Brucea javanica oil (BJO) is widely used in traditional Chinese medicine to treat various types of cancer and inflammatory diseases. There is significant interest in understanding the medicinal activities of BJO and its molecular components, especially quassinoids, and in exploring how they can be incorporated into nanomedicine delivery strategies for improved application prospects. Herein, we cover the latest progress in developing different classes of drug delivery vehicles, including nanoemulsions, liposomes, nanostructured lipid carriers, and spongosomes, to encapsulate BJO and purified quassinoids. An introduction to the composition and medicinal activities of BJO and its molecular components, including quassinoids and fatty acids, is first provided. Application examples involving each type of drug delivery vehicle are then critically presented. Future opportunities for nanomedicine delivery strategies in the field are also discussed and considered within the context of translational medicine needs and drug development processes. The field of traditional Chinese medicine (TCM) often incorporates natural products from plant, animal, and mineral sources as medicinally active ingredients for applications such as anticancer, anti-inflammatory, and antimicrobial therapy . Given tBrucea javanica (L.) Merr is a species of evergreen plant shrub from the Simaroubaceae family that grows abundantly throughout Southern China and Southeast Asia [B. javanica is oval-shaped, solid, and has a typical length and diameter of around 8 mm and 5 mm, respectively [B. javanica fruit is often used to treat medical illnesses such as intestinal inflammation, diarrhea, malaria, and different types of cancer [As shown in ast Asia . The bitectively  —contain medicinally active components, including quassinoids as well as fatty acids such as oleic acid and linoleic acid [The fruit and especially its seed oil—termed eic acid ,8. The ceic acid . Notablyeic acid , a featuB. javanica oil emulsion (BJOE) systems that enable improved solubility [B. javanica fruit together with a naturally sourced lipid emulsifier [While BJO and its molecular components are medicinally active, they have a poor solubility in aqueous solutions, which has motivated the development of lubility . BJOE saulsifier . To dateulsifier  and in culsifier  or chemoulsifier  to improulsifier . Howeverulsifier ,18.Such challenges have spurred ongoing efforts to develop innovative nanomedicine strategies that harness the medicinal activity of BJO and its molecular components with nanoparticle carriers. Key examples of promising nanostructures in various stages of development include nanoemulsions, liposomes, nanostructured lipid carriers, and spongosomes, which can help to improve bioavailability, controlled release, physicochemical stability, drug loading, and encapsulation efficiency. The objective of this review is to cover the latest progress in the field, starting with an overview of the pharmacological properties of BJO and its molecular components, followed by a critical presentation of various types of cutting-edge nanomedicine strategies and how they are being utilized to deliver BJO and purified quassinoids.B. javanica seeds, and the major molecular components are various fatty acids and fatty acid derivatives, including around 63% oleic acid and 21% linoleic acid in free and mono-, di- and triglyceride forms, along with medicinally active quassinoids in lower concentrations [BJO is mainly extracted from trations . While otrations . CertainSince BJO has been shown to have clinical prospects as a cancer treatment option, there have been intensive experimental efforts to investigate the anticancer activity of BJO. Accordingly, BJO has been shown to inhibit leukemia , liver , and lun50) values down to the nanomolar level [Various molecular components of BJO are believed to contribute to anticancer activity. For example, linoleic acid is reported to inhibit the cyclooxygenase-2 (COX-2) enzyme that is related to tumor cell invasiveness and angiogenesis ,29. In aar level , and henar level ,39.Inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis can affect the small and large intestines and cause a wide range of symptoms such as bloody diarrhea, vomiting, and abdominal pain . Hence, Huang et al. reported that BJOE could have therapeutic benefits in an experimental rat model of Crohn’s disease and inhibited the activation of the NF-κB pathway, resulting in decreased levels of proinflammatory cytokines and increased levels of anti-inflammatory cytokines . In a moWhile it has long been suggested that BJO is useful for treating diabetes mellitus , more reNoorShahida et al. extracted various fractions of medicinally active compounds from BJO and observed that the oral or intraperitoneal administration of BJO fractions could reduce blood glucose levels in normoglycemic mice . FurtherTMV) infection and replication [TMV, could be used as a foliar spray to inhibit the systemic TMV infection of plants, and also inhibited potato virus Y (PVY) and cucumber mosaic virus (CMV) [Plant viruses are a major threat to agricultural production  and havelication . A follous (CMV) . It has us (CMV) .PepMOV), which affects pepper plants (Capsicum spp.) and can impair pepper production quality and yield [PepMOV infection of plant leaves based on a fluorescence reporter assay, in which case PepMOV-infected leaves exhibited fluorescence under ultraviolet (UV) light. In marked contrast to this result, leaves treated with BJO extract or brucein A quassinoid did not exhibit fluorescence, indicating an effective antiviral activity. Real-time polymerase chain reaction (RT-PCR) and Western blot data further indicated the lack of viral coat protein for the treated samples, in alignment with the negative control data without virus and in contrast to the virus-only positive control data.Using a leaf-based assay, Ryu et al. further investigated the antiviral properties of various quassinoids to inhibit pepper mottle virus (nd yield . It was HIV) along with herpes simplex, Semliki forest, and Coxsackie viruses [In addition to plant viruses, certain quassinoids have also been reported to inhibit certain human viruses, such as human immunodeficiency virus  have also been explored and have an imperfect crystalline structure that enables high drug loading . Hence, Lv et al. developed an NLC carrier based on a combination of solid- and liquid-phase lipids and other materials to encapsulate BJO . The mixNLC loading markedly slowed the BJO release, and slightly more than 50% of BJO was gradually released over a roughly four-day period, as opposed to a rapid release within 10 h for free BJO. In vitro cell cytotoxicity testing against human lung cancer cells further demonstrated that NLCs without BJO did not have an inhibitory activity, while free and NLC-encapsulated BJO exhibited a dose-dependent inhibition B. The NLExpanding on this concept, Li et al. developed pH-responsive lipid nanoparticles with liquid crystalline properties, which were loaded with BJO and doxorubicin (DOX), a widely used anticancer medication . The lipThe pH range around which the LCNPs exhibited morphological changes was relevant to the conditions found near and in tumor microenvironments, and the rate of in vitro release of DOX from the dual-loaded DOX-BJO LCNPs depended on the solution pH. An appreciably quicker release rate was observed at pH 5.3 vs. pH 7.4, and it was noted that the presence of a liquid crystalline structure tended to slow down the drug release D. In vitThere has also been an ongoing exploration of sponge-type nanocarriers that are called spongosomes, which are liquid crystalline lipid nanocarriers that exhibit a high encapsulation efficiency and controlled release, and that can protect active ingredients from degradation. Spongosomes typically lack a solid inner core structure, which allows them to function as spongy materials, and they are endowed with stabilizing molecules on the outer surface to support colloidal stability . Compare50 values of free BJO and spongosomes containing 2 mg/mL BJO were around 68 and 28 μg/mL, respectively, while the spongosome without BJO was nontoxic.Zou et al. reported the fabrication of BJO-loaded spongosomes and conducted a detailed structural characterization using cryogenic TEM (Cryo-TEM), SAXS, and dynamic light scattering (DLS) techniques . The spoIn another effort, Chen et al. reported the development of poly(ethylene glycol)-coated spongosomes that encapsulated both BJO and baicalin (BAI), which is a small-molecule flavonoid with anticancer properties . The colWhile there has long been interest in harnessing the medicinal properties of BJO and its molecular components in order to treat a wide range of medical diseases, recent progress in nanomedicine strategies has greatly advanced the field by realizing the potential to not only improve the solubility of BJO and its molecular components but also to achieve greater performance outcomes in terms of treatment efficacy and pharmacological properties. To date, a large number of proof-of-concept studies have been performed with many different types of nanoparticle-like delivery vehicles, and the results have set important precedents, especially with nanoemulsions. At the same time, there is an outstanding need to continue translational efforts by focusing on specific nanomedicine strategies that have the best potential for targeted applications and envisioned administration routes. For example, a suitable nanoparticle delivery vehicle for oral administration possibly has distinct properties from a suitable one for intravenous administration. Future efforts directed at creating nanomedicine delivery strategies that incorporate molecular components of BJO in a biomimetic manner would be useful, along with a deeper focus on characterizing and optimizing the physicochemical properties of specific nanoparticle-like delivery vehicles. Such work is needed in order to ultimately translate promising nanomedicines into viable clinical treatment options, and additional preclinical research directed at defining acute toxicity and other relevant pharmacological parameters through dose-escalation studies in animal models would also be beneficial as a key step towards eventual human clinical trials. Considering the clinical importance of BJO and emulsion-type BJOE systems already demonstrated in certain parts of the world, there is an enormous worldwide potential to continue pursuing nanomedicine delivery strategies to enhance the prospects of BJO and its molecular components as viable treatment options for a wide range of medical conditions, including various types of cancer and anti-inflammatory diseases.There is also a significant opportunity to further explore how nanomedicine formulations of quassinoids might be used treat additional types of diseases, since quassinoids have been shown potentially useful in treating neurodegenerative diseases such as spinal muscular atrophy , in stim


