BH pharmacokinetic data were studied in human volunteers, following transdermal delivery of F7, using Phoresor® Unit II iontophoresis device, compared to conventional oral tablets. F7 showed 1.9- and 2-fold higher values of Cmax and AUC0–40, respectively compared to Concor® tablets, as well as a smaller Tmax (2.00 ± 2.00 h). The relative bioavailability of F7 was found to be 201.44%, relative to Concor® tablets, demonstrating the significantly enhanced transdermal permeation of BH from the selected hydrogel by iontophoresis, in human volunteers. Finally, results showed the successful utility of permeation enhancers combined with iontophoresis in significantly enhanced transdermal permeation of BH, despite its large molecular weight and hydrophilic nature. Therefore, this strategy could be employed as a successful alternative route of administration to conventional oral tablets.Bisoprolol hemifumarate (BH) is an antihypertensive drug that is used as first-line treatment for chronic hypertension and angina pectoris. Our study was performed to enhance the transdermal delivery of BH, a hydrophilic drug active with high molecular weight, through differently prepared hydrogels. The synergistic effect of permeation enhancers and iontophoresis was investigated via both ex vivo and in vivo permeation studies. Ex vivo iontophoretic permeation studies were performed by using male albino Wistar rat skin. Cellosolve Hypertension is a significant and a major risk factor for both morbidity and mortality, especially in geriatrics, which is the most rapidly growing population sector in the world. It is a chronic and asymptomatic cardiovascular disorder that is often associated with the risk of stroke, congestive heart failure, kidney failure, and dementia . CollectBisoprolol hemifumarate (BH), chemically known as 1-[4-(2-isopropoxyethoxymethyl) phenoxy]-N-isopropyl-3-aminopropan-2-ol fumarate, is a selective β1 receptor blocker . As repoTransdermal route is considered an effective alternative to oral drug administration . It helpAs a matter of fact, only few drug candidates can allow successful transdermal delivery. This could be attributed to the barrier properties of the stratum corneum (SC) which allows easy permeation of small molecules having moderate lipophilicity . On the Iontophoresis is the permeation of ionized molecules through skin membranes using an external electric current ,14. It c® oral tablets.Thus, the aim of the present study was to evaluate the potential of transdermal delivery of BH via iontophoresis. This involved evaluating the role of different permeation enhancers, within BH hydrogels, in improving the measured ex vivo iontophoretic parameters compared to drug solution. Moreover, BH pharmacokinetic (PK) parameters were investigated after iontophoretic transdermal application to human volunteers, in comparison with the commercially available Concor® sodium carboxymethylcellulose (7HF) (SCMC) was obtained from Hercules, Wilmington, DE, USA. NatrosolTM 250 HHX Pharm hydroxyethylcellulose (HEC) was supplied from Hercules, Zwijndrecht, The Netherlands. Carbomer (Carbopol 940) (Cp940), DermarolTM 6CC and EMAROLTM 80 (Tween® 80) were purchased from CISME, Milan, Italy. 2-ethoxyethanol (Cellosolve®) was obtained from Oxfordlab, Gujarat, India. Triethanolamine was obtained from El Nasr chemical company, Cairo, Egypt. Propylene glycol was purchased from Fluka Chemie GmbH CH-9471, Buchs, Switzerland. Ethyl alcohol (95%) was purchased from El-Goumhouria chemical company, Cairo, Egypt. Distilled water was used throughout the whole study. All solvents and reagents were used as received.Bisoprolol hemifumarate (BH) was supplied as a gift from Global Napi Pharmaceuticals, 6th of October, industrial zone, Cairo, Egypt. Aqualonmax) and the flow rate were 227 nm and 1 mL per minute (min), respectively. Sample injections were performed in triplicate, where the average peak area of the three injections were plotted against the concentration. The mean correlation coefficient (R2) was equal to 0.999. The procedural constant (K) was calculated from the reciprocal of the slope of the linear regression line representing the calibration curve.A stock solution of BH in phosphate buffer (pH 7.4) was prepared to obtain a concentration of 4 mg/mL. Serial dilutions of BH in phosphate buffer (pH 7.4) containing 1.2, 1.6, 2, 2.4, and 2.8 mg/mL were prepared. HPLC procedure was performed by using ACME 9000 HPLC , which was equipped with 3.9 mm × 30 cm Zobrax column packed with octyl silane chemically bonded to porous silica particles (5 µm in particle size). The mobile phase was a mixture of 20 mL triethyl amine with 1000 mL distilled water and 680 mL methyl alcohol, adjusted to pH 4. The injection loop had a volume of 50 µL. The detection wavelength (λw/w) was dissolved in an alcoholic solution (5% w/w) under continuous stirring until a clear solution was obtained. Propylene glycol (10% w/w) was added to BH solution while stirring. It served as a humectant in all formulations [BH gels were prepared by using cellulose polymers as gelling agents, namely HEC and SCMC, as well as polyacrylic acid polymers, such as Cp940. BH . The obtained solutions were filtered, using a 0.45 µm Millipore filter, and filtered samples were assayed for drug content, using a validated HPLC method, employing the previously obtained calibration curve equation .−1), “N” is the Farrow’s constant, “S” is the shear stress (dyne/cm2), and “η” is the viscosity in centipoises (cps). Flow is said to be dilatant (shear thickening) or plastic/pseudo-plastic (shear thinning) for “N” values smaller than or greater than one, respectively.Rheological parameters of the prepared BH hydrogels were determined using Cone and Plate Viscometer . All experiments were done at room temperature. Both ascending and descending parts of flow curves were assessed, where determinations were performed from 0.5 up to 100 revolutions per minute (rpm). Rheological behavior of all gel formulations was evaluated via plotting the shear rate values versus shear stress. Farrow’s equation was calculated in order to determine the flow behavior of the prepared gels, as follows :(1)Log DHairless albino Wistar rat skin was employed as a skin membrane model. Newly born male albino Wistar rats were sacrificed and full thickness skin was excised and inspected for any irregularities. Remaining adherent debris were removed by a scalpel. Fresh skin pieces were washed with tap water and preserved in glycerin solution (10%) at −20 °C. The study performed in this section was approved by Research Ethics Committee, Faculty of Pharmacy, Cairo University . Prior to experimentation, skin pieces were allowed to thaw at room temperature. For hydration, they were soaked in phosphate buffer (pH 7.4) for 1 h, at room temperature [The desired electric current was produced from a custom-made iontophoretic device , that wa2 in all cases. Experiments were carried out for a period of 3 h and extended for 1 h post-iontophoresis process. Permeation experiments were done by loading 5 mL of BH solution (2 mg/mL) or 5 mL of the selected BH gel into the donor compartment. The receptor solution contained 75 mL phosphate buffer (pH 7.4). Silver and silver chloride electrodes were employed as anode and cathode electrodes, respectively. Anodal iontophoresis (2) was applied across the rat skin. The receptor compartment was continuously stirred at 500 rpm, using Teflon-coated magnetic stirrers (3 cm) [Ex vivo iontophoretic transport studies were performed, using a vertical two compartment glass diffusion apparatus. It consisted of a double open sided tube. Each piece of rat skin was placed between the two compartments, where the SC faced the donor compartment (having a diameter of 2 cm) in all experiments. The surface area of skin membrane exposed to the donor solution was 3.14 cmphoresis  was perfs (3 cm) , in orde2) was plotted versus time (h). The steady state flux, Jss (µg·h/cm2), was calculated from the slope of the linear portion of the plot [The mean cumulative amount of BH (Q) permeated into the receptor compartment, per unit surface area of rat’s skin  for BH permeation was denoted by using the following equation :FER = JsData were statistically analyzed by one-way analysis of variance (ANOVA), followed by Tukey-Kramer test, with the significance level set at 0.05. Data were expressed as mean ± SD (Graph pad INSTAT 3.01).®, in 5 mg tablets. The study was performed according to a blind, single-dose, two-period, random crossover design and adopted under fasting condition. Six adult, healthy, male human volunteers contributed in the study, having average weight of 76.66 ± 7.39 kg, aged between 35 and 40 years old and height from 170 to 180 cm. All volunteers were non-smokers. Their biochemical examination revealed normal liver and kidney functions. The nature and the aim of the study were completely explained to them. In addition, an informed written consent was obtained from each volunteer, where they were totally informed with the nature of the study. The clinical study protocol was reviewed and approved by the human ethical principles of Human Research Ethics Committee at the Faculty of Pharmacy, Cairo University in Egypt.The study was performed to determine the PK parameters of BH after iontophoretic delivery from the selected BH hydrogel compared to the oral delivery of the market product Concor® tablets, at 5 mg (Treatment A), with 200 mL water for ingestion. Group II received the selected BH gel (Treatment B). Forearm areas of each volunteer were first swabbed with alcohol to remove any contaminants then, left to dry. The swabbed area was then marked with a circle (diameter = 2 cm). Using a syringe, 2.5 mL of the selected BH gel was applied on the marked area of the skin, using an iontophoresis device, Phoresor® Unit II , having TransQe iontophoresis electrodes. The active area of diffusion was 13.4 cm2 and the current intensity was adjusted to be 0.5 mA /cm2 for two hours. Both treatments were administered to fasted volunteers overnight, where only drinking water was allowed. Lunch and dinner were provided only after 4 and 10 h of drugs administration, respectively. A period of 10 days separated the two treatments as a washout period. In the second period, group I received treatment B and group II received treatment A.Prior to any treatment, 10 mL venous blood were withdrawn from each volunteer to measure the baseline. The study was done over two periods. In the first period, group I received the market product ConcorOur study was carried out under the supervision of a physician. Volunteers’ safety and samples collection were his responsibility throughout the whole study. Then, 5 mL blood samples were withdrawn from each volunteer, using indwelled cannulas at specified time intervals 0 min (pre dose), 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 9, 12, 18, 24, 36, and 40 h post-administration of each treatment. All withdrawn blood samples were filled into heparinized glass tubes, centrifuged at 3000 rpm for 10 min at 4 °C to separate plasma. Plasma was immediately stored frozen in plastic tubes at −20 °C, prior to drug analysis.All samples were thawed to room temperature. A total of 50 µL of acetonitrile was added to all human plasma samples (1 mL), as the internal standard (IS). All samples were mixed for 1 min, using a vortex mixer, and then centrifuged at 3000 rpm for 10 min. The supernatant was filtered through Millipore filters (0.45 µm), and then samples were evaporated until dryness at 40 °C via centrifugal vacuum concentrator. Residues were reconstituted with the mobile phase (100 µL), where 20 µL was injected to the column via an auto sampler.v/v) was used as the mobile phase. Metoprolol was used as (IS).Plasma determination of BH was performed using a sensitive, selective and accurate liquid chromatographic method associated with electrospray ionization mass spectrometry (LC-ESI/MS), described by Ding et al. . Ammoniu® program . Maximum drug plasma concentrations  and their corresponding time  were obtained from individual plasma concentration-time curves. T1/2 (h) was calculated as 0.693/k. The area under the curve AUC0–40 (ng·h/mL), determined as the area under the plasma concentration-time curve was calculated using the linear trapezoidal rule, up to the last measured sampling time. AUC 0−∞ (ng·h/mL) was calculated from AUC0–40 + Ct/K, where Ct is the last measured concentration at time (t) [Non-compartmental analysis for determination of BH PK data, for each volunteer, was performed via Kineticatime (t) .