Despite their great complexity, there is decidedly limited clinical data on botanical medicine. In fact, of the 28 botanicals that were examined, only 2 (Citrus paradisi and Rhodiola rosea) show in vivo pharmacological interactions in human subjects. On the contrary, for the other botanicals, there is only weak evidence of dubious clinical significance or potential interactions shown in animal models or in vitro without clinical confirmation. This study provides a rational assessment of the most widely used products, including those used in self-medication, to simplify patient management during the COVID-19 health emergency.In this review, we examined the top 10 nutraceutical products sold in Italian pharmacies and parapharmacies as well as hypermarkets and supermarkets; in the first, three product categories saw the greatest increase in sales  for the 12-month period between October 2019 and October 2020 (including first pandemic wave of SARS-CoV-2). We are investigating their respective formulas and isolating the botanicals that are used to make them. Many of these products have undergone preclinical and clinical studies. We performed a systematic literature search in the MEDLINE database using PubMed and Google Scholar from November 15, 2020 to December 15, 2020 (including studies carried out between 1980 and 2020). The search terms that were used included the complete name of the medicinal plant in English or Latin and the terms “cytochrome” or “drug interactions,” crossing, respectively, the Latin name and English common names with “cytochrome” and “drug interactions.” The search included  Containment measures, including quarantine and social isolation, are among the main factors that can negatively impact mental health and may play a role in acute stress disorders, anxiety, irritability, poor concentration, and indecisiveness, deteriorating work performance, post-traumatic stress disorders, high psychological distress, depressive symptoms, and insomnia. A recent survey carried out in Italy between April 19, 2020 and May 3, 2020 showed a rising incidence of mental health issues, with 24.7% of the interviewed subjects suffering from depression, 23.2% reporting symptoms of anxiety, and 42.2% suffering from sleep disorders, with 17.4% reporting moderate/severe insomnia.1 This also had repercussions for the nutraceuticals industry. Indeed, an analysis carried out by the National Association of Health Products Manufacturers and Distributors  in December 2020 showed a 38% increase in the sales volume of vitamins and minerals as well as immunostimulants, and a 28.8% increase in the sales volumes of sleep products.3 The products that were assessed are freely sold at pharmaceutical, parapharmaceutical, and supermarket and hypermarket sales points (which require the presence of personnel able to provide advice on the purchase and use of the products). These products may contain vitamins, minerals, and botanicals.The SARS-CoV-2 4 While many of these products have undergone preclinical and clinical studies, due to different national drug regulatory rules, it is not mandatory to study the pharmacokinetics of every botanical product. Hence, not much is known about the pharmacokinetic properties of botanicals or whether their assumption could impair the effects of other ingested drugs or vice versa. Indeed, the concomitant use of botanicals and prescription drugs that share the same metabolic pathway could theoretically pose a serious risk, leading to the inefficacy of many lifesaving drugs, or, even worse, leading to supratherapeutic drug exposure that could even cause fatal levels of toxicity.Botanicals and their derivatives are being used more and more in the Western world, as their standalone use or in synergy with prescription drugs is becoming widely accepted in the management of common illnesses. According to the latest WHO global report on traditional and complementary medicine, at least 39% of the German and British population use or have used botanicals.5 The P450 cytochrome system is composed of many different isoforms, each one responsible for the metabolism of a specific class of molecules. CYP3A4, CYP2D6, CYP2C9, CYP1A2, and CYP2E1 are the P450 cytochrome isoforms that are most involved in drug metabolism. They are not present in equal proportions, and their representation, which is influenced by many parameters, including age, sex, and health status, is different in every individual. These isoforms can also be induced or inhibited by many exogenous molecules, including prescription drugs and botanicals. Hence, the clearance of the same amount of a given drug could vary greatly from individual to individual.6Among drug metabolism and excretion components, one common pathway for many compounds is the P450 cytochrome system, a hepatic microsomal enzyme complex involved in the phase 1 reaction of the metabolic pathway. The P450 cytochrome system oxidizes not only many compounds, including drugs and botanical medicines, but also many other xenobiotics, allowing their clearance.7–13 have investigated this issue. In our investigation of reviews, systematic reviews, and metanalyses that examine botanicals and their possible interactions, several distinct approaches emerged: based on the functional classification of the botanicals14 or investigating several botanicals or whole herbal extracts from laboratory, animal, human subjects for their impact on cytochrome P450 isoenzymes or assessing specific drug–herb interactions within or not a clinical trial, or in case reports and case series,15–18 focusing in some works on sources, causes, and clinical relevancy of pharmacokinetic herb–drug interactions,19–22 or examine the top botanicals sold in the United States.22Several studiesThe present review is the first of its kind to focus specifically on botanicals that are commercially available in Italy in a specific public health context, namely the pandemic, an already complex situation for clinicians, in which any further confounding elements could pose significant challenge.in vitro studies carried out using hepatic tissue containing cytochromes may raise concerns, they do not carry the same weight as clinical trials on human subjects because of the complexity of human metabolic pathways. Furthermore, many of the available data may prove to be inconclusive. For example, Piperine, one of the principal alkaloids of Piper nigrum, has been shown in vitro to be an inhibitor of CYP3A4 and a less potent inhibitor of CYP1A2 and CYP2D6.23 Old studies conducted on human volunteers seem to confirm these findings, showing an increase in the levels of rifampicin and phenytoin in the bloodstream with administration of 20 mg/day of Piperine, but specific involvement of P450 cytochrome was not specified.24 Shedding light on this issue is of considerable importance because numerous botanical derivatives could be added to the physician's toolbox expanding the therapeutic options and allowing them to save conventional pharmacological tools for severe cases. It is therefore essential to have rationally organized and quickly readable data to be able to use these additional tools in complete safety.Interactions between botanical products and conventional drugs are frequently described in the medical literature. Moreover, while results from 2 we consulted the IQVIA data for the 12-month period between October 2019 and October 2020. The data analyzed come from the panels of pharmacies  and parapharmacies  that IQVIA manages, collects, produces, and makes available to its customers, and from the panels of super and hypermarkets managed by Nielsen, which sell botanicals with dedicated corners. This allowed us to identify the top 10 products sold, respectively, in the pharmaceutical, parapharmaceutical, and super and hypermarket channels (which require the presence of personnel able to provide advice on the purchase and use).Based on the analysis carried out by the National Association of Health Products Manufacturers and Distributors ,In the pharmaceutical channel, the top 10 products represent, respectively, 32% of the value and 28% of the volume of sedative-sleeping pills, 38% of the value and 34% of the volume of polyvitamins/minerals, and 33% of the value and 24% of the volume of immunostimulants. In the parapharmaceutical channel, the top 10 products represent, respectively, 22% of the value and 20% of the volume of sedative-sleeping pills, 29% of the value and 25% of the volume of polyvitamins/minerals, and 36% of the value and 27% of the volume of immunostimulants. Finally, in the super and hypermarket channel, the top 10 products account, respectively, for 46% of the value and 54% of the volume of sedative-sleeping pills, 46% of the value and 30% of the volume of polyvitamins/minerals, and 81% of the value and 78% of the volume of immunostimulants.e.g., different formats of the same product or the same product present in multiple distribution channels) and isolating the botanicals used  and human cytochromes or equivalent study models.We performed a systematic literature search in the MEDLINE database via PubMed and in the Google Scholar portal from November 15, 2020 to December 15, 2020. The search terms that were used included the complete name of the medicinal plant in English or Latin and the terms “cytochrome” or “drug interactions,” crossing, respectively, the Latin name and English common names with cytochrome and drug interaction. The search included in vivo effects on humans or animal models or in vitro effects on equivalent human or animal cellular study models were excluded. Out of a total of 246 articles that were found, 104 met the requirements described above. Case reports or articles which lacked data but provided information relevant to the topic in sections dedicated to the individual botanicals were also taken into consideration.The search encompassed studies carried out between 1980 and 2020, but focused on investigations involving human clinical trials and recent reviews. All the articles that did not include an evaluation of the All the works that were retrieved were read, and described interactions were reported in In the Boswellia serrata, also known as Indian olibanum, is a species of plant of the Burseraceae family, native of India and the Punjab region. The dried gum-resin is the best known botanical derived from the tree; the resin and its extract contain a large amount of boswellic acid and its derivatives, which have been used traditionally as analgesics to treat joint pain, arthritis, and other forms of rheumatism.25 A single in vitro study showed how boswellic acid and its derivatives can inhibit various P450 cytochromes, namely CYP1A2, CYP2C9, CYP2C19, and CYP2D6.26 However, since there are no clinical studies in the literature, the significance of these interactions is questionable.Camellia sinensis (or green tea) is a species of shrub in the flowering plant family Theaceae, and its leaves are commonly used to produce tea. Indeed, it may be one of the most widely used plants. The main substances released by its oral ingestion are teine and epigallocatechin gallate, substances with renowned stimulating and antioxidant properties.27 CYP3A4, CYP2D6, and CYP2C9 interactions have been described in preclinical studies using in vitro and animal models, pointing to possible interactions with commonly used drugs such as warfarin, midazolam, irinotecan, and