Rats with chronic hypoxia-induced non-inflammatory pulmonary hypertension (PH) are resistant to ventilator-induced lung injury. We investigated the effect of high tidal volume ventilation in another model of PH, monocrotaline (MCT)-induced PH, which is a type of inflammatory PH.2) and Evans blue dye (EBD) extravasation were measured. Hypertensive pulmonary vascular remodeling was assessed morphometrically by the percentage of muscularized peripheral pulmonary arteries (%Muscularization) and the media wall thickness to external diameter ratio, namely percentage medial wall thickness (%MWT). To assess inflammation, lung IκB protein and cytokine mRNA expression levels were assessed.PH was induced in rats by subcutaneous injection with 60 mg/kg MCT. Normal control rats, rats at 2 weeks after MCT injection (MCT2), and rats at 3 weeks after MCT injection (MCT3) were ventilated with low tidal volume  or high tidal volume  for 2 h with room air without positive end-expiratory pressure. Arterial oxygen pressure , and IL-10 mRNA levels in MCT3 rats. %Muscularization, %MWT, and the expression of lung elastin were significantly higher in MCT3 rats than in normal and MCT2 rats.Baseline mean pulmonary arterial pressure was significantly higher in MCT rats . After 2-h ventilation, PaOWe found that HV-associated damage might be reduced in MCT-induced PH rats compared with normal rats. The results of this and earlier studies suggest that hypertensive pulmonary vascular structural changes might be protective against the occurrence of ventilator-induced lung injury, irrespective of the etiology of PH.The online version contains supplementary material available at 10.1186/s12890-022-01867-6. Mechanical ventilation is used under general anesthesia in patients with and without lung disease and patients with respiratory failure. Injurious ventilation, such as high tidal volume ventilation, might worsen preexisting lung damage or initiate new lung injury in healthy lungs, termed ventilator-induced lung injury (VILI) –6, whichPulmonary hypertension (PH) occurs in patients with congenital heart disease with a left-to-right shunt –15, chro®, Kyoritsu Seiyaku Corporation, Tokyo, Japan) via intraperitoneal injection. After obtaining no consciousness with respiratory depression and no response to the stimulation, which took about 5–10 min and showed deep anesthesia, rats were put under mechanical ventilation through tracheostomy. Then the abdomen was incised and the rats were exsanguinated by aortic incision and heart and lung samples were removed.The Animal Experiments Committee of the Mie University School of Medicine, Mie, Japan approved the study protocol . Seven-week-old male Sprague–Dawley rats  were subcutaneously injected with 60 mg/kg MCT to induce PH , 33, andEighty-five rats were randomly assigned to one of six groups: (1) rats ventilated with low tidal volume of 6 mL/kg  (n = 14); (2) rats ventilated with high tidal volume of 35 mL/kg  (n = 22); (3) rats ventilated with low tidal volume of 6 mL/kg at 2 weeks after a single subcutaneous injection of 60 mg/kg MCT (MCT2/LV) (n = 11); (4) rats ventilated with high tidal volume of 35 mL/kg at 2 weeks after a single subcutaneous injection of 60 mg/kg MCT (MCT2/HV) (n = 15); (5) rats ventilated with low tidal volume of 6 mL/kg at 3 weeks after a single subcutaneous injection of 60 mg/kg MCT (MCT3/LV) (n = 11); and (6) rats ventilated with high tidal volume of 35 mL/kg at 3 weeks after a single subcutaneous injection of 60 mg/kg MCT (MCT3/HV) (n = 12). The number of rats used in each experiment are presented in each figure. Rats with mean pulmonary arterial pressure less than 25 mmHg in the MCT3/LV and MCT3/HV group were excluded. Since pulmonary hypertension is a condition defined on condition that the mean pulmonary arterial pressure at rest is 25 mmHg or higher, rats with a mean pulmonary arterial pressure of less than 25 mmHg were excluded in order to examine respiratory management for MCT3 PH rats, ie, severe PH in the present study.Under intraperitoneal pentobarbital sodium (45 mg/kg) anesthesia, a right internal carotid and a pulmonary artery catheter  were inserted by using the closed-chest technique, as described previously , 30, 33.2 tension was kept between 40–50 mmHg.The rats were ventilated without positive end-expiratory pressure using an SN-480-7 volume cycle ventilator  . All groAfter 15 min stabilization of ventilation with a tidal volume of 6 mL/kg, baseline arterial blood gas analysis was performed by a portable blood gas analyzer , which was followed by experimental tidal volume (6 or 35 mL/kg) assignment  with conAt the end of the protocol, lung tissue was obtained to measure lung water content and pulmonary microvascular permeability , 33. TheTo estimate right ventricular hypertrophy (RVH), the heart was removed and fixed in buffered neutral formaldehyde solution , 33. The2O and fixed in 10% formalin for 72 h, as described previously [For western blotting and real-time PCR analyses of the lung, the hilum of the right lung was ligated and the right lung was excised and put into liquid nitrogen. To assess hypertensive pulmonary vascular structural changes, the left lung was injected with a hot barium-gelatin mixture at a pressure of 36 cmHeviously , 30, 33.eviously , 30, 33.To assess the magnitude of hypertensive pulmonary vascular remodeling, light microscope  slides were analyzed at ×400, without previous knowledge of the treatment group, which was described previously , 30, 33.Western blotting for IκB, high-mobility group box 1 (HMGB-1), and elastin in whole lung tissue was performed as described previously , 24, 25.The TaqMan Gene Expression Assay was used for analysis of IL-6 (Rn01410330), IL-10 (Rn01644839), C-X-C motif chemokine ligand 1 (CXCL-1/KC/MIP-1) (Rn00578225), MCP-1 (Rn00580555), HMGB-1 (Rn02377062), Elastin (Rm01499782) and β-actin (Rn00667869), as described previously .In the other sets of normal, MCT2 and MCT3 rats , bronchoalveolar lavage (BAL) was performed to show the magnitude of lung inflammation without mechanical ventilation. The rats were anesthetized by the intraperitoneal administration of 50 mg/kg pentobarbital sodium. The BAL fluid (BALF) was obtained by cannulating the trachea with plastic tube and by infusing the lungs with 7-ml of PBS. The recovered fluid (BALF) was centrifuged  using a cytofuge , and the cells were stained with May Grunwald Giemsa .p < 0.05 was considered to be significant.All values are expressed as the mean ± standard error. Analysis of variance with repeated measures was used for arterial blood gas, hemodynamic and PIP analysis. EBD extravasation, the percentage of lung water content, right ventricular hypertrophy, cytokine and chemokine mRNA levels, IκB and HMGB-1 protein expressions were performed by two-way analysis of variance. Morphologic parameters and elastin protein expression levels were  performed by one-way analysis of variance. When significant variance was found, Fisher’s predicted least significant difference test was used to establish which groups were different. 2) between the LV and HV groups in normal, MCT2, and MCT3 rats before the administration of high tidal volume ventilation       rats compared with normal and mild PH (MCT2) rats.The main focus of this study was VILI in PH rats. Since VILI is associated with the release of inflammatory mediators, the presence of inflammation in the lung before mechanical ventilation might exacerbate VILI. So, the inflammatory component of the present MCT-induced PH model deserve comments, in which reduction of IκBα protein levels were confirmed in the present study. Although the exact mechanism through which MCT causes PH is not known, inflammation is considered to have an important role . NeutropThe severity of lung injury was similar between normal and mild PH (MCT2) rats with high tidal volume ventilation because there was no difference in the lung wet/dry weight ratio, EBD extravasation, and arterial oxygenation. In contrast, EBD extravasation and arterial oxygenation were less impaired in severe PH (MCT3) rats compared with normal and mild PH (MCT2) rats, suggesting resistance to high tidal volume ventilation-induced lung injury in this group, which was unexpected. We had anticipated that lung injury caused by high tidal volume ventilation might be more severe in MCT-induced PH rats compared with normal rats, and that MCT3 rats would have more severe lung injury than MCT2 rats, because MCT-induced PH is a type of inflammatory PH and inflammatory cytokines are involved in the development of VILI , 28–30. High tidal volume ventilation tended to increase the expression of proinflammatory cytokines in normal and PH rats, suggesting an increased inflammatory response with high tidal volume ventilation, which is consistent with earlier reports , 2, 7–10Overinflation and the increase in surface forces induced by high tidal volume ventilation both lead to an augmentation of vascular transmural pressure , partly HMGB-1 is normally present as a nuclear protein and is passively released from damaged cells . The redGiven that elevated vascular wall stress in PH increases the production and accumulation of elastin , we dete2 [The first limitation of this study is that it was observational and not mechanistic. Although observational, this study suggested a relationship between hypertensive pulmonary vascular remodeling and the occurrence of VILI. Most of the previous studies have used control rats and/or rats treated with HCl , lipopol2 , which a2 , 19 and 2 , the pre2 . Fourth,In summary, the results reported here and those from a previous study  showed tAdditional file 1. The original gels in Fig. 7a, 7b, and 7c.


