have been found at any time of pregnancy studied.PPARG has anti-inflammatory properties and its ligands have been demonstrated to suppress production of monocyte/macrophages inflammatory cytokines such as TNF, IL-6, and IL-1β through inhibiting the activity of transcription factors such as nuclear factor κB, activator protein-1, and signal transducers and activators of transcription , 40, 41.PPARG Ala12 allele has been related to increased long-term weight gain [vs. 30.51%), although not reaching statistical significance, it has been reported that subjects carrying Pro allele have increased diabetes risk [PPARG polymorphism on lymphocytes subsets observed in our study. In this regard, as subject number involved in the current study is limited, replication in a larger population is needed since reduced transcriptional activity of the PPARG gene result of Pro to Ala aminoacid substitution has been demonstrated [PPARG (rs1801282) was significantly associated with increased/decreased risk of GDM in Asian population, mostly from China, but not in Caucasian population [ght gain , and alsght gain . In our tes risk , 43–45. pulation .CC carriers vs. CG + GG, as described above. These results are in line with a study in a large German cohort including mother/child pairs showing that PPARG Pro12Ala polymorphism is not relevant for intrauterine growth, although effects developed later in life depending on predisposition or environmental factors could not be discarded [PPARG gene is associated with high weight and ponderal index at birth [PPARG mRNA and protein levels have been observed in subcutaneous adipose tissue from obese gestational diabetic women, suggesting that this change might be part of the molecular mechanism to accelerate fat catabolism and thereby ensure fetal nutrition in late gestation [Birth weight is slightly higher in the offspring of heterozygous mothers compared to homozygous pregnant women. However, no differences in newborn weight were found when comparing newborns iscarded . Howeverat birth . Since Pestation .Some limitations in the present study should be noted, as the sample size is relatively low, although sample characteristics are quite homogeneous: all the mother–child pairs where from Spanish origin and no statistically significant differences were found among groups depending on rs1808212 genotype distribution in clinical or demographic characteristics. No data is available about PPARG expression in our study population. Moreover, blood counts and cytokine levels data were not available at the beginning of the pregnancy. Thus, the study results should be taken with caution, and should be replicated in a larger population. Given the pivotal role that PPARG plays in lipid storage, systemic energy homeostasis and in the control of inflammation as well, more research is warranted in this field.PPARG Pro12Ala polymorphism might be one of the factors that would determine higher CD19 cells during pregnancy in homozygous PPARG rs1808212 SNP mothers, especially at delivery, compared to heterozygous participants. Additionally, CD4/CD8 ratio was also slightly higher in mothers with Pro12Pro genotype at second and third trimester of pregnancy, but not at the time of delivery (40 weeks). Besides, our investigation reveals that no other immune molecular and cellular components seems to be altered by the Ala allele of rs1808212 PPARG polymorphism in mothers at 24 and 34 weeks of pregnancy. However, as newborn innate immune system does not seems to be influenced by PPARG Pro12Ala polymorphism in cord blood, more research is warranted to clarify the role of PPARG on immune parameters and their relationship with inflammatory response during pregnancy.In summary, results suggest that