tamoxifen. However, clinical studies on healthy volunteers found no interaction with CYP2C9 and CYP2D6 metabolized drugs, showing only a minor decrease in CYP3A4 activity in one study and no decrease in its activity in another investigation.29 Camellia sinensis leaf extract, was found to have CYP1A, CYP2B, and CYP3A induction capacity in animal models, while several other potential interactions have been reported in vitro, but the clinical significance of such reports remains unclear.23Carica papaya , a tropical fruit plant belonging to the Caricaceae family, produces spherical or cylindrical berries, 15–45 cm long, commonly eaten as a whole fruit, or used to make a juice. It is used as a botanical in its whole fruit form, or in the form of papain, a proteolytic enzyme derived from the dried fruit latex, which is used as a digestive and edema-reducing agent.27 Papaw juice has shown modest CYP3A4 inhibition properties in vitro,30 while whole fruit consumption could inhibit CYP2E1, as shown in an in vivo animal study.31 Although a few case reports hypothesize important interactions with drugs such as warfarin,32 no data derived from human clinical trials are available.Chamaemelum nobile (Roman chamomile or English chamomile), a low growing perennial plant of the Asteraceae family, has been traditionally used as an infusion or as an extract for its renowned relaxing properties and as a sleep adjuvant.25 Our search did not find case reports or relevant P450 cytochrome interaction studies, neither in vivo nor in vitro. In fact, since C. nobile is widely used, drug interactions seem unlikely.34Citrus aurantium (bitter orange or Seville orange), indigenous to tropical Asia, is a citrus tree whose fruit is commonly used as a food. The bitter orange is also used as a botanical for its neurostimulating properties, attributable to one of its constituents, the alkaloid synephrine, which has adrenergic effects.27 The whole fruit also contains flavonoids and furanocoumarins, which may be responsible for the CYP3A4 inhibition observed in a small clinical trial, which found a rise in cyclosporine blood levels in healthy volunteers.35 Moreover, a significant CYP3A4 and CYP1A2 induction was also observed in an in vivo animal study, showing a possible mixed effect on cytochromes.36 Therefore, since large human clinical trials are still not available, caution should be exercised with the concomitant administration of C. aurantium derivatives and CYP3A4 metabolized drugs.Citrus paradisi, or grapefruit, is a subtropical citrus tree known for its fruit.37 It is traditionally used as a botanical in various forms: whole fruit, juice, extract, essential oil, or dried seed extract. All these derivatives are known for their antioxidant and antimicrobic properties.38 As this fruit is rich in furanocoumarins, such as bergamottin and bergaptene, it has a renowned capability to irreversibly inhibit intestinal and hepatic CYP3A4 with consequent significant interactions. In fact, it is recommended not to drink grapefruit juice with warfarin, benzodiazepines such as alprazolam, statins such as atorvastatin, immunosuppressives such as cyclosporine, antiplatelet agents such as ticagrelor, and antivirals such as indinavir because these drugs could raise the blood levels to dangerous levels, even resulting in fatal toxicity events.39–45 The dried seed extract was also reported to have a potent CYP3A4 and CYP2C9 inhibitory activity in a study carried out after the publication of a case report, which displayed a rise in warfarin blood levels in a patient who was also consuming grapefruit seed extract.46Crataegus (or Hawthorn) is a genus of shrubs and trees in the Rosaceae family. Crataegus monogyna, the most common species of the genus, is a flowering plant whose leaf and berry extracts are used for their antioxidant, cardioprotective, and cardiotonic properties. Crataegus oxyacantha is the name, still used in some products but no longer recognized by the botanical community, of a species of Northern European hawthorn, which was subsequently identified as a variant of C. monogyna and Crataegus laevigata.25 While a few case reports hypothesized the possible interaction of hawthorn with commonly used drugs such as digoxin, a clinical trial rejected this hypothesis47 and our search did not retrieve reported interactions with the P450 cytochrome system.Echinacea angustifolia (narrow-leaved purple coneflower or blacksamson echinacea), of the Asteraceae family, is a North American plant whose root extract, rich in flavonoids, has been traditionally used as a botanical for its antioxidant properties.25 The presence of flavonoids could also be responsible for the plant's reported in vitro CYP3A4 inhibition.49 However, there is a lack of reliable drug interaction reports or clinical trials in the literature regarding this inhibition.Echinacea purpurea is an herbaceous flowering plant belonging to the Asteraceae family. Growing in Eastern and Central North America, it is traditionally used for the prevention and treatment of acute upper respiratory tract infections, thanks to its immunostimulant properties.25 The E. purpurea root extract shows repressive capacity on CYP2D6, CYP3A4, CYP2C19, and CYP2C9 in vitro and on CYP1A2 and CYP3A in vivo in humans, while E. purpurea whole plant extract shows CYP3A1 and CYP3A2 inhibition and in vivo CYP1A1 and CYP2D1 induction in animal models. Given the widespread consumption of this botanical, the real clinical significance of these interactions remains questionable.51Eleutherococcus senticosus (devil's bush or Siberian ginseng), a small woody shrub native of north-eastern Asia, has traditionally been used as a botanical, as its root extract is used to make a renowned tonic and antistress agents.25 While its root extract has shown CYP2C9 induction capability in vitro,52 clinical trials on human volunteers have shown only modest competitive CYP2D6 and CYP3A4 inhibition with no significant clinical consequences.53Eschscholzia californica  is a flowering plant of the Papaveraceae family, whose hydroalcoholic extract, rich in morphine-like isoquinoline alkaloids, is used for its sedative, analgesic, spasmolytic, and hypnotic properties.27 These alkaloids, such as escholtzine and allocryptopine, could play a role in the CYP3A4, CYP2C19, and CYP2C9 inhibition observed in vitro, while protopine could be responsible for the CYPD26 inhibition in vitro reported in the same study.54 However, no significant drug interactions have been reported in humans.Griffonia simplicifolia (Bandiraea simplicifolia or African bean) is a West and Central African native woody climbing shrub of the Fabaceae family, with greenish flowers and black pods. The plant seeds are used as botanicals because they contain 5-hydroxytryptophan, a serotonin precursor, with consequent antidepressant properties.55 Our search did not find interactions of this substance with the P450 cytochrome system.Lavandula angustifolia, also called English lavender, is a flowering plant native to the Mediterranean, belonging to the Lamiaceae family. The essential oil extracted from fresh flowers, extracts derived from flowers collected just before opening and dried, fresh flowers, and dried flowers are used as botanicals. The main compounds found in these botanicals are terpenes, such as linalool, tannins, and hydroxycoumarins. English lavender has traditionally been used as an antimicrobic or as an adjuvant in mood disorders, such as restlessness or insomnia.27 While an in vivo study on rats showed CYP2D6 inhibition and CYP2A induction,56 a small in vivo study on humans did not report significant P450 cytochrome interactions after the administration of an oral extract of lavender for 11 days.57Malpighia punicifolia var. lancifolia  is a Central and Southern American native evergreen shrub or small tree with bright red fruits 1–3 cm long. These fruits are often used as botanicals for their high antioxidant content and are traditionally administered for treatment of upper respiratory symptoms.55 Our search has not found reports of significant drug interactions associated with this botanical.Matricaria chamomilla  of the Asteraceae family, an annual plant, whose structure and properties are very similar to C. nobile, has also traditionally been used as an infusion (chamomile tea) or as an extract for its relaxing properties and as a sleep adjuvant.25 Its derivatives contain apigenine, a natural flavonoid with antispasmodic effects, which could also be responsible for the observed in vitro inhibition of CYP3A4 and CYP1A1.58 Chamomile tea has also shown in vivo CYP1A2 inhibition capability in rats.34 The crude essential oil shows moderate in vitro inhibition of CYP2C9, CYP2D6, and CYP3A4 and strong inhibition of the CYP21A2 isoform.59 The chamomile leaf extract has also shown in vitro CYP3A4 inhibition properties.49 CYP3A4 inhibition was also observed in a case report on a subject [in a group of 300] who drank chamomile tea regularly while consuming cyclosporine for a renal transplant and showed consequent elevated levels of the immunosuppressant.60 Although limited data are available, caution should be exercised with regard to the consumption of CYP3A4-metabolized drugs with chamomile tea.Melaleuca alternifolia (tea tree) is a small tree of the Myrtaceae family, native of Australia. Its essential oil has traditionally been used for the treatment of upper respiratory symptoms.25 Caution should be exercised if the botanical is assumed regularly because one of its main constituents, p-cymene, has shown broad competitive in vitro inhibition of the P450 cytochrome system.61 However, widespread use of this product with no reported consequences casts doubt on the real clinical significance of these interactions.Melissa officinalis , native to South-Central Europe, is a perennial herbaceous plant belonging to the Lamiaceae family. Its leaves are commonly used in teas and their extract is commonly used as a botanical for its properties as a sleep and digestive aid.25 Our search did not find significant drug interactions for M. officinalis derivatives.Ocimum tenuiflorum, commonly known as holy basil, is an aromatic plant in the Lamiaceae family. Extracts from its leaves are used as botanicals, often as essential oils. The main substance contained in the essential oil is the guaiacol eugenol, which has antiseptic properties.27 While an in vivo study on rats showed a potential inhibition of CYP3A1 and CYP3A2,62 studies on drug interactions involving humans have yet to be conducted; therefore, the real clinical significance of the animal study remains questionable.Olea europaea (olive or European olive) of the Oleaceae family, although native to the Mediterranean area, is a tree that is grown all over the world. Its fruits are commonly used as food or for olive oil production, while its leaves are a source of essential oil, or subjected to extraction of derivatives, such as oleuropein and hydroxytyrosol, which have shown preclinical neuroprotective properties.27 Oleuropein is also thought to be responsible for in vitro CYP1A2 and CYP3A4 inhibition properties64 and in vivo CYP2E1 induction capability observed in rats.65 Maslinic acid, a compound derived from the olive fruit, has also shown broad in vivo (in rats) and in vitro P450 cytochrome system inhibition properties.66 However, since olive oil and olive fruits are commonly used in the human