Deep endometriosis most commonly involves the rectosigmoid junction and its management often requires a colorectal resection. Anastomotic leakage is a severe complication after resection and affects 1-6% of the cases.To evaluate the risk factors related to anastomotic leakage following endometriosis sur-gery, its prevention techniques and the role of protective stomas.A comprehensive literature review was carried out for English-language publications in Pubmed and Google Scholar. We included all studies including the following MeSH terms and key words: Anastomotic leakage AND bowel surgery OR Endometriosis OR Colorectal surgery OR Bowel endometriosis. Two authors independently made a selection and analysed relevant abstracts according to the aim of this review.Risk factors and preventive measures were categorised considering the patient condition, the intra- operative setting and the surgical procedure itself. Level I and II recommendations include modifiable risk factors such as the use of stapled or handsewn anastomosis; intra-operative air leak test to check the integrity of the anastomosis; systematic use of pelvic and trans-anal drainage; application of protective or ghost ileostomy in low rectal resections; vaginal closure before the bowel resection; use of oral antibiotics the day before surgery and performing partial mesorectal resection near the bowel wall. Diverting stomas may decrease the morbidity and the clinical consequences of leakage over 65% of low rectal resections but may cause significant adverse effects.Evidence-based protective actions are crucial to reduce clinical consequences of anastomotic leakage and to minimise the use of protective stomas in endometriosis surgery. Bowel endometriosis is defined as the presence of endometrial-like glands and stroma infiltrating the bowel wall and affects 5% to 12% of patients with deep infiltrating endometriosis (DIE) . When suA comprehensive review of the literature was carried out for English publications in Pubmed and Google Scholar relating to bowel anastomotic leakage following endometriosis surgery. We included all studies found under the search of following MeSH and the keywords terms: Anastomotic leakage AND Bowel surgery OR Endometriosis OR Colorectal surgery OR Bowel endometriosis.Initially, a structured investigation question was created using the PICO strategy as shown in AL is defined as the leakage of luminal contents from the surgical join between two hollow viscera . The lumDiagnosis frequently encompasses clinical, biochemical and imaging exams. Leakage may present itself as pain, fever or feculent discharge from a drain. Clinical signs may include tachycardia, abdominal tenderness and signs of peritonitis . LaboratBruce et al.  proposedRadiological: No clinical signs.Clinical-Minor: Leakage of luminal contents through the drain/wound - local inflammation - fever (>38°C) - tachycardia - leukocytosis (over 10.000/ litre) - faecal purulent discharge from drain/wound (abscess).Clinical-Major: Same as minor plus severe disruption of the anastomosis.There are known risk factors for leakage inherent to the patient condition, intra-operative setting and surgical technique . A summaA thorough analysis of the principal risk factors will be presented later (See Prevention of Risk Factors).Bowel endometriosis, the most common extragenital endometriosis location, is defined as endometriotic infil-tration of the intestinal muscle layer and affects between 5% to 12% of patients with DIE . It is uSurgery is indicated in symptomatic cases or when an intestinal obstruction is present. The therapeutic options include nodulectomy  and segmental resection. Shaving is defined as the superficial serosal and subserosal bowel excision, not requiring suturing or closure. The mucosal skinning  includes excision of the rectal muscularis without opening the mucosa, requiring suturing. Finally, disc excision encompasses a full-thickness resection of the entire anterior rectal wall, while segmental resection includes removal of a whole bowel segment .The correct treatment choice is selected after an assessment of the disease variables such as nodule diameter, number, location, depth of infiltration, and presence/absence of luminal stenosis . SegmentDeep endometriosis is prevalent and commonly a multi-organ disease. With these characteristics, the lack of a universal standard operative method to report it becomes a significant disadvantage. Standardisation of a procedure helps to eliminate errors due to omission or admission, provides benchmarks to determine when corrective actions are required, facilitates training by providing regular steps that can be taught, practised and evaluated, creates a common language to describe a specific process that can be understood and commu-nicated between surgical teams and preserves the knowledge in time. Therefore, maximal effort must be put in to correctly classify DIE as a very first step in each surgical case.The most commonly used system, the American Society of Reproductive Medicine (revised) classification, has the drawback of not considering deep endometriosis and does not correlate the severity of clinical symptoms (pain) with the extent of the disease . RecentlAfter the first laparoscopic bowel resection for endometriosis was performed in 1988, several well designed prospective studies have demonstrated the advantages of laparoscopy versus laparotomy in the treatment of bowel DIE, including less blood loss and hospital stay, post-operative complications and higher pregnancy rate, without significant differences in the long term post-operative symptoms control . Thus, lAnastomotic leakage is a complication that can occur after any of these bowel procedures. Even when segmen-tal resections tend to show a higher absolute risk of leakage than nodulectomies in retrospective data, this is not necessarily true and must be carefully interpreted. So far, the only randomised controlled trial (RCT) available comparing functional outcomes after conservative  or radical  bowel DIE treatment did not find significant differences in “complications related to stoma repair”  between the groups (7.4% vs 3%) .Segmental resection implies complete resection of the affected bowel segment with subsequent primary end-to-end, end-to-side or side-to-side anastomosis, usually requiring extensive dissection of para-rectal spaces where important vascular and nerve structures are located. If they are damaged, severe morbidity including bowel ischemia, fistulas and anastomotic leakage can develop . IndicatAbo et al.  comparedDonnez and Roman  reviewedUp until now, 30 series with over 5500 cases of bowel resection have been published . All stuBertocchi et al.  publisheRuffo et al.  presenteSimilarly, in 357 bowel resections and end-to-end anastomoses , Minelli et al.  reportedRoman et al.  evaluateMalzoni et al.  analysedSimilarly, Keckstein and Wiesinger  analysedFinally, Dousset et al.  analysedThe systematic review of De Cicco et al.  includesSimilarly, Meuleman et al.  reviewedFor low and ultra-low colorectal anastomosis at high risk of developing fistula and leakage, the use of temporary protective ileostomy is usually recommended in order to prevent these complications. However, it is asso-ciated with stoma-related risks, such as hernia, retraction, stenosis, sinus formation, dehydration, prolapse and necrosis . RandomiConsidering that endometriosis is a benign disease in young and healthy people, adequate case selection and patient counselling is crucial . TreatmeThe stoma can significantly affect patients’ quality of life and sense of well-being, also burdening the health care system . ComplicWhether to perform a bowel stoma or not depends on the surgical team preferences, but must be guided by some specific factors:The site and height of anastomosis are crucial. It is well accepted that the serosal layer has a critical role in anastomotic healing. Since the lower rectum is lacking in this layer, a higher risk of leakage is expected at this level . FurtherThe level of the anastomosis can be classified into three types according to their distance from the anal verge (AV) :High/Medium: Equal or more than 8 cm.Low: Less than 8 cm but more than 5 cm.Ultra-low: 5 cm or lower.Even though initial studies did not show a significant relationship between the rate of fistulas and level of anastomosis, probably due to the bias of using protective ileostomy in low and ultra-low anastomosis , recent Total meso-rectal excision (TME) is the standard treatment for locally advanced rectal cancer, reducing the risk of recurrence and improving global prognosis , while bCurrently, an air leak test is one of the most frequent intra-operative tests for evaluation of mechanical anastomosis competence, as well as diagnosis and treatment of occult disruptions . A positStudies in colorectal cancer suggest that the nodule diameter may be a predicting factor for AL. With the in-crease in lesion size, intra-pelvic manipulation becomes restricted and rectal transection is more challenging, starting from 3 to 4 cm diameter . Thus, wAs previously noted, AL events can occur in any of these surgical modalities, but predominantly in the low/ultra-low segmental bowel resection (See anastomotic leak in bowel endometriosis treatment).Concerning RVF, concomitant vaginal resection appears to be a predisposing factor. According to Abo et al  RVF was Surgeon’s experience is still one of the significant factors in deciding whether to perform a stoma or not. Colo-rectal surgery is an advanced and difficult procedure which expose patients to a relatively high risk of severe complications. Thus, it should be performed in expert centres to reduce the instances of such complications . The French group of Bendifallah et al.  analysedUp to now many techniques have been proposed to prevent or reduce the rate of leakage, but many of them do not have a corresponding