® software. The level of significance was α = 0.05, where p-values less than or equal to 0.05 were considered statistically significant.The obtained PK data for the selected BH hydrogel compared to that obtained from the oral treatment were analyzed for statistical significance by one-way analysis of variance (ANOVA) adopting Kinetica® 80. Mean percent of BH content ranged from 98.00 ± 0.13 to 103.00 ± 0.76 in all formulations. pH of the prepared hydrogels ranged from 7.36 ± 0.01 to 7.44 ± 0.03. All results are shown in All the prepared BH hydrogels exhibited homogenous texture without showing any clumps. Some of the prepared hydrogels were clear, others were turbid. Moreover, all hydrogels were colorless except F8 had a slight yellow color, which may be due to the presence of Tweeny-axis represented the shear rate (s−1) and x-axis represented the shear stress (dyne/cm2)). Hydrogels showed coincidence of the ascending and the descending curves without any hysteresis loops indicating no or negligible thixotropic breakdown. Calculated Farrow’s constant (N) for all formulations gave a value >1  while, F6 exhibited the highest viscosity (5361 cp) . The effvalue >1 , confirm180 and Q240 = 5.24 ± 0.06 and 5.77 ± 0.05 µg/cm2), respectively, with a flux of 1.44 ± 0.01 (µg·h/cm2). This could be attributed to the high molecular weight of BH, where drug molecules were unable to penetrate the skin via the main routes of passive diffusion, such as trans-cellular and/or para-cellular routes. On the other hand, transdermal permeation of BH solution via iontophoresis was significantly enhanced (p < 0.0001) compared to passive diffusion, during the same time intervals  value of 0.01040 ± 0.00011 cm/h. Enhanced transdermal permeation of BH solution could be attributed to disarrangement of the lamellar lipid bilayer that is produced within the SC by the action of iontophoresis. Similar results were reported by Nair and Panchagnula in their study on the effect of iontophoresis on permeation of Arginine Vasopressin through rat skin [The measured permeation parameters of BH through rat skin are represented in ntervals , showingrat skin . Moreoverat skin . The apprat skin .180), from BH hydrogels (not containing permeation enhancers), can be arranged in the following ascending order: F3 (598.99 ± 5.24) < F1 (652 ± 8.06) < F2 (706.41 ± 5.14) < F4 (742.04 ± 5.67) < F5 (900.57 ± 8.79) (µg/cm2). Their fluxes were 171.28 ± 1.64, 179.86 ± 0.16, 198.10 ± 2.36, 212.62 ± 1.34, and 245.65 ± 1.76, µg·h/cm2, respectively. It is clear that iontophoretic permeation of BH was significantly enhanced from these hydrogels compared to BH solution (240 (982.61 ± 7.04 µg/cm2) with Kp value equivalent to (0.02457 ±0.00018). HEC is a characteristic cellulose ether derivative that is widely used for its safety and biocompatibility [The cumulative amount of BH permeated across rat skins after 3 h . These hydrogels can be arranged in the following ascending order: F6 (1058.78 ± 18.37) < F8 (1186.55 ± 12.00) < F9 (1307.44 ± 4.26) < F7 (1390.68 ± 3.65) (µg/cm2). Their corresponding fluxes were 286.67 ± 1.26, 323.00 ± 1.67, 353.98 ± 1.18, 367.71 ± 2.37 (µg·h/cm2), and their ascending kp values were 0.02867 ± 0.00013, 0.03230 ± 0.00017, 0.03540 ± 0.00012, and 0.03677 ± 0.00024 cm/h, respectively. Enhanced permeation profiles could be attributed to the addition of chemical permeation enhancers which leads to further modification in the structure of the lipid skin bilayer, resulting in widened intercellular spaces. Consequently, the transport of drug molecules via iontophoresis was effectively facilitated through the SC, where transdermal permeation was synergistically facilitated by physical and chemical enhancement techniques.Results indicated significantly improved permeation pattern of BH from HEC hydrogels containing permeation enhancers, namely F6, F7, F8, and F9, compared to F5 (lacking permeation enhancer) and BH solution . The cumTM 6CC is chemically defined as a mixture of mono-, di-, and triglycerides of capryl/capric ethoxylates . It is a derivative of PEGylated alkyl glycerides, namely polyethylene glycol-6 caprylic/capric glycerides (PEG-6-CCG). PEGylated alkyl glycerides are a sub-fraction of PEGylated oils. PEGylated oils were known to be used in cosmetics and PEGylated alkyl glycerides have been reported for their skin surfactant, as well as emulsifying properties. Its use has been reported in several research studies [180, Jss and ER compared to plain HEC hydrogel (F5). This could be attributed to the skin surfactant properties of PEG-6-CCG, as previously mentioned. Being a non-ionic surfactant, enhanced permeation is because it fluidizes lipids of the SC on two steps: first, by penetrating through the intercellular regions and solubilizing its lipid content, and second, by binding to keratin within the intercellular matrix, producing disruption within corneocytes [Dermarol studies , which i studies . Moreove studies . In addi studies . To the neocytes . Thus, t180, Jss and ER compared to plain HEC hydrogel (F5). This could be attributed to the skin surfactant properties of Tween® 80. Being a non-ionic surfactant, its mechanism of action would be similar to PEG-6-CCG, as previously discussed. Moreover, an additional mechanism for the permeation enhancing properties of Tween® 80 could be attributed to its chemical structure, containing ethylene oxide group, which helps in permeating hydrophilic drugs, such as BH. This can be confirmed by the fact of being a hydrophilic, water soluble surfactant, having high hydrophilic/lipophilic balance (HLB) value, equal to 15. This is in addition to its ability to emulsify sebum, allowing higher power of drug penetration into the skin as previously mentioned. Similar results were obtained by Nabarawi et al. in their study on transdermal delivery of BH, where Tween® 80 changed the skin’s barrier properties [® 80 is among the widely used and pharmaceutically acceptable permeation enhancers used for transdermal delivery of a wide variety of drug actives.Results also showed that F8 exhibited 1.3-fold increase in Qoperties , allowin® hydrogels, namely F7 and F9, significantly enhanced skin permeation of BH through hairless rat skin. Compared to plain HEC hydrogel (F5), F7 exhibited a 1.5-fold increase in Q180, Jss, and ER and F9 showed a 1.5-fold increase in Q180, as well as a 1.4-fold increase in Jss and ER. They also exhibited higher permeation compared to F6 and F8, as well as significantly enhanced permeation parameters compared to BH solution (p < 0.05).In addition, Cellosolve® is the ethylene glycol monoethyl ether, that is often known as 2-ethoxyethanol. It has been used in cosmetic preparations, such as skin lotions and oral care and nail products, as well as cleansing preparations. Moreover, it has been reported for its rapid percutaneous absorption [®) in enhanced ex vivo transdermal permeation of each of clonazepam through rabbit ear skin [Cellosolvesorption . This cosorption , being aear skin , diclofeear skin , and baiear skin .®. Referring to 2-ethoxyethanol, as an imparted moiety within Transcutol® chemical structure [®. Similarly, enhanced transdermal permeation of Cellosolve® could be attributed to improving the fluidity of skin bilayers resulting in reduced skin barrier and facilitated pathway of BH towards the skin. In our study, it is worth to say that enhanced transdermal permeation was achieved by using Cellosolve® with a concentration that is less three- and two-fold than its maximum allowable dose per day, within F7 and F9, respectively. It was also noted that F9 exhibited decreased values of permeation parameters, however non-significant, compared to F7. This could be attributed to its higher viscosity (981 cps) (owing to the higher Cellosolve® concentration), which consequently lowered drug permeation. These results cohere with the previously performed rheological measurements. Thus, F7 was selected for further in vivo studies.The literature lacks data about enhanced BH skin permeation using Cellosolvetructure , clarifi® tablets (5 mg) to six healthy human volunteers are shown in max determined from the selected hydrogel, was 1.9-fold higher and statistically significantly different (p = 0.0001) compared to Concor® tablets. In addition, the mean determined Tmax was smaller (2.00 ± 2.00 h) and statistically significantly different (p = 0.002) compared to the conventional tablets. The AUC0–40 values after transdermal and oral treatments were 43.19 ± 3.31 and 21.44 ± 3.60 ng·h/mL, respectively. Statistically significant difference was found between the two values (p = 0.0001), where the calculated relative bioavailability of BH after transdermal iontophoresis was about 201.44%, relative to Concor® tablets. The significant higher values of both Cmax and AUC0–40 together with the significant lower value of Tmax compared to the conventional oral tablet confirm the enhanced transdermal transport of BH through combined action of permeation enhancers and iontophoresis. This could be due to the loosened structure of SC layers caused by their dual effect, which correlates well with the previous ex vivo permeation results. On the other hand, the mean elimination half-life (T1/2) and the mean residence time (MRT) were found to be insignificantly different (p > 0.05) for F7, compared to oral tablets. These results strongly cohere with the PK theory, where drug elimination is not affected by enhanced drug permeation and absorption. Similar findings were reported by Fouad et al. in their study on sublingual and intranasal delivery of dapoxetine hydrochloride versus commercial oral tablets [BH mean plasma concentration time profiles following transdermal iontophoretic delivery of F7 and administration of single oral dose of Concor tablets .Based on the previous findings, it can be concluded that F7 enhanced transdermal iontophoretic delivery of BH through the human’s skin. However, owing to the minute number of volunteers participating in the study, results can be preliminary. Therefore, further studies should be performed to prove the applicability of utilizing chemical enhancers combined with iontophoresis for transdermal permeation of BH.® hydrogel (F7) exhibited significant increase in transdermal permeation parameters, including Q180, Jss, and FER, through albino Wistar rat skin. It also showed significantly enhanced bioavailability compared to traditional Concor® tablets in human volunteers. The methodology described in our study can be applied to other hydrophilic drug actives, in order to achieve alternative enhanced transdermal delivery. Further clinical studies should be performed, employing a larger number of human volunteers to prove the clinical efficacy of F7 in hypertension.To conclude, our study showed enhanced transdermal delivery of BH into human skin through the utility of combined strategy of both iontophoresis and permeation enhancers. These findings pointed to a successful alternative to conventional oral tablets which can, in return, solve the problem of dysphagia in elderly patients. Overcoming the hydrophilic nature of BH along with its high molecular weight via dual action of iontophoresis and permeation enhancers is the main reason for enhanced BH transdermal delivery. These results were confirmed via both ex vivo permeation and in vivo pharmacokinetic studies. Cellosolve