diet, significant drug interactions seem unlikely.Panax ginseng, belonging to the Arialaceae family, is a species of plant whose root is used in foods and beverages. It is also used as a botanical medicine to boost memory or to treat fatigue, menopause symptoms, and diabetes.25 Ginsenosides, the major active constituents of the root, have been studied for their antioxidants and antiproliferative effects.67 Drug interaction studies conducted in vitro have reported different results, yielding inconclusive data and suggesting only a weak interaction with the P450 cytochrome system, with a modest inhibition of CYP3A4, CYP1A2, CYP2C9, CYP2C19, and CYP2D6. However, two studies conducted in healthy human volunteers using a midazolam probe found significant CYP3A induction; therefore, it is advised that people taking CYP3A4 substrates with narrow therapeutic indices, such as cyclosporine, tacrolimus, irinotecan, and sirolimus, should avoid coadministration of P. ginseng, as they could be at risk of therapeutic failure.P. ginseng and warfarin coadministration are described in the literature, with an observed reduction in therapeutic action; overall adverse drug effects with anticoagulants appear to be rare, as CYP2C9, one of the two P450 cytochromes involved in warfarin metabolism, is not affected by P. ginseng. However, close clinical monitoring is still suggested for patients taking warfarin with P. ginseng.68 For P. ginseng root extract, CYP3A induction and CYP2D inhibition were described in an in vivo animal model69; Anderson et al. described no clinically significant effects for CYP3A in a human in vivo model.70 Numerous potential interactions are described in in vitro study models, but no clinically proven effects have been found. The widespread use of this botanical suggests that it may have no clinically significant effects as Anderson et al. found in their investigation.Two case reports involving Passiflora incarnata, also called purple passionflower, is a perennial vine native to the Southeastern United States belonging to the Passifloraceae family. Traditionally, it has been used for the treatment of neuralgia, generalized seizures, hysteria, nervous tachycardia, spasmodic asthma, spasmodic visceral symptoms, and insomnia.71 Despite the absence of data on possible interactions between passionflower consumption and cytochromes, two case reports provide data that deserve to be taken into consideration regarding P. incarnata consumption. Fisher et al. reported the case of a 34-year-old female patient who developed severe nausea, vomiting, drowsiness, prolonged QTc, and episodes of no sustained ventricular tachycardia following self-administration of P. incarnata at therapeutic doses. Her condition required hospitalization for cardiac monitoring and intravenous fluid therapy. The authors hypothesized that this patient may have had polymorphic variation of xenobiotic metabolizing genes such as CYP2D6.71et al. reported the case of a 40-year-old male patient being treated with lorazepam for generalized anxiety disorder. The patient's self-medication with Valeriana officinalis and P. incarnata was correlated with the onset of shaking hands, dizziness, throbbing and muscular fatigue .72 Since there are only a very limited number of cases of rare episodes, it is difficult to evaluate the real clinical impact of P. incarnata consumption; however, pending further studies, it is recommended that an accurate medical history be taken before evaluating its use, especially at full dosage.Carrasco Polygonum cuspidatum, also called Asian or Japanese knotweed, is an herbaceous perennial plant of the Polygonaceae family native to East Asia, Japan, China, and Korea and subsequently exported to North America and Europe. It is generally used for its content of resveratrol, a polyphenol widely used for its anti-inflammatory, antioxidant, and immunomodulator potential.55 Resveratrol has also been used in clinical trials to test its antitumoral activity. One of the possible underlying mechanism of its action is P450 cytochrome inhibition, with consequent reduced cell exposure to activated carcinogens. In fact, resveratrol has shown in vivo and in vitro CYP3A4 inhibition capabilities, even in humans. Chow et al.73 observed an increase in buspirone blood levels after coadministration with resveratrol in a clinical trial. The same authors observed a weak in vivo CYP1A2 induction in a study, in which healthy volunteers were administered 1 g of resveratrol per day for 4 weeks. The ratio between caffeine and the metabolite, paraxanthine, was then measured. A modest decrease in CYP2D6 and CYP2C9 activity was also observed in the same study.74 In addition, Chi et al., using an in vivo animal model, found that the decoction from the crude drug significantly increased systemic exposure and brain concentration of Carbamazepine, likely through the inhibition of CYP3A4 activities.75 Further clinical trials are required to confirm these interactions; however, caution should be exercised in the concomitant administration of P. cuspidatum with CYP3A4 metabolized drugs such as carbamazepine.Rhodiola rosea  is a perennial flowering plant native of wild Arctic regions of Europe, Asia, and North America. R. rosea has been used as an adaptogen and nutraceutical for the treatment of minor anxiety and depression.55 Two of its isolable components, Rhodiosin and Rhodionin, were identified by Xu et al. as in vitro inhibitors of CYP2D6,76 while Hellum et al. identified the rhizome extract as capable of in vitro CYP3A4 inhibition.77 Spanakis et al. reported that R. rosea significantly alters the pharmacokinetic properties of losartan after concurrent oral administration in an in vivo rabbit model.78 Maniscalco et al. reported the case of a 68-year-old female patient with recurrent moderate depressive disorder with somatic syndrome being treated with paroxetine. The patient developed a serotonergic syndrome with vegetative symptoms, a feeling of restlessness and trembling after she began taking a nonspecified formulation of R. rosea.79 Thu et al. identified R. rosea rhizome extract as an in vitro and in vivo CYP2C9 inhibitor in a human model, excluding in vivo inhibition of CYP3A4, CYP2D6, and CYP1A2.80–82 In light of these findings, considerable caution should be exercised in the concomitant administration of R. rosea with drugs metabolized by CYP2C9.Salvia officinalis, also called sage, is a perennial evergreen subshrub native to the Mediterranean region. The plant is a member of the Lamiaceae family. The medicinal parts of the plant comprise the fresh leaves and the fresh flowering aerial parts, the dried leaves, and the oils extracted from the flowers and stems. Extracts contain flavonoids, thujones, camphor, and rosmarinic acid. S. officinalis is traditionally used to treat upper respiratory symptoms and as an antiseptic.27 In one study, a 5-week sage infusion in rats was associated with upregulation of CYP2E183; however, the absence of clinical studies on humans and the widespread use of S. officinalis, also as a food, cast doubt on the real clinical significance of this study.Sambucus nigra, commonly called elderberry, is a species of flowering plants native to Europe and North America belonging to the Adoxaceae family.25S. nigra berry extract has been described in only a very limited number of in vitro studies as a very weak inhibitor of CYP1A2, CYP2D6, and CYP3A485 ; hence, the true clinical significance of this action appears dubious.Uncaria tomentosa, commonly called cat's claw (due to the claw-shaped thorns), is a vine native to the tropical areas of Central and South America belonging to the Rubiaceae family. The bark is used as a nutraceutical in support of the body's natural defences and joint function.25U. tomentosa bark extract has been identified in vitro as a potent CYP3A4 inhibitor in human cell lines.87 Quílez et al., using an in vivo mouse model, reported an increase in the effects attributable to diazepam, a drug metabolized mainly by CYP3A4.88 López Galera et al. reported the case, later cited by Müller and Kanfer, of an HIV-positive female patient with hepatitis C-related cirrhosis, who had a C.min of the protease inhibitors 1.5- and 3- and a more than 10-fold higher than the recommended target ; these values normalized after withdrawal of U. tomentosa.90 Although this is an isolated case report, involving a severely physically compromised patient on polytherapy, these data suggest that caution should exercised in the administration of U. tomentosa with drugs metabolized by CYP3A4, at least in conditions of polytherapy and/or hepatic impairment.V. officinalis, belonging to the Caprifoliaceae family, is a perennial flowering plant native to Europe and Asia traditionally used for anxiolytic and sedative purposes.25 Valeric acid, which is believed to be responsible for its activity, has been identified in vitro to interact with CYP3A4, CYP2C9, and CYP2C19. For V. officinalis root extract, there are reports of in vitro interactions with CYP3A4 , CYP3A5, CYP219, and, with a dynamic dose-dependent mechanism, both induction and modest inhibition of CYP2D6.92 Donovan et al.92 reported a very modest inhibition of CYP2D6 and CYP3A4 in an in vivo human model, concluding that typical doses are unlikely to produce clinically significant effects. Kelber et al. reported the absence of clinically relevant effects in vivo in a review.93 Regarding both the case reports and the articles on reported or potential interactions, drugs active on the central nervous system, such as lorazepam and haloperidol, could account for the development of neurological symptoms, likely attributable to an additive effect.94–9899 This class of secondary metabolites is found abundantly in a variety of medicinal plants, fruits, and vegetables. Indeed, onions, kale, lettuce, tomatoes, apples, grapes, and berries are considered to be rich sources of flavonoids.100 Of the flavonoids, flavonols constitute the most studied compounds against coronavirus.101 Indeed, kaempferol, quercetin, and myricetin, along with their derivatives, are likely some of the most documented molecules with antiviral activities against SARS-CoV-2. Nonetheless, in our assessment of the most widely sold botanicals, we did not find any products whose flavonol content was highlighted, or that were promoted for their possible anticoronavirus effect in terms of prevention or treatment; for example, the pulp of the fruit of P. incarnata contains on average 158.0 mg/mL of total flavonoids, 16.2 mg/mL of isoorientin, and 0.42 mg/g of quercetin102; similarly S. nigra contains flavonoids such as quercetin and anthocyanidins103; and P. cuspidatum contains flavonoids such as quercetin and catechin.104Our analysis, contrary to our original expectations, did not reveal any products developed specifically to counteract the current pandemic. Perhaps we should have extended our analysis to include a greater number of nutraceuticals. In fact, one of the promising therapeutic strategies against COVID-19 virus infection are the enzyme inhibitors found in natural compounds. Thoroughly investigated using molecular docking, natural compounds, such as the flavonoid class, constitute a possible target to identify antiviral drugs, thanks to their wide range of biological properties .P. nigrum, where p-glycoprotein may play an important role in elevated drug blood levels as a consequence