evidence-based background .After evaluating the literature, we were able to classify the general and specific techniques for the prevention of leakage. Following the Canadian Task Force Levels of Evidence, we present the classification of these techniques based on the level of evidence .Modification of adjustable risk factors is important. Phillips  gives siSince the intake of over 105g/week is associated with an increased risk of leakage, prompt discontinuation of alcohol intake is recommended in the pre- operative period .Malnutrition impairs anastomotic healing by affecting collagen synthesis and fibroblast proliferation. Protein deficit  and pre-operative weight loss are associated with low collagen levels and reduced bursting strength in the colonic anastomosis, leading to significantly higher rates of AL .Enteral supplementation significantly decreases the risk of surgical complications (including leakage) , especiaSmoking increases the risk of leakage up to 4 times. Several studies have independently cited this as a factor, including both the active and previous smoking history (more than 40 pack-year) . Since sTheoretically, NSAIDs increase the risk of leakage by generating a downregulation of prostaglandin expression and corresponding hydroxyproline levels, harming the normal healing process . Alt-hough initial studies were contradictory , a systeSeveral studies report that BMI ≥ 35 kg/m2 is independently associated with AL , signifiPre-operative workup of colorectal surgery usually includes mechanical and enteral bowel preparation. In the last decade, there have been several studies which show no benefits of mechanical procedures in the prevention of leakage. Furthermore, this procedure carries risks of electrolyte disturbances and clostridium difficile infection. The classic French GRECCAR III RCT compared bowel preparation versus no preparation in 178 rectal cancer surgeries, and demonstrated that mechanical methods reduce the overall rate of septic complications, but not anastomotic leakage . Hence, In contrast, the policy of using non-absorbable oral antibiotics on the day prior to surgery is still highly rec-ommended since there is plenty of evidence of their benefits in reducing surgical site infections and AL in colonic surgery . In a reAppropriate fluid administration should be part of intra-operative and post-operative care since there is evi-dence that both over-hydration and restriction are directly associated with a high risk of AL after colecto-my/gastrointestinal surgery . The NatPatients with prolonged diastolic blood pressure drops have a 3-fold higher risk of AL . SimilarHigher intra-operative blood loss is associated with increased risk of AL  by reducInadequate perfusion and partial O 2 pressure are responsible for impaired wound healing. Normal pre- operative levels of haemoglobin must be checked and corrected when needed. A retrospective analysis of over 1200 major abdominal surgeries found that haemoglobin levels under 8g/dl independently increase the rate of AL 1.91 times .Duration of surgery is positively correlated with postoperative morbidity in both major and minor proce-dures . The retIt has been suggested that poor perfusion of anastomotic site as demonstrated by indocyanine green (ICG) may increase the risk of AL. Apart from the use of fluorescence-guided surgery for the detection of superficial endometriosis, intra- operative ICG assessment of the bowel wall (after shaving procedure) or the anastomotic line (after bowel DIE resection) vascularisation is a potential tool that might be helpful in confirming com- plete macroscopic resection of the disease and reducing the rate of bowel perforations. Therefore, the two theoretical benefits are choosing the adequate transecting line and evaluating the rectal vascularisation after mechanical anastomosis . ICG ideGeneral surgical principles and technical points for bowel closure must be followed and maintained to avoid anastomotic leakage  Table VTable V.Excluding ileocolic and oesophago-gastric anastomosis evaluations, three significant meta- analyses have been published in recent years. Naumann et al.  evaluateThe use of bio-absorbable staple-line reinforcing material is appealing to some. Although studies have shown that these reinforcements are safe and there have been several RCTs on the subject, to date, there have been no compelling studies which have demonstrated a decrease in leakage rates when they are used .Reinforcement sutures are typically placed around the anastomosis, but intra-luminal reinforcement has also been carried out . To dateTo date, one RCT has failed to show a decrease in leakage rate with the use of fibrin glue . At the Decades ago, several materials, such as silk, linen, catgut, polyglactin 910, and nylon, were commonly used for colorectal anastomosis. Nowadays, it is evident that absorbable sutures are safe, leaving no channel for luminal microbial migration once absorbed. Most gastrointestinal anastomoses, including colorectal, are constructed with polydioxanone sutures.Absorbable sutures compared with non- absorbable or slowly absorbable sutures cause more tissue reaction and dissolve too rapidly, reducing the anastomotic strength .Multifilament, compared to monofilament sutures, cause more tissue damage and easier adherence of material within the interstices of the multifilament , creatinPolydioxanone thread possesses all characteristics considered important; monofilament, little tissue reaction, slowly absorbable with long preservation of strength and low bacterial adherence risk. Based on experimental studies, non-absorbable, or slowly absorbable monofilament sutures seem to be the first choice for colorectal anastomosis .Continuous suture provides a tighter seal than an interrupted one. The main fear is that if this suture breaks, the entire suture line could open. RCTs investigating interrupted and continuous sutures for colorectal anastomosis are lacking. Only one small, non-randomised, comparative clinical study found no significant differences . Two cliLembert described the construction of intestinal anastomoses in dogs using suture bites with 5-mm distance to the cut edge nearly two centuries ago . An RCT Lembert reported a distance of approximately 1 cm between sutures . NeitherIn routine clinical practice, two undefined schools of thought seem to exist. The first believes that sutures should be tightened to prevent dehiscence of the anastomosis, and the second considers that sutures should be applied more loosely, allowing maximal perfusion of the cut edges. The bowel is highly supplied with blood and may become oedematous and hardened when tight stitches are used. Only one rat study investigated this, with moderate tension giving the best histological and micro-angiographic results . WhetherThe main strength of the gastrointestinal tract is in the muscularis and submucosa. Thus, effective closure involving at least these two layers is needed. Additionally, avoiding entering into the mucosa could help to prevent leakage. Cohort studies report low rates of AL for both serosa-submucosal and full-thickness suture types . We can Both everting and inverting (as well as end-to-end) techniques have been performed, but both have drawbacks. An RCT showed a 5-fold increased incidence of AL in patients receiving an everting compared with those receiving an inverting suture. Hence, there seems to be an advantage of inverting over everting colorectal anastomosis .The classic technique is based on a double-layer inverting anastomotic method. One RCT  matched A pelvic drain may prevent haematomas or seromas that constitute a medium for bacterial infection which can involve the anastomosis, thereby causing dehiscence. Moreover, a pelvic drain may help control leaks if they do occur, leading to a less severe clinical course . HoweverThe evidence is contradictory. One RCT showed no reduction in AL when trans-anal stents were used in 194 patients subjected to anterior rectal resection . NeverthTheoretically, the interposition of omental graft on a vascular pedicle, covering the area of the anastomosis, offers two main benefits :Re-enforcement of the anastomotic line during the first post-operative days  acting as a biologically viable plug which can seal microscopic leaks.Increased angiogenesis and neo-vascularisation at the anastomotic site by providing vascular endo- thelial growth factor, promoting microvascular anastomosis between the omentum and the bowel wall  and 3.9% (0-10.3%), evidence is predominantly retrospective and AL definitions are heterogeneous among the different studies. Careful monitoring is essential after any of these procedures. Control of modifiable risk factors, together with strictly following surgical principles such as avoidance of anastomotic tension, tissue ischemia and overlapping remain paramount in general prevention.Results of this evidence-based analysis lead us to recommend the following peri-operative modifiable measures; the use of either stapler or handsewn (single layer closure) anastomosis construction; intra-operative use of air leak test to check the mechanical integrity of anastomotic line; systematic use of pelvic  and trans-anal drainage; application of further preventive interventions (protective or ghost ileostomy) when the nodule is located under 8 cm from the anal verge and in high-risk patients; closure of the vagina before performing bowel resection (when colpotomy is required); systematic use of non-absorbable oral antibiotics one day before surgery and performing partial mesorectal resection near the bowel wall .Temporary defunctioning stomas may decrease the morbidity and clinical consequences of the leakage in over 65% of low colorectal anastomosis, but are associated with significant side effects that must be balanced against the risk of leakage. The treatment, considering the benign nature of endometriosis, must always be tailored according to the patient’s disease, desires and expectations, with comprehensive case-by-case selection and patient counselling.Finally, readers must be aware that the majority of the studies on this topic come from colorectal surgeons’ experience. This is relevant since colorectal oncology patients usually have a different demographic to the young, healthy patients in the endometriosis setting. However, the large endometriosis series, including more than 5500 segmental resections, support the conclusions presented here.