The presentation of multiple intestinal perforations is a severe complication of enteric cytomegalovirus (CMV) infection, sometimes associated with immune reconstitution inflammatory syndrome (IRIS) after the initiation of antiretroviral therapy (ART) in patients with human immunodeficiency virus (HIV). Here we reported a rare case of a patient with HIV infection who developed multiple perforations in the small bowel shortly after ART initiation without any prodromal gastrointestinal symptoms. We also reviewed the literature of reported cases to clarify their clinical characteristics for early diagnosis and rapid intervention.A patient with HIV presented with fever after 16 days of ART initiation and was admitted to our hospital. He was treated with intravenous ganciclovir due to persistent CMV viremia. The fever resolved 10 days later. However, he reported persistent left lower abdominal pain.The patient was diagnosed with multiple small bowel perforations, CMV-related IRIS, and acquired immune deficiency syndrome. An upright abdominal x-ray in a tertiary level hospital revealed bilateral moderate intraperitoneal free air. We performed a pathological examination and metagenomic next-generation sequencing. CMV enteritis was confirmed by immunohistochemical staining and other opportunistic infections were excluded by metagenomic next-generation sequencing.The patient was treated with intravenous ganciclovir and 24 hours later, the patient underwent exploratory laparotomy. Partial resection and surgical repair of the small intestine were performed.The patient ultimately died from intestinal obstruction and septic shock 55 days after surgery.Perforations due to CMV-related IRIS are very rare, and usually appear shortly after ART initiation. Most cases lack the prodromal symptoms of abdominal pain and diarrhea. Intestinal perforations are lethal, and early detection and surgical treatment are lifesaving. Although it is very rare, bowel perforation could be the presenting feature of CMV-related immune reconstitution inflammatory syndrome (IRIS) in HIV-infected patients.–6 This is a case report of a 28-year old man with HIV infection who presented with multiple small bowel perforations 26 days after the initiation of antiretroviral therapy (ART), without any prodromal gastrointestinal symptoms.In patients infected with human immunodeficiency virus (HIV), cytomegalovirus (CMV) retinitis is the most common clinical manifestation of CMV end-organ disease, followed by gastrointestinal tract involvement. Although CMV infections can affect the entire gastrointestinal tract, it frequently involves the esophagus and the colon.2+ T-cell count was 25 cells/μL. After a skin lesion biopsy, the patient was diagnosed with Kaposi's sarcoma. The CMV-DNA load was 6.0 × 104 copies/mL, CMV antibody (IgM) was <8 U/mL (0–18 U/mL), and CMV antibody (IgG) was 12.3 U/mL (0–12 U/mL). No abnormalities were found on funduscopic examination. The patient was found to have specific antibodies to Treponema pallidum and rapid plasma reagin test titer was positive with a titer of 1:2. The patient was treated with benzathine penicillin for 3 weeks. The patient was started on antiretroviral therapy (ART) with a regimen of lamivudine 300 mg daily, tenofovir disoproxil 300 mg daily, and nevirapine 200 mg twice daily.A 28-year-old man presented with several purple-blue nodules on the face and neck of one-month duration. HIV antibody testing was positive and his nadir CD49 cells/L (3.5–9.5  × 109 cells/L), lymphocyte of 1.29 × 109 cells/L (1.1–3.2 × 109 cells/L), granulocyte of 4.04 × 109 cells/L (1.8–6.3 × 109 cells/L), hemoglobin (Hb) of 87 g/L (130–175 g/L), and platelet (PLT) of 348 × 109 cells/L (125–350 × 109 cells/L). The level of C-reactive protein was 88.7 mg/L (0–8 mg/L), and procalcitonin was 0.66 ng/mL (0–0.05 ng/mL). Toxoplasma IgG and IgM antibodies were negative. Liver and kidney function tests were within the normal range. The HIV RNA load was 2.46 × 103 copies/mL and his CD4+ T-cell count had already increased to 75 cells/μL. The CMV-DNA load was 1.3 × 104 copies/mL. The patient was treated with intravenous ganciclovir for persistent CMV viremia.After 16 days of ART initiation, the patient presented with fever and was admitted to our hospital. His highest recorded temperature was 38.9°C, accompanied by chills. He denied abdominal pain, diarrhea, or hematochezia. Routine blood tests showed a leukocyte count of 5.76 × 10Ten days later, the fever resolved. However, the patient reported persistent left lower abdominal pain which was spastic and tolerable. He denied abdominal distension, nausea, or vomiting. The patient was conscious and had a normal blood pressure of (BP) 115/65 mmHg, respiratory rate (R) of 16 bpm, pulse rate (P) of 88 bpm, and body temperature (T) of 36.8°C. Physical examination showed a soft and flat abdomen, left lower abdominal tenderness, without rebound pain or rigidity. Bowel sounds were 4/min. An upright abdominal x-ray in a tertiary level hospital revealed bilateral moderate intraperitoneal free air  and creatinine was 92 μmol/L (59–104 μmol/L). An exploratory laparotomy was performed. A total of 1000 mL of suppurative peritoneal fluid was cleared. Multiple perforations at 40, 50, 65, 140, and 240 cm proximal to the terminal ileum were found at the anti-mesenteric border of the small bowel, the largest diameter of which was 1 cm. Furthermore, multiple localized discolorations on the serosal surface of the small intestine were observed indicating multiple deep ulcers . Partial enterectomy (35 cm) and surgical repair of the small bowel were performed. Histopathological analysis showed destruction of the mucosa, submucosa, and muscular layers with neutrophilic infiltration and granulation tissue formation (Fig. + plasma cells (Fig. + tissue cells (Fig. + lymphocytes (Fig. + lymphocytes (Fig. Twenty-four hours later, the vital signs were BP 110/70 mmHg, R 18 bpm, P 130 bpm, and T 38.4°C. The patient had left upper and right lower abdominal tenderness, rebound pain throughout the entire abdomen with rigidity. A repeat of the upright abdominal x-ray showed massive bilateral intraperitoneal free air (Fig. 9/L, lymphocyte of 0.38 × 109/L, granulocyte of 4.35 × 109/L, Hb of 88 g/L, and PLT of 158 × 109/L. Amylase, lipase, myocardial enzymes, liver, and renal function tests were within the normal range. An upright abdominal x-ray showed visibly dilated intestines and gas-fluid levels (Fig. One month after discharge, the patient was re-admitted for abdominal pain and vomiting. The patient was conscious. He had discontinued ART without consulting his physician. The vital signs were T 36.5°C bpm, 110/76 mmHg, P 103 bpm, and R 16 bpm. An upright abdominal x-ray did not show any free gas or liquid gas levels (Fig. 3+ T-cell count after ART initiation.In this case report, the multiple small bowel perforations were associated with CMV-related IRIS. Although the HIV RNA load before ART initiation was unavailable, this patient met the criteria for IRIS in terms of presentation of new signs and symptoms of CMV-associated perforations in the small bowel, a low HIV RNA load, and an obvious increase in CD4–4 and are summarized in Table ,8 and the common causes include tuberculosis, Crohn disease, and malignancies. The most common cause among HIV-infected individuals is CMV infection, although there has been a dramatic decrease since the development of ART. Kaposi sarcoma and lymphoma are also occasionally associated with small bowel perforations.Only five CMV-associated IRIS bowel perforations, including ours, have been reported in the literature, CMV colitis could also be a manifestation of unmasking or paradoxical IRIS.–13 The pathogenesis of gastrointestinal CMV disease is believed to be through submucosal vasculitis with thrombosis resulting in ischemia, ulcers, thinning of the intestinal wall, subsequent perforation, and gangrene. Histologic examination showed multiple areas of mucosal ulceration with acute and chronic inflammation, in addition to transmural inflammation and necrosis at the perforation sites. The criterion standard for diagnosis is the observation of cytomegalic cells with viral inclusion bodies in epithelial, endothelial, smooth muscle, and inflammatory cells.,16 CMV infection is confirmed by immunohistochemical testing for an immediate early antigen. Real-time polymerase chain reaction and CMV culture are alternative laboratory methods. An important diagnostic tool, metagenomic next-generation sequencing using formalin-fixed and paraffin-embedded samples of lesions was significant in this case to establish differential diagnoses among various pathogens such as tuberculosis, non-tuberculous mycobacteria, histoplasmosis, and salmonella, which could all be associated with bowel perforations in patients with HIV.The most common presenting symptoms of CMV gastroenteritis are fever, abdominal pain, and diarrhea, whereas disease limited to the small bowel may be asymptomatic. CMV enteritis presenting with perforation in HIV-infected patients has been associated with high mortality due to postoperative complications including reperforation, bowel obstruction, severe sepsis, and multi-organ failure.,18 Routine enteroscopy and capsule endoscopy screening have not been recommended. In order to prevent life-threatening CMV colitis, early ART initiation and maintenance of high CD4+ T-cell counts are necessary. Being aware of atypical manifestations of the disease and rapid initiation of individualized therapy is also crucial. Last but not least, identifying high-risk individuals and using preemptive anti-CMV therapy may be lifesaving. Assessment and monitoring of patients should be done by HIV specialists and those with increased risk factors of mortality may benefit from early optimal interventions.In conclusion, bowel perforations shortly post-ART initiation could be considered as CMV-related IRIS in HIV-infected individuals. Preemptive anti-CMV therapy is not recommended in HIV-infected individuals with asymptomatic viremia. However, they have been proven effective in advanced cases.Data curation: Xuyong Lin, Yuji Li.Formal analysis: Xuyong Lin, Yuji Li.Writing – original draft: Yanli Wang.Writing – review & editing: Ying Wen.