of enhanced absorption.105In this study, we focused on the analysis of possible interactions strictly related to the action of cytochromes, excluding from our analysis known mechanisms of action of the investigated substances, which are not directly related to cytochromes. Future studies examining these mechanisms could further enrich our knowledge of the safety of these substances. Furthermore, the actual effectiveness of each botanical, taken individually or in combination, was not evaluated, since as market data suggest, botanicals are assumed regardless of their demonstrated effectiveness, and it is therefore possible that potential interactions may occur. Another limitation of the present study is its focus on drug interactions involving only the P450 cytochrome system, while other significant interactions have been recognized in the literature, for example, for C. paradisi and R. rosea) show in vivo pharmacological interactions in humans. For the other botanicals that were examined, there is weak evidence of interactions or only potential interactions shown in vitro or in animal models and a lack of clinical drug interaction reports; therefore, these findings are of dubious clinical significance. Clearly, clinicians should exclude products with proven interactions, and this review provides them with an additional tool to make decisions based on a rational approach, evaluating the patient's whole therapeutic plan and considering his or her overall condition, therefore, prescribing substances based on a rational risk-benefit ratio. Finally, this study provides a rational assessment of the most used nutraceuticals, including popular self-medication products, to simplify patient management even in complex health care situations such as the current pandemic.In conclusion, our analysis of the data on the investigated plant species reveals a highly complex constellation of potential effects deriving from their use as extracts or as derived substances. Although several studies point to potential complex interactions, clinical evidence remains decidedly limited; in fact, of the 28 sources examined, only 2 (


The present work aimed to evaluate the effect of farming practices and season on the fat and protein content and fatty acid (FA) profile of milk and Halloumi cheese produced in Cyprus. Over a year, raw bulk-tank milk samples from cow, goat, and sheep farms were collected seasonally from all organic (11) and representative conventional (44) dairy farms, whereas Fresh Halloumi cheese samples were collected monthly from retail outlets . The different farming practices did not affect the milk fat content of ruminants, while protein levels were decreased in organic bovine and caprine milk. Under organic farming practices, milk and cheese contained increased values of total mono-unsaturated FA (MUFA) and poly-unsaturated FA (PUFA), and specific FA, such as oleic, conjugated linoleic, linoleic, and α-linolenic acids. Total saturated FA (SFA) levels were particularly decreased in organic samples and, consequently, the atherogenic indices of milk and cheese were decreased. Season influenced milk and Halloumi cheese FA profile; spring samples had lower SFA and higher PUFA and MUFA concentrations. Overall, the organic farm practices improved the lipid profile of milk and Halloumi cheese, which is more likely attributed to the different feeding strategies applied in organic dairy farms. Milk fatty acid (FA) composition can fluctuate widely according to several factors, including animal breed and species, season, stage of lactation, management, and diet , with thThe different farming systems used in organic production have been reported to induce a desirable effect on milk FA composition of dairy animals  ,17,18. WDuring a whole year (2014), bovine, caprine, and ovine milk samples from all organic farms of Cyprus were collected . Conventional milk samples were also collected from 24 cow, 10 goat, and 10 sheep farms that were considered to be representative of the country’s dairy farming according to their annual milk yield. As a standard sampling practice, raw bulk-milk samples from each farm were seasonally collected throughout the year (one sample per farm and season). Thus, a large set of bulk-milk samples accumulated, as follows: 118 bovine , 60 caprine , and 52 ovine . In Cyprus, caprine, ovine, and bovine milk is mainly derived from Damascus goats, cross-bred Chios ewes, and Holstein Friesian cows, respectively. After their collection, the milk samples were transferred in cool opaque boxes to the laboratory into 30 mL sterile, screw-top plastic bottles and then stored at −20 °C until further analysis. Measurements for total fat and protein were performed by the application of combined thermo-optical procedures  calibrated previously for protein with the Lowry protein assay and fat with the Gerber method 989.05 . –1 to provide a firm coagulum in about 60 min. Pasteurised sheep and goats milk the primary component, with cow’s milk used in less quantities [Moreover, four organic and four conventional fresh Halloumi cheeses were sampled every month from one specific brand, which is the largest of the island, available in supermarkets and other retail outlets, summing a total of 96 samples , which were also stored at −20 °C until further analysis. The chemical composition of fresh Halloumi cheese collected was standardized by the manufacturer following the Protected Designation of Origin (PDO) regulations  . In Cyprantities . g for 30 min. at 4 °C. The resulting separated fat globules (lipid cake) were removed, placed in new tubes, and then allowed to melt at room temperature for 20 min. The samples were then recentrifuged at 19,500× g for 20 min. at room temperature, and 20 mg aliquots of the resulting lipid cake were removed to new tubes and dispersed in 1 mL of n-hexane by shaking. Lipids from milk were extracted, as described by Tzamaloukas et al. . Briefly2SO4 was added to 30 to 40 g of cheese samples and mixed vigorously. After 15 min., the mixture was filtered through filter paper  from a funnel. The filtrate was centrifuged for 2 min. at 2500× g at room temperature to remove the undesired particles that originated from cheese. The liquid phase of diethyl ether and oil was taken into a centrifuge test tube and diethyl ether was removed using a rotary evaporator at 40 ± 1 °C. Subsequently, the sample was flushed with nitrogen to remove the remaining ether from oil. The cheese fat was extracted with diethyl ether according to the method described by Neofytou et al. . BrieflyFatty acid methyl esters (FAME) of milk and Halloumi cheese lipids were prepared by transesterification with methanolic potassium hydroxide according to the ISO  method. p < 0.05, and p-values within 0.05 and 0.10 were regarded as tendencies.All of the data on the composition of milk and Halloumi cheese were analyzed based on mixed-effects models with 2 × 4 factorial treatment design representing the farming system (F) and season (S) as the main fixed effect factors, using the SPSS 20 software . The results are presented as least square means ± standard error of the means (SEM). Because of the lack of significant interactions, the main effects for the season were compared when appropriate using Tukey’s Honestly Significant Difference (HSD) test. Significant differences for both fixed main effects were considered at p < 0.01) and caprine (p < 0.001) milk, respectively, while it was similar in the organic and conventional ovine milk samples. Seasonally, there were no significant differences in the proportion of protein milk of cows and ewes, while the caprine milk samples collected during the winter had a significantly higher (p < 0.01) percentage of protein when compared with the other three sampling periods. The fat percentage was neither affected by the farming system nor by season in the milk produced from all species . On the p < 0.001), while that of C18:0 was higher in organic as compared with conventional milk samples, with the exception of some FA in ovine milk. For instance, there were no significant differences in C12:0 and C18:0 levels, while a tendency for decreased C14:0 concentration was observed in organic ewe milk. The levels of total MUFA were increased (p < 0.001) in organic milk of cows and goats, while, in ovine milk, no statistical differences were observed. Regarding the concentrations of individual 18-C MUFA, such as OA (C18:1 cis-9), they were increased in organic milk of cows (p < 0.001), goats (p < 0.001), and ewes (p < 0.05), while the content of vaccenic acid  and other C18:1 isomers was only enhanced in organic bovine and ovine milk. In contrast, the contents of other MUFA, i.e., C10:1 cis-9, C12:1 cis-9, C14:1 cis-9, and C16:1 cis-9 acids, were decreased in organic bovine and caprine milk fat, while, in ovine milk, the levels of these FA were not affected by the applied farming practices. p < 0.001). Additionally, the concentrations of RA and ALA were significantly increased in all types of organic as compared with conventional milk samples (p < 0.05). At the same time, the levels of linoleic acid  were elevated between 36, 40, and 30% in organic bovine, caprine, and ovine milk. No significant differences due to the applied farming system in the percentage of arachidonic acid (C20:4n-6) were observed. The LA:ALA ratio was significantly decreased in bovine (p < 0.05), tended to be decreased in caprine (p < 0.1), and was not affected in ovine organic milk. Additionally, the atherogenic index was significantly diminished within 26 and 33% in organic compared with the conventional milk of cows, goats, and ewes. The content of total PUFA was enhanced between 43 and 67% in bovine, caprine, and ovine milk as a result of the organic farming practices (p < 0.05). Concentrations of total PUFA were increased by 13 and 17% in cow and goat milk collected during spring, respectively. The average percentage of RA was higher in the milk of cows and ewes collected during spring when compared with the other seasons, while the LA concentrations were only statistically enhanced in the caprine milk during spring. Additionally, the percentages of VA and other C18:1 were significantly higher in all types of milk collected during spring, while the highest values for the major MUFA, like OA (C18:1 cis-9), were observed in autumn for cows (p < 0.001) and in winter for goats (p < 0.01). Nevertheless, the milk atherogenic index was decreased during spring, while the LA:ALA ratio did not differ across seasons in all types of milk. A considerable variation in bovine, caprine, and ovine milk FA content was observed due to the season of production , particularly short- and medium-chain SFA between C6:0 to C14:0. Furthermore, the concentrations of total MUFA and the OA, VA, and other C18:1 isomers were enhanced in retail organic Halloumi cheese (p < 0.001). It is also noteworthy that the concentrations of total PUFA were higher by 26% in organic as compared with the conventional cheese (p < 0.001), with the average percentages of individual PUFA, namely LA, ALA, and RA, being particularly enhanced, by 25%, 20%, and 28%, respectively. No farming effect in the levels of C20:4n-6 as well as in the LA:ALA ratio of cheese fat was observed. The atherogenic index of Halloumi cheese was also affected and diminished by 15% in the organic samples when compared with the corresponding conventional.p < 