Adolescence is a developmental period associated with rapid age-specific physiological, neural, and hormonal changes. Behaviorally, human adolescents are characterized by age-typical increases in novelty-seeking and risk-taking, including the frequent initiation of alcohol and drug use. Alcohol use typically begins during early adolescence, and older adolescents often report high levels of alcohol consumption, commonly referred to as high-intensity drinking. Early-onset and heavy drinking during adolescence are associated with an increased risk of developing alcohol use disorders later in life. Yet, long-term behavioral consequences of adolescent alcohol use that might contribute to excessive drinking in adulthood are still not well understood. Recent animal research, however, using different exposure regimens and routes of ethanol administration, has made substantial progress in identifying the consequences of adolescent ethanol exposure that last into adulthood. Alterations associated with adolescent ethanol exposure include increases in anxiety-like behavior, impulsivity, risk-taking, and ethanol intake, although the observed alterations differ as a function of exposure regimens and routes of ethanol administration. Rodent studies have also shown that adolescent ethanol exposure produces alterations in sensitivity to ethanol, with these alterations reminiscent of adolescent-typical ethanol responsiveness. The goal of this mini-review article is to summarize the current state of animal research, focusing on the long-term consequences related to adolescent ethanol exposure, with a special emphasis on the behavioral alterations and changes to ethanol sensitivity that can foster high levels of drinking in adulthood. Initiation of alcohol use is commonly reported during early (11–15 years of age) adolescence Faden, , and thiThe developing adolescent brain is thought to be particularly vulnerable to alcohol  paradigm , intraperitoneal injections (IP), and ethanol vapor inhalation (VI). Findings from studies evaluating the effects of adolescent ethanol exposure on later ethanol intake are presented in Different animal models of ethanol exposure have been used to determine whether adolescent experience with ethanol influences subsequent intake. Voluntary ethanol consumption in laboratory rodents is commonly assessed using a two-bottle choice (2BC) paradigm, in which animals are given free access to water and ethanol, with this paradigm also used for adolescent ethanol exposure. Intake levels in the 2BC paradigm generally do not produce blood ethanol concentrations (BECs) in the binge range (80 mg/dl and higher), but rather in the low-to-moderate range. The original 2BC paradigm has been modified to increase ethanol consumption, via a 2BC paradigm during mid-adolescence, and Pandey et al.  that used voluntary ethanol consumption, such as 2BC or similar models of adolescent exposure, demonstrated increased ethanol intake later in life. Very few studies reported BEC data during the voluntary consumption exposure phase, and those that did generally observed low BECs that ranged from 0 to 100 mg/dl with averages around 25–35 mg/dl , exposure duration, exposure mode (voluntary vs. forced), exposure levels, BECs achieved, and strain. Given their respective effects and contributions, all these factors should be considered in future studies.In general, adolescent laboratory rodents are less responsive than their adult counterparts to adverse effects of ethanol that may curb ethanol intake. These adverse effects of ethanol include social inhibition  have been assessed in laboratory rodents. For instance, adult male rats exposed to ethanol during adolescence  and challenged with a hypnotic ethanol dose regained their righting reflex more rapidly than did their non-exposed counterparts . Diaz-Granados and Graham  was also evident in adult male and female Wistar rats, as well as in alcohol-preferring (P) male rats  of Sprague–Dawley male rats resulted in precipitation of adolescent-typical responding to acute ethanol challenge with social facilitation  when these males were tested in adulthood , and this exposure regimen resulted in decreased anxiety, as indexed by increased open arm behavior evident in adult rats (Gass et al., via self-administration increased percent open arm time on the EPM when these males were tested in adulthood (Gilpin et al., However, Torcaso et al. , exposinvia decreases in social investigation and social preference (Varlinskaya et al., Anxiety-like alterations associated with adolescent ethanol exposure were reported for other tests of anxiety as well. Experiments using the light/dark box have shown that adolescent ethanol exposure increases time spent in the dark portion of the apparatus and decreases entries into the light side in male Sprague–Dawley rats (Pandey et al., Increased anxiety-like behavior following adolescent ethanol exposure may also contribute to increases in ethanol intake due to the anxiolytic properties of ethanol. Although links between anxiety and alcohol consumption have been commonly reported in humans (Vorspan et al., Although adolescent alcohol exposure is associated with behavioral alterations and changes in ethanol sensitivity, it is still not clear whether these alterations contribute to increases in ethanol intake. Considering that very few studies assessing changes in ethanol intake following adolescent ethanol exposure included both sexes, the question of whether responding to ethanol exposure during adolescence differs in males and females remains unanswered. Furthermore, among the studies that assessed changes in ethanol intake, only a limited number included both adolescent and adult ethanol exposure conditions see , produciTT and EV contributed equally.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Comparative genomics studies are growing in number partly because of their unique ability to provide insight into shared and divergent biology between species. Of particular interest is the use of phylogenetic methods to infer the evolutionary history of cis-regulatory sequence features, which contribute strongly to phenotypic divergence and are frequently gained and lost in eutherian genomes. Understanding the mechanisms by which cis-regulatory element turnover generate emergent phenotypes is crucial to our understanding of adaptive evolution. Ancestral reconstruction methods can place species-specific cis-regulatory features in their evolutionary context, thus increasing our understanding of the process of regulatory sequence turnover. However, applying these methods to gain and loss of cis-regulatory features historically required complex workflows, preventing widespread adoption by the broad scientific community.MapGL simplifies phylogenetic inference of the evolutionary history of short genomic sequence features by combining the necessary steps into a single piece of software with a simple set of inputs and outputs. We show that MapGL can reliably disambiguate the mechanisms underlying differential regulatory sequence content across a broad range of phylogenetic topologies and evolutionary distances. Thus, MapGL provides the necessary context to evaluate how genomic sequence gain and loss contribute to species-specific divergence.MapGL makes phylogenetic inference of species-specific sequence gain and loss easy for both expert and non-expert users, making it a powerful tool for gaining novel insights into genome evolution. Comparative genomics uses sequence-level differences between species to gain insights into how genomes function and evolve . AccordiHowever, ancestral reconstruction is a complex process and, while tools exist to reconstruct ancestral protein and DNA sequences –5 and anMapGL applies a simple phylogenetic inference approach based on Wagner parsimony  to inferIn cases where the root state cannot be resolved, we set the root state equal to the unambiguous descendant state supported by the subtree with the most leaves. In the case of a single terminal outgroup species, where the right subtree has only one leaf, this is equivalent to pruning the most-terminal outgroup at the branch directly above the root Fig. c-d. WhenMapGL takes as inputs a set of genomic query features in BED format, a set of liftover chains  correspohttps://pypi.org/project/newick/). MapGL is compatible with MacOS and most Unix-like operating systems, and is available for installation through PyPI and Bioconda, with source code available from the GitHub repository (https://github.com/adadiehl/mapGL).MapGL is free and open-sourced under the MIT License. It is based on bnMapper , which mD. melanogaster. Human data were compared to mouse, a distant target species, in the mammalian analysis .Consistent with the relatively small evolutionary distance between human and chimpanzee, we observed a substantially lower rate of turnover in CTCF binding sequence content in the primate analysis relative to the mammalian analysis Fig. SC. Howevens Fig. SF was verns Fig. SD despiteComparative genomics relies on the ability to infer the creative mechanisms and downstream effects of lineage-specific sequence divergence. However, even sequences with similar properties can have very different evolutionary histories. This ambiguity compromises our ability to determine how lineage-specific gain and loss of short sequence features relates to conserved and divergent functions. Placing species-specific sequence features in their proper evolutionary context enables a deeper understanding of functional divergence but requires application of phylogenetic methods that have not been previously integrated into a single piece of software. MapGL answers this need, opening these methods to a much broader user base.While query and target species will have generally been chosen prior to the decision to apply MapGL, the choice of appropriate outgroups should also be considered carefully, as it is critical in optimizing the performance of MapGL. Thus, we offer some general guidelines for outgroup selection. First, we recommend using a minimum of two outgroups, while three or more is better. Because MapGL utilizes a known, fixed phylogeny to describe species relationships, errors in the tree topology may lead to erroneous gain/loss inferences. Therefore, we advise choosing species for which a reliable phylogeny is available. In order to ensure sufficient data to resolve between competing gain/loss scenarios, the amount of evolutionary distance captured within the outgroup subtree  should equal or exceed the distance between the query/target clade. Likewise, to avoid biasing gain/loss predictions toward either the query or target branch, the overall distance between the nearest outgroup and the root of the query/target clade should be at least as long as the shorter of the query or target branch. Finally, the mechanisms involved in creating indel sequences of interest should be considered as they may dictate the evolutionary distances necessary to accurately resolve competing gain/loss scenarios. For example, sequence turnover that occurs gradually, such as accumulation of small indels over many generations, may require relatively distant outgroups for reliable inference. By contrast, processes that occur within a relatively short evolutionary timespan, such as individual transposon dispersal events, may be resolvable using more closely related outgroups. It is also important to note that MapGL depends on the accuracy of chain files used to determine sequence presence/absence in target and outgroup species. Intuitively, alignment errors can create erroneous gaps in alignment chains, leading to erroneous gain/loss predictions. Since these errors are more prevalent in alignments between low-coverage/low-quality genome assemblies, these metrics should be considered in selecting outgroup species.Similarly, inferences within difficult-to-align regions, including simple sequence repeats and repetitive elements, may be of lesser quality than those elsewhere. In some cases, difficult-to-align sequences may manifest themselves as regions with many apparent substitutions and indels. As discussed in the Implementation section, predictive accuracy within such regions may be improved through careful tuning of the –gap (−g) and –threshold (−t) options. We note several use cases where it may be beneficial to use non-default values for the -g and -t parameters, but this list is far from exhaustive: 1) comparisons between closely-related query and target species ; 2) labelling query sequences that fall within difficult-to-align regions with high rates of alignment error; 3) classifying sequences created by indel processes prone to creating multiple, closely-spaced indels; 4) maximizing the specificity of ortholog predictions at the expense of decreasing predictive accuracy for gain/loss events.MapGL was designed to resolve the evolutionary histories of indels between a pair of query and target species. Therefore, by design, its resolution in placing gain and loss events in evolutionary time is inherently limited. While the program does not prohibit intermediate species in the phylogeny, beyond providing additional data from which to predict the ancestral state, their inclusion will not improve the temporal resolution or quality of gain/loss predictions. However, we show that the current implementation of MapGL performs well in the specific tasks for which it was designed and should be sufficient for most anticipated use cases. We show that MapGL performs well over a wide range of evolutionary distances and tree topologies in both mammalian and invertebrate datasets, establishing MapGL as a simple and robust method to infer the evolutionary histories of short sequence features.Project name: MapGLhttps://github.com/adadiehl/mapGL. Project home page: Operating systems: MacOS, LinuxProgramming language: PythonOther Requirements: NoneLicense: MITRestrictions: NoneAdditional file 1: Table S1. Datasets. Figure S1. Phylogenetic trees and gain/loss statistics for CTCF binding sites in mammalian, primate, and invertebrate phylogenies. Figure S2. Representative gain and loss predictions from the primate and invertebrate analyses.