This work was assisted by glyceryl palmitostearateand diethylene glycol monoethyl ether-based nanostructured lipid carriers(NLC) to render 2m bioavailable and effective after itsoral administration. The loading of 2m in NLC drasticallyenhanced its intestinal permeability and provided plasmatic levelshigher than its effective concentration (IC50). In L. infantum-infected BALB/c mice, 2m-NLC reducedthe parasite burden in the spleen, liver, and bone marrow by at least95% after 5 doses, demonstrating similar efficacy as intravenous Fungizone.Overall, compound 2m and its formulation merit furtherinvestigation as an oral treatment for visceral leishmaniasis.Leishmaniasis urgently needs neworal treatments, as it is oneof the most important neglected tropical diseases that affects peoplewith poor resources. The drug discovery pipeline for oral administrationcurrently discards entities with poor aqueous solubility and permeability such as the diselenide  Leishmania.1 Among the different clinical manifestations,visceral leishmaniasis (VL) is the most severe form of the disease,and it is principally caused by the species L. infantum and L. donovani. This parasite resides in hostmacrophages, mainly from liver, spleen, bone marrow, and lymph nodes,and it causes anemia, leukopenia, hepatosplenomegaly, hypoalbuminemia,weight loss, and ultimately death if untreated.1 300,000 out of more than 1 million reported cases of leishmaniasisevery year belong to VL, which is responsible for around 40,000 deaths.2Leishmaniasiscomprises a groupof parasitic diseases caused by protozoa of the genus 4 All of them have limitations in terms of toxicity, variable efficacy,price, and inconvenient treatment schedules.5 Moreover, they are parenteral drugs, with the exception of miltefosine,which is orally administered. Nevertheless, miltefosine includes severallimitations such as gastrointestinal toxicity, teratogenicity, highcost, and long elimination half-life, leading to subtherapeutic levelsover several weeks and facilitating the appearance of resistances.6 Due to the geographical distribution of the diseaseand the poor access to drugs for those people infected in low-middleincome countries, it is a priority the search of new drugs and/orformulation strategies to be orally administered.7The range of drugs available for the treatmentof VL is limited,and it includes pentavalent antimonials, amphotericin B deoxycholate,lipid formulations of amphotericin B, miltefosine, and paromomycin.8 Drug discovery algorithms for oraladministration currently recommend discarding entities with poor aqueoussolubility and/or permeability because they lead to low bioavailabilityand suboptimal efficacy. However, lipid-based systems have shown markedincrease in the oral absorption of this type of compound.10 Among the different kinds of lipid systems, nanostructured lipidcarriers (NLC) stand out over other types of nanocarriers becausethey offer many encouraging advantages.11 Compared with liposomes or nanoemulsions, NLC present higher stabilityin the physiological conditions found in the gastrointestinal tract.12 Moreover, they overcome the inherent disadvantagesof their parent solid lipid nanoparticles (SLN), such as low drugpayload and drug expulsion during storage.13Although the combination therapy of drugs has improvedthe VL outcomeof current therapeutic arsenal, the long-term goal is to identifynew active compounds and develop an entirely new generation of oraldrugs.in vitro antileishmanial activity of several series of selenocompounds.19 Two of these derivatives, N,N′-)bis-furan-2-carboxamide(2h) and N,N’-)bisnaphthamide(2m)  activity couldimplicate alterations in the parasite redox metabolism. TR is a validatedtarget for the designing of antileishmanial drugs.20 It is essential for parasite survival, but it is absentin the host, in which TR is replaced by glutathione reductase.21 Moreover, antimonials currently in clinicaluse inhibit TR and evidence that TR is a druggable target. Althougha large number of TR inhibitors have been identified by virtual andhigh-throughput screening,23 few of them have furtherprogressed because of the need for strong and irreversible inhibitionin the presence of endogenous substrates and suitable drug-like properties.24In the past years, our group has reported the synthesis and ide(2m) 1, could ectively1.19 Thei2h and 2m wereput through the screening cascade for VL therapy.26 The in vitro absorption, distribution, metabolism,and excretion (ADME) properties to predict their oral bioavailability,namely, intestinal permeability, metabolic stability, and in vivo pharmacokinetics, were determined. Besides, to circumvent 2h and 2m poor water solubility and to enhancetheir oral permeability and bioavailability, their encapsulation inNLC was carried out. Finally, their efficacy was assayed in a murinemodel of established VL when administered orally.In this study, compounds in vitro antileishmanial activity of novel selenocompounds whose mechanismof action mainly involved parasite redox processes.19 Compounds 2h and 2m were chosenfor further profiling because they exhibited the highest selectivityindex, calculated as the ratio between the activity against intramacrophageparasites and the cytotoxicity against host macrophages (THP-1 cells)(invitro IC50 < 10 μM and good selectivityindex.26We recently published the synthesis and 1 cells)1, among in vivo studies, the in vitro ADME properties of 2h and 2m likely to be predictive of their oralbioavailability were measured. Both compounds exhibited low aqueoussolubility in fasted simulated intestinal fluid (SSIF) and very poor ex vivo intestinal permeability  and intrinsic clearance (Clint) values of 1.7h and 24.9 mL·min–1·kg–1 for 2h and 1.2 h and 33.7 mL·min–1·kg–1 for 2m, respectively . In general, compounds with t1/2 > 1 h are defined as stable in liver microsomal metabolism,and Clint values between 15 and 45 mL·min–1·kg–1 are indicators of intermediate clearancerate.27Prior to eability 2. Howeve2h  and especially 2m (logP 519)are poorly water-soluble compounds and, thus, a priori suitable candidates for their loading into NLC.28 Moreover, these types of nanocarriers have widely demonstratedpotential to increase solubility, oral bioavailability, and even lymphaticabsorption of hydrophobic drugs.11 Thepoor solubility of 2h and 2m compounds inmany common organic solvents and lipids restricted NLC fabricationand composition to the hot high-shear homogenization (HHSH) processwith glyceryl palmitoestearate (Precirol) and diethylene glycol monoethylether (Transcutol) as solid and liquid lipids, respectively. Transcutolwas the solvent that presented the highest solubilizing capabilityfor 2h and 2m compounds, being the onlyone able to dissolve up to 6 mg of compound in 0.4 g. The compositionof the optimized NLC is shown in 2h-NLC and 2m-NLC, respectively. Although they are below the criticalzeta potential for stability in terms of purely electrostatic repulsions,the sterically stabilizing effect of polysorbate 80 (Tween 80) couldexplain the low PDI of the NLC suspensions. In fact, a relativelyhigh concentration of Tween 80 was required to obtain monodisperseNLC of around 100 nm by the HHSH method with a total lipid concentrationof 17%. In general, NLC sizes and PDI tended to increase with lipidconcentration.30Homogeneous NLC with a meansize around 100 nm and high encapsulation efficiencies (between 85%and 95%) were obtained 4 for bot30 Thermal curves and X-ray diffractionpatterns of pure 2h, pure 2m, empty NLC, 2h-NLC, 2m-NLC, and their physical mixtures inequivalent concentration (0.3% w/w) as in the final optimized formulationsare shown in 2h and 2m compounds showedmelting endothermic peaks at approximately 222 and 252 °C, respectively.Empty NLC showed a sharp melting peak at 60 °C correspondingto Precirol. 2h-NLC .TG-DTA andX-ray diffraction studies were performed to analyzethe degree of crystallinity, the possible polymorphic modifications,and the drug incorporation into the lipid matrix, as these parameterscould modify their release profile and affect the stability of thesystem over time.. 2h-NLC 2a and 2m  probably reflect the incorporationof the compounds into an amorphous or molecularly dispersed stateinside the lipid matrix.Overall, the absence of peaks correspondingto the melting transitionsof pure compounds or XRD patterns in In order to prolong their storage,aqueous dispersions of NLC werelyophilized. Trehalose (at a ratio 1:1.5 to lipid) was suitable forprotecting NLC of aggregation, as lyophilized NLC with this cryoprotectorshowed similar size, drug loading, and acceptable PDI (between 0.2and 0.3).2h and 2m compounds from NLC as a function of time when incubatedin simulated gastric fluid  and later in simulatedintestinal fluid  in order to mimicthe oral administration. Both formulations exhibited a slow and incompleterelease profile. 2h was released from NLC slightly fasterthan 2m, although the differences were not significant.About 5% of the compounds (5.5 ± 3.1% for 2h and3.9 ± 0.3 for 2m) was released in the first 2 hof incubation in SGF (mean residence time in the stomach). Then, theslow release continued in SIF. After 4 h of incubation in SIF, NLCreleased around 13% and 10% of 2h and 2m, respectively. The rapid degradation of several types of Precirol-basedNLC has been previously described,31 althoughthe correlation between degradation and drug release has not beenproperly evaluated. We could hypothesize that the degradation of NLCinto mixed micelles solubilizing the drug and unable to cross thedialysis membrane may be responsible for the low amount of compound(around 15%), expressed as selenium (Se) content, released at theend of the study (24 h).2h and 2m into NLC produced a higher increase in the intestinalpermeability compared to free drugs. At the end of the study, theamount of Se in the receptor compartment (RC) for the nanoparticleformulations was 20 times higher than for the free compounds, withapparent permeability (Papp) values of(7.67 ± 1.85) × 10–5 and (6.44 ±1.08) × 10–5 cm/s for 2h-NLC and 2m-NLC, respectively. Estimated Papp values for the free compounds were 100-fold lower, around (7.17± 2.62) × 10–7 and (5.91 ± 1.08)× 10–7 cm/s for 2h and 2m, respectively  parameters, calculatedby noncompartmental analysis, are summarized in tmax) and higher values of elimination half-times (t1/2). Apparent clearance (Cl) values, after the p.o. administration,were referred to total administered dose because mice immediatelydied after i.v. bolus of the compounds. These Cl values allowed thedetermination of hepatic clearance rates (<0.3) suitable for furtherbiological evaluation, except for i.p. 2m compound thatpresented an extraction ratio (ER) higher than 0.7 or loaded into NLC (p.o.) displayed several peaks in the plasma Seconcentration–time curve (A single i.p. or p.o. dose of compounds than 0.7 5. Howeveme curve 6. After of 1 μM)1.L. infantum stationary-parasites inoculation. Mice received 5 p.o. administrationsof compounds loaded into NLC (2h-NLC and 2m-NLC) at a dose of 4 mg/kg every 2 days. Fungizone was used as positivecontrol i.v. administered at 1 mg/kg during 10 consecutive days. Asshown in 2m-NLC but not 2h-NLC p.o. reduced the parasiteburden similarly to Fungizone. Healing rates of 96.9% in the liver, 79.1% in the spleen, and 86.4% (p < 0.01) in the bone marrow for 2h-NLC treated miceand cure rates of 99.9% (p < 0.001) in the liver,98.5% (p < 0.001) in the spleen, and 94.8% (p < 0.05) in the bone marrow for 2m-NLCtreated mice were found. On the other hand, unloaded NLC (around 12mg of lipids/mice) did not shown any effect, probably because theirprincipal ingredients were either nonabsorbed or digested to compoundswithout activity after their oral administration.31The short-term efficacy of the formulations was tested in a micemodel of established VL infection 4 weeks