0.001), while no seasonal effect in OA or other C18:1 isomers of cheese fat was observed (p < 0.001) than the content in all other seasons, showing, on average, a 15% increase. Regarding the results of individual PUFA, the average percentages of LA, ALA, and RA in Halloumi cheese produced during spring was significantly increased when compared with that produced during the other seasons. There was no difference in the cheese LA:ALA ratio and the concentration of arachidonic acid across seasons. Finally, the atherogenic index was significantly lower in Halloumi cheese that was collected during spring compared with the other seasons.The effect of season on the FA composition of retail Halloumi cheese was similar to that on milk FA composition of cows, goats, and ewes. In particular, the concentrations of total SFA and individual FA, such as C14:0 and C16:0, were lower in the fat of cheese produced in spring as compared with that manufactured during the other three seasons. In accordance, the average percentages of 18-C FA, like VA, were the highest in spring products  due to heat stress during the summer months. Nonetheless, this was not observed in the present study, since fat and protein content was similar across seasons in all species, with the only exception of protein content of goats, which was found to be elevated in milk that was collected during winter. Increased milk protein content has been reported in the winter season, in temperate climates, when the concentrate-to-forage ratio of diets is higher as compared with grazing season , and this has been associated with higher propionic acid production in the rumen and increased microbial protein supply . NeverthThe content of total PUFA, MUFA, including RA and VA, was higher, while the SFA concentrations were lower in milk that was collected from all species during spring when compared with other seasons. Similar effects have been reported in the milk that was collected during summer months from cows ,31,41,44Interestingly, the effects of organic farming system on the FA composition of retail Halloumi cheese were similar to those that were observed in the organic bovine, caprine, and ovine milk. More specifically, a reduction in the concentration of SFA and in the atherogenic index was demonstrated similarly to the organic milk FA results of all ruminant species, while increased levels of PUFA and MUFA were observed in organic Halloumi cheese, as compared with conventional ones that were collected throughout the year, a result that is line with the results observed in organic cow and goat milk. Organic Halloumi cheese had increased content of 18-C FA, including OA, VA, RA, LA, and ALA, agreeing with the results of Prandini et al.  and BergThe current work is the first large-scale study undertaken in a semi-arid, Mediterranean type of climate, such as that of Cyprus, investigating the effects of farming system  and season on milk composition and lipid quality of milk and Halloumi cheese. The major finding of the present work is related to organic milk and cheese produced. Although similar fat content was observed in organic and conventional milk, the protein percentage was slightly decreased in organic milk. Regarding FA profile, organic milk from all species and organic Halloumi cheese had improved quality, since they contained lower total SFA levels and individual ones, like palmitic acid, while the only elevated SFA was stearic acid compared with conventional milk and cheese. Additionally, higher MUFA, PUFA, and individual FA levels, such as oleic, vaccenic, rumenic, linoleic, and linolenic acids, all related with beneficial effects for human health, were observed in both organic milk and Halloumi cheese samples. Furthermore, milk and cheese that were collected during spring months contained lower saturated and higher unsaturated lipids than the other sampling seasons.


Moreover, combined oxidation and crosslinking assays and molecular dynamics simulations support that zinc ions interact with Cys328 in its thiolate form, whereas Glu329 and Asp331 stabilize zinc coordination. Vimentin oxidation can induce disulfide crosslinking, implying the close proximity of Cys328 from neighboring dimers in certain vimentin conformations, supported by our computational models. Notably, micromolar zinc concentrations prevent Cys328 alkylation, lipoxidation, and disulfide formation. Moreover, zinc selectively protects vimentin from crosslinking using short-spacer cysteine-reactive but not amine-reactive agents. These effects are not mimicked by magnesium, consistent with a lower number of magnesium ions hosted at the cysteine region, according to molecular dynamics simulations. Importantly, the region surrounding Cys328 is involved in interaction with several drugs targeting vimentin and is conserved in type III intermediate filaments, which include glial fibrillary acidic protein and desmin. Altogether, our results identify this region as a hot spot for zinc binding, which modulates Cys328 reactivity. Moreover, they provide a molecular standpoint for vimentin regulation through the interplay between cysteine modifications and zinc availability.The intermediate filament protein vimentin is involved in essential cellular processes, including cell division and stress responses, as well as in the pathophysiology of cancer, pathogen infection, and autoimmunity. The vimentin network undergoes marked reorganizations in response to oxidative stress, in which modifications of vimentin single cysteine residue, Cys328, play an important role, and is modulated by zinc availability. However, the molecular basis for this regulation is not fully understood. Here, we show that Cys328 displays a low pK Vimentin is a type III intermediate filament protein that forms robust filaments in mesenchymal cells. The intermediate filament network maintains an intimate crosstalk with the other cytoskeletal systems (microfilaments and microtubules) and modulates essential cell properties, from cell size to migration, division, and elasticity ,2,3. In The cellular architecture of vimentin filaments is still not completely understood. In contrast to the polarity observed in microfilaments and microtubules, vimentin filaments are non-polar and can grow at both ends. Moreover, they are highly dynamic in cells and can exchange subunits along their length and undergo severing and reannealing ,13. NeveVimentin monomers consist of three domains, head, rod, and tail, of which the rod is mainly alpha-helical, whereas the head and tail domains lack a defined structure. Being a coiled-coil protein for a high proportion of its sequence, the full crystal structure of vimentin has not been solved yet. Using “divide and conquer” approaches, the structure of some vimentin segments in different degrees of association, i.e., dimers or tetramers, has been obtained  was from Cayman Chemical. No unexpected or unusually high safety hazards were encountered throughout this work.Dimethylsulfoxide (DMSO), monosodium and disodium phosphates, Tris, Pipes, DTT, PMSF, Zincon, diamide, and dibromobimane  were frhttps://www.rcsb.org, accessed on 28 June 2021), with PDB IDs 3KLT and 1GK4. The sequences of PDBs 3KLT and 1GK4 structures share seven amino acids . One monomer of each PDB structure was kept and superimposed onto the dimer from the other PDB structure by this overlapping region. Thus, two vimentin dimer models comprising residues Asp264 through Gly406, but differing in their geometry, were constructed: 1GK4 superimposed onto 3KLT model (Model A) and 3KLT superimposed to 1GK4 model (Model B). Only Model B was considered for further computational studies.PyMol software  was usedModel B of vimentin dimer from the molecular dynamics (MD) simulations was submitted to HADDOCK  and ZDOC−1·A−2 harmonic potential constraint was applied on both the proteins and the ligand. In the four subsequent steps, the harmonic potential was progressively lowered, respectively, to 10, 5, and 2.5 kcal·mol−1·A−2 for 600 steps of conjugate gradient algorithm each time, and then the whole system was minimized uniformly. In the following step, the system was heated from 0 to 100 K using the Langevin thermostat in the canonical ensemble (NVT) while applying a 20 kcal· mol−1·A−2 harmonic potential restraint on the proteins and the ligand. The next step heated up the system from 100 to 300 K in the isothermal−isobaric ensemble (NPT) under the same restraint condition as in the previous step. In the last step, the same parameters were used to simulate the system for 100 ps, but no harmonic restraint was applied. At this point, the system was ready for the production run, which was performed using the Langevin thermostat under the NPT ensemble, at a 2 fs time step. All production runs were performed for 100 or 150 ns. Zinc atoms were added to the system using leap. Of note, no secondary structural changes were observed during MD simulations. All the studied structures (Asp264-Gly406 region) were quite stable, as can be inferred from the RMDS values (Supporting Information). Nevertheless, changes in other regions of the protein cannot be excluded.MD simulations were carried out using Amber14. The system was solvated with the TIP3P water molecules model, and the simulation box was built such that the edges were at least 10 Å distant from any atom. Eight steps of preparation were performed before running MDs. The first one consisted of 1000 steps of the steepest descent algorithm followed by 7000 steps of conjugate gradient algorithm; a 100 kcal·mola of Cys328 at physiological pH (pH = 7).The models of vimentin dimer B and the corresponding vimentin dimer of dimers assembly were used for calculations. PROPKA ,56 was e280 nm, using an extinction coefficient of 22,450 M−1cm−1. Aliquots of the protein were kept at −80 °C until use.Recombinant human vimentin wild type (wt) and Cys328Ser in 8 M urea, 5 mM Tris-HCl pH 7.6, 1 mM EDTA, 10 mM 2-mercaptoethanol, 0.4 mM PMSF, and approximately 0.2 M KCl, purified essentially as described , were puo). The ratio of alkylated protein at time t (Ft) vs. Fo was calculated and used to obtain the ln(Ft/Fo). The slopes of the representation of ln(Ft/Fo) against time provide the kobs values, which were represented against the final reaction pH to obtain the pKa (the inflexion point of the curve). Three independent experiments were carried out for both vimentin wt and Cys328Ser using different protein preparations.Buffers were prepared by mixing 5 mM concentrations of each of the following to obtain the desired pH: citric acid/disodium phosphate (pH 3–5.5), disodium phosphate/monosodium phosphate (pH 6–8), Tris/HCl (pH 8.5–9), and sodium carbonate/sodium bicarbonate (pH 9.5–10.8). Purified vimentin wt or Cys328Ser was thawed and subjected to five cycles of dilution with 5 mM Pipes/Na pH 7.0 (10–12 mL) and ultrafiltration on Amicon Ultra filter units for DTT elimination, immediately before monobromobimane (MBB) modification. The last flowthrough was kept, and the protein concentration was measured on a Nanodrop (Thermo Fisher). MBB 50 mM was prepared fresh in DMSO and later diluted with 5 mM Pipes/Na pH 7.0 to 333 μM, the concentration required to obtain a final 1:4 (mol/mol) ratio protein:MBB in the modification protocol. MBB reactions were carried out in triplicate in the dark on black-polystyrene NBS-treated multiwell plates  in a final volume of 100 μL/well. Each well