The metabolome of an organism depends on environmental factors and intracellular regulation and provides information about the physiological conditions. Metabolomics helps to understand disease progression in clinical settings or estimate metabolite overproduction for metabolic engineering. The most popular analytical metabolomics platform is mass spectrometry (MS). However, MS metabolome data analysis is complicated, since metabolites interact nonlinearly, and the data structures themselves are complex. Machine learning methods have become immensely popular for statistical analysis due to the inherent nonlinear data representation and the ability to process large and heterogeneous data rapidly. In this review, we address recent developments in using machine learning for processing MS spectra and show how machine learning generates new biological insights. In particular, supervised machine learning has great potential in metabolomics research because of the ability to supply quantitative predictions. We review here commonly used tools, such as random forest, support vector machines, artificial neural networks, and genetic algorithms. During processing steps, the supervised machine learning methods help peak picking, normalization, and missing data imputation. For knowledge-driven analysis, machine learning contributes to biomarker detection, classification and regression, biochemical pathway identification, and carbon flux determination. Of important relevance is the combination of different omics data to identify the contributions of the various regulatory levels. Our overview of the recent publications also highlights that data quality determines analysis quality, but also adds to the challenge of choosing the right model for the data. Machine learning methods applied to MS-based metabolomics ease data analysis and can support clinical decisions, guide metabolic engineering, and stimulate fundamental biological discoveries. Metabolomics is a rapidly emerging field aiming to identify and quantify cellular metabolites. Together with genomics, transcriptomics and proteomics, metabolomics provides valuable insights into the composition of organisms. Mass spectrometry (MS)-based metabolomics is frequently used because of a wide analyte coverage, high sensitivity, high selectivity and high throughput. Metabolomics raw data are inherently complex, and continuous improvements of analysis pipelines are necessary for optimal information retrieval. The complexity stems from the systemic linear and nonlinear interactions among metabolites and the structure of spectrographic data. The challenges associated with the structure of MS data include features  typically outnumbering the samples, high noise levels, batch effects during measurements, and missing values. Hence, the metabolomics community has always been eager to adopt new mathematical and computational tools to improve data analysis. Here, we will focus on the potential of machine learning (ML) to support metabolomics data analysis and show the ability of ML to resolve nonlinear relationships and process large heterogeneous datasets. Moreover, we will focus on supervised ML approaches that provide quantitative predictions and are suitable for hypothesis-driven research .In ML, statistical models are trained on data to make reasonable predictions on unknown data. The ML tools use different algorithms and Nonlinear data analysis was applied to metabolomics since its origins. Among the first ML methods applied were ANNs: in 1990, Curry and Rumelhart published ‘MSnet’ to distinguish metabolite composition classes  and ANNsML has made rapid progress and now supplies a diverse spectrum of algorithms. Most of the ML tools developed have user-friendly interfaces and enable chemometricians to test various ML solutions and improve the applications for spectral analysis. For example, the Scikit-learn environment in Python provides functions for implementation, along with excellent documentation . The PytIn this review, we give a summary of the recent developments of supervised machine learning for metabolomics analysis. Specifically, we focus on random forest (RF), support vector machine (SVM), artificial neural network (ANN), and genetic algorithm (GA). Data processing, including baseline correction, noise filtering, peak detection, and alignment, is used for precise metabolite annotation and quantification . There aPrior to statistical analyses, all data should be normalized to exclude sample-to-sample variations, especially when integrating results from different batches or different analytical instruments. In the following, we discuss the different data processing steps, including (i) peak picking and integration, (ii) metabolite annotation, (iii) normalization , and (iv) missing data imputation. Data scaling or transformation are frequently used to adjust biases among various metabolites and to reduce heteroscedasticity in high-dimensional metabolomics data . AfterwaSeveral ML-based approaches were developed to process and select chromatogram peaks . Peaks hWe give a brief update adding to recent illustrative reviews on ML-assisted metabolite annotation, including fragmentation prediction ,50. A prFor high-quantity samples, metabolite-specific degradation dynamics and instrument sensitivity declines lead to nonlinear signal variations. Quality control/quality assurance (QC/QA) samples measured throughout the analysis are used to exclude inter- and intra-batch variations while preserving biological information. ML-assisted normalization methods mainly employ SVM- and RF-based regression. Normalization based on SVM has shown mixed performance. SVM outperformed linear and polynomial regression for PCA; however, OPLS-DA showed clear signs of overfitting . The limIf quality control samples are absent, they can even be simulated from the data. The procedure is performed by ‘pseudoQC’ with the goal to reduce data variation . SVM, RFMS-based analytical methods have a significant advantage in metabolite coverage, but a significant proportion of data are missing values. Notably, LC–MS missing data could be even in the range of 30–50% ,72. DiffThe imputation of missing values is necessary because most statistical data analysis approaches cannot process null information and a reasonable imputation strategy introduces less bias compared to feature removal. Various strategies exist to replace missing values with realistic estimates; however, the optimal strategy depends on the missing value type and also on the subsequent statistical analysis. Thus, the ability to discriminate between the missing value origins is advantageous, although often not known a priori. Shah et al.  used a BIn this section, we will discuss various topics for data analysis such as biomarker detection, classification and regression, pathway inference, the combination with mechanistic models, and multi-omics integration. The results will testify to the impressive predictive capacities of ML approaches, but will also reiterate that there is no predefined route to data analysis. Our examples provide broad coverage of the field; for more clinically oriented ML-assisted metabolomics analysis; see the review by Lee and Hu . The ML Given the multitude of ML approaches, we are provoked to ask: ‘are there guidelines to select appropriate ML approaches?’ The following sections will reveal the complexity of the question, and it is instructive to clarify the relations among the different ML approaches to judge their performance and requirements. The PLS approach is fundamentally an ANN with one hidden node and linear activation functions . The nonWe start by introducing the concepts of this section with an illustrative example of the microbes and metabolites in the digestive system. Two studies on the relation of gut microbes and ambient metabolome reveal how microbes predict metabolite concentrations, and how the latent variables of an ANN provide interpretable biological information. The data sources combined are metabolite feature concentrations and microorganism abundance. Le et al.  trained While metabolomics data are dense, the information-rich features are only a small subset of all detected features; moreover, the features frequently outnumber the sample size greatly. With too many features, training of the ML algorithm takes longer, the risk of overfitting increases, and model interpretability is compromised. Feature selection or feature extraction are dimensionality reduction strategies to alleviate the dense data problem . FeatureThe sample size is an important parameter that determines how well statistical interactions can be resolved, and detailed guidelines are available for spectrographic experiments . TypicalBinary classification problems are often simple enough that conventional statistical approaches outperform machine learning. Mendez et al. (2019)  tested eEach ML tool applies a distinct strategy for statistical analysis and yields best performance when fit to appropriate data structures. These data structures include frequency distributions or data types like canonical or linear data, connected or independent data, which are often not known in advance. Because each dataset is unique, and any data property can affect the performance of the different statistical approaches, it is advisable to test multiple ML tools on the data. Notably, linear multivariate analysis approaches like PLS need to be included as many reports showed their competitiveness. The crucial consequence is that any model is just as good as the data, and careful experimental design remains the strongest indicator for a good model .ML is excellently positioned to analyze metabolomics data and has provided impressive predictive competencies, but the knowledge gain, in general, is limited. The biological and chemical disciplines preferably use mechanistic models to enable the testing of hypotheses and extrapolation to experimentally inaccessible regimes. The most popular mechanistic models for metabolomics data analysis are kinetic models and stoichiometric constraint-based models. The integration of ML with constraint-based models was recently discussed ,105. KinThe ability of ML to predict pathway properties based on targeted metabolite information has contributed to improving strains in metabolic engineering. Costello and Martin  simulateDuring pathway enrichment, metabolomics data are interpreted in the biological context to identify active pathways. Pathway reconstruction is typically performed with genomic information of cataloged enzyme activities and represents the general metabolic capabilities of an organism. With metabolomics, pathway activities represent conditions after post-translational effects, like enzyme modifications or allosteric regulation, thus providing much more representative information compared to genomics or proteomics approaches. Current statistical approaches include MetScape or Mummichog . A compaToubiana et al.  used an 13C isotopically labeled substrates within the metabolites, in combination with cellular physiology, allows for computing intracellular metabolic rates and global flux distributions [Metabolic flux analysis (MFA) based on targeted metabolomics of labeling experiments allows an understanding of metabolic network properties. In MFA, the accumulation of ibutions . Machineibutions  is basedibutions  is an alStudies are no longer limited to a single omics level with the advent of increasingly faster and cheaper high-throughput technologies. The integration of multiple omics levels will enhance our understanding of the interactions among the different biological layers. The review by Noor et al.  gives anWe defined five categories, namely, ‘data,’ ‘model,’ ‘integration method,’ ‘dimensionality reduction,’ and ‘model organism.’ The ML approaches used for analysis are listed under the category ‘models’ . Since mMulti-omics integration increases the number of features with the addition of each omics level, stressing the importance of dimensionality reduction. Cellular features are highly correlated, and models assuming feature independence might perform poorly. Acharjee et al.  used RF Multi-omics analysis is more potent if mechanistic knowledge is used to connect the biological layers, a procedure well suited for Bayesian models. The Bayesian model ‘iSchrunk’ samples metabolite concentrations based on kinetic parameters and served to generate surrogate samples for training an RF-like classifier to estimate control coefficients ,123. A BMany tools integrating multi-omics datasets have been published and implemented in other research areas with the potential to be used with metabolomics data. ‘AutoOmics’ finds ANN for each omics layer and converts the input into the latent variables. The final layers from each omics technology are concatenated and used to train a final ANN model. ‘MixOmics’ is an R package with tools for univariate, multivariate, and multi-omics analysis. Other tools use matrix decomposition ,125, graWith an unprecedented accumulation of information, the relevance of machine learning intensifies and new algorithms and tools mushroom. According to the No Free Lunch Theorem, no general best-performing optimization algorithm can exist and thus there will always be competing algorithms streamlined to sets of special problems . While n