after 33 although the evolution time seemed to be affectedby the experimental conditions.34 Fouror six weeks after i.v. administration of 108 stationary-phasepromastigotes, untreated mice showed similar parasite burden in theliver , spleen and bone marrow .The detailed results are summarized in the Supporting Information .The genotoxicity of compounds 2m-NLCat 4 mg/kg. No signs of toxicity were found in the stomach and intestinesof treated mice  with IC50 values of 2 and 1 μM, respectively, against intramacrophageamastigotes and selectivity index higher than 20 and 50 vs THP-1 macrophages.In the current study, they were progressed for ADMET profiling. Accordingto ADMET prediction,19 neither compound 2h nor 2m fulfilled Lipinski’s rule of5. However, there are many approved drugs that are outside the cutoff,36 and this issue is especially clear for antimicrobials,probably due to their intracellular target.37 The ex vivo intestinal permeation assay  with the highest purity, can also be foundin a number of oral prescription drugs, although the Scientific Committeeon Consumer Products (SCCP) did not advise its use because recentstudies indicated side effects by this administration route.49 However, the toxicity could be produced by impuritiesand only at higher exposure levels than the ones used in the currentstudy.50 In fact, the amount of administeredNLC corresponded to 200 mg/kg of Transcutol every other day for 10days, whereas the no observed adverse effect level (NOAEL) was about850–1000 mg/kg/day after a daily oral administration for 90days. On the other hand, the NLC composition used in the current studyalso included a relatively high proportion  still remainsthe most successful strategy for the identification of antileishmanialcompounds,on assay 5 and inectively 2. Thus, ion 20%, 3 of Twee kidneys 8a–c, and kidneys 6 did notex vivo intestinal permeation study  were used for this purpose.However, permeability limitations of 2h and 2m compounds and their probable enterohepatic circulation complicatedtheir PK profile and their correlation with the in vitro release studies.The PK parameters obtained after the oral administrationof NLC5 confirmon study 5 would i2m-NLC formulation was higher than its leishmanicidalIC50 value, an in vivo evaluation of itsefficacy was performed in a mice model of established VL after theadministration of 4 mg/kg. 2h-NLC p.o. administered werealso tested for comparison. The reduction of parasite burden in theliver, spleen, and bone marrow of mice receiving 2m-NLCwas similar to that obtained after the i.v. administration of Fungizone,whereas 2h-NLC significantly reduced the parasite loadin the liver and bone marrow but not in the spleen , 2m-NLCreached its IC50 at around 8 h (2h or 2m either free or loaded into NLC). Althoughmultiple peaking can occur as a consequence of different mechanisms,the characteristic enterohepatic circulation shown for organic Secompounds has been previously described as a way to control theirbody levels53 and the most common causeof several peaks in blood.54 However, aslower lymphatic absorption, especially for 2m compoundand its formulation, cannot be discarded in view of its highly lipophiliccharacter (logP of 5). Furthermore, we should quantifythe compounds (and/or their metabolites) in the main VL affected organs, as the antileishmanial efficacyof common drugs has been more closely correlated with their organaccumulation than with their plasmatic levels.55The higher efficacyof ound 8 h 6. Howeve2m intoNLC was performed, and this formulation given orally showed similarefficacy to i.v. Fungizone. Further studies are necessary to elucidateits mechanism of hepatic clearance and perform the identificationof metabolites in order to optimize the compound. Finally, we wouldalso consider the formulation of 2m in simpler and cheaperlipid-based delivery systems  for better translation of an accessible medicine to low-resourcescountries.Overall, the loading of the selenocompound 2m (low solubility and low permeability)would be currently discarded for further evaluation according withthe most common decision algorithm for oral drug discovery againstleishmaniasis. NLC drastically modified its drug-likeness propertiesand rendered it bioavailable and effective after oral administration.This work is a good example of the importance of introducing deliveryapproaches in early drug discovery.Despite its good antileishmanial activityand selectivity index,the BCS type IV compound 2h )bis-furan-2-carboxamide)and 2m )bisnaphthamide)were synthesized as previously described.19 Briefly, compound 2h was obtained by the reductionof N-(4-selenocyanatophenyl)furan-2-carboxamide withsodium borohydride in ethanol and stirring for 2 h. Compound 2m was synthesized from bis(4-aminophenyl)diselenide and naphtoylchloride in dry chloroform with stirring for 24 h. For their purification, 2h was recrystallized from ethanol, whereas 2m was only washed with diethyl ether. Both compounds were obtainedwith high yields  and with a purity >95%confirmed by infrared spectroscopy (FTIR), nuclear magnetic resonance, mass spectrometry, and elementalanalysis.19 Polyethylene glycol 400 and20,000 (PEG) were obtained by Fluka . Phosphate-bufferedsaline (PBS) was obtained by ThermoFisher Scientific . Glyceryl palmitoestearate (Precirol ATO5) and high purity diethyleneglycol monoethyl ether (Transcutol HP) were kindly gifted by Gattefossé. Polysorbate 80 (Tween 80) was purchased from Panreac. l-Glutamine was obtained from Acros , and dimethyl sulfoxide (DMSO) was purchased from VWR prolabo. d-α-Tocopherol polyethyleneglycol 1000 succinate, sucrose, β-nicotinamide adenine dinucleotidephosphate reduced tretra(cyclohexylammonium) salt (NADPH), glycerol,sodium phosphate dibasic (Na2HPO4), and sodiumtaurocholate were purchased from Sigma , and monosodiumphosphate (NaH2PO4) from BDH Prolabo . Lipoid S100 (soybean lecithin) was kindly giftedby Lipoid GMBH . Acetonitrile (ACN) and water(HPLC grade) were obtained from Sigma and VWR Prolabo Chemicals, respectively.All other reagents were of analytical grade and were employed withoutfurther purification.Compounds L. infantum infective promastigotes(strain BCN-150) were maintained at 26 °C in a continuously stirredSchneider’s modified medium (Sigma) supplemented with 20% FBSand 40 mg/mL of gentamicin (Sigma). Parasites were grown until theyreached the stationary phase. Then, they were washed twice with PBSand were harvested for inoculation in BALB/c mice. Parasite infectivitywas maintained by constant passages in BALB/c mice.Female BALB/c mice weighting between 20 and25 g and female Wistar rats weighing 200–250 g (Harlan) werekept under normal conditions with free access to food and water. Theywere housed in groups of four or five in plastic cages in controlledenvironmental conditions (12/12 h light/dark cycle and 22 ± 2°C). The study was conducted according to ethical standards approvedby the Animal Ethics Committee of the University of Navarre in strictaccordance with the European legislation in animal experiments .Salmonella typhimurium (TA1535/pSK1002strain)  was employed to determine the genotoxic potential of thecompounds 2h and 2m and some of their possiblemetabolites, as previously described.56 Bacteria were exposed to different concentrations of compounds eitherwith or without metabolic activation  to determinethe bacterial survival percentage, calculated by measuring the absorbanceat 600 nm. Afterward, the β-galactosidase activity was conductedusing a colorimetric method employing o-nitrophenyl-β-d-galactopiranoside (ONPG) as a substrate. Genotoxicity activitywas measured in terms of β-galactosidase activity calculatingan induction factor (IF) relative to the negative control  and positive controls .A compound was considered genotoxic for IF values ≥2 at noncytotoxicconcentration . The experimentwas considered valid for positive controls if IF ≥ 2.57 Mouse livers were removedand rinsed with 0.9% (w/v) NaCl solution. Then, livers were homogenizedin ice-cold 0.25 M sucrose in 0.1 M PBS (pH 7.5). After 20 min ofcentrifugation at 9000 × g, the supernatantwas ultracentrifuged at 100,000 × g for 1 hat 4 °C. The obtained microsome pellet was washed with 0.05 MPBS (pH 7.5) followed by another ultracentrifugation at 100,000 × g for 1 h at 4 °C. Microsomes were resuspended in 20%glycerol–0.1 M PBS and stored at −80 °C until use.Protein concentration was measured by microBCA kit (Thermo Scientific).For the metabolic assay, compounds were incubated with liver microsomes(0.5 mg protein/mL) for several time points. Stock solutions wereprepared in DMSO, and the final concentration in the mixtures didnot exceed 1% (v/v). The tested compounds 2h and 2m, at a final concentration of 500 μM, along with themicrosomes were mixed in 0.05 M potassium phosphate buffer and incubatedat 37 °C with continuous shaking in a final volume of 300 μL.Reaction was initiated by adding NADPH (1 mM). Controls without hepaticcofactor and blanks without compounds were also analyzed. Metabolismwas determined at 0, 0.1, 0.25, 0.5, 1, 1.5, and 2 h by the additionof an equal volume of ice-cold ACN, and samples were centrifuged at9000 × g for 10 min. Supernatants were storedat −20 °C until high liquid performance chromatography(HPLC) quantification analysis at 290 nm, as described in the Supporting Information. Results are expressedas mean ± SD of at least three replicates. In vitro half-time  was establishedfrom the slope of linear regression of the percentage of remainingparent compound against time. The rate of microsomal intrinsic clearance was then calculated following t1/2 wasthe half-life of the tested compound (h), vol was thevolume of incubation (0.3 mL), and mg was the amount of protein (0.15mg.).Liver microsomes were isolated as previously described with minormodifications.int,micr was used for theprediction of in vivo intrinsic hepatic clearance(Clint) using 27The calculated Cl58 In brief, 1 mL of FaSSIF (3 mM sodiumtaurocholate and 0.75 mM lecithin in sodium phosphate buffer at pH6.5) was added to 2 mg of the solid compounds. The resulting suspensionswere shaken at 900 rpm for 4 h at r.t. Then, they were centrifugedat 12,600 × g for 10 min. The amount of selenocompounds,as selenium (Se) content, remaining in the supernatant was quantifiedby graphite atomic absorption spectrometry, as described in the Supporting Information. Results are expressedas mean ± SD of three replicates.This experimentdetermines the solubility of a solid compound in fasted simulatedintestinal fluid (FaSSIF) at pH 6.5 after 4 h of equilibration atr.t.2h and 2m were formulated inNLC. The preparation of NLC was made using a modification of a previouslydescribed hot high shear homogenization (HHSH) method.12 Briefly, 0.35 g of Precirol ATO5 was meltedat a temperature 10 °C above its melting point and then mixedalong with 0.15 g of Transcutol HP and 0.6 g of Tween 80. Then, 3mg of the compounds 2h and 2m were addedto the melted lipid mixture until complete solubilization. After that,12 mL of purified water, previously heated at the same temperature,was added to the lipid phase and the mixture was homogenized usinga high-shear laboratory mixer  in the heated waterbath at 12,300 rpm for 10 min to maintain the melting temperatureof the lipids. The NLC dispersions were finally obtained by allowingthe hot nanoemulsion to cool in an ice bath with gentle agitationfor 5 min. After 24 h, samples were purified by centrifugation at1500 × g for 5 min and Sephadex G-25/PD-10 columnsand concentrated until 3 mL by dialysis using PEG 20,000. The finaldispersions were lyophilized  with 10%(w/v) of trehalose and stored at 4 °C until further use.Compounds Themean hydrodynamic diameter of the formulations and the zeta potentialwere determined before and after freeze-drying by photon correlationspectroscopy and electrophoretic laser Doppler