contained 2.5 μM vimentin wt or Cys328Ser, 77 μL of buffer at the desired pH, and 10 μM MBB. Controls lacking the protein included the flowthrough of ultrafiltration to establish the background or reduced glutathione  as a positive control of MBB modification. Kinetics of modification were followed by measuring fluorescence emission at 480 nm every 3 min for up to 90 min, upon excitation at 380 nm using Varioskan Flash (Thermo Fisher). The final pH for each reaction was measured with a pH-meter using equivalent mixtures in a final volume of 2 mL. Fluorescence data for each well were adjusted to a sigmoidal curve using GraphPad Prism v.5 to obtain the value of the bottom plateau corresponding to the initial non-alkylated protein . Biotin incorporation was assessed by incubating blots with streptavidin-HRP  and vimentin levels estimated by western blot with anti-vimentin V9 monoclonal antibody , followed by enhanced chemiluminescent (ECL) detection . Signal intensities were evaluated by image scanning and analysis with ImageJ.Additional verification of the results obtained with MBB was achieved by subjecting vimentin to modification with Iac-B at different pHs for 30 min at room temperature, in the dark. Reactions contained 2.5 μM vimentin wt or Cys328Ser, 50 μM Iac-B, and buffers at different pHs near the pK2, MgCl2, or NaCl for 1 h at room temperature, as indicated. Incorporation of 15d-PGJ2-B or Iac-B was analyzed by SDS-PAGE followed by blot and biotin detection as described above. For detection of crosslinked products, samples were run on 7.5% SDS-polyacrylamide gels, which were fixed with 40% (v/v) methanol and 10% (v/v) acetic acid for 60 min, stained with Sypro Ruby overnight, washed with 10% (v/v) methanol and 6% (v/v) acetic acid, and visualized under UV light on a Gel-Doc XR Imaging System (Bio-Rad).Modification of vimentin was assessed by gel-based techniques. Briefly, vimentin at 3.8 μM in 5 mM Pipes-Na, pH 7.0, was incubated for 1 h at room temperature in the presence of the indicated compounds. Given the fact that DTT can react with electrophilic compounds and chelate metals , its fin2. The last filtrate was used as control. Absorbance at 620 nm was measured as an index of the formation of the Zincon-Zn2+ complex.The ability of vimentin to compete for zinc binding in the presence of Zincon was assessed by a modification of a previously reported method . Immediat-test was used. Differences were considered significant for p < 0.05.All experiments were performed at least three times using different protein batches. Results are presented as average values ± standard error of mean (SEM). Statistical analyses were carried out using GraphPad Prism v5. For comparisons of different sets of values, the Student’s Identification of zinc binding proteins based on sequence analysis and database searching is often unsuccessful. Indeed, vimentin is not identified using these resources. Moreover, analysis of zinc binding to vimentin by some techniques, including mass spectrometry, has proven not viable due to the ability of zinc to induce oligomeric forms of the protein and compromise its solubility. To overcome these difficulties, we undertook a computational approach. Zinc binding sites in proteins often involve cysteine residues or carboxylic amino acids ,62. BaseTherefore, Model B was used to interrogate the hypothesis of a potential interaction of zinc atoms in this region A. FirstlOther proteins have been found to interact with multiple zinc atoms, mainly through carboxylic residues, which can play a structural role stabilizing subunit interactions . Interes−) forms depending on their pKa and the environmental pH. In our initial Model B, we considered the neutral form of Cys328 thiol group  or thiolate , values of 9.3 and of 10.0 were obtained for cysteines facing outwards and towards the interdimer space, respectively    C, left. 0.07249) C, right. bonding . Cysteinerfamily . Howeveroctamers ,23,74. T protein . In ordeed assay . This sea of Cys328 falls within the physiological pH range, suggesting that a significant proportion of cysteine residues in vimentin molecules will occur in thiolate form in cells and supporting the behavior of this residue as a hot spot for oxidative modification and therefore redox sensing [Taken together, these results indicate that the pK sensing ,76.2  [a of cysteine residues, stabilizing the negatively charged thiolate anions and making them more reactive towards oxidants and electrophiles [a implies a higher fraction of reactive thiolates, but it also may imply a lower nucleophilicity [2S [To substantiate the importance of the interaction of zinc with the Cys328 region, we assessed the extent of modification by Iac-B after preincubating the protein with ZnCl2 D. Micromn plasma  or in cen plasma , i.e., an plasma , zinc ca 214 nM)  indicate 214 nM) ,81. Therrophiles ,82. In ahilicity . Therefohilicity ,84, but city [2S .Mutation of cysteine to serine in proteins is a widely used strategy to assess the importance of thiol moieties in protein function. Regarding vimentin, we have previously shown that a Cys328Ser mutant is competent for filament formation in vitro and confers resistance to the alterations of vimentin filaments induced by oxidants and electrophilic agents , althougThe relative position of vimentin tetramers within filaments is not completely understood yet. Importantly, oxidative conditions have been reported to induce the formation of oligomeric species of vimentin by disulfide bonding ,41, whic2O2 induce disulfide bonds, and therefore reversible dimerization [This electrophoretical mobility, although lower than theoretically expected, is compatible with the position of previously reported vimentin dimeric species ,41. Of nrization , whereasrization . Formatirization , preincurization . Moreoverization ,86,87. Arization .a of the cysteine residues. In the dimer model, thiol groups appear oriented outwards and are more accessible and surrounded by the solvent. In contrast, in addition to external thiol groups, the dimer of dimers presents thiol groups facing the dimerization interface. These internal Cys-SH groups are buried by the vicinal side chains. Moreover, one of these internal thiol groups establishes a hydrogen bond with the Gln324 CO group, which may favor a putative decrease of its pKa, as has been described for peroxiredoxin active site Cys [In view of the above considerations, we addressed the modeling of a dimer of dimers to study whether cysteine crosslinking would be geometrically possible D. For thsite Cys .Next, the interaction of the putative “nearby-CYS” tetrameric arrangement with zinc ions was studied . To thisA closer look at the region surrounding Cys328 showed stable interactions of zinc ions with the vicinal Glu329 and Asp331 residues, resembling those observed in the thiol-form of the dimer A.Interactions equivalent to those found in the thiolate form of the dimer can also be deduced, as well as for several combinations of cysteine species (thiol or thiolate) that could occur. Therefore, we cannot exclude other dimer–dimer association patterns involving thiol and thiolate species of vimentin simultaneously, in which the coordination of zinc ions will occur through the predominant participation of the carboxylate or the cysteine residues, respectively. In fact, zinc atoms are observed to interact at the interface between both dimers. As more detailed structural information on vimentin filaments becomes available, it will be possible to predict whether zinc atoms participate in protein–protein interactions at the dimer or tetramer levels under physiological conditions or even in filament bundling, as suggested by recent evidence ,45.Next, the Cys328Ser dimer of dimers was constructed as a control. This structure revealed a significant difference with the wt tetrameric assembly consisting in the formation of a H-bond between the OH groups of Ser328 residues from the different dimers, since their side chains fall in the correct orientation and distance . This co2 in a concentration-dependent manner. Moreover, crosslinking by DBB was markedly attenuated in the presence of zinc  [D in the nanomolar to micromolar range have been measured [In summary, based on our cysteine modification assays and computational models, we propose that the region surrounding the single cysteine residue of vimentin behaves as a hot spot for zinc binding, possibly modulating vimentin assembly. The proposal of this zinc-binding motif is compatible with the binding of this cation at multiple vimentin sites, and also with the potential contribution of distant segments of the protein, such as the head or tail domains, or of other dimers to zinc binding at the Cys328 region once the protein is organized in filaments. Indeed, results of the competition assay with the zinc chelator Zincon suggest the existence of several sites for zinc interaction in vimentin, with affinities in the nanomolar to micromolar range. These affinities would be clearly lower than those of some well-known zinc binding proteins including metallothionein or Keap1, which possess K10−11 M) . Neverth10−11 M) , includimeasured ,62. Thesmeasured , in contmeasured ,97, althmeasured . This asmeasured  through measured . Importameasured . Thus, iThe pathophysiological consequences of the exposure of vimentin to zinc could be diverse depending on its location. We have previously shown that the vimentin network is thinner and more susceptible to oxidative damage in fibroblasts from patients with genetic zinc deficiency, i.e., acrodermatitis enteropathica , and theCompared to the other main cytoskeletal systems, microfilaments and microtubules, the regulation of the assembly and reorganization of intermediate filaments is insufficiently understood. Apart from posttranslational modifications, interaction of intermediate filaments with “cofactors”, and specifically with divalent cations, is arising as a potential regulatory mechanism in vitro and in cells. In previous works, we identified the interaction of micromolar concentrations of zinc with vimentin and its impact on vimentin network organization and filament morphology, in vitro as well as in cellular models of disease. In addition, zinc, but not other divalent cations, affected the response of vimentin to oxidants and electrophiles, for which the presence of its single cysteine residue is an important factor. The findings herein reported provide a biochemical basis for the behavior of vimentin and, possibly, other members of the Type-III intermediate filament family, as redox sensors and potentially as drug targets, relying on the particular characteristics of the cysteine residue. Moreover, the identification of a hot spot for zinc binding in the region of the cysteine residue supports the role of zinc in vimentin organization and redox regulation and offers a working hypothesis to assess the role Type-III intermediate filaments in the pathogenesis of diseases associated with oxidative or electrophilic stress and/or zinc deficiency. Finally, given the central position of intermediate filaments in cytoskeletal crosstalk and in the homeostasis of cellular organelles, this interplay opens interesting avenues for the study of intermediate filaments as mediators of zinc availability and redox signaling, both in cells and in the extracellular medium, which has important implications in health and disease.