Theobroma grandiflorum) to develop an innovative skin regeneration formulation based on chitosan-coated nanocapsules. Cupuaçu seed extract significantly stimulated cell proliferation and migration. A reparative gene expression profile could be verified following extract treatment, which included high levels of MKI67, a cellular proliferation marker, and extracellular matrix genes, such as ELN and HAS2, which code for elastin and hyaluronic acid synthase 2. Formulations with Cupuaçu seed extract successfully entrapped into nanocapsules (EE% > 94%) were developed. Uncoated or coated nanocapsules with low-molecular-weight chitosan presented unimodal size distribution with hydrodynamic diameters of 278.3 ± 5.0 nm (PDI = 0.18 ± 0.02) and 337.2 ± 2.1 nm (PDI = 0.27 ± 0.01), respectively. Both nanosystems were physically stable for at least 120 days and showed to be non-irritating to reconstructed human epidermis. Chitosan coating promoted active penetration into undamaged skin areas, which were still covered by the stratum corneum. In conclusion, the present study demonstrated for the first time the biotechnological potential of the frequently discarded Cupuaçu seed as a valuable pharmaceutical ingredient to be used in regenerative skin products.Scarless skin regeneration is a challenge in regenerative medicine. Herein, we explore the regenerative potential of a Cupuaçu seed extract ( Theobroma grandiflorum (Willd. ex Spreng.) K. Schum) presents a taxonomic relationship with cocoa (Theobroma cacao L.) and is one the most popular native fruits cultivated in the Brazilian Amazon region. The fruit pulp is used in a wide variety of food products, holding considerable economic importance for the Brazilian state of Pará, the largest producer and exporter [Cupuaçu , marginal acid (>95%), sodium methoxide, Span 60, polycaprolactone polymer, low-molecular-weight chitosan , Vivaspin , dimethyl sulfoxide (DMSO), and mucin (type II) were all purchased from Sigma-Aldrich . Ethyl ether, acetic acid, ethanol, and monobasic and dibasic sodium phosphate were acquired from Vetec . The solvents methanol and n-hexane 95% were supplied by J.T. Baker . Dulbecco’s Modified Eagle Medium (DMEM) was obtained from Thermo–Fisher Scientific , and fetal bovine serum (FBS) was acquired from Gibco . Soy lecithin was purchased from Lipoid . Polysorbate 80 was acquired from Merck , whereas acetone and sodium chloride were from Serva . Sodium hydroxide was purchased from Contemporânea Chemical Dynamics . All analyses were performed using ultrapure water provided by Millipore . Formvar resin and uranyl acetate were used for nanocapsules morphological analysis were purchased from Electron Microscopy Science . The reconstructed human epidermis test method kit was donated by EpiSkin™ . The skin used in permeation analyses was obtained from porcine ears that had been generously provided by Bonasa  and stored at −4 °C.m/z. The components of the extract were identified by comparing their retention indices and mass spectra (molecular mass and fragmentation pattern) with data stored in the NIST [Cupuaçu seeds were dried in an oven at 40 °C for 24 h, which was the lowest temperature capable of reducing seeds humidity to lower than 20% in a relatively short time. Then, the seeds were ground, and fatty acids were extracted by a Soxhlet using hexane as a solvent for 3 h. The sample was analyzed in a GC-MS-QP2010 Ultra system , equipped with an auto-injector (AOC-20i). The parameters of analysis were: silica capillary column Rxi-5ms  ; injector temperature: 250 °C; oven temperature programming: 60–240 °C (3 °C/min); helium as a carrier gas, adjusted to a linear velocity of 36.5 cm/s (1.0 mL/min); splitless mode injection of 1 μL of sample (oil 5 μL: hexane 500 μL); ionization by electronic impact at 70 eV; ionization source and transfer line temperatures at 200 and 250 °C, respectively. The mass spectra were obtained by automatically scanning every 0.3 s, with mass fragments in the range of 35–400 the NIST .v/v) fetal bovine serum (FBS). The cells were maintained at 5% CO2, 37 °C, and 95% humidity.For cell viability, proliferation, and migration tests, fibroblasts used in this study were isolated from the dermis of healthy donors. They were produced as pilot batches that had been kindly provided by CellSeq Solutions , and cultured using Dulbecco’s Modified Eagle Medium (DMEM), and supplemented with 10% , which were maintained in cell culture media throughout the experiment and treated with vehicle only, being considered as having 100% cell viability; and treated with Cupuaçu extract . After 48 h incubation, the cells were washed with PBS, and fresh DMEM supplemented with FBS was added to each well. At this time, 10 μL of 3--2,5-diphenyl-2H-tetrazolium bromide  were also added , followed by 4 h plate incubation in the dark. Then, the MTT solution was removed, and DMSO was added to dissolve the formazan crystals formed. A microplate spectrophotometer  was used to determine the absorbance at 570 nm. A minimum of three independent experiments was executed.Different concentrations of Cupuaçu extracts and analytical standards were previously tested to select the proportions that could ensure in vitro cell viability. Pure extracts were tested in dilutions of 10, 50, 80, 100, 150, and 200 µg/mL in DMEM. For this, 1 × 104 cells were cultured in 96-well microplates. These were left untreated for 24 h for complete attachment to the culture surface in their respective cell culture media. Afterward, the cells were incubated with Cupuaçu extract for 1, 4, and 7 days. At each point in time, an MTT assay was performed. Untreated fibroblasts were kept in DMEM without FBS supplementation as negative cell proliferation control. A minimum of three independent experiments was executed.The MTT assay was also executed to assess cellular proliferation, using the highest non-toxic concentration of Cupuaçu extract (100 μg/mL) and longer incubation time-points. Unlike in the cell viability assay, fibroblasts were trypsinized and harvested, and 1 × 10The Cupuaçu extract’s effect on the migratory behavior of fibroblasts was analyzed as described in Liang et al.’s protocol . BrieflyThe sequences of the target genes were obtained from Genbank and exported to Primer Express v.3.3 programs . The transcripts assessed for cultured cells were B-cell CLL/lymphoma 2 (BCL-2), elastin, basic fibroblast growth factor (FGF-2), MKI-67 proliferation marker (MKI-67), matrix metalloproteinase-1 (MMP-1), hyaluronic acid synthase-2 (HAS-2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). After 48 h of treating the cells for migration assay, the total RNA within each group was isolated using Trizol reagent, following manufacturer instructions. The cells cultured wells had the medium removed, and 1 mL of TrizolTM was added . After successive pipetting to promote cell lysis, Trizol was added to 1.5 mL DNA-RNAse-free Eppendorf, and 200 μL of chloroform was added to each tube, which was vigorously stirred. After that, the tubes were incubated at room temperature for 2–3 min and centrifuged for 15 min at 4 °C and 13,000 rpm. The upper (aqueous) phase was collected, and the RNA precipitated for 10 min with the addition of isopropyl alcohol. Then, another centrifugation was performed at 4 °C and 13,000 rpm for 10 additional min and washed with 75% ethanol, followed by another 10-min centrifugation at 4 °C and 7500 rpm. The RNA was diluted in autoclaved ultrapure water. RNA concentration was determined by measuring its absorbance at 260/280 nm in a Nanodrop device . RNA samples were reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit, according to the manufacturer’s instructions. In summary, reverse transcription of the isolated total RNA was performed using 500 ng of the total RNA from each sample, reverse transcriptase , Oligo dT (12–18) primer , and RNAse inhibitors, reaching a final volume of 20 μL. Incubation steps were taken according to the time and temperature recommended by the manufacturer. Amplification by quantitative polymerase chain reaction (qPCR) was performed in duplicates. Reactions were prepared with standardized reagents for real-time PCR , using the following primers: GAPDH F-ACATCGCTCAGACACCATG, GAPDH R-TGTAGTTGAGGTCAATGAAGGG; BCL2 F-CAAAGCTGCAGGCTGTTTAAG, BCL2 R-GTCTGTCTGTGTGTGTGATGT; MKI67-AACACCATCAGCAGGGAAAG, MKI67 R-CTGCACTGGAGTTCCCATAAA; MMP1-GAGCTTCCTAGCTGGGATATTG, MMP1 R-ACTGGCCTTTGTCTTCTTTCT; FGF2 F-CAAGGACCCCAAGCGGCTGT, FGF2 R-AGCTTGATGTGAGGGTCGCTCTT; ELN F-AAGGCTGCCAAGTACGGAGT, ELN R-CAAACTGGGCGGCTTTGG; HAS2 F-CTCGCAACACGTAACGCAAT, HAS2 R-CAGTGCTCTGAAGGCTGTGT. Amplifications were performed by StepOne Plus equipment , and the data were analyzed by StepOne Software v2.3, Foster City, CA, USA .The polymeric lipid-core nanocapsules were prepared by the interfacial deposition method of preformed polymers, as previously described by Bender et al. . Brieflyw/v) chitosan solution in 1% acetic acid was added dropwise under moderate magnetic stirring over the colloidal solution. The reaction medium continued to be stirred for 4 h at room temperature. In this way, two types of nanoparticles were produced: (i) polymeric nanocapsules containing the Cupuaçu extract (NC) and (ii) polymeric nanocapsules containing the Cupuaçu extract coated with chitosan (NC-CH).For the coating procedure, 10 mL of a 0.3% (v/v) diluted in water. The pH of the nanocapsule suspensions was measured by a calibrated potentiometer  with the direct immersion of the electrode in the nanoformulation. The entrapment efficiency EE (%) of the biomarker methyl oleate in the nanoparticles was determined by separating the free drug (FD) from the nanoparticles’ dispersions. Thus, 1 mL of each sample was centrifuged for 30 min at 2150× g using Vivaspin to increase the samples’ concentration. The filtrate was collected and analyzed by GC-MS, according to the method described in The nanocapsules’ hydrodynamic diameter, polydispersity index (PdI), and zeta potential were evaluated using Zetasizer Nano equipment . Before being analyzed, the samples were 1:10 (v/v), placed in gold gratings to be covered with “formvar” resin, and left drying at room temperature. Next, the excess formulation was removed with filter paper. After this step, 3 μL of 3% (w/v) uranyl acetate solution was added and allowed to dry, protected from light for 5 additional min at room temperature. Again, the excess solution was removed with filter paper, and the sample was analyzed in the