anemometry, respectively,using a Zetamaster analyzer system . The resuspension of freeze-dried NLC was performed in type Iwater with vigorous mechanical agitation. The diameter of the NLCand polydispersity index (PDI) were determined after dispersion inultrapure water (1:100) and measured at 25 °C by dynamic lightscattering angle of 90 °C. The zeta potential was determinedafter dilution of 200 μL of the samples in 2 mL of KCl solution(0.1 mM). Results are expressed as mean ± SD of at least threereplicates.g for 20 min. The amount of drug remainedin the supernatant was then quantified by HPLC at 290 nm, as describedin the Supporting Information. The drugloading (DL%) was calculated as μg of compound per mg of lipids.The entrapment efficiency (EE) was determined as percentage of drugloaded into NLC from nominal initial amount used for their preparation.The drug loading in both aqueous and lyophilizedNLC was determinedafter digesting and vortexing for 10 min the NLC with ACN, which promotedthe precipitation of the lipid phase. Samples were centrifuged at4200 × –1 of N2 as purge gas. Measurements were performed in triplicate.The response to heating was studiedemploying a simultaneous TGA/sDTA 851e Mettler Toledo thermoanalyzer. Experimental thermogravimetric and differentialthermal analysis curves were obtained by monitoring about 1–5mg of the different samples in an alumina crucible at a heating rateof 10 °C/min from 25 to 575 °C. The thermal analyses wereperformed under static air atmosphere using 20 mL min2h and 2m and their formulations. For this purpose,samples were placed in powder form on a plastic plate in a diffractometerBruker D8 Advance ECO , with a LYNXEYE-XE-T 1Ddetector, using Cu Kα1 radiation of 1.54060 Å,a voltage of 40 kV, and a current of 30 mA, with primary and secondarysoller slits. The diffraction patterns were carried out from 5°to 40° 2θ, 3 s per step, and a step size of 0.02°.X-ray diffraction studieswere performed in order to study the crystalline state of compounds 2h and 2m were performedat 37 °C using simulated gastric fluid ) and simulated intestinal fluid ) with 2% v/v PEG 400, 5% w/v d-α-tocopherol polyethyleneglycol 1000 succinate and 2.5% v/v DMSO added to ensure sink conditions.The dialysis membranes (with 100 kDa pore size) were filled with 30mg of lyophilized formulations either 2h-NLC or 2m-NLC (corresponding to around 60 μg of the selenocompounds)dispersed in 1 mL of distilled water. The loaded dialysis membraneswere placed into a beaker containing 25 mL of SGF. The simulated fluidwas kept under magnetic stirring at 150 rpm, and at fixed times 0.250mL samples were withdrawn and replaced with an equal volume of SGF.Two hours after the incubation in SGF, the device was transferredto another beaker containing 25 mL of SIF magnetically stirred at150 rpm. As previously, at fixed times, samples were withdrawn andreplaced with free SIF. The in vitro release studyfinished 24 h after the experiment began. The amount of Se releasedfrom the nanocarriers was determined by graphite atomic absorptionspectrometry, as described in the Supporting Information. Results are expressed as mean ± SD of at least three replicates.The releasestudies of compounds 59 Wistar female rats were fasted for 16 h with free access to waterbefore the experiment. Rats were anesthetized with isoflurane andsacrificed by cervical dislocation. Jejunum was aseptically extracted,excised in 2 cm segments avoiding Peyer’s patches, and placedin cold 0.9% NaCl solution. Jejunum segments were anchored into theUssing chambers. All experiments were realized from the apical sideor donor compartment (DC) to the basolateral or receptor (RC) direction.For 2h and 2m compounds experiments, 5 mLof deionized water at pH 7.4 containing 4% (w/v) d-α-tocopherolpolyethylene glycol 1000 succinate, and 5% (w/v) PEG 400 was addedto both compartments to ensure sink conditions. In the case of NLC,5 mL of PBS was used in both compartments. Chambers were continuouslyoxygenated and maintained at 37 °C. After 10 min of preincubation,the solution in the DC was replaced by 5 mL of either 2h and 2m solutions  or theirlyophilized NLC diluted in PBS at a 1 mM (in selenocompound concentration).Samples of 500 μL were collected from the RC every 20 min upto 2 h and replaced by an equal volume of the RC solution. Sampleswere frozen at −20 °C until analysis. The membrane integritywas evaluated by the addition of yellow lucifer to the DC and thedetermination of its fluorescence at the end of the experiment.60 Experiments were carried out in triplicate foreach compound and formulation. Se content in the RC was analyzed byflame atomic absorption spectrometry, as explained in the Supporting Information. The apparent permeabilitycoefficient per unit membrane surface area (Papp (cm/s)), expressed as mean ± SD, was calculated accordingto eq Q/dt was the steady-flux (mg/s), C0 was the initial compound concentration in the DC (mg/mL), and A was the surface area of the membrane (cm2).It has been stablished that, in general, compounds with Papp > 1 × 10–6 cm/s are well-absorbedin the human intestinal tract whereas Papp < 1 × 10–6 cm/s are correlated with poorabsorption.32Intestinal permeation experiments were performed by using the Ussingchamber method as previously described.61 Three mice were treated with a dose of 17.5 mg/kg of eachcompound, 2h and 2m, administered by i.p.route. If all the mice died, 1/3 of the dose was administered to othernew three mice. If all of them survived, the dose was tripled. Ifone or two of them died, the same dose was administered to one ortwo mice more. Both compounds were solubilized in PEG 400:H2O:d-α-tocopherol polyethylene glycol 1000 succinate.Acute toxicity was carried out followingthe Up-and-Down procedure proposed by the OECD 425.2m-NLC were p.o. administered at 4 mg/kg every other day for5 days. Two days after the last dose, animals were sacrificed, andliver and kidneys were fixed in 4% (w/v) paraformaldehyde for 24 h,embedded in paraffin, and stained with hematoxylin-eosin (Merck) forhistological observation analysis. Furthermore, body weights of 2m-NLC treated mice were recorded prior to day 1 and priorto necropsy. Serum biochemistry was also analyzed after 2m-NLC p.o. administered at 25 mg/kg every other day for 5 days. Bloodsamples were collected 2 days after the last dose. For that, bloodsamples were kept at r.t. for 30 min and then centrifuged at 3500× g for 10 min. Serum was harvested from eachblood sample, and albumin (ALB), glucose (GLU), alanine aminotransferase(ALT), aspartate aminotransferase (AST), cholesterol (CHO), creatinine(CREA), total protein (TP), and blood urea nitrogen (BUN) were measuredin a Cobas biochemistry analyzer . Serumlevels of treated mice were compared to untreated ones. Results wereexpressed as mean ± SD (n = 5).For the 5-day repeated dose toxicity experiments, 2h, 2m, 2h-NLC, and 2m-NLC were evaluated in mice after i.p. (free drugs) or oraladministration (free drugs and loaded into NLC). The dose (4 mg/kg)was selected taking into account LD50 obtained after i.p.administration of the compounds. Blood was collected from the submandibularplexus at different time points: 0, 0.25, 0.5, 1, 2, 4, 8, 24, and48 h. Collected whole blood samples were centrifuged at 3000 × g for 10 min at 4 °C. The obtained plasma samples werestored at −80 °C until quantification of the drug by graphiteatomic absorption spectroscopy, as described in the Supporting Information. Experiments were carried out in triplicatefor each compound and formulation. Pharmacokinetic parameters wereestimated by fitting the experimental data to a noncompartimentalmodel (NCA) using PKsolver Excel program.62 The analyzed parameters were as follows: Area under concentration–timecurve (AUC), maximum serum concentration (Cmax), clearance (Cl), time of maximum concentration (tmax), mean residence time (MRT), and product half-life(t1/2). The extraction rate (ER) was calculatedas the ratio between systemic blood clearance and mouse hepatic bloodflow (considered 90 mL·min–1·kg–1). Extraction ratio can be generally classified as high (>0.7),intermediate(0.3–0.7), or low (<0.3), and these values are currentlyused for new chemical entities triaged during drug discovery.63A single-dose pharmacokineticstudy of 8 stationary-phasepromastigotes of L. infantum in the tail vein. Fourweeks after infection, mice were divided into the following groups:(i) negative control , (ii) unloaded NLC (p.o.), (iii) 2h-NLC , (iv) 2m-NLC , and (v) positive control .Fungizone was daily administered for 10 consecutive days, whereasoral treatments were given every other day for 10 days (5 treatments).BALB/c mice were i.v. infected with 1064 Briefly, the liver, spleen, and bone marrow samples perfusedfrom the femur cavities of each mouse were individually homogenizedin Schneider medium supplemented with 10% heat-inactivated FBS andantibiotics and filtered through 40 μm cell strainers to obtain a cell suspension. Cells were serially diluted (1/3) in96-well flat-bottomed microtiter plates (Thermo Fischer Scientific)containing the same medium employed for homogenization (6 replicates).The number of viable parasites was calculated as the geometric meanof the titer obtained from 6 replicates cultures × reciprocalfraction of the homogenized organ added to the first well. The titerwas the reciprocal value of the highest dilution at which promastigoteswere observed with an inverted light microscope after 7 days of incubationat 26 °C. Results are indicated per mg of spleen and liver, orparasites per 1 × 106 BM cells.65Levels of parasite burden in liver, spleen, and bone marrow weredetermined at the beginning and the end of the treatments by limitingthe dilution assay (LDA), as previously described.t test. GraphPad Prism7 version  was used to perform the analyses. Significance was establishedfor a p value <0.05.Statisticalanalyses between multiplegroups were performed by using Kruskal–Wallis (nonparametric)followed by Dunn’s multiple comparisons tests. Differencesbetween the two groups were analyzed by an unpaired


Ubb is required for fertility in both male and female mice. In particular, the Ubb-null male mice showed an azoospermia phenotype due to arrest of spermatogenesis at the pachytene stage. Here, we analyzed the whole testis proteome at postnatal day 20 to define the molecular mediators of the male-infertility phenotype caused by Ubb knockout. From the identified proteome, 564 proteins were significantly and differentially expressed in Ubb-knockout testes and, among these, 36 downregulated proteins were involved at different stages of spermatogenesis. We also found that levels of piRNA metabolic process-related proteins, including Piwil2 and Tdrd1, were downregulated in Ubb-null testes through functional gene ontology analysis. Further, protein–protein interaction mapping revealed that 24 testis development-related proteins, including Hsp90aa1, Eef1a1, and Pabpc1, were directly influenced by the depletion of ubiquitin. In addition, the reduced mRNA levels of these proteins were observed in Ubb-knockout testes, which closely resembled the global downregulation of piRNA-metabolic gene expression at the transcriptional and post-transcriptional levels. Together with proteomic and transcriptional analyses, our data suggest that Ubb expression is essential for the maintenance of testicular RNA-binding regulators and piRNA-metabolic proteins to complete spermatogenesis in mice.Testis development, including early embryonic gonad formation and late postnatal spermatogenesis, is essential for the reproduction of higher metazoans to generate fertile gametes, called sperm. We have previously reported that the polyubiquitin gene  Uba52 and Uba80) encode a single Ub fused with each ribosomal protein, and two polyubiquitin genes (Ubb and Ubc) comprise tandem repeats of 4 or 9 Ub-coding