Parity and season are two prominent variants which influence buffalo reproductive performance and synchronization outcome. The present study compared modified new Ovsynch synchronization (GPGMH) protocol with conventional Ovsynch (OVS) in reproductive parameters of buffaloes with different parity (nulliparous and multiparous) in different seasons (peak and low breeding seasons). Compared to the conventional Ovsynch, the modified GPGMH protocol was more effective to enhance the estrus rate, ovulation rate and pregnancy rate in multiparous crossbred buffaloes. In addition, the application of GPGMH protocol improve the reproductive performance during the peak breeding season than low breeding season.n = 128; GPGMH = 94, OVS = 34) and multiparous  buffaloes during the peak  and low breeding  seasons. Buffaloes were monitored for follicular dynamics, estrus response, ovulation, and pregnancy rates. The results showed that protocol, parity, and season had significant effects on estrus, ovulation, and pregnancy variables, and interactions among parity and protocol, season and protocol, and season and parity were observed for few of reproductive indices in the crossbred buffaloes. There were no significant (p > 0.05) interaction for protocol, parity and season. In multiparous buffaloes, the application of the GPGMH protocol significantly (p < 0.05) increased the interaction to the interval to estrus onset after the second GnRH, estrus response, ovulation rate, and pregnancy rate, and lowered (p < 0.05) the silent estrus when compared with the conventional OVS protocol. During the peak breeding season, the application of the GPGMH protocol significantly (p < 0.05) improved the interaction to the estrus response, ovulation rate, and pregnancy rate, while it lowered (p < 0.05) the silent estrus incidence when compared to the conventional OVS protocol. In conclusion, the GPGMH protocol, in comparison to the OVS protocol, improves the follicular dynamics, estrus response, ovulation, and pregnancy rates in crossbred multiparous buffaloes during the peak breeding seasons.The present study was conducted to examine the effect of conventional the Ovsynch protocol (OVS) and a modified Ovsynch synchronization (GPGMH) protocol on the follicular dynamics, estrus, ovulation, and pregnancy in nulliparous and multiparous crossbred (swamp × riverine) buffaloes during different seasons. GPGMH or OVS protocols were used to synchronize nulliparous ( Buffalo productivity is influenced by the seasonality pattern of breeding. The delayed attainment of puberty in heifers and prolonged inter-calving intervals in multiparous buffaloes are major determinants for low fertility ,2. Poor 2α [In recent decades, several estrus synchronization protocols have been applied aiming to manipulate ovarian activity in nulliparous and multiparous buffaloes during peak and low breeding seasons . Synchro2α ,19, prog2α ,20, insu2α , and an 2α  in the ODecreased progesterone concentrations at the time of AI and greater CL volume are prerequisites for future pregnancy establishment in buffaloes . Our preThe study was approved by the tab of animal experimental ethical inspection of the Laboratory Animal Centre, Huazhong Agriculture University . All experimental protocols were performed in accordance with the guidelines of the Committee of the Animal Research Institute, Huazhong Agricultural University, China.n = 128) and multiparous (n = 154) crossbred buffaloes , maintained at a private buffalo farm in Hubei Jinniu Co., Ltd., Hubei province, China, during peak breeding  and low breeding  seasons. The nulliparous buffaloes varied from 2.5 to 3.5 years old, with a moderate body condition score (BCS 2–3.5) and average body weight (BW) 433 ± 72 kg, whereas selected multiparous buffaloes had 2.5–3.5 BCS, 5–8 years of age, and 588 ± 89 kg BW at the beginning of the experiment.In the present study, cyclic nulliparous  . Machine milking was in practice twice a day (6:00 and 18:00). Ad libitum fresh and clean water was provided routinely. Buffaloes were housed in an open shed with a cemented roof top and a two-sided wall fenced by galvanized wire mesh. The multiparous buffaloes were clinically and physically healthy with a normal calving history and reproductive soundness. Additionally, the nulliparous buffaloes were also screened through ultrasonography on a monthly basis to monitor cyclicity or any reproductive anomaly prior to the start of the trial.2α  on day 7, the 2nd GnRH and mifepristone (0.4 mg/kg) simultaneously on day 9, and hCG  on day 5 after AI. OVS protocol was used as described by  [In this experiment, GPGMH protocol was shown as n day 9) .n = 128) and multiparous (n = 154) buffaloes were selected. The selected buffaloes were synchronized through OVS (n = 66) and GPGMH (n = 216) protocols, keeping the categories of parity  and multiparous ) and seasons , low breeding seasons ).A total of two hundred and eighty-two nulliparous , starting from day 6 to day 12 of the protocol for follicular dynamics. Ultrasonography was performed twice a day and follicular dimensions were calculated using follicle diameter recordings at different intervals. The ovulation was defined as the time of the sudden disappearance of dominant follicles (>10 mm) following the 2nd GnRH treatment. Ovulatory follicle diameter was recorded on the basis of reciprocal measurements from ovulation to the initial follicular scan. Anovular follicles were defined as the presence of a follicle (>15 mm) persisting from the last scan at day 12 of the protocol.Estrus response was detected by daily visual observations . Buffaloes were inseminated at a fixed-time AI schedule (16 h after the 2nd GnRH dose) by a skilled AI technician using frozen-thawed semen. Pregnancy diagnosis was confirmed by transrectal ultrasonography at day 40 post AI and confirmed by the presence of amniotic fluid with a viable embryo.p < 0.05 was considered as statistically significant.Data were analyzed using statistical software . The values are expressed as mean ± SD. Repeated measures analysis of variance (ANOVA) was used to compare the effect of season, protocols, and parity for follicular diameters and durations to estrus or ovulation and pregnancy rate. In addition, the influence of parity status, protocols, and season on the estrus response, silent estrus, ovulation rate, follicular cyst incidence, and pregnancy rate were compared using logistic regression with the model consisting of parity (multiparous or nulliparous), season , and treatment (GPGMH or OVS) as main factors and their interactions. The values p < 0.05) of different protocols on the follicle diameter at day 6, interval to estrus after the second GnRH, estrus response, estrus duration, silent estrus rate, interval to ovulation after the second GnRH, ovulation rate, and pregnancy rate were observed. There was a significant effect of parity on the follicle diameter at day 6, estrus response, estrus duration, interval to ovulation after the second GnRH, ovulation follicle diameter, ovulation, and pregnancy rate in buffaloes. Also, a significant effect (p < 0.05) of season on the interval to estrus or ovulation after the second GnRH, estrus response, and ovulation and pregnancy rate was observed.p < 0.05) for the interval to estrus after the second GnRH, estrus response, silent estrus rate, interval to ovulation after the second GnRH, ovulation follicle diameter, and ovulation rate in buffaloes. Significant interactions (p < 0.05) between season and protocol were observed in relation to the interval to estrus after the second GnRH, estrus response, silent estrus rate, ovulation rate, and pregnancy rate in the crossbred buffaloes. In addition, season and parity interacted significantly (p < 0.05) for ovulation rate in the crossbred buffaloes. However, there were no significant interactions for protocol, parity, and season for any reproductive variable in the buffaloes  increased the interaction with the interval to estrus onset after the second GnRH, estrus response, ovulation rate, and pregnancy rate and decreased the silent estrus rate (p < 0.05) when compared with the conventional OVS protocol. However, the application of GPGMH did not promote reproductive performance in nulliparous buffaloes. The pregnancy rate was influenced by parity (multiparous) and protocols (GPGMH), but no interaction was found between the parity and used protocols  was found between the influencing factors parity and protocol for follicle diameter at day 6, estrus duration, or the incidence of follicular cysts  the interaction to the estrus response, ovulation rate, and pregnancy rate, and decreased (p < 0.05) the silent estrus rate when compared with the conventional OVS protocol. However, the application of GPGMH did not promote reproductive performance during the low breeding seasons. The pregnancy rates were influenced by season (PBS) and protocol (GPGMH) with a significant interaction (p < 0.05) between the influencing factors (seasons and protocols).The effects of seasons and synchronization protocols on the reproductive indices of the synchronized crossbred buffaloes are presented in p < 0.05) was found between the influencing factors parity and protocol for follicle diameter at day 6, estrus response, estrus duration, interval to ovulation after the second GnRH, ovulation follicle diameter, or the incidence of follicular cysts  compared to the estrous duration (~18 h) of swamp buffaloes synchronized through OVS ; this vaAnother plausible reason for the success of GPGMH in multiparous buffaloes could be the use of hCG post-AI. Under such conditions, hCG, seeming to act as a substitute of LH as it haves the biological activity of LH, was used at the time of AI or 5 days later to promote the accessory CLs to maintain an optimum progesterone level. Because of its long-standing action in circulation, its dissociation from the LH receptor is gradual, which helps the granulosa cells to transform into luteal cells. It has been reported that CL formed after hCG use has a similar steroidogenic activity on the progesterone hormone . AdditioAlthough, the buffalo is a polyestrous animal, a seasonality pattern in breeding has been reported ,18,28,29In conclusion, the GPGMH protocol enhances the estrus, ovulation, and pregnancy traits in multiparous buffaloes. Moreover, the modified Ovsynch protocol (GPGMH) could be applicable during peak and low breeding seasons for promising reproductive results in crossbred multiparous buffaloes.