equipment.The morphology of polymeric nanocapsules was analyzed by transmission electron microscopy . The samples were diluted in a ratio of 1:200 (w/v). The pH was then adjusted to 7.4 with 1.0 mol/L NaOH. The solution was diluted in HEPES buffer (pH 7.4) with a final mucin concentration equal to 1% (w/v) and was sonicated with a tip sonicator  to produce mucin nanoparticles with a hydrodynamic diameter smaller than 500 nm. Mixtures of the NC and NC-CH with mucin nanoparticles were prepared, mixing equal volumes of colloidal dispersions and homogenizing  for different periods . As described above, nanoparticle dispersions hydrodynamic diameter and zeta potential were measured before and after the mixture with mucin. The section size statistics were calculated in triplicates using the Zetasizer Nano Series device.Mucoadhesiveness of chitosan nanoparticles was evaluated in vitro as previously described . Porcinen = 3) were stored at temperatures of 8 °C ± 3.0 and 25 °C ± 2.0 and possible changes in the average hydrodynamic diameter, PdI, zeta potential, and pH were monitored.The physical stability of the nanocapsules was determined at 0, 7, 15, 30, 70, and 120 days after their production, in which samples of NC and NC-CH , and potentially non-irritating negative control  (n = 3). Cell viability was measured by MTT (1 mg/mL) reduction in a microplate spectrophotometer at 570 nm . For this protocol, a substance was determined as irritating when the percentage tissue viability—assessed by MTT assay—was less than or equal to 50% and as non-irritating when this percentage was above 50% [Skin irritation was also evaluated using reconstructed human epidermis (RHE) provided by EpiSkin™, according to  OECD guibove 50% .2). The burnt skin sample simply consisted of porcine skin that had been burnt for 7 sec by a hot plate at 85 °C [In vitro penetration was evaluated in intact and burnt porcine skin samples following a modified Saarbrücken method . Brieflyat 85 °C . PermeatOleic acid was chosen as a biomarker of the extract for skin penetration studies. For this, samples from the permeation studies were previously submitted to the esterification process described by Bannon et al.  adapted ® Software, version 7.02 . The significance level was set at p < 0.05.All experiments were performed as technical and biological triplicates. Permeation experiments were performed in quintuplicates. Data were analyzed considering the analysis of variance and using GraphPad PrismOleic acid was identified as the principal constituent of the Cupuaçu seed extract, comprising 41.81% of the extracted content. The second main constituent is stearic acid (35.79%), followed by palmitic acid (8.67%), as shown in p < 0.05). To the best of our knowledge, such biotechnological potential of the frequently discarded Cupuaçu seed is demonstrated here for the first time, aggregating value to its production chain.The skin wound healing process includes an inflammatory response, the formation of granulation tissue, and matrix remodeling. In this context, several different cellular processes must occur in a controlled manner to guarantee optimal wound healing. Among such processes, cellular proliferation, migration, and contractility have been pointed out as critical events for regeneration, cell proliferation and migration necessary to populate the wound bed, and cell contractility contributing to wound closure . Fibrobl®). As depicted in p < 0.05). Therefore, it is possible to suggest that the Cupuaçu extract holds promising skin regenerative potential for treating different skin lesions, including burns.Cell proliferation and cell migration are relevant events for the tissue regeneration process, especially in the context of extensive wound healing, in which it is imperative to prevent water loss and tissue infection. Currently, most treatment regimens in this context focus on preventing infection and protecting the lesion bed with dressings containing expensive cell proliferation and migration-inducing recombinant factors, such as PDGF , which corroborate the observations made in the proliferation assay and support the notion that Cupuaçu extract might contribute to the reestablishment of a functional skin ECM that supports skin elasticity [Experimental results showed that, after 48 h treatment of dermal fibroblasts with Cupuaçu extract, it was possible to observe a significant induction of mRNA expression of MKI67, Elastin, and HAS2 . Although the wound healing potential of the Cupuaçu seed extract could be demonstrated in cell culture experiments, the actual wound healing therapeutic potential can only be fully explored if the phytotherapeutic can reach the target cells from a safe topical formulation. Therefore, when evaluating the skin penetration of the molecules of interest, considering their purpose, i.e., to promote wound healing to damaged skin tissue, the state of the skin is a crucial factor. For this, the penetration of the Cupuaçu biomarker  was evaluated in two skin models, intact and burnt skin .2) (p < 0.05) when compared to NC (9.3 ± 0.3 μg/cm2). However, such a penetration enhancing effect is not observed in burnt skin samples, in which there were no differences in penetration amount (~15.0 μg/cm2) despite the coating (p > 0.05). These are entirely plausible results when considering the mechanism by which chitosan promotes skin penetration, i.e., chitosan acts by transiently widening the tight junctions between cells [Results demonstrated, as expected, that the chitosan coating almost doubled the oleic acid penetration into the intact skin (21.5 ± 3.6 μg/cmen cells . Togetheen cells ,45. Besien cells . Hence, en cells  and evenen cells  that couIn the present study, we have demonstrated, for the first time, the potential biotechnological value of Cupuaçu almonds, which are often discarded as a food industry waste, as a promising pharmaceutical ingredient for the development of skin regenerative products. Cupuaçu seed extract showed to be non-toxic and to induce cell proliferation, migration, and a reparative gene expression profile in primary human dermal fibroblast cultures. Furthermore, when incorporated in a nanoparticulated system, the extract was non-irritant. Additionally, chitosan coating the nanocapsules promotes active penetration into undamaged skin areas, which are still covered by the stratum corneum. Hence, chitosan-coated nanocapsules loaded with Cupuaçu seed extract represent promising delivery systems for skin regeneration.


Leishmania infantum is a protozoan parasite that causes a vector borne infectious disease in humans known as visceral leishmaniasis (VL). This pathology, also caused by L. donovani, presently impacts the health of 500,000 people worldwide, and is treated with outdated anti-parasitic drugs that suffer from poor treatment regimens, severe side effects, high cost and/or emergence of resistant parasites. In previous works we have disclosed the anti-Leishmania activity of (-)-Epigallocatechin 3-O-gallate (EGCG), a flavonoid compound present in green tea leaves. To date, the mechanism of action of EGCG against Leishmania remains unknown. This work aims to shed new light into the leishmanicidal mode of action of EGCG. Towards this goal, we first confirmed that EGCG inhibits L. infantum promastigote proliferation in a concentration-dependent manner. Second, we established that the leishmanicidal effect of EGCG was associated with i) mitochondria depolarization and ii) decreased concentration of intracellular ATP, and iii) increased concentration of intracellular H2O2. Third, we found that the leishmanicidal effect and the elevated H2O2 levels induced by of EGCG can be abolished by PEG-catalase, strongly suggesting that this flavonoid kills L. infantum promastigotes by disturbing their intracellular redox balance. Finally, we gathered in silico and in vitro evidence that EGCG binds to trypanothione reductase (TR), a central enzyme of the redox homeostasis of Leishmania, acting as a competitive inhibitor of its trypanothione substrate. Leishmania infantum and L. donovani that affects 500,000 people and is fatal in over 95% of cases if left untreated. It is estimated that 50,000 to 90,000 new VL cases occur worldwide each year  is a neglected tropical disease caused by ach year . The disach year . In the ach year . The intach year . Neverthach year  renderinach year .O-gallate (EGCG), the most abundant flavonoid constituent of green tea, was recently demonstrated to display activity against different species of Leishmania, including visceral leishmaniasis species .in vitro, against intracellular amastigotes with a selectivity index over 100, and in vivo, in a murine model of infection also without evident toxicity towards treated animals 2] at expenses of NADPH. Trypanothione is a dithiol, composed of two molecules of glutathione linked by one spermidine bridge. It is the major thiol in trypanosomatids, where it acts as electron donor for a multitude of biological reactions (including the elimination of hydroperoxides), and thus assumes the functions of glutathione in other organisms. The recycling of trypanothione back to its reduced, active form is carried out by the enzyme trypanothione reductase (TR). This process is absolutely critical for parasite redox balance and overall cell viability, as demonstrated by the essential character of TR , trypanothione, JC-1, horseradish peroxidase (HRP), digitonin, NADP+, NADPH, DTNB, FCCP, HEPES, Schneider’s Drosophila medium, fetal calf serum and RPMI 1640 medium were obtained from Sigma Aldrich . Amplex Red, and alamarBlue were obtained from Invitrogen Molecular Probes . Other reagents were purchased from Merck . Deionized distilled water was obtained using a Milli-Q system of resins  and was used to prepare all solutions.(−)-Epigallocatechin 3-Leishmania infantum was used throughout this study. Leishmania infantum amastigotes were isolated from BALB/c mice and maintained as promastigotes at 26°C in Schneider’s medium supplemented with 20% fetal bovine serum (v/v), 100 U/ml penicillin and 100 μg/ml streptomycin. Parasite maintenance was achieved by passaging every 3 days. Female BALB/c mice (8−10 weeks) were provided by the Instituto Ciências e Tecnologia em Biomodelos (ICTB/FIOCRUZ). All animals were bred and maintained at the Instituto Oswaldo Cruz according to Guide for the Care and Use of Laboratory Animals of the Brazilian National Council for Control of Animal Experimentation (CONCEA). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Instituto Oswaldo Cruz .The MHOM/MA/67/ITMAP263 strain of L. infantum (106/mL) promastigotes were incubated with different concentrations of EGCG (0.015–1 mM) or vehicle (PBS) for 72 h. The cell density was estimated with the addition of alamarBlue (10% v/v) for 3 h at 26°C. The absorbance was measured at 570 nm using a spectrophotometer. The growth curve was initiated with 1 x 106 cells/ml. The 50% inhibitory concentration (IC50) was determined by logarithmic regression analysis using GraphPad Prism 6 . The experiments were run three times in triplicate.7 cells/mL) were incubated with HBSS containing 10 μM Amplex Red reagent, 64 μM digitonin (to permeabilize parasites) and 10 U/ml HRP for 30 min at 26°C. Fluorescence was monitored at excitation and emission wavelengths of 560 and 590 nm, respectively, in a spectrofluorometer. H2O2 concentrations were calculated from an H2O2 standard curve.Hydrogen peroxide production was measured using Amplex Red and horseradish peroxidase (HRP) . Promastm) as described previously (6 cells/ml) were cultured for 72 h in the absence or presence of EGCG (125 – 500 µM). Cells were harvested and resuspended in HBSS. The cell number was obtained by counting in a Neubauer chamber. Promastigotes (107 cells/ml) were incubated with JC-1 (10 μg/ml) for 10 minutes at 37°C. After washing twice with HBSS, fluorescence was measured using a spectrofluorometer at 530 nm and 590 nm using an excitation wavelength of 480 nm. The ratio of the values obtained at 590 nm and 530 nm was plotted as the relative ΔΨm. The mitochondrial uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone  was used as control.The cationic probe JC-1 was used to determine the mitochondrial membrane potential (ΔΨeviously . PromastL. infantum promastigotes were measured using a CellTiter-Glo luminescent assay (Promega), where the signal was proportional to ATP concentration. Briefly, promastigotes were treated for 72 h in the absence or presence of EGCG (125 – 500 μM). Cultures were washed three times, and parasite concentration was adjusted to 107 cells in 200 μl of PBS. A 50-µl aliquot of each sample was transferred to a 96-well plate and mixed with the same volume of CellTiter-Glo. Plates were incubated in the dark for 10 min, and bioluminescence was measured using a GloMax-Multi Microplate Multimode Reader (Promega). ATP concentrations were calculated from the ATP standard curve.Intracellular ATP concentrations in www.rcsb.org) (www.nbcr-222.ucsd.edu) using the AMBER forcefield, and the protonation state was determined using PROKA at pH 7.4. All proteins were converted to PDBQT format using Autodock Tools. A cubic grid map with 80 Å of side and spacing of 0.375 Å was built centralized on active site residues  of TR on both states (oxidized and reduced) using Autodock Tools.  were downloaded from the RCSB Protein DataBank (csb.org) . The atok Tools. .L. infantum TR was performed with AutoDock (The docking of EGCG to AutoDock . The griAutoDock . Grid boAutoDock .The docking procedure was performed using 500 runs of Lamarckian Genetic Algorithm (LGA). Ligand binding position and interaction analysis were performed using PyMOL software and the 2D interaction was visualized with BIOVIA Discovery Studio 2020 .LiTR (tritryp ID: LINF_050008500) was PCR-amplified from the genomic DNA of L. infantum and cloned into the NdeI/XhoI sites of the pET23a(+) plasmid. The resulting construct was introduced in Escherichia coli BL21(DE3) for expression of a modified version of LiTR containing a carboxy-terminal six-histidine tag (LiTR.6His), as follows. Bacteria were grown at 37 °C, in Luria Bertani (LB) medium supplemented with 50 μg/ml ampicillin and 0.1% (w/v) glucose, until an OD600nm of 0.6. LiTR.6His expression was induced by addition of 2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and further incubation at 37 °C, for 4 h. Bacteria were pelleted and suspended in 20 mM Tris-HCl pH7.6, 500 mM NaCl, 10 mM imidazole, at a ratio of 1 g wet weight per 5 ml buffer, followed by disruption by sonication and centrifugation at 30,000 ×g, for 20 min, at 4 °C. LiTR.6His was obtained as a soluble protein in the supernatant of lysed bacteria and purified by passage through a 1 ml HisTrap column , followed by elution in a 25-500 mM imidazole gradient, at 4 °C and a flow rate of 0.5 ml/min, using an ÄKTAprime plus equipment . Fractions confirmed by SDS-PAGE to contain LiTR.6His were pooled, passed through a PD-10 desalting column packed with Sephadex G-25 resin , and recovered in 50 mM HEPES pH 7.6, 1 mM EDTA. LiTR.6His quality and purity was confirmed by SDS-PAGE, and its concentration estimated using the bicinchoninic acid assay (Pierce) with bovine serum albumin as standard.Full-length 2] by TR is coupled with the chemical oxidant 5,5’-dithiobis-(2-nitrobenzoic acid)  , 0.1 mM DTNB, 0.15 mM NADPH and increasing concentrations of EGCG (7.8 – 500 µM). Reactions were started by addition of 7 nM recombinant LiTR.6His , purified from Escherichia coli by nickel chromatography and quantified by the bicinchoninic acid (BCA) protein assay (Pierce), using bovine serum albumin (BSA) as standard. Upon addition of LiTR.6His, the rate of TNB formation was monitored at 410 nm at 25°C for 5 minutes using a UV-2401PC UV–vis recording spectrophotometer (Shimadzu Scientific).This assay, in which generation of reduced trypanothione : i) the cluster with lowest energy, and ii) the cluster containing the most prevalent conformation.Our observation that EGCG triggers a Hnothione . Moleculox or TRred, respectively, which translated into theoretical iK values of 3.85 and 6.40 µM  values of -7.39 or -7.09 kcal/mol between EGCG and TR 6.40 µM Table 1.in silico models.From this analysis, we obtained theoretical support to our premise that EGCG interacts with TR. To gain insight into the binding site of EGCG within the TR quaternary structure, we moved on to carry out a detailed structural analysis of EGCG/TR in silico structural analysis of EGCG/TR interaction, we wondered which of these TR sites are preferentially targeted by EGCG. To assess this, we looked into high confidence molecular models of EGCG interaction with both TRox and TRred, taking into account both the lowest energy and the most prevalent clusters  EGCG interaction with residues of the Z-site , purified from E. coli. Our assay consisted in monitoring TR activity based on the DTNB-based colorimetric test originally described by Hamilton et al. (2 to T(SH)2, and re-oxidation of the T(SH)2 product back to TS2 coupled to reduction of DTNB, forming a yellow thionitrobenzoate (TNB) ion. This method maintains a constant substrate concentration, saving on reagents, and enables the linearity of the assay for at least 60 min.To provide experimental demonstration to the n et al. . Briefly50 of EGCG against LiTR.6His is 193.8 µM.We started by testing the impact that increasing concentrations of EGCG have on TR activity. The results, plotted in LiTR.6His and varying concentrations of EGCG  and trypanothione (2 - 60 µM). The velocities estimated for these set of reactions were analyzed in a Lineweaver-Burk plot (1/V vs. 1/[T(SH)2]) across the varying concentrations of the inhibitor   .Next, we moved on to characterize the type of inhibition that EGCG exerts over TR. This was done by performing a set of reactions with a fixed amount of nhibitor Figure 6L. infantum and L. donovani parasites, against which new therapeutic formulas are in urgent demand to overcome the limitations of the currently-in-use, leishmanicidal drugs. Natural products, with their enormous chemical diversity, are a major resource for the discovery and development of novel therapeutics , the most abundant flavanol constituent of green tea (Camellia sinensis (L.) Kuntze; Theaceae), is widely studied and has generated considerable interest as a biologically active compound with a wide range of potential therapeutic activities ] -Epitivities . In a pr(167.8)] . The preLeishmania species  is a central molecule of the H2O2 metabolism in Leishmania spp., we hypothesized that the EGCG-triggered boost of H2O2 resulted from TR inhibition. Our hypothesis was validated by 1) in silico analyses, where we found evidence that EGCG can bind to TR with iK values spanning between 3.85 and 20.49 µM . High confidence models of EGCG/TR interaction predicted that the flavonoid could act as a competitor for the trypanothione substrate of TR by occupation of its binding pocket. Importantly, these predictions were confirmed by 2) in vitro kinetic assays of purified recombinant TR in the presence of EGCG, revealing a Ki of 293.9 ± 66.3 μM, this Ki value is higher when compared with other TR inhibitor such trivalent antimony (Sb+3), clomipramine and chalcone-based compounds (L. infantum promastigote proliferation and both EGCG-induced TR inhibition (Pearson correlation r = 0.9521) and increased intracellular H2O2 concentration in L. infantum promastigotes (Pearson correlation r = 0.9476)These parasite forms then set the basis for us to characterize the physiological alterations imposed by EGCG on ompounds . PositivLeishmania spp. and related trypanosomatids, EGCG impairs the antioxidant response of the parasites, leading to the development of oxidative stress and resulting in the destruction of cellular macromolecular components promoting parasite death. This mechanism is also the basis of various antiprotozoal medications used to combat parasites in infected cells .Conceptualization: JI and EA-A. Data curation: JI, MF, GL-S, HC, and EA-A. Formal analysis: JI, MF, GL-S, AT, HC, and EA-A. Investigation: JI, GL-S, AT, HC, and EA-A. Methodology: JI, MF, GL-S, AT, HC, and EA-A. Writing – original draft: JI, AT, HC, and EA-A. Writing – review and editing, JI, GL-S, AT, HC, and EA-A. All authors contributed to the article and approved the submitted version.This work was supported by Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Programa Estratégico de Apoio a Pesquisa em Saúde (PAPES/FIOCRUZ);, and Fundação Oswaldo Cruz (FIOCRUZ). EA-A is the recipient of a research scholarship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). JI was supported by a postdoctoral fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). JI is a recipient of a postdoctoral fellowship from Fundação Oswaldo Cruz (FIOCRUZ). HC is funded by Fundação para a Ciência e Tecnologia  through the “Investigador FCT” contract IF/01244/2015.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.