In eukaryotes, proteasomes are abundant proteolytic complexes that direct the degradation of numerous short-lived proteins whose function is required within a limited time frame, or proteins whose structure is damaged. If these unwanted proteins are not cleared by proteasomes, cells become susceptible to challenging environmental conditions such as nutrient starvation and physical or chemical stress. Failure to overcome these challenges can accelerate aging and cause diseases such as cancer and neurodegeneration, thus providing strong medical relevance to proteasome research.Kors et al..To degrade proteins at the right time and in the right place, proteasomes must be highly dynamic, not only with regard to their intracellular localization and subunit composition, but also in their specificity for particular substrates, which permits them to serve diverse protein degradation pathways. All proteasomes contain the proteolytic core complex, named the 20S core protease (CP), which can interact with different individual regulatory proteins or multi-subunit regulatory complexes, the most common of which is known as the 19S regulatory particle (RP) that caps one or both ends of the CP. This 26S CP-RP holo-complex is composed of at least 33 different subunits, along with numerous accessory proteins that transiently interact with the particle to help identify appropriate substrates and ensure that proteasomal degradation is tightly regulated, as reviewed by Müller and Weber-Ban.Proteasome substrates are often targeted for degradation by the covalent attachment of poly-ubiquitin chains, which are recognized by intrinsic and extrinsic ubiquitin receptors associated with the proteasomal regulatory complex. In bacteria, a ubiquitylation-like event termed pupylation exists as an archaic degradation signal that allows specific substrates to access proteasome-like proteases, indicating that the delivery of substrates into proteasomes is conserved in prokaryotes and eukaryotes. Deubiquitylation or depupylation peptidase activities associated with regulatory subunits of the proteasome typically release this signal prior to substrate degradation so it can be reused. Disordered or unstructured protein substrates can additionally be degraded without being first ubiquitylated or pupylated, confirming that proteasomes possess a dynamic interactome with a diversity of substrates, as outlined by Marshall and Vierstra. Precise regulation of proteasomal gene expression is also crucial for both the assembly and dynamic rearrangement of proteasome complexes. The transcription factors Rpn4 (in yeast) and NRF1  augment proteasomal gene expression in response to proteasomal dysfunction and nutrient-induced mTORC  activation, while mammalian NRF2 performs this role in response to oxidative stress. The regulation of proteasomal gene expression on a post-transcriptional level through MAP (mitogen-activated protein) kinases facilitates subunit incorporation during proteasome assembly, as reviewed by Motosugi and Murata. A plethora of post-translational modifications of the 26S complex then also modify proteasome activities, influence its intracellular localization, and ultimately help trigger its turnover .Since the proteolytic active sites that cleave potentially toxic substrates into harmless peptides are secluded inside the CP cavity, proteasomes are essentially a self-compartmentalized garbage disposal. This self-compartmentalization begins during proteasome maturation, as subcomplexes are assembled from inactive precursors bearing inhibitory propeptides that must be auto-catalytically cleaved within the nascent proteolytic cavity to fully complete CP assembly, as reviewed by Schipper-Krom et al.. For example, using activity-based probes mimicking proteasomal peptides, a prominent nuclear localization of the CP was apparent within the mammalian cancer cell line MelJuSo, as shown by Gan et al.. To co-localize activity-based probes with GFP-labeled proteasomal subunits, the immuno-proteasome CP subunit β5i is an ideal recipient for the GFP fusion, as its incorporation into the CP is highly efficient upon release of cytokines. Almost 90% of the GFP-labeled immuno-CP is nuclear in U2OS and Hela cells. After photobleaching of the entire cytoplasm, proteasomes remain in the nucleoplasm, indicating the presence of large holo-proteasome complexes .Due to the intricate assembly mechanism of proteasomes, the labeling of a proteasomal subunit with fluorescent reporters such as green fluorescent protein (GFP) can be problematic, and thus should be used as a reliable reporter of the active holo-enzyme only if the GFP fusion protein is fully incorporated into the mature proteasome complex. Since proteasome subunits also exist as part of inactive precursor complexes, activity-based probes have been developed to specifically target the active sites of the CP. These probes penetrate the plasma membrane of mammalian cells and illuminate active proteasomes, thus permitting their localization. In combination with either indirect fluorescence microscopy using proteasome-specific antibodies or direct fluorescence microcopy using GFP-labeling techniques, the mature CP can now be localized with high fidelity, as shown by Wendler and Enenkel.In proliferating yeast cells, GFP labeled proteasomes are also mainly nuclear, an observation independent of which CP or RP subunit is fused with the GFP reporter protein. These observations provide evidence that proteasome localization is highly conserved in eukaryotes. Studies in yeast have also provided insights into the intracellular movement of proteasomes, which are imported into the nucleus as either immature precursor complexes or mature holo-enzymes, depending on their availabilities under different growth conditions. The nuclear import of proteasome complexes uses the classical import receptor importin/karyopherin αβ, together with transiently associated proteins to either assist in proteasome assembly or confer classical nuclear localization signals to mature proteasomes. Proteasome-associated regulatory proteins such as yeast Blm10  also facilitate nuclear localization of the CP, as summarized by Karmon and Ben-Aroya. Functional proteasomes within proteasome storage granules are highly mobile and remain ready for reimport back into the nucleus upon exit from quiescence and the resumption of growth (Wendler and Enenkel).Upon nutrient starvation, proteasomes surprisingly exit the nucleus into the cytoplasm. When yeast cells are deprived of glucose, decreasing energy sources [in the form of reduced adenosine triphosphate (ATP) levels] and acidosis cause them to transition into quiescence and sequester proteasomes into motile and reversible cytoplasmic condensates known as proteasome storage granules (PSGs). These membraneless PSGs are considered to serve as sorting compartments in which functional proteasomes are saved and separated from non-functional proteasomes as summarized by Karmon and Ben-Aroya). A sophisticated sorting mechanism seems to distinguish active and functional proteasome complexes from their inactive and non-functional counterparts. Comprehensive studies in yeast, plants, and mammalian cells have revealed that inactive and non-functional proteasomes are first ubiquitylated and sequestered by proteasome-specific receptors for autophagic elimination, while active and functional proteasomes are protected from vacuolar or lysosomal degradation in PSGs . New therapeutic schemes can thus be envisioned to enhance proteasome rehabilitation.Environmental challenges leading to proteasome inhibition also induce the spatial reorganization of proteasomes into nucleoplasmic or cytoplasmic aggregates (The life cycle of proteasome homeostasis, starting with regulated proteasomal gene expression, proteasome assembly into interchangeable proteasome complexes with different configurations, altered proteasome localization in response to changing growth conditions and environmental challenges, and finally autophagic turnover of excess or inactive proteasomes offer interesting starting points for therapeutic interventions. The significance of future work on proteasome dynamics can be envisioned for age-related proteasome movements in mammalian cells in which old proteasomes are cytoplasmic, while newly produced proteasomes are primarily nuclear. Differences in the localization and proteolytic capacity of proteasomes may have significant impact on protein homeostasis in health and disease.