Thromboembolic events (TEs) are the second cause of death in cancer patients. Two forms of thromboembolic events may arise: arterial, such as ischemic stroke or myocardial infarction; and venous, such as deep vein thrombosis or pulmonary embolism. Bevacizumab is a monoclonal antibody directed against vascular endothelial-derived growth factor, and is widely used in advanced ovarian cancer. However, whether bevacizumab increases the risk of thromboembolic events in ovarian cancer is matter of debate since studies have shown conflicting results. In our systematic review and meta-analysis, we included 14 trials with bevacizumab in ovarian cancer. We found that the risk of arterial thromboembolic events more than doubled with a risk ratio of 2.45. Also the risk of venous thromboembolism increased 30% with bevacizumab treatment. Bevacizumab, therefore, can be considered an additional risk factor for selecting patients for primary prophylaxis with anticoagulants.p = 0.008). VTE incidence was reported in 9 trials (5121 patients) where the absolute risk was 5.4% with bevacizumab vs. 3.7% without . Our analysis showed that the risk of arterial and venous thromboembolism increased in patients treated with bevacizumab. Thrombolic events (TEs) are probably underreported, and studies should discriminate between ATE and VTE. Bevacizumab can be considered as an additional risk factor when selecting patients for primary prophylaxis with anticoagulants.Thromboembolic events are the second cause of death in cancer patients. In ovarian cancer, 3–10% of patients present with venous thromboembolism (VTE), but the incidence may rise to 36% along the disease course. Bevacizumab is a monoclonal antibody directed against vascular endothelial-derived growth factor, and in in vitro studies it showed a predisposition to hemostasis perturbation, including thrombosis. However, in vivo and clinical studies have shown conflicting results for its use as a treatment for ovarian cancer, so we conducted a systematic review and meta-analysis on the risk of arterial thromboembolism (ATE) and VTE in ovarian cancer patients treated with bevacizumab. The review comprised 14 trials with 6221 patients: ATE incidence was reported in 5 (4811 patients) where the absolute risk was 2.4% with bevacizumab vs. 1.1% without (RR 2.45; 95% CI 1.27–4.27,  Thromboembolic events (TEs) frequently occur during malignancy and are the second leading cause of mortality in cancer patients ,2,3,4. FIn ovarian cancer, 3–10% of patients present with a VTE at diagnosis, but the incidence increases up to 36% during treatment . Risk faFédération Internationale de Gynécologie et d’Obstétrique) stage-III (EMA IIIB and IIIC only) and stage-IV disease based on the GOG-0218 [Angiogenesis is one of the hallmarks of malignancy, and the vascular endothelial growth factor (VEGF) is one of the key promoting factors because it alters the tumor micro-environment and its cancer cells. Bevacizumab is a recombinant humanized monoclonal antibody against VEGF and is widely used in the treatment of different cancer types, either in monotherapy or in addition to chemotherapy or immunotherapy ,13,14,15GOG-0218  and ICONGOG-0218 , both ofGOG-0218 ,17,18,19GOG-0218  and GOG-GOG-0218 .Overall, bevacizumab has a manageable safety profile and is well tolerated. Typical adverse events include hypertension , proteinuria  and minor mucocutaneous bleeding (7–36%) ,25. Of sThe in vitro inhibition of the VEGF pathway led to endothelial dysfunction and facilitates vasoconstriction which are predispositions for disturbed hemostasis and vascular thrombosis . FurtherA systematic literature search was performed using MEDLINE, EMBASE and Cochrane Central Register of Controlled Trials (CENTRAL), with final update on 2 April 2021. The search string was built with the help of a specialized librarian using search terms related to “ovarian cancer” and bevacizumab. The detailed search syntaxes for all search engines are provided in the https://rayyan.qcri.org, accessed on 1 October 2020) for further screening and selection. Screening was done by two authors (MS and NV) based on title and abstract. Full-text analysis and study selection was done by authors MS and HK. Any disagreement was resolved by consensus. When the same patient source was included in different publications , the most recent and relevant was used.The studies that met the following criteria were eligible for inclusion in our meta-analysis: (1) prospective randomized controlled trials; (2) available in English; (3) patients with OC, including primary peritoneal cancer or cancer of the fallopian tube; and (4) randomization to treatment with or without bevacizumab, alone or in combination with chemotherapy, targeted therapy, or immunotherapy. Titles and abstracts that did not clearly meet the inclusion criteria were excluded. Full texts were obtained for the remaining records. In case a study had multiple reporting articles, the most recent and most relevant was used. Articles reporting subanalyses or exploratory analyses were excluded if they were not relevant to the assessed outcome parameter . The results from all search engines were merged using RAYYAN ; patient and tumor characteristics ; treatment characteristics ; and outcome parameters , OS, ATE, VTE). In case of missing or confounding data, the corresponding author was contacted.The primary outcome was the incidence of venous and arterial thromboembolism in patients treated with bevacizumab versus without treatment. We extracted the thrombo-embolic risk for individual studies when provided and made a separate analysis for venous and arterial thrombo-embolic risk. In case of a three-arm study, we pooled the data of all bevacizumab-treated patients. Missing or confounding data were addressed to the corresponding author. Data that were not provided were left blank. https://training.cochrane.org/online-learning/core-software-cochrane-reviews/revman, accessed on 9 April 2021). The summary estimates were generated using a fixed-effect model (Mantel–Haenszel method). Statistical heterogeneity was assessed with the Q-test and the I2 statistic. I2 values of 25, 50 and 75% were considered to indicate low, moderate, and high heterogeneity, respectively [p < 0.05 was regarded as statistically significant, and all tests were two-sided. If >10 studies were included, a funnel plot was generated to evaluate the publication bias, and Egger’s regression method was used to test the symmetry of funnel plots. Risk of bias was assessed by two reviewers (MS and HK) using the Cochrane RoB2-tool [We pooled data from individual trials and calculated the risk ratio (RR) for developing a thrombo-embolic even using RevMan 5.3, provided by the Cochrane collaboration .Details of the protocol for this systematic review were registered with PROSPERO . Heterogeneity between studies was moderate (I2 = 35%). Based on subgroup analysis, this risk was present in all treatment settings: either frontline treatment, relapsed platinum-sensitive or platinum-resistant  reported on the incidence of arterial thromboembolism see . With a esistant . Nine studies, with a total of 5121 patients , reported on the incidence of venous thromboembolism. With a total number of 155 events in the bevacizumab group and 83 in the control group, the absolute risk of development of VTE was 5.4 and 3.7%, respectively.p = 0.04) compared to no bevacizumab treatment  and 1.32 , respectively. However, we also found that the absolute risk of ATE and VTE was lower than expected in both groups as the incidence of ATE and VTE was, respectively, 2.4 and 5.4% in the bevacizumab group and 1.1 and 3.7% in the control group. Our study is the largest meta-analysis to address the thrombogenic risk of bevacizumab in ovarian cancer. We selected 14 studies with a total of 6119 patients and made a subgroup analysis according to disease setting. This was particularly interesting as the incidence of TEs may vary among tumor types and disease setting. However, we used pooled data from the trials as we did not have access to individual patient data. We could not account for possible confounding factors according to disease and patient characteristics , surgical factors , or prophylactic use of LMWH. Furthermore, we were confronted with scarce and heterogeneous data because ATE and VTE incidence was only reported in 5 and 9 studies, respectively, of the 14 studies overall. We decided to select all prospective trials for qualitative review, including those not reporting ATEs and VTEs, to minimize the risk of selective outcome reporting and to illustrate the fact of inadequate TE reporting: five of the studies did not report any incidence of ATE or VTE see . Of the This retrospective series showed thromboembolic events in 12–36% of ovarian cancers ,42. In t2 Q3w), whereas the result in the low-dose group (7.5 mg/m2) was not significant [Previous meta-analyses showed conflicting results for the risk of TE with bevacizumab see . In 2007nificant .In ovarian cancer, six meta-analyses investigated the benefit and harm of angiogenesis inhibitors, including thromboembolic events see . Five foThe burden of thromboembolic complications in ovarian cancer is high, and there is a clinical need to investigate relevant risk factors for TEs to define possible prevention strategies for TE development in OC. The Khorana risk score (KRS) was the first risk assessment model to identify ambulatory patients at risk for VTE, with a score of >3 as high risk . Several2, hemoglobin < 10 g/dL, leukocytosis > 11 × 106/µL or thrombocytosis > 350,000/µL. When weighing risks and benefits, treatment with bevacizumab can be considered as an additional risk factor besides others such as ascites, clear cell histology or a prior history of VTE [Current guidelines recommend primary prophylaxis for hospitalized patients and prolonged prophylaxis after surgery. In ambulatory settings, prophylaxis is recommended for high-risk patients (Khorana risk score > 2) ,74,75,76y of VTE .In ovarian cancer patients, an increased risk of ATE and VTE was observed in treatment with bevacizumab. The incidence of TEs was probably underreported, and arterial and venous TE should be described separately. Caution should be made when initiating bevacizumab in patients at risk for TE. Primary prophylaxis of VTE with LMWH or DOACs, based on the Khorana risk score, may reduce the TE burden in OC. When selecting patients for primary prophylaxis, treatment with bevacizumab should be considered as an additional risk factor for VTE development.