UA). Further studies on FEUA in hypouricemic individuals are needed for a more accurate diagnosis of RHUC. Methods: In 30,685 Japanese health-examination participants, we genotyped the two most common nonfunctional variants of URAT1 (NFV-URAT1), W258X (rs121907892) and R90H (rs121907896), in 1040 hypouricemic individuals (SUA ≤ 3.0 mg/dL) and 2240 individuals with FEUA data. The effects of NFV-URAT1 on FEUA and SUA were also investigated using linear and multiple regression analyses. Results: Frequency of hypouricemic individuals (SUA ≤ 3.0 mg/dL) was 0.97%  and 6.94%  among 30,685 participants. High frequencies of those having at least one allele of NFV-URAT1 were observed in 1040 hypouricemic individuals. Furthermore, NFV-URAT1 significantly increased FEUA and decreased SUA, enabling FEUA and SUA levels to be estimated. Conversely, FEUA and SUA data of hypouricemic individuals are revealed to be useful to predict the number of NFV-URAT1. Conclusions: Our findings reveal that specific patterns of FEUA and SUA data assist with predicting the number of nonfunctional variants of causative genes for RHUC, and can also be useful for practical diagnosis of RHUC even before genetic tests.Background: Renal hypouricemia (RHUC) is characterized by a low serum uric acid (SUA) level and high fractional excretion of uric acid (FE UA). Although most hypouricemia patients are normally asymptomatic and are found by chance in health examinations, RHUC is sometimes accompanied by severe complications, such as exercise-induced acute kidney injury (EIAKI) and urolithiasis /[SUA (mg/dL) × urinary creatinine (mg/dL)]  .URAT1, we genotyped the two most common variants (W258X and R90H). We used custom TaqMan assay probes designed for R90H, VIC-CCGCCACTTCCGC and FAM-CGCCGCTTCCGC, and for W258X, VIC-CGGGACTGAACACTG and FAM-CGGGACTGGACACTG. Direct sequencing was performed with a 3130xl Genetic Analyzer (Life Technologies) to confirm all the heterozygotes and homozygotes of NFV-URAT1, using the following primers [The genomic DNA of each participant was extracted from whole peripheral blood cells. For genotyping, we performed the TaqMan method  using a LightCycler 480 , as described previously . For NFV primers : for R90URAT1 on FEUA or SUA. We also carried out multiple regression analysis in a stepwise method using the following equation: y  =  β0  +   β1x1  +  β2x2, where y is FEUA or SUA levels, x1 is a dummy variable representing whether the number of alleles of NFV-URAT1 is one , x2 is a dummy variable representing whether the number of alleles of NFV-URAT1 is two  and β0, β1 and β2 are partial regression coefficients for each covariate. We used SPSS v. 22  for all calculations in the statistical analyses carried out in this study.Linear regression analyses were performed to evaluate the influence of the allele of NFV-URAT1. The characteristics of these hypouricemic individuals (SUA ≤ 3.0 mg/dL) are shown in URAT1 and hypouricemic populations. As shown here, those with two NFV-URAT1 alleles were seen only in severe hypouricemia in both sexes. For mild hypouricemia, the largest population was males with one NFV-URAT1 allele, although it was those with 0 alleles in females. Of the mild hypouricemia individuals, at least two thirds of the males and one third of the females were assumed to be the “mild” RHUC type 1 due to having only one NFV-URAT1 allele.As displayed in URAT1 and FEUA or SUA in 2240 Japanese individuals  and decreased SUA . The mean FEUA levels of those with 0, 1 and 2 alleles for NFV-URAT1 were 3.94% ± 0.06%, 6.57% ± 0.39% and 42.6% ± 12.8% in males, and 5.37% ± 0.10%, 6.43% ± 0.67% and 45.9% ± 3.81% in females, respectively (URAT1 were 6.10  ±  0.03 (mg/dL), 4.17 ± 0.11 (mg/dL) and 0.75 ± 0.04 (mg/dL) in males, and in females were 4.56 ±  0.04 (mg/dL), 3.31 ± 0.19 (mg/dL) and 0.65 ± 0.11 (mg/dL), respectively  among those 2240 individuals whose FEUA data were available  and extremely low SUA levels of ≤1.0 mg/dL (severe hypouricemia). On the other hand, the other 48 individuals  with one or no NFV-URAT1 alleles displayed a mean FEUA level of 8.01% (range: 2.05–16.86%). Most of them were mild hypouricemia  markedly elevated FEUA in both sexes by approximately 40% . One allele of NFV-URAT1 (β1) gave significantly elevated FEUA in males , but the variance of one allele of NFV-URAT1 was eliminated for females in this multiple regression analysis. Conversely, two alleles of NFV-URAT1 (β2) markedly reduced SUA levels . One allele of NFV-URAT1 (β1) also significantly reduced SUA levels . In other words, these results indicate that FEUA and SUA levels can be estimated from the genotyping results of NFV-URAT1 (W258X and R90H), and, vice versa, from the clinical data (FEUA and SUA levels), we can predict the presence and number of NFV-URAT1, which can reveal whether or not a patient is RHUC type 1.URAT1 in hypouricemic individuals  and mild hypouricemia (2.0 < SUA ≤ 3.0 mg/dL) in a general Japanese population , the freividuals  and the  and SUA ; Table 5The prevalence of hypouricemia (SUA ≤ 2.0 mg/dL) was 0.18% for males and 0.54% for females in the present study , which iURAT1 is high in Japanese [URAT1 (W258X and R90H) in the general population of hypouricemia and mild hypouricemia individuals (SUA ≤ 3.0 mg/dL).Although the frequency of NFV-Japanese ,15, thisURAT1  are observed among hypouricemia , as the clinical practice guideline for RHUC recommends in its clinical algorithm [URAT1 alleles (W258X and R90H). Further studies to identify other known dysfunctional variants [URAT1 to be able to more accurately elucidate the frequency of RHUC type 1 in hypouricemic individuals (SUA ≤ 3.0 mg/dL).High frequencies of NFV-lgorithm . This stvariants ,18,19,20URAT1 significantly increases FEUA and decreases SUA, using 2240 individuals whose FEUA data were available . We also suggest that it is possible to predict the presence and number of NFV-URAT1 alleles from laboratory data (FEUA and SUA levels). Of 52 hypouricemic individuals, 4 individuals  menopausal status was not considered, and (2) the influence of environmental factors such as alcohol intake and medications were not adjusted. Further analyses will be necessary to elucidate the effects of these factors.UA and SUA data   (GLUT9 (UA data are not high and urinary uric acid (UA) levels are nearly zero in severe hypouricemia patients    25–90%) c, these  25–90%) h because) (URAT1 f. (2) Wh > 100%) d, these  > 100%) i because) (GLUT9 g. 3) Whpatients e, they apatients j. With mpatients k, their ; 5–15%) l, and thor GLUT9 m. Detect9 , the third and recently reported renal urate reabsorption transporter [As shown in nsporter , at the UA and SUA data. A more accurate diagnosis of RHUC will not only enable clinicians to prevent complications of RHUC such as EIAKI and urolithiasis, but will also lead us to a better understanding of the mechanism of urate handling and hypouricemia.We believe that our findings will assist with a more practical diagnosis of RHUC based on the specific distribution patterns of FEURAT1 (W258X and R90H) in 1040 hypouricemia and mild hypouricemia individuals (SUA ≤ 3.0 mg/dL). Third, the presence and number of NFV-URAT1 alleles assists with estimating the FEUA data of hypouricemic individuals (SUA ≤ 3.0 mg/dL). This suggests that the FEUA data of hypouricemic individuals should be very useful for predicting the presence and number of NFV-URAT1 alleles, and also assists with diagnosis of RHUC type 1. Fourth, we were able to propose how to make a more reliable diagnosis of RHUC based on the distribution patterns of FEUA and SUA data.In summary, we have demonstrated four important findings. First, we investigated the prevalence of hypouricemia (SUA ≤ 2.0 mg/dL) and mild hypouricemia individuals (2.0 < SUA ≤ 3.0 mg/dL) among 30,685 Japanese participants, and discovered the prevalence of hypouricemic individuals (SUA ≤ 3.0 mg/dL) to be 0.97% in males and 6.94% in females. Second, we revealed a very high frequency of NFV-These findings have the potential to lead to a more practical diagnosis of RHUC based on specific patterns of laboratory data, and therefore a revision of the next edition of the clinical practice guideline for RHUC.


The optimum progressive motility of boar spermatozoa was observed in extender with 0.5% and 0.6% BH addition. Afterward, sperm quality parameters, bacterial profile and composition, total antioxidant (T-AOC), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) levels of control, BH supplementation, antibiotics supplementation, and incorporated supplementation were compared during liquid storage period, to further investigate antibacterial and antioxidant properties of BH. The results showed that BH supplementation significantly improved sperm motility, acrosome integrity, plasma membrane integrity, inhibited opportunistic bacterial growth, and altered microbial compositions at the end of preservation. Additionally, T-AOC, SOD, and CAT levels were significantly higher in the BH supplementation group than those in the control and antibiotic supplementation group, whereas MDA level exhibited opposite change pattern. Importantly, BH addition to the extender was able to exert a synergistic effect in combination of antibiotic use. Our findings suggested that the appropriate concentrations (0.5% and 0.6%) of BH were added to the extender could act antibacterial and antioxidant roles in liquid preservation of boar semen.In the present study, we hypothesized that buckwheat honey (BH) should be regarded as a potential alternative to antibacterial and antioxidant agent in liquid storage of boar semen. To this end, boar semen was firstly studied for  Artificial insemination (AI) is the most important biotechnological application in swine reproduction which brings about rapid genetic improvement in pigs by spreading the use of males with high productive characteristics . The semin vitro tolerability of boar sperm to BH was determined. In addition, sperm motility, bacterial concentrations, bacterial compositions, acrosome integrity, plasma membrane integrity, total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD), and micromalondialdehyde (MDA) levels in different extenders with or without BH addition during liquid storage period were investigated. The aim of our study was to evaluate the antibacterial and antioxidant effects of BH on boar sperm liquid preservation at 17°C, which would provide a basic knowledge of application of BH as an alternative to antibiotic and antioxidant agent in the extender.In this study, we first introduced buckwheat honey (BH) into the extender, and Natural pure buckwheat honey (BH) was harvested using the sterilized container in beekeeping cooperative in Inner Mongolia province, China, according to standard quality GH/T 18796-2012. The major physicochemical parameters of BH including total sugar, moisture content, pH, and color were verified according to the national standard method  in College of Bee Science of Fujian Agriculture and Forestry University Table .ad libitum. Before semen collection, the penis was disinfected using a sponge soaked with 75% ethanol, and all collection wares were sterilized. Three semen ejaculates were collected from each boar by using gloved hand technique. Gelatinous protein was filtered by double layers of sterile gauze to obtain the sperm-rich fraction of the ejaculate. Only semen samples with volume ≥ 100 ml, concentration ≥ 0.5 billion sperm/mL, and motility ≥ 70% were retained. To minimize the effects of individual differences, a 50 mL qualified semen sample from each boar was pooled for further experimental analysis (two pools). Semen samples were stored at 17°C for transportation. All animal experiments were conducted according to the guidelines for the care and use of experimental animals issued by the Ministry of Agriculture and Rural Affairs of China. The project was approved by Animal Care and Use Committee (ACUC) in Fujian Agriculture and Forestry University.Six Landrace boars were raised in Yongcheng Agriculture Animal Husbandry Technology Co. . The commercial formula feeds and water were offered v/v) at 0, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1%, the osmolarity of each group was shown in Table To evaluate tolerability of boar semen to different concentrations of BH, pooled semen samples were aliquoted and diluted to a final concentration of 45 million sperm/mL in the sodium citrate buffer  which containing BH (Beltsville thawing solution (BTS) was prepared by dissolving  in 1 L distilled water. Experimental extenders were prepared as follows: control (BTS), E1 (BTS +0.6 g penicillin and 1 g streptomycin), E2 (BTS+0.25% BH +0.3 g penicillin and 0.5 g streptomycin), E3 (BTS+0.3% BH+0.3 g penicillin and 0.5 g streptomycin), E4 (BTS+0.5% BH), and E5 (BTS+0.6% BH). Pooled semen samples were aliquoted and diluted (45 million sperm/mL) with different experimental extenders. During liquid storage at 17°C within 1-5 days, sperm motility, plasma membrane integrity, acrosomal integrity, bacterial concentration, bacterial compositions, T-AOC, CAT, SOD, and MDA levels were measured.