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The chloroquine family of antimalarials has a long history of use, spanning many decades. Despite this extensive clinical experience, novel applications, including use in autoimmune disorders, infectious disease, and cancer, have only recently been identified. While short term use of chloroquine or hydroxychloroquine is safe at traditional therapeutic doses in patients without predisposing conditions, administration of higher doses and for longer durations are associated with toxicity, including retinotoxicity. Additional liabilities of these medications include pharmacokinetic profiles that require extended dosing to achieve therapeutic tissue concentrations. To improve chloroquine therapy, researchers have turned toward nanomedicine reformulation of chloroquine and hydroxychloroquine to increase exposure of target tissues relative to off-target tissues, thereby improving the therapeutic index. This review highlights these reformulation efforts to date, identifying issues in experimental designs leading to ambiguity regarding the nanoformulation improvements and lack of thorough pharmacokinetics and safety evaluation. Gaps in our current understanding of these formulations, as well as recommendations for future formulation efforts, are presented. Chloroquine (CQ) and hydroxychloroquine (HCQ) have been used for decades in the prevention and treatment of malaria and in the treatment of some autoimmune diseases such as lupus erythematosus and rheumatoid arthritis due to their immunomodulatory properties ,2,3. DesCQ and HCQ have also recently received worldwide attention due to their potential use in treating coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Previous studies showed in vitro efficacy of these drugs against Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory coronavirus (SARS-CoV), and a recent study demonstrated CQ could effectively inhibit viral infection of SARS-CoV-2 in vitro ,11,12. ACQ and HCQ are both basic amphiphiles that concentrate in the lysosome and inhibit lysosomal function as their primary mechanism of action . While CNanoparticle drug delivery is one promising strategy to overcome drug liabilities such as poor PK and toxicity while improving site-specific drug delivery. Nanomedicines can provide a variety of benefits, such as improving the solubility of hydrophobic drugs, protecting drugs from degradation, and altering tissue distribution through passive or active targeting mechanisms . Indeed,P. berghei-infected animals  or targeting moieties , can result in decreased uptake by the mononuclear phagocytic system (MPS) and site-specific delivery, respectively ,24. Lipo animals  [25,26,2C. neoformans murine model, liposomal CQ in combination with fluconazole provided better antifungal prophylaxis and treatment compared to free drug controls due to enhanced liposomal drug uptake by macrophages [P. falciparum-infected mice [It is worth noting that these studies utilized non-PEGylated liposomes, which are known to be rapidly cleared by resident macrophages in MPS organs such as the liver and spleen . Althougrophages ,34. Mostted mice . Overall® , which reduced the drug’s dose-limiting cardiotoxicity and increased tumor exposure due to the enhanced permeability and retention (EPR) effect [Liposomes initially found clinical success as drug carriers in cancer treatment with the development of Doxil) effect . The EPR) effect ,38. In p) effect ,40. HoweSeveral HCQ-loaded liposomes have also been developed for cancer indications. For example, Wang et al. combined HCQ-loaded liposomes with TAT-Beclin 1 peptide to induce autophagy catastrophe in a 4T1 breast cancer model . The comTo further improve the delivery of HCQ to tumors, liposomes have been modified with various targeting ligands to enable tumor-specific drug delivery. For example, liposomes decorated with pH-sensitive RGD peptides for targeting ITGAV-ITGB3/integrin αvβ3 receptors were used for HCQ delivery to melanoma tumors . Both unIn addition to integrin αvβ3 receptors, Yin et al. also targeted neuropilin-1 receptors on melanoma cells for co-delivery of HCQ and PTX . This taUltrasound (US) is another method that has been investigated as a means to improve nanoparticle delivery across the BBB and is also involved in sonodynamic therapy ,71. ThisDrugs that are not suitable for remote loading into the liposomal aqueous core or are not sufficiently lipophilic to associate with the lipid bilayer can be conjugated to a lipid anchor to facilitate loading within the lipid bilayer . AlthougPolymer-based nanoparticles have been used to improve the solubility of hydrophobic drugs and facilitate enhanced tumor distribution through the EPR effect. Polymeric micelles, one of several different types of polymer-based nanoparticles, generally consist of amphipathic polymers that co-precipitate with drugs to form a hydrophobic core surrounded by a hydrophilic shell. These formulations have been shown to have low critical micelle concentrations (CMC) and have better stability than traditional surfactant micellar systems due to hydrophobic interactions between the drug and polymer . CQ is aIn addition to micelles, polymeric nanoparticles can be formed through emulsion techniques. This approach can be used to encapsulate hydrophilic drugs and biologics within the polymer matrix and do not require amphipathic polymers. For example, Yang et al. developed a nanoparticle composed of poly(lactic-co-glycolic acid) (PLGA) for co-delivery of CQ and pDNA expressing the mSurvivin-T34A protein . In thisHCQ has also been formulated with biologics to aid in lysosome escape. For example, Liu et al. developed a PLGA nanoparticle co-loaded with HCQ and ovalbumin (OVA) as a model antigen for a proof-of-concept vaccine delivery formulation . This foSimilar to liposomes, polymeric nanoparticles can be coated with antibodies to enable tumor-specific drug delivery, but few have been developed for CQ or HCQ. In one example, a cd20-antibody-targeted poly(caprolactone)/PLA nanoparticle was co-loaded with HCQ and chlorambucil and evaluated for efficacy in a Burkitt lymphoma animal model . The tarAlthough most polymers used in drug delivery are biodegradable, some non-biodegradable polymers such as acrylamide-based polymers have shown success for small molecule and oligonucleotide delivery ,77. One Overall, due to a lack of appropriate controls, there is limited data to support the utilization of polymeric nanoparticles for improving the delivery of either CQ or HCQ.P. yoelii-infected mice compared to CQ control (80%), indicating the formulations significantly reduced the antimalarial efficacy of CQ. One possible explanation for this decrease in efficacy could be due to a reduction in systemic drug exposure. Previously, dendrimers composed of PEG and poly(lysine) with and without galactose terminal groups significantly reduced the maximum concentration (Cmax) and area under the concentration-time curve (AUC) of CQ in comparison to unformulated CQ [Dendrimers are repetitively branched molecules generally constructed as macromolecular polymers with variable cores and terminal groups to facilitate drug encapsulation and drug delivery . Propertlated CQ . A simillated CQ .Alternatively, Panagiotaki et al. designed dendrimers composed of poly(ethylenimine) with triphenylphosphate terminal groups to facilitate mitochondrial delivery of DXR and CQ for improved cancer therapy . DendrimPolyelectrolyte complexes, also sometimes referred to as polyplexes and coacervates, are formed by mixing oppositely charged polyionic species in an aqueous medium, and various ionic polymers have been investigated extensively for their ability to complex with nucleic acids . HoweverP. yoelii-infected mice treated with the polymer/CQ complexes achieved 100% survival 30-days post-infection compared to 0% survival in the unformulated CQ control group. Although the polymers alone were shown to reduce parasitemia in vitro, polymer-only controls were not included in the in vivo efficacy study. Therefore, it is unclear whether the improved survival is due to an improvement in CQ delivery or rather due to additive or synergistic effects of the drug and polymers. Furthermore, the formulations provided no statistically significant improvement in survival compared to CQ alone in P. yoelii-infected mice when administered orally [CQ is positively charged at physiological pH due to its two ionizable amine groups, and because of this, researchers have attempted to load the drug into complexes containing ionic polymers. In one example, Urban et al. developed poly(amidoamine) polymers that formed ~10 nm complexes when mixed with CQ . Drug red orally .P. berghei-infected mice [Another CQ-polyelectrolyte complex, composed of chitosan and tripolyphosphate, was shown to reduce parasitemia to a greater extent than unformulated CQ in several efficacy studies in ted mice ,61,62,63In addition to liposomes, there are a variety of other lipid-based nanoparticles including solid lipid nanoparticles (SLN), nanoemulsions, and niosomes. These formulations are generally used for improving the solubility and delivery of hydrophobic drugs and are highly biocompatible and biodegradable due to their physiological lipid compositions.Unlike other lipid-based carriers, SLN contain a solid lipid core and are often utilized as oral formulations to improve solubility and intestinal absorption of hydrophobic drugs ,81. CQ iP. berghei-infected mice 5 days post-infection. However, the blank lipid emulsion reduced parasitemia by 35.35%, indicating the lipid emulsion alone inhibited malarial infection. Therefore, it is unclear if the efficacy from the CQ nanoemulsion is due to an improvement in drug delivery or simply additive or synergistic effects with the lipid emulsion and drug.In addition to loading drugs, lipid-based carriers have been shown to inhibit malarial parasitemia in erythrocytes . Due to Niosomes are another class of drug delivery vehicle capable of loading both hydrophobic and hydrophilic drugs. Niosomes are similar to liposomes in that they also contain a bilayer and an aqueous core. Unlike liposomes, which typically utilize phospholipids, niosomes are formed from mixtures of non-ionic surfactant molecules and cholesterol. Niosomes have been used for transdermal drug delivery due to their ability to improve drug penetration through the skin and provide local and sustained drug release . This stMetallic nanoparticles have been successfully implemented as contrast agents and many are being investigated as therapeutic agents and drug delivery vehicles ,87. One HCQ has also been used to enhance sonodynamic therapy of metallic nanoparticles through autophagy disruption. For example, Feng et al. designed HCQ-loaded hollow mesoporous titanium dioxide nanoparticles that are coated with a cancer cell membrane to allow homologous targeting to the tumor . HCQ relThere is new interest in repurposing CQ and HCQ for novel applications such as cancers, as well as improving therapy for their traditional indications such as infectious and inflammatory diseases. Nanomedicines have been evaluated for their ability to improve the safety and efficacy of chloroquines. There are a variety of nanoparticle types, with each having their own advantages and disadvantages, and it is important to understand the liabilities and physicochemical properties of the drug being formulated in order to select the most appropriate platform. In the case of CQ and HCQ, off-target toxicities can be reduced, and efficacy enhanced using a combination of site-specific drug delivery and controlled release; the balance between delivery and release kinetics being a crucial factor in improving therapeutic index . In orde® PM, a polymeric nanoparticle formulation of PTX that is approved as a cancer therapy in South Korea, has been shown to completely release its drug within 10 min after exposure to plasma [Polymeric nanoparticles, which have been successful in formulating hydrophobic drugs in preclinical and clinical studies, are typically unstable formulations that release their drug immediately after injection, thereby eliminating any potential benefits of nanoparticle distribution and essentially acting as solubilizing formulations. For example, Genexolo plasma . Dendrimo plasma ,92. MetaOn the other hand, liposomal formulations appeared to provide a clear benefit to the delivery of CQ and HCQ in various malaria and tumor models, respectively. With its ionizable amine groups, CQ can be actively loaded into the aqueous liposomal core, and erythrocyte-specific targeting ligands on the surface of the liposomes improve drug uptake within red blood cells, a target for malaria. Since lipids have been shown to inhibit Plasmodium infection, combining CQ with lipid-based carriers may provide not only better drug delivery to uninfected and infected red blood cells, but also synergistic efficacy. Liposomes also make a good choice for improving the delivery of these drugs to tumors. With their ~100 nm size and good stability, liposomes are able to accumulate within the tumor microenvironment via the EPR effect and deliver their therapeutic cargo . With thOne notable absence in the above nanotechnology formulation discussion of chloroquines is polymer prodrug systems, a major drug delivery class that has scarcely been evaluated for these drugs and may offer an ideal balance of targeting and stability. Polymer prodrugs can be designed to be biodegradable, provide site-specific targeting, and enable controlled drug release through the polymer-drug linker chemistry . This stIt should be emphasized that despite the promising preclinical data for some of the formulations presented in this review, none of the formulations have made it to the clinical stage. The lack of clinical development is likely due to poor intellectual property protections and uncertain commercial promise for the formulation platforms presented, many of which rely on generic formulation strategies. However, it is expected that the recent commercial success of novel nanotechnology-based delivery platforms and renewed interest in chloroquine drugs for novel indications, such as cancer, will fuel future clinical development of chloroquine nanoformulations ,99. Over