units, respectively  in 90% [vol/vol] acetonitrile). The gradient was as follows: 0–7 min, 0% B; 7–13 min, 16% B; 13–73 min, 40% B; 73–77 min, 44% B; 77–82 min, 60% B; 82–96 min, 60% B; 96–110 min, 0% B , and 128 N TMT reagents, and the three peptide samples from For LC–MS analysis, a nanoACQUITY UPLC system  was coupled to a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer  and equipped with a trap column  for cleanup followed by an analytical column . The peptides were separated using a mobile phase comprising solvent A (0.1% formic acid and 2% acetonitrile in water) and solvent B (0.1% formic acid in 90% acetonitrile). For global proteome-digested peptides, the labeled peptides were trapped using 100% solvent A for 4 min at a flow rate of 3 μL/min, and the optimized linear-gradient elution program was set as follows (T min:% of solvent B): 0:3, 4:3, 7:12, 143:40, 144:80, 154:80, 155:5, and 180:5. The flow rate was 300 nL/min throughout the run time. Full MS scans were acquired for the mass range of 350–1,800 m/z at a resolution of 70,000 in MS1 level and an automatic gain control (AGC) target of 1e6, and MS/MS analysis was performed by data-dependent acquisition in positive mode. The electric potential of electrospray ionization was set to 2.2 kV, and the temperature of the capillary was set to 250 °C. The top 12 precursor peaks were selected from the MS1 scan and separated for fragmentation. For MS2 acquisition with high-energy collisional dissociation (HCD), the resolution was set to 35,000 with a fixed first m/z of 100 m/z, an AGC target of 1e6, an isolation window of 2.0 m/z, an isolation offset of 0.5 m/z, and a normalized collision energy (NCE) of 32%. The charge states of unassigned, 1, or >6 were discarded, and dynamic exclusion for 30 s was enabled.Mus musculus reference  using the Sequest HT search engine. The search was restricted to fully tryptic peptides, allowing up to two missed cleavage sites. In modification, carbamidomethylation, alkylation of disulfide bonds in cysteine (+57.021 Da), TMT modification of lysine, and N-termination (+229.163 Da) were noted as static modifications. Oxidation of methionine (+15.995 Da) was used as a variable modification. The false-discovery rate (FDR) for peptide level was set to 0.01 for removing false-positive data and to quantify each reporter ion, using the “peptide and protein quantifier” method in Proteome Discoverer 2.4 .The MS/MS spectra were searched against a composite database of Uniprot Ubb, Piwil1, Piwil2, Piwil4, Dync2h1, Dynlrb2, and Thbs3, were normalized to Gapdh levels. The sequences of the primers used for qRT-PCR are listed in Supplementary table Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) was performed as previously described, with slight modifications . BrieflyUbb-knockout using g:Profiler [For the enrichment pathway analysis of all identified proteins, we classified proteins with more than ±1.2-fold expression changes in a two-tailed Student’s t-test as differentially expressed proteins (DEPs) with a p-value < 0.05. Annotation enrichment processes were performed for the significantly upregulated or downregulated DEPs. Gene Ontology (GO) searches were performed to explore the biological processes and cellular components affected by Profiler . The GO Profiler . The netSupplementary table 1 & informationSupplementary table 2Supplementary table 3Supplementary table 4Supplementary table 5


Lupinus albus and at assessing its efficacy in vitro and in vivo. The protein was isolated using chromatographic and 2-D electrophoretic procedures and sequenced by using MALDI-TOF TOF and MS/MS analysis. In vitro MMP-2 and 9 inhibitions were determined on colon adenocarcinoma (HT29) cells, as well as by measuring the expression levels of genes related to these enzymes. Inhibitory activities were also confirmed in vivo using a model of experimental TNBS-induced colitis in mice, with oral administrations of 15 mg·kg−1. After chromatographic and electrophoretic isolation, the L. albus MMP-9 inhibitor was found to comprise a large fragment from δ-conglutin and, to a lower extent, small fragments of β-conglutin. In vitro studies showed that the MMPI successfully inhibited MMP-9 activity in a dose-dependent manner in colon cancer cells, with an IC50 of 10 µg·mL−1 without impairing gene expression nor cell growth. In vivo studies showed that the MMPI maintained its bioactivities when administered orally and significantly reduced colitis symptoms, along with a very significant inhibition of MMP-2 and -9 activities. Overall, results reveal a novel type of MMPI in lupine that is edible, proteinaceous in nature and soluble in water, and effective in vivo, suggesting a high potential application as a nutraceutical or a functional food in pathologies related to abnormally high MMP-9 activity in the digestive system.Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) are regarded as important clinical targets due to their nodal-point role in inflammatory and oncological diseases. Here, we aimed at isolating and characterizing am MMP-2 and-9 inhibitor (MMPI) from  Lupinus albus seeds exhibited strong inhibitory activity on MMP-2 and MMP-9 in HT29 cells + of insulin (5734.51 m/z), ubiquitin I (8565.76 m/z), cytochrome c , and myoglobin ; [M + 2H]2+ of cytochrome c (6180.99 m/z) and myoglobin (8476.65 m/z).The intact isolated MMPI was analyzed by MALDI TOF MS using an Ultraflex II MALDI-TOF TOF Bruker-Daltonics equipped with a LIFT cell and N2 laser. The mass spectrometer was operated with positive polarity in linear mode and spectra were acquired in the range of v/v) aqueous formic acid containing 3% (v/v) acetonitrile before loading onto an EASY-nLC II equipped with an EASY-column, 2 cm, ID 100 μm, 5 μm, C18-A1 (Thermo Fisher Scientific) and an EASY-column, 10 cm, ID 75 μm, 3 μm, C18-A2 (Thermo Fisher Scientific). Chromatographic separation was carried out using a multistep linear gradient at 300 nL/min , 0–90 min linear gradient from 0% to 35% of mobile phase B, 90–115 min linear gradient from 35% to 90% of mobile phase B, and 115–120 min isocratic flow at 90% of mobile phase B.Prior to ESI MS/MS analysis, all samples were diluted with 100 μL of 0.1% (®) in information-dependent acquisition (IDA) mode. Peptides were resolved by liquid chromatography  on a MicroLC column ChromXPTM C18CL reverse phase column  at 5 μL·min−1. Peptides were eluted into the mass spectrometer with a multistep gradient: 0–2 min linear gradient from 5 to 10%, 2–45 min linear gradient from 10% to 30% and, and 45–46 min to 35% of acetonitrile in 0.1% (v/v) TFA. Peptides were eluted into the mass spectrometer using an electrospray ionization source  with a 50 μm internal diameter stainless steel emitter (New Objective). Protein identification was obtained using Protein Pilot™ software .Selected isolated peaks were analyzed on a 5600 TripleTOF mass spectrometer  were copolymerized with 1% (w/v) gelatin. HT29 cancer cell culture supernatants treated with a non-reducing buffer containing 62.6 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, and 0.01% (w/v) bromophenol blue were loaded into each well of the SDS-gel. Electrophoresis was carried out as described before [w/v) acrylamide resolving gel and a 5% (w/v) acrylamide stacking gel, performed in a vertical electrophoresis unit at 200 V and 20 mA per gel. After electrophoresis, gels were washed three times in 2.5% (v/v) Triton X-100 for 60 min each and were incubated 48 h with a specific buffer solution , stained with Coomassie Brilliant Blue G-250 as described by Lima et al. [In order to determine specific metalloproteinase activities, gelatin-zymography was performed according to standard methods ,37, withd before  in a 12.a et al. . White ba et al. .The human colon adenocarcinoma cell line, HT29 (ECACC 85061109), obtained from a 44-year-old Caucasian female was used. HT29 cells were maintained according to Lima et al. .4 cells/well) and MMPI sample was added to the growth medium at different concentrations  concentration and incubated for 48 h. The extracellular medium was collected, and the wells washed with PBS to remove unattached cells. Cell proliferation and viability was determined using the standard MTT assay as described before, with few alterations [HT29 cultured cells were seeded in 96-well plates . For reverse transcription, the RevertAid reverse transcriptase priming with oligo-d(T) kit was used according to the manufacturer’s recommendations .® Supermixes  using an iQ5 Real-Time Thermal Cycler . Reaction conditions for cycling were as follows: 95 °C for 3 min followed by 40 cycles at 95 °C for 10 s, 61 °C for 25 s, and 72 °C for 30 s. Each analysis was performed in triplicate reactions of three biological replicates. The corresponding quantification cycles (Cq) were determined by the iQ5 optical system software  and exported to a MS Excel spreadsheet  for further analysis. Cq values of each gene of interest were normalized with respect to actin (Act) [For each gene studied , a set of specific primers was designed and used to amplify HT29 cDNA resulting from the transcription of 2 µg of total RNA by using conventional PCR and gel agarose electrophoresis. When amplification was observed, confirming the expression, the transcripts were quantified by real-time PCR (RT-qPCR), which was performed in 20 μL reaction volumes composed cDNA derived from 2 µg RNA and 0.5 μM gene-specific primers (Online Resource 1) in SsoFast™ EvaGreenin (Act) .Male CD-1 mice, 25 to 30 g in weight and 5 to 6 weeks of age , were housed in standard polypropylene cages with ad libitum access to food and water inside a controlled environment room kept at 22 °C ± 1 °C with a 12 h light and 12 h dark cycle at the Faculty of Pharmacy Central Animal Facility, University of Lisbon, Lisbon, Portugal.Experiments were performed by agreeing to the most recent rules and recommendations for the care and processing of laboratory animals, namely to the presently adopted European Commission regulations (Directive 2010/63/EU). In addition, the studies were performed in agreement with the ARRIVE Guidelines for Reporting Animal Research. The Ethics Committee of the Faculty of Pharmacy (University of Lisbon) also endorsed the experimental protocol .−1) and xilazine (10 mg·kg−1). Then, 100 µL of TNBS solution was administered with a catheter inserted 4.5 cm into the colon. Mice were kept for 30 min in a Trendelenburg position to avoid reflux. Four days after induction, mice were euthanized by cervical dislocation and necropsied. The colon was removed, freed from surrounding tissues, and opened longitudinally for observation and classification of diarrhea severity. Subsequently, the colon was washed with PBS for macroscopical observation of the tissue.TNBS was instilled as an intracolonic single dose as previously described . On induAnimals were randomly allocated into four experimental groups as described below.n = 6): Animals were subjected to the procedures described above except for intracolonic administration which comprised 100 µL of saline solution. During the 4 days of the protocol, the animals were administered orally with 10 mL·kg−1 of distilled water.1. Control group (n = 6): Animals were subjected to the procedures described above except for intracolonic administration with 100 μL of 50% (v/v) ethanol. During the 4 days of the protocol, the animals were administered orally with 10 mL·kg−1 of distilled water.2. Ethanol (EtOH) group (n = 10): Animals were administered with 100 µL of 2.5% (w/v) TNBS in 50% (v/v) ethanol. During the 4 days of the protocol, the animals were administered orally with 10 mL.kg−1 of distilled water.3. TNBS group (n = 9): Animals were administered with 100 µL of 2.5% (w/v) TNBS in 50% (v/v) ethanol. During the 4 days of the protocol, the animals were administered orally with the isolated MMPI (15 mg·kg−1).4. TNBS + Lupinus MMPI p.o. .w/v acrylamide) were copolymerized with 1% (w/v) gelatin. Colon polypeptide extracts previously treated with a denaturing, non-reducing buffer containing 62.6 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, and 0.01% (w/v) bromophenol blue were loaded into each well of the SDS-gel. Electrophoresis was carried out as described before . SigmaPlot software (version 12.5) was used for comparing different treatments, using one-way and two-way analysis of variance (ANOVA). Tukey’s test was used to compare differences among groups and the statistical differences with Lupinus and other legume seeds as possible nutraceutical or functional foods in the prevention and treatment of diseases related to MMP-9 activity.Here, we have shown novel MMP-2 and MMP-9 inhibitory activity from protein components of lupine, which were found to be active in vivo when administered orally, whilst alleviating the symptoms of induced colitis in mice. These results open novel perspectives on the role of storage proteins in