μL) of semen sample was injected into a counting chamber. For three replicates of each sample, at least 250 spermatozoa in five separate and random fields were analyzed using a computer-assisted semen analysis (CASA) system (HT CASA II Version 3.0). The setting parameters of CASA were described as follows: temperature was 37°C; minimum contrast was 45; both low and medium velocity average pathway (VAP) cut-off were 30; 60 frames/s; straightness threshold was 30%.The percentages of total motility spermatozoa (TM) and progressive motility spermatozoa (PM) were assessed by using a modified protocol according to Bucci et al. . In brieμL of semen sample was mixed with 1 mL hypo-osmotic solution , and the mixture was incubated at 37°C for 30 min. Then, place a drop (10 μL) of mixture onto a microscopic slide which covered with a coverslip. The count of spermatozoa with swollen tails out of at least 200 sperm in each field was recorded by a phase-contrast microscope at 400× magnification.The hypo-osmotic swelling test (HOST) method was used to evaluate the membrane integrity of the sperm as previously described by Ramu and Jeyendran . BrieflyμL Wright's-Giemsa solution was added on a prepared sperm smear and incubated at room temperature for 2 min. 1.8 mL phosphate-buffered saline (PBS) was thoroughly mixed with Wright's-Giemsa and hold for 10 min. After that, the slide was rinsed with running distilled water and naturally air-dried. The stained sperm smear was randomly selected, and at least 200 sperms in five different fields of each slide were measured under a phase-contrast microscope at 1000× magnification.Sperm acrosomal membrane integrity was tested by using modified Wright's-Giemsa staining assay . 600 μL The bacterial concentrations and compositions were investigated to evaluate the antibacterial effects of BH and antibiotics in diluted boar sperm as previously described by Shaoyong et al. . In brieThe SOD, T-AOC, CAT, and MDA levels of 1, 3, and 5 days preserved semen samples were determined by antioxidant assay kits . The extracts of sperm were prepared using Triton X-100 (1%) by centrifuging at 5000 × g for 10 min. Supernatants were collected and mixed with corresponding reaction buffer from different kits. Subsequently, SOD, T-AOC, CAT, and MDA levels were measured by a spectrophotometer (UNICO UV-2100) at 560, 520, 520, and 532 nm wavelengths, respectively. Every parameter of each group was measured for three times. Due to Raoultella, bacteria are able to produce catalase, and to explore the source of Raoultella in the E3 group, the CAT activity of extender E3 which with or without semen addition was measured.p < 0.05.Data processing and analysis were accomplished by Statistical Package for the Social Sciences software, version 25 . Shapiro–Wilk test and Levene test were performed to analyze normality of data and homogeneity of variances, respectively. Normalization of data was achieved by log2 transformation. The effects of BH and different experimental extenders on sperm motility, plasma membrane integrity, acrosomal integrity, bacterial concentration, bacterial compositions, T-AOC, CAT, SOD, and MDA levels were detected by one-way ANOVA. Multiple comparisons were performed by using Duncan's method and significance threshold was set at p < 0.05). Interestingly, the highest sperm progressive motility was observed when supplemented with 0.5% and 0.6% of in comparison with the control group.As shown in p < 0.05).Effects of BH and antibiotics supplementation on sperm motility are shown in p < 0.05).The morphological characteristics of sperm after the hypo-osmotic swelling test (HOST) were shown in Figure p < 0.05, p < 0.05) in both E2 and E3  compared with extender E1 (without BH supplementation). When comparing both extenders without antibiotics, acrosome integrity was significantly higher (p < 0.05) in E4 (with 0.5% BH) than E5 (with 0.6% BH).The morphological characteristics of sperm after boar semen stained with Wright's-Giemsa solution were shown in Figure p < 0.05) in extender E2 (1.02 ± 0.10), E3 (1.53 ± 0.16), E4 (1.33 ± 0.07), and E5 (0.91 ± 0.07) compared with extender control (0.61 ± 0.07) and E1 (0.65 ± 0.13d). The CAT and SOD levels in all extenders showed the similar change pattern with T-AOC, while MDA level that increased with the preservation time and lower values was observed in extender E2 (8.74 ± 0.08), E3 (7.76 ± 0.12), E4 (7.60 ± 0.03), and E5 (8.99 ± 0.16) than extender control (9.45 ± 0.08) and E1 (9.38 ± 0.08) at the end of preservation.The antioxidant parameters of semen samples including T-AOC, CAT, SOD, and MDA levels were determined on days 1, 3, and 5 of preservation . Interes3 ± 2.95 × 103 CFU/ML) followed by E4 (2.53 × 104 ± 1.30 × 104 CFU/ML), E2 (1.10 × 105 ± 5.78 × 104 CFU/ML), E5 (3.30 × 105 ± 9.48 × 104 CFU/ML), E1 (7.88 × 105 ± 4.37 × 105 CFU/ML), and control (8.15 × 106 ± 4.09 × 106 CFU/ML).As shown in p > 0.05, Table p < 0.05, Table To further investigate the bacterial composition in the semen samples at the end of preservation, we performed 16S rRNA gene sequencing. At the phylum level, Proteobacteria, Firmicutes, and Actinobacteria were the most dominant phyla in the microbial communities of all preserved semen . AlthougAdditionally, LEfSe analysis was performed to detect more differential enriched microbial taxa by using relative abundances of OTUs. Fourteen differential enriched OTUs were identified, which consist of nine OTUs enriched in control, one OTU augmented in E1, and two OTUs more abundant in each of E2 and E3, respectively . These ONowadays, antibiotics are routinely used in boar semen liquid storage to inhibit bacterial growth and to improve semen quality. However, the extensive use of antibiotics clearly drives the evolution of antibiotic resistance. Accordingly, it is necessary to identify appropriate substitutes for antibiotics. On the other hand, ROS is generated by sperm during the process of in vitro preservation, which results in oxidative stress in the sperm plasma membrane and functional impairment of the sperm cells. Thus, antioxidant agent supplementation to decrease the impact of different oxidants and protect the sperm cells from oxidative damage is also crucial for the successful liquid preservation of boar semen. In this study, the antibacterial and antioxidant effects of buckwheat honey (BH) were firstly evaluated during boar semen liquid storage to pave the way for using BH as an alternative to antibiotics and antioxidant agents.It is well known that the suitable levels of supplements for boar semen preservation are very important . Thus, tSperm quality is regarded as one of the most important indices to assess the efficiency of semen preservation. To compare BH and antibiotics affect sperm quality, sperm motility, plasma membrane integrity, and acrosome integrity were evaluated during liquid preservation. We found that extender only with BH supplementation had better protective effects on all sperm quality parameters than that only with antibiotics supplementation. Additionally, we also observed the beneficial effects of the incorporation of BH in combination with antibiotics on sperm quality parameter improvement. These findings suggested that BH could maintain sperm quality alone or exert a synergistic effect with the use of antibiotics. Although antibiotics are essential components of semen extenders for the control of bacterial contamination and growth, inflict negative impacts on sperm quality have been reported , 19. RecAntioxidant characteristic of boar semen intimately related to sperm quality has been noted in previous studies. Pipan et al. found that SOD level was significantly associated with progressive motility and viability during short-term preservation and suggested that SOD in fresh boar semen can be used as a predictor of semen quality after storage . Gloria While performing sperm preservation, the scope is not only to improve sperm antioxidant capacities but also to control bacterial growth simultaneously. Therefore, bacterial concentrations and compositions were compared between BH and antibiotics supplemented extenders to evaluate the antibacterial roles in semen preservation. At the end of preservation, the bacterial concentrations were higher in the antibiotics group than those in the BH group, and the lowest bacterial concentration was observed in the BH and antibiotic incorporated group. BH maintained bacterial concentrations at low level can be attributed to hydrogen peroxide (byproduct of glucose oxidation), bee peptides, lysozyme, phenolic acids, flavonoids, and other nonperoxide bacterial inhibitory components , 31. Flap < 0.001). This finding implied that Raoultella derived from boar semen was nourished by BH which may affect sperm antioxidant capacities by catalase secretion. However, further investigations should be performed to verify this speculation.With regard to bacterial compositions, we found that the phyla Proteobacteria, Firmicutes, Actinobacteria, genera Raoultella, Stenotrophomonas, and Finegoldia were the most dominant microbial taxa. Although we did not observe any major differences in relative abundances of the predominant phyla, the significantly higher relative abundances of Raoultella, Finegoldia, and Stenotrophomonas were found in extender E3, E2, and control, respectively. The genus Raoultella is a gram-negative bacterium which has been isolated from humans, animals, and natural environments , 35. UnsAt the OTU level, 14 OTUs showed significantly different abundances among different extenders. Among these, OTUs annotated to the genus Stenotrophomonas, Blastococcus, Sphingomonas, Massilia, Roseiarcus, and Enterobacter were more abundant in the control group. Most members of these genera are antibiotic-resistant bacteria and detrimental to sperm quality. For example, bacteria from genus Sphingomonas and Stenotrophomonas were frequently detected in bovine semen exhibited a wide range of antimicrobial resistance phenotypes which further impaired sperm motility and suppressed early embryonic development , 40. SpeOur study is limited by small sample size but provides important evidence that incorporation of BH in extender could improve sperm quality, enhance seminal antioxidant capacities, and inhibit the growth of detrimental bacteria. Nevertheless, future studies aiming to elucidate the underlying mechanisms are needed.Our results indicated that a suitable concentration (0.5% and 0.6%) of BH as added to extender exert antibacterial and antioxidant roles in boar semen liquid preservation, which highlighted BH should be regarded as a potential substitute for antibiotics and antioxidant agent.