The Washington Post, et al., et al., et al., In the absence of any vaccine or effective pharmaceutical treatment options for COVID-19, the likelihood of long-term necessity of social restrictions and confinement practices like isolation and quarantine among older adults—the most visible victims of COVID-19—remains high —the most common and major health deterrent combustion product of solid fuels, can easily translocate to the central nervous system through the olfactory-neural pathway and the cardio-respiratory system to induce neuroinflammation, oxidative stress, microglial activation, cerebrovascular dysfunction, and alterations in the blood–brain barrier  for a 1-μg/m3 increase in PM2.5 concentration (Oudin et al., Experimental evidence suggests that air pollutants, particularly fine particulate matter (PMTherefore, given the plausibility of exacerbation of adverse mental health outcomes due to HAP exposure among older adults, primary prevention should be enabled at the household level itself. The need for maintaining maximum ventilation at homes, particularly whilst undertaking activities involving solid fuel use, should be mass communicated along with improving awareness around HAP and associated mental health risks among older adults and their caregivers. Further, ensuring affordability and robust door-step-delivery mechanisms for clean fuels and systems for older adults would be critical, considering their limited mobility and financial capacities that have been worsened by the pandemic. This can be challenging, especially in developing countries that are highly populated and where social welfare is suboptimal. Strengthening of psychosocial care by optimizing existing mental health services for older adults, alongside preventing their HAP exposures by considering the drivers and barriers of sustained clean fuel use should be prioritized. This would require the engagement of multidisciplinary stakeholders, as it may help prioritize context-specific strategies to meet the mental health and clean fuel needs of the geriatric populace. Lastly, from a research perspective, building psychogeriatric evidence in relation to COVID-19 should incorporate modeling for environmental risk factors, including household air quality and trends in fuel usage. Such research efforts should be contextualized by accounting for personal environmental exposures, socio-economic and ethnocultural differences, and the implications of various psychosocial support interventions extended for older adults during the global crisis.


Gas hydrate technology holds great potential in energy and environmental fields, and achieving efficient gas hydrate formation is critical for its industrial application. Graphene is a novel carbon-based nanostructured material with excellent thermal conductivity and a large specific surface area. Therefore, the use of graphene-based materials for the promotion of gas hydrate formation might be feasible and has aroused a lot of interests. Accordingly, to evaluate the current research on graphene-based promotion of gas hydrate formation, this work presents a review of existing studies involving graphene-based promoters of gas hydrate formation. Here, the studies applying various types of graphene-based promoters for gas hydrate formation are listed and detailed, the peculiar properties of graphene-based promoters are discussed, and the promotion mechanisms are analyzed. Through this review, comprehensive insight into graphene-based promotion of gas hydrate formation can be obtained, which can guide the design and applications of novel graphene-based promoters and might contribute to achieving efficient gas hydrate formation. Gas hydrates have captured an increasing amount of attention during the past decades because of their great potential for energy storage and environmental conservation  reduced the hydrate formation period by 45–80% and improved the hydrate formation rate and the storage capacity by 190–660% and 45–70%, respectively, compared with pure water. The results implied that the graphene nanosheets not only increased heterogeneous nucleation in the system and provided abundant active sites for hydrate nucleation but also produced a high transfer efficiency that could remove the heat generated by hydrate formation from the system, consequently improving the efficiency of methane hydrate formation. Due to similar promotion mechanisms, graphite nanoparticles also have positive effects on gas hydrate formation. Zhou et al.  compared in a pure water system. Adding SDBS into GN nanofluid could inhibit GN aggregation and greatly reduce the surface tension of the solution, making gas molecules dissolve in water more easily, which favored CO2 hydrate formation.In addition to the direct promotion of gas hydrate formation, graphene can also be mixed with surfactants to generate surfactant-stabilized nanofluids. Graphene usually exhibits poor dispersity and stability during gas hydrate formation, causing weak performance and cyclability during promotion. Therefore, a mixture of graphene and surfactants has been employed, where the surfactants function as both stabilizer and co-promoter. Hosseini et al.  used SDS−1 of SGO, and the storage capacity reached 140–150 v/v  onto surfaces of graphene/GO, aiming to obtain novel graphene-based promoters with desirable properties to significantly promote gas hydrate formation. Moreover, because of its nanostructure and remarkable electrical conductivity, graphene might serve as a nano-sized electric rotor under an electric field that could effectively stir within nano-sized confinement spaces, which might possibly be applied for HFP promotion via improving mass transfer.The existing studies on graphene-based promotion of gas hydrate formation were implemented in lab-scale experiments, so the promotion effects, stability, and cyclability of graphene-based promoters in gas hydrate formation need to be investigated in pilot tests, which could be conducted in future work. Additionally, further research can focus on grafting graphene/GO with functional groups to produce exceptional CMG or introducing functional nanoparticles (e.g., Ag and FeM-TS wrote and revised the manuscript. FW and G-DZ supervised and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


COLQ gene in a CMS patient born to consanguineous parents of Pakistani origin. Electromyography (EMG), electroencephalography (EEG), clinical exome sequencing (CES), and single nucleotide polymorphism (SNP) array analyses were performed. The subject was born at term after an uneventful pregnancy and developed significant hypotonia and dystonia, clinical pseudoseizures, and recurring respiratory insufficiency with a need for mechanical ventilation. CES analysis of the patient revealed a homozygous deletion of the COLQ gene located on the 3p25.1 chromosome region. The SNP-array confirmed the presence of deletion that extended from exon 11 to the last exon 17 with a size of 19.5 Kb. Our results add new insights about the underlying pathogenetic mechanisms expanding the spectrum of causative COLQ mutations. It is relevant, considering the therapeutic implications, to apply suitable molecular approaches so that no type of mutation is missed: “each lost mutation means a baby treated improperly”.Congenital myasthenic syndromes (CMSs) are caused by mutations in genes that encode proteins involved in the organization, maintenance, function, or modification of the neuromuscular junction. Among these, the collagenic tail of endplate acetylcholinesterase protein  has a crucial role in anchoring the enzyme into the synaptic basal lamina. Here, we report on the first case of a patient with a homozygous deletion affecting the last exons of the  Congenital myasthenic syndromes (CMSs) are a group of genetically and clinically heterogeneous disorders with impaired neuromuscular transmission and symptoms such as fatigable skeletal muscle weakness generally confined to ocular, bulbar, or limb-girdle muscles . CMSs caCMSs differ from myasthenia gravis, an autoimmune disorder that affects the same anatomic region, because of early age of onset, positive family history, rarity, and lack of response to immunomodulatory drugs. Clinical presentation of CMSs is highly heterogeneous and ranges from mild symptoms to severe manifestations, sometimes with life-threatening respiratory episodes especially in the first decade of life . All subOnset can be prenatal, during infancy, childhood, or adolescence (rare). Clinical onset may sometimes be in adulthood with EMG changes not present in all muscles and intermittent ., or in protein glycosylation   in the N-terminal region, heparan sulfate proteoglycan-binding domain (HSPBD) in the collagen domain [exons 4–14], and the C-terminal region [exons 15–17]. The causative mutations would exert their effects by different mechanisms resulting in prolonged synaptic currents and different action potentials due to expanded residence of acetylcholine in the synaptic space. Most of the assembly A 3]..COLQ genCOLQ gene. Twenty years later, Wang et al. [COLQ gene in compound heterozygosity with the IVS16 + 3A > G variant.To date, multiexon COLQ deletion has been described only in the two following cases. In 1998, Ohno et al.  reportedg et al.  identifiCOLQ gene  was obtained but after the molecular diagnosis, pyridostigmine was stopped and salbutamol was given, leading to a significant reduction in apnea and respiratory insufficiency. This finding highlights the importance of timely genetic counselling because the final diagnosis is crucial for correct CMSs treatment. Non-pharmacological treatments rely on physiotherapy, speech therapy, and occupational therapy. Sometimes invasive treatments are needed such as nasal intermittent positive pressure ventilation (NIPPV) during the night or the entire day in case of respiratory failure. PEG is used in the case of dysphagia, failure-to-thrive, or a nutritional disturbance. Surgical corrections are performed for severe deformities .Prognosis and outcome of CMSs derive only from observational studies, case studies, and case reports because prospective outcome studies are not available. Due to the clinical variability, outcome and prognosis may vary considerably accordingly to various CMS types and infections. Fever or psychosocial stress could also have a negative effect on the outcome of these patients . Our cas