Klebsiella pneumoniae (hvKp) bacteria are more virulent than classical K. pneumoniae (cKp) with resultant differences in clinical manifestations and management. It is unclear whether all hvKp isolates share a similar pathogenic potential. This report assessed the utility of siderophore production, mucoviscosity, and murine infection for defining the virulence spectrum of hvKp. Three strain cohorts were identified and defined based on the CD1 mouse subcutaneous (SQ) challenge model: (i) fully virulent hvKp strains (fvhvKp), lethal at a challenge inoculum (CI) of ≤103 CFU; (ii) partially virulent hvKp strains (pvhvKp), lethal at a CI of >103 to 107 CFU; (iii) classical K. pneumoniae, not lethal at a CI of 107 CFU. Quantitative siderophore and mucoviscosity assays differentiated fvhvKp and pvhvKp strains from cKp strains but were unable to differentiate between the fvhvKP and pvhvKP strain cohorts. However, SQ challenge of CD1 mice and intraperitoneal (IP) challenge of CD1 and BALB/c mice, but not C57BL/6 mice, were able to discriminate between an fvhvKp and a pvhvKp strain; SQ challenge of CD1 mice may have the greatest sensitivity. cKp was differentiated from hvKp both by SQ challenge of CD1 mice and IP challenge of all three mouse strains. These data identify a means to define the relative virulence of hvKP strains. It remains unclear whether the observed differences of hvKp virulence in mice translates to human infection. However, these data can be used to sort random collections of K. pneumoniae strains into fvhvKp and pvhvKp strain cohorts and assess for differences in clinical manifestations and outcomes.Hypervirulent IMPORTANCE The pathogenic potential of hvKp strains is primarily mediated by a large virulence plasmid. The minimal set of genes required for the full expression of the hypervirulent phenotype is undefined. A number of reports describe hvKp strains possessing only a portion of the virulence plasmid; the clinical consequences of this are unclear. Therefore, the goal of this report was to determine whether virulence among hvKp strains varied and, if so, how to best identify the relative virulence of hvKp isolates. Data demonstrate hvKp pathogenic potential varies in CD1 and BALB/c murine infection models. In contrast, measurements of siderophore production and mucoviscosity were unable to discriminate the differences in hvKp isolate virulence observed in mice. This information can be used in future studies to determine the mechanisms responsible for differences between fully virulent hvKp and partially virulent hvKp and whether the differences observed in mice translate to disease in humans. Klebsiella pneumoniae is widely known for its propensity to cause health care-associated and hospital-acquired infections (HAI) and has become increasingly resistant to antimicrobials (K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp)  , and sele (hvKp) . In stare (hvKp) . A monomhalmitis .iucA, iroB, peg-344, prmpA, and prmpA2 genes which reside on the canonical hvKp virulence plasmid, albeit imperfectly, most accurately predicts whether a strain possesses the hypervirulent phenotype. This result was validated in the outbred CD1 murine infection model, which was highly discriminatory for differentiating hvKp-rich and cKp-rich strain cohorts that were developed based on clinical criteria from human infection  and a collection of isolates from the Walter Reed Army Institute of Research Multidrug-Resistant Organism Repository and Surveillance Network (MRSN) that were identified by possessing all of the hvKp-specific biomarkers iucA, iroB, prmpA, prmpA2, and peg-344 (n = 13) underwent screening. The following three K. pneumoniae strain cohorts were defined by their phenotype after SQ challenge of outbred CD1 mice; fully virulent hvKp strains (fvhvKP) were lethal at a challenge inoculum (CI) of ≤103 CFU; partially virulent hvKp strains (pvhvKp) were lethal at a CI >103 to 107 CFU; and classical K. pneumoniae (cKp) were not lethal at a CI of 107 CFU. Of the 100 hvKp strains in our strain library that consists of isolates from otherwise healthy, ambulatory patients with a clinical syndrome of tissue invasive infection , 11/100 (11%) possessed a pvhvKP phenotype. Of the 13 MRSN isolates, 7/13 (54%) possessed a pvhvKP phenotype. For this study, 15 fvhvKp strains, 14 pvhvKp strains, and 15 cKp strains were chosen for further study. Quantitative lethality data for these strains is listed in fvhvKp strains and 13/14 pvhvKp strains (hvKp42 lacks iucA and rmpA) possess all of the hvKp-specific virulence plasmid genes iucA, iroB, prmpA, prmpA2, and peg-344, which have been shown to accurately identify hvKp strains (fvhvKp), hvKp94 (pvhvKp), and cKp1 (cKp) were selected for additional detailed studies in CD1, BALB/c, and C57BL/6 mice. These data support that not all hvKp strains share a similar pathogenic potential in mice.The CD1 subcutaneous challenge (SQ) infection model has been validated for differentiating hvKp from cKp pathotypes, and it is extraordinarily sensitive as evidenced by at least a 5-log-unit window for detecting differences in lethality after bacterial challenge with fully virulent hvKp and cKp strains. Therefore, this model was initially used to assess for potential differences in virulence between hvKp strains . Our hvK strains , whereas strains . hvKp2  and hvKP94 (pvhvKp)  more lethal after IP challenge with approximately 102 CFU and was significantly more lethal with approximately 103 to 105 CFU compared to hvKP94 at similar titers; resultant mortality rates for these CIs were 70%, 90%, and 100% for hvKP2 compared to 0%, 0%, and 30%, for hvKP94, respectively  challenge of C57BL/6 or BALB/c mice is commonly used to determine the relative virulence of (pvhvKp) . When CD(pvhvKp) . For CD1ectively . For BALectively . By contectively . Exact tfvhvKp) and hvKp94 (pvhvKp), SQ challenge was more numerically discerning than IP challenge  and 60% (P = 0.381), respectively (P = 0.095) and 70% (P = 0.077), respectively  Quantitative siderophore assay. Previous data have shown that total siderophore production of >30 μg/ml is able to accurately differentiate between hvKp and cKp strains  fvhvKp strains (n = 15), (ii) pvhvKp strains (n = 14), and (iii) cKp strains (n = 15) . Results from individual strains are depicted in fvhvKp strains and 12/14 pvhvKp strains produced siderophore levels of >100 μg/ml. Although 2/14 pvhvKp strains produced siderophore levels of 15.85 ± 2.57 and 34.51 ± 4.9, these data do not support the use of quantitative siderophore measurement as a means to differentiate fvhvKp strains from pvhvKp strains. strains . Therefo(n = 15) . The mea(ii) Quantitative mucoviscosity assay. Mucoviscosity, which is due to RmpA regulated production of RmpD, is a phenotype that is associated with hvKp strains  values were 0.44 ± 0.17, 0.47 ± 0.21, and 0.09 ± 0.06 for the fvhvKp, pvhvKp, and cKp strain cohorts, respectively, when grown in LB and 0.37 μg/ml ± 0.13, 0.46 ± 0.18, and 0.10 ± 0.04 when grown in c-M9-te . Results from individual strains are depicted in fvhvKp and pvhvKp strains had a post/prespin OD600 ratio of >0.15 when grown in c-M9-te broth; 15/15 and 13/14 of the fvhvKp and pvhvKp strains had a post/prespin OD600 ratio of >0.15 when grown in LB. By contrast, 1/15 and 2/15 cKp strains had a post/prespin OD600 ratio of >0.15 when grown in LB and c-M9-te broth, respectively. Interestingly, the mean mucoviscosity values for the index pvhvKp strain hvKp94 were 0.51 ± 0.10 and 0.54 ± 0.05 when grown in LB and c-M9-te broth, respectively, similar to fvhvKp strains. These data demonstrate that the quantitative mucoviscosity assay on average is able to differentiate hvKp from cKp strains but is unable to differentiate fvhvKp from pvhvKp strains. strains –29. TheriucA, iroB, peg-344, prmpA, and prmpA2 or siderophore production of >30 μg/ml have been shown to accurately differentiate the hvKp and cKp pathotypes  resulted in multiple sites of infection causing meningitis and brain abscess. However, this strain was from Taiwan and shipped unfrozen to the United States for study. Therefore, a genetic alteration due to handling and storage cannot be excluded. The pvhvKp strain hvKp94 caused prostatic abscesses, but it was likely directly introduced as a result of transrectal biopsy. Therefore, since pvhvKp strains can cause community-acquired infection in otherwise healthy hosts, clinical data support enhanced virulence compared to cKp strains, but it remains unclear whether they are equally virulent to fvhvKp strains and if differences exist, what the management and infection control implications would be, if any.The clinical data available for the ine hvKp , and incine hvKp . Four isiucA, iroB, peg-344, prmpA, and prmpA biomarkers. Twenty-eight of 29 hvKp strains in this study possessed all five of these markers (iucA and rmpA2 were absent in one strain), which is not surprising since they are usually linked on the canonical hvKp virulence plasmid  was isolated from the blood of a patient who presented from the community in Buffalo, New York, with bacteremia and endophthalmitis (iucA+iroB+peg-344+prmpA+prmpA2+) was isolated from the blood of a patient from Buffalo, New York, post-transrectal prostatic biopsy that was complicated by multiple prostatic abscesses. cKp1  was isolated from the blood of a patient from Montreal, Quebec, Canada  to precipitate protein and cellular debris. The gDNA was extracted using isopropanol, followed by two washes with 70% ethanol before resuspension in 10 mM Tris (pH 8.5). DNA concentration and purity were assessed using a Nanodrop One spectrophotometer with a minimum requirement of a 260/280 ratio of Taq Mix, 12.5 ng gDNA, 0.1 nM each primer and nuclease-free water in a 10-μl reaction mixture. PCR conditions were as follows: 2 min at 95°C; 35 cycles, with 1 cycle consisting of 15 s at 95°C, 30 s at 60/53°C, and 1.5 min at 72°C, with a hold at 4°C. The PCRs were analyzed on a 1.5% agarose gel. Four multiplex PCRs were performed with the following primer pair combinations ; (ii) peg589 and peg1631; (iii) peg-344 and prmpA; (iv) iucA pair #2, prmpA2 and terB. The hypervirulent strain hvKP1 (Genomic DNA (gDNA) was extracted from 300 μl of a strain grown overnight in lysogeny broth (LB) . Cells underwent centrifugation, and the resultant pellet was resuspended and incubated for 15 min in a Tris-EDTA (TE) lysing buffer containing 0.5% sodium dodecyl sulfate (SDS) and 0.16 mg/ml proteinase K, followed by a 30-min incubation at 37°C with DNase-free RNase (0.033 mg/ml). Next, 36 μl of 5 M sodium chloride was added, and the solution was vortexed vigorously and centrifuged  iuc Tris pH .5. DNA c10.1128/mSphere.00045-21.2TABLE S2Table S2, XLSX file, 0.01 MB.Multiplex PCR primers. Download Copyright © 2021 Russo et al.2021Russo et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the For the quantitative siderophore assay, strains were grown individually overnight at 37°C in iron-chelated M9 minimal medium containing Casamino Acids (c-M9-CA) , and cul2, 1 μg/ml CoCl2, 20 μg/ml MgCl2, 10 μg/ml MnCl2) (c-M9-te) to a starting OD600 of approximately 0.2. Pilot experiments compared the mucoviscosity of selected strains grown to either an OD600 of 1.0, grown for 6 h, or grown for 24 h. It was determined that the 24-h cultures demonstrated higher mucoviscosity values and allowed greater differentiation between strains; therefore, mucoviscosity was measured after growth for 24 h. Strains were grown shaking (275 rpm) for 24 h at 37°C, and the OD600 was normalized to 1.0 (prespin OD600) with either LB or c-M9-te minimal medium as appropriate. The OD600-adjusted culture (1.5 ml) was then centrifuged at 1,000 × g for 5 min. Seven hundred microliters was carefully withdrawn from the top of the supernatant without disturbing the pellet, and the OD600 was measured (postspin OD600). Mucoviscosity was recorded as the post/prespin OD600 ratio. Three independent assays were performed on different days for each strain, and the data were reported as means ± SD.Overnight cultures grown in LB broth were used to inoculate 10 ml of either LB or iron-chelated M9 minimum medium plus added trace elements (5 μg/ml CaClin extremis state)/death, which was recorded as a dichotomous variable. Signs that were monitored and that resulted in immediate euthanasia using methods consistent with the recommendations of the American Veterinary Medical Association Guidelines included hunched posture, ruffled fur, labored breathing, reluctance to move, photophobia, and dehydration. Challenge inocula for all animal experiments are listed in Animal studies were reviewed and approved by the Veterans Administration Institutional Animal Care Committee and the University at Buffalo-SUNY and were carried out in strict accordance with the recommendations in the guidelines delineated in the "NIH Guide for the Care and Use of Laboratory Animals" (revised 1985) and the "Ethics of Animal Experimentation Statement"  as monitored by the Institutional Animal Care and Use Committee (IACUC). All efforts were made to minimize suffering. Veterinary care for the animals was supplied by the staff of the Veterans Administration Animal Facility under the direction of a fully licensed veterinarian. BALB/c, C57BL/6, and CD1 male mice, 4 to 6 weeks old, were obtained from Charles River Laboratories and quarantined for 5 days before use. Bacteria were grown overnight in LB and were subsequently serially diluted to the required titers in 1× phosphate-buffered saline (PBS). A 100-μl bacterial suspension was injected either subcutaneously (SQ) or intraperitoneally (IP) using a 0.5-ml insulin syringe. The bacterial titer was enumerated by serial dilutions of the inoculum. The animals were closely monitored for 14 days after challenge for the development of the study endpoints, survival or severe illness  was performed for the mucoviscosity assays to compare strain cohorts when grown in LB and c-M9-te medium. Bonferroni posttests were used to account for multiple comparisons. A P value of <0.05 was considered statistically significant. P values for all animal experiments are listed in Statistical analyses were performed using GraphPad Prism 9.0. A log rank (Mantel-Cox) test was performed to analyze 10.1128/mSphere.00045-21.4TABLE S4P values for animal experiments. Download Table S4, XLSX file, 0.01 MB.Copyright © 2021 Russo et al.2021Russo et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


This study aimed to identify the major determinants of longitudinal change of HbA1c in non-diabetic patients. This study included subjects from the 104,451 participants enrolled between 2012 and 2018 in the Taiwan Biobank. We only included participants with complete data at baseline and follow-up . Patients with diabetes at baseline or follow-up (n = 3983) were excluded. Finally, 23,226 participants without diabetes at baseline and follow-up were selected in this study. △Parameters was defined as the difference between the measurement baseline and follow-up. Multivariable linear regression analysis was used to identify the major determinants of HbA1c longitudinal change (△HbA1c). During a mean 3.8 year follow-up, after multivariable analysis, new-onset hypertension , high △heart rate , high △BMI , high △fasting glucose , low △creatinine , high △total cholesterol , high △hemoglobin , high △GPT , and low △albumin  were significantly associated with high △HbA1c. In non-diabetic population, strategies to decrease the development of new-onset hypertension, resting heart rate, body mass index, fasting glucose, total cholesterol, and GPT and increase serum albumin level might be helpful in slowing the longitudinal change of HbA1c. In addition, increased hemoglobin and decreased serum creatinine over time also had an impact on the HbA1c elevation over time in non-diabetic population.Although many cross-section studies have assessed the determinants of glycated hemoglobin (HbA HbAdiabetes .1c is not only a helpful diagnostic and prognostic parameter in patients with diabetes, but is also useful in predicting future diabetic risk in non-diabetic patients [1c could be used as a predictive biomarker for coronary artery disease in non-diabetic low-risk patients [1c level was correlated with the severity of coronary artery disease as measured by the SYNTAX score. However, no correlation was found between admission glucose or fasting glucose levels and severity of coronary artery disease [1c level was associated with cardiovascular and overall mortality in non-diabetic hemodialysis patients, whereas a lower HbA1c level was not [HbApatients . Ewid etpatients . Arbel e disease . In addi was not .1c concentration was found to be accompanied with an increase in body mass index (BMI) regardless of diabetes status [1c increased as renal function declined [1c in a large cohort study with repeated measurements of above data over a mean of 3.8 years of follow up and identify the major determinants of longitudinal change of HbA1c in non-diabetic patients.Many cross-section studies ahve assessed the relationship between clinical important parameters and glucose metabolism in non-diabetic patients. Higher resting heart rate in individuals without diabetes was reported to be associated with future unfavorable changes in insulin levels and insulin sensitivity. Resting heart rate might be a risk marker for future pathophysiological changes in glucose metabolism . Systolis status . A U-shas status . High chs status . Previous status . Kang etdeclined . Howeverhttps://www.twbiobank.org.tw/new_web_en/; accessed on 14 April 2020). Written informed consent was acquired from all enrolled participants and all investigations in this study were conducted according to the Declaration of Helsinki. The present study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (KMUHIRB-E(I)−20180242), which was approved on 8 March 2018.The data contained in this study were collected from the Taiwan Biobank (TWB), a general population-based research database comprised of cancer-free residents aged 30−70 years included from 31 recruitment stations in Taiwan since 2008. The data source of TWB has been described previously ,18. The 1c is not only a helpful diagnostic and prognostic parameter in patients with diabetes, but is also useful in predicting future diabetic risk in non-diabetic patients were collected. All of theabove data were obtained at baseline and at around 3.8-year follow-up. We only included participants with complete data at baseline and 3.8-year follow-up . Patients with diabetes at baseline or follow-up (n = 3983) were excluded. Finally, 23,226 participants without diabetes at baseline and follow-up  were selected in this study.The present study included study subjects from the 104,451 participants enrolled between 2012 and 2018 in TWB. The demographic information including age, gender, and history of diabetes and hypertension, was acquired from a face-to-face interview with TWB investigators. BMI, systolic and diastolic blood pressures, resting heart rate, and overnight fasting blood chemistry parameters including fasting glucose, total cholesterol, triglycerides, uric acid, serum creatinine, hemoglobin, glutamic-oxalocetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), albumin, and HbA1c was less than 6.5% were considered not to have diabetes.Participates who did not take anti-diabetic medication, had no past history of diabetes, and whose fasting glucose was less than 126 mg/dL and HbAt-test. △Parameters was defined as the difference between the measurement baseline and follow-up. Statistically significant variables in univariable linear regression analysis were selected into the multivariable analysis. Multivariable linear regression analysis was used to identify the major determinants of baseline HbA1c and HbA1c longitudinal change (△HbA1c). A p value of less than 0.05 was considered to be significant.We used SPSS 22.0 for Windows  to perform statistical analysis. Data were expressed as mean ± standard deviation and percentage. The differences between longitudinal changes of clinical and laboratory data during the 3.8-year follow-up were checked by paired 1c, serum creatinine, total cholesterol, triglyceride, GOT, and GPT were increased and uric acid and albumin were decreased at follow-up. 1c. After the multivariable analysis, old age , female , high BMI , high fasting glucose , low creatinine , high uric acid , high total cholesterol , high triglyceride , low GOT , high GPT , and low albumin  were significantly associated with high baseline HbA1c. 1c longitudinal change and longitudinal changes of other parameters in the univariable and multivariable analyses. In the multivariable analysis, new-onset hypertension , high △heart rate , high △BMI , high △fasting glucose , low △creatinine , high △total cholesterol , high △hemoglobin , high △GPT , and low △albumin  were significantly associated with high △HbA1c. 1c longitudinal change and longitudinal changes of other parameters. In participants with fasting glucose <100 mg/dL , new-onset hypertension , low △systolic blood pressure , high △diastolic blood pressure , high △BMI , high △fasting glucose , low △creatinine , high △total cholesterol , high △hemoglobin , high △GPT , and low △albumin  were significantly associated with high △HbA1c.We have further performed two subgroup analyses (group of fasting glucose <100 mg/dL and group of 100 ≤ fasting glucose < 126 mg/dL) to survey the correlation between HbAn = 3078), high △heart rate , high △BMI , high △fasting glucose , low △creatinine , low △uric acid , high △total cholesterol , high △hemoglobin , low △GOT , high △GPT , and low △albumin  were significantly associated with high △HbA1c.In participants with 100 ≤ fasting glucose < 126 mg/dL  when heart rate increased by 1 beat/min. Kim et al. found an increase in resting heart rate over a two-year follow-up period was significantly associated with a risk of diabetes, independently of baseline resting heart rate and glycol-metabolic parameters in 7,416 adults without diabetes [1c change over a 3.8-year follow-up in non-diabetic population. Hence, control of resting heart rate might be beneficial to slow HbA1c longitudinal change.Although baseline resting heart rate had no correlation with baseline HbAsmall 0.0% when he1c and longitudinal change of BMI had also a strong positive correlation with HbA1c longitudinal change in the multivariable analysis. Maw et al. found higher BMI might have influenced HbA1c trends during one to two-year follow-up [1c longitudinal change. It suggested good body weight control was effective in slowing HbA1c change in non-diabetic patients.In the present study, baseline BMI had a strong positive correlation with baseline HbAollow-up . In this1c and longitudinal change of fasting glucose also had a strong positive correlation with HbA1c longitudinal change in our multivariable analysis, which was consistent with previous studies [1c longitudinal change.Baseline fasting glucose had a strong positive correlation with baseline HbA studies ,23,24. H1c in the univariable analysis, but such association became negative after multivariable adjustment. Hence, baseline serum creatinine was not a major determinant of increased HbA1c at baseline. In addition, longitudinal change of blood creatinine was negatively correlated with HbA1c longitudinal change in our multivariable analysis. Hu et al. examined a prospective association between serum creatinine level and diabetes in 31,343 male workers without diabetes and found low serum creatinine was associated with an increased risk of diabetes [1c increase over time during 3.8-year follow-up in non-diabetic population. Hence, decreased blood creatinine over time might reflect decreased skeletal muscle mass over time [1c increase over time. Baseline serum creatinine was positively associated with baseline HbAdiabetes . Our prever time . Skeletaver time  and decrver time ,28, whic1c, which was consistent with the previous study [1c longitudinal change after multivariable analysis. Hence, longitudinal change of uric acid may only have no or a minor role in HbA1c longitudinal change in our present study. In this study, baseline uric acid was positively associated with baseline HbAus study . However1c, which was similar with previous studies [1c longitudinal change in our multivariable analysis. Hence, control of total cholesterol should be useful in slowing HbA1c longitudinal change.Our result showed baseline total cholesterol and triglyceride were positively associated with baseline HbA studies ,31. In a1c increased with decreasing hemoglobin levels [1c. Recently, Sakamoto et al. demonstrated that elevated HbA1c among anemic people with mean corpuscular volume (MCV) < 80 fL or MCV 80–90 fL, and decreased HbA1c among anemic people with MCV > 90 fL, suggesting that different types of anemia might influence HbA1c differently [1c. In the present study, baseline hemoglobin was not associated with baseline HbA1c, but longitudinal change of hemoglobin had a positive correlation with HbA1c longitudinal change after multivariable analysis. The temporal association between hemoglobin and HbA1c over time might be more informative than that found in the cross-sectional studies. Hence, hemoglobin elevation with time might cause HbA1c to increase concurrently. Previous study reported that genetic variation potentially affects HbA1c. Wheeler E. et al. [1c. Nineteen glycemic and twenty-two erythrocytic variants were associated with HbA1c at genome-wide significance. However, in our study, is it difficult to assess the influence of genetic variation on HbA1c, which represents a technical limitation. Several previous studies reported HbAn levels ,33,34. Hn levels  or no con levels  between ferently . The inc. et al.  used gen1c, but longitudinal change of GOT had no significant correlation with HbA1c longitudinal change after multivariable analysis. Hence, longitudinal change of GOT may only have a minor role in HbA1c longitudinal change . However, baseline GPT was positively associated with baseline HbA1c and longitudinal change of GPT also had a positive correlation with HbA1c longitudinal change in the multivariable analysis. Previous studies have demonstrated a relationship between elevated liver enzymes and incident type 2 diabetes [1c change over time. Hence, lowering GPT should be helpful in slowing HbA1c longitudinal change.Baseline GOT was negatively associated with baseline HbAdiabetes ,39. In fdiabetes . It suggdiabetes . In the 1c and longitudinal change of albumin also had a negative correlation with HbA1c longitudinal change in the multivariable analysis. Hence, decreased albumin with time might result in HbA1c increase concomitantly.Reduced albumin was reported to be associated with an unfavorable metabolic profile, characterized by increased adipose tissue inflammation, adiposity, and glucose and with an increased risk for type 2 diabetes mellitus . In this1c. Hence, our results could not apply to diabetic patients. Second, the information of anti-hypertensive medication and lipid lowering agents were similarly lacking. These medications undoubtedly influenced the values of blood pressures, resting heart rate, and lipid profile. Additionally, information about diet during the follow-up, anemia-related data including mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, serum iron, transferrin, and ferritin were lacking, which might influence the interpretation. Therefore, we could not exclude the impact of such medication on our present results. Third, according to the statistics of TWB, the proportion of participants coming back to track is about 50%. Therefore, selection bias may affect the interpretation of our results. Finally, we had no information concerning smoking status and exercise habit, so we could not analyze the impact of these parameters on the longitudinal change of HbA1c.There were several limitations to this study. First, our study population were selected from TWB and no medication data were available in this data bank. We excluded patients with diabetes mellitus s most of them received anti-diabetic mediation therapy, which certainly had an impact on the longitudinal change of HbA1c longitudinal change in the multivariable analysis. Hence, strategies to decrease the development of new-onset hypertension, resting heart rate, BMI, fasting glucose, total cholesterol, and GPT and increase serum albumin level might be helpful in practice for slowing the longitudinal change of HbA1c in non-diabetic population. In addition, increased hemoglobin and decreased serum creatinine over time also had an impact on the HbA1c elevation over time in non-diabetic population.In this study including 23,326 subjects with non-diabetes, we found new-onset hypertension and longitudinal changes of resting heart rate, BMI, fasting glucose, total cholesterol, hemoglobin, and GPT were positively and longitudinal changes of serum creatinine and albumin were negatively correlated with HbA


Clostridioides difficile, mediates the release of the large clostridial glucosylating toxins (LCGTs), TcdA and TcdB, and TpeL from C. perfringens uses another holin-like protein, TpeE, for its secretion; however, it is not yet known if TcdE or TpeE secretion is specific to these proteins. It is also unknown if other members of the LCGT-producing clostridia, including Paeniclostridium sordellii (previously Clostridium sordellii), use a similar toxin-release mechanism. Here, we confirm that each of the LCGT-producing clostridia encode functional holin-like proteins in close proximity to the toxin genes. To characterise the respective roles of these holin-like proteins in the release of the LCGTs, P. sordellii and its lethal toxin, TcsL, were used as a model. Construction and analysis of mutants of the P. sordellii tcsE (holin-like) gene demonstrated that TcsE plays a significant role in TcsL release. Proteomic analysis of the secretome from the tcsE mutant confirmed that TcsE is required for efficient TcsL secretion. Unexpectedly, comparative sample analysis showed that TcsL was the only protein significantly altered in its release, suggesting that this holin-like protein has specifically evolved to function in the release of this important virulence factor. This specificity has, to our knowledge, not been previously shown and suggests that this protein may function as part of a specific mechanism for the release of all LCGTs.Protein secretion is generally mediated by a series of distinct pathways in bacteria. Recently, evidence of a novel bacterial secretion pathway involving a bacteriophage-related protein has emerged. TcdE, a holin-like protein encoded by toxigenic isolates of  Protein secretion, defined as the transport of proteins from the cell cytoplasm to the extracellular milieu or into a target cell , is a crClostridioides difficile, TcsH (haemorrhagic toxin) and TcsL  from Clostridium sordellii (renamed Paeniclostridium sordellii), TpeL from Clostridium perfringens, and Tcnα  from Clostridium novyi . As. AsC. diand tcdE ,17, wherand tcdE ,19, althand tcdE . As for and tcdE , althougand tcdE . Furthern system . Holins n system . Despiten system ,24, the on-lytic ,25.Escherichia coli  or carrying a deletion in holin and endolysin genes [λCmrΔ(SR)] were used as hosts for the plasmid constructs. These plasmids included pJN5, carrying the gene encoding the λ holin S105, and pBRQ(ΔRBS), used as positive and negative controls, respectively. Both λ(Sam7) and λCmrΔ(SR) encode a thermo-sensitive CI repressor (cI857) and are induced upon shifting the culture temperature from 30 °C to 42°C. Strains were grown in LB broth at 30 °C until the OD600 reached 0.15–0.25 before the thermo-induction of the λ prophage at 42 °C for 15 min. Bacterial growth and lysis at 37 °C were then followed by monitoring the absorbance at 600 nm at 15 min intervals. The curves in The d by PCR , digestetcsE, based on predictions from the Perutka Algorithm [http://clostron.com/clostron2.php, accessed on 10 March 2017). The correct insertion of the intron and the loss of the plasmid were confirmed using PCR and Southern hybridisation as previously described [P. sordellii strain referred to in the parB plasmid partitioning gene, followed by subculture [Mutagenesis using the TargeTron system and subsequent complementation was performed using a published method . For mutlgorithm  via the lgorithm  design tescribed . Primersescribed . Similarbculture .tcsE. PCR products were cloned into pRPF185 using restriction enzymes BamHI and SacI. The relevant primers are listed in For complementation, genes including their RBS were PCR amplified using ATCC9714 genomic DNA as a template, with primers DLP525 and DLP526 for P. sordellii cultures in HIS broth were diluted to an OD600 of 0.3 before a 1/100 dilution into 20 mL TY  broth in tissue culture flasks. The TY cultures were incubated for 22 h at 37 °C in an anaerobic environment. At T = 5 h, anhydrous tetracycline (50 ng/mL) was added to all cultures to induce the complementation vectors. At T = 10 h, cells were collected and RNA extracted for transcript analysis. At T = 10 h and T = 22 h, culture supernatants were collected by pelleting cells by centrifugation at 2500× g, followed by filtering the supernatant through a 0.22-micron filter to remove all bacterial cells. The supernatants were analysed for TcsL levels using a C. difficile TcdB-specific ELISA detection kit (tgcBiomics), which is cross-reactive to TcsL  nm of 0.05, for 24 h, and the cells pelleted by centrifugation. The supernatants were filter sterilized through 0.22 µ filters and stored at 4 °C prior to use. Cells were cultured in minimum essential medium  containing 10% heat-inactivated fetal calf serum (HI FCS), 100 units/mL penicillin, and 100 µg/mL streptomycin in culture flasks at 37 °C in 5% CO2. The cells were grown to a confluent monolayer and then sub-cultured by incubating in 2 mL of 0.1% trypsin in 1 mM EDTA. The cells were counted and resuspended in fresh medium at a concentration of 1 × 105 cells/mL, and 100 µL aliquots of the cell suspension were seeded into each well of 96-well plates. The plates were then incubated for 20 h, followed by the removal of the culture medium. Serial two-fold dilutions of the P. sordellii culture supernatants were prepared in MEM alpha medium supplemented, with 1% HI FCS and 100 µL added to each well. Negative controls, which were cells treated with 100 µL MEM alpha medium supplemented with 1% HI FCS, were included. Plates were then incubated at 37 °C in 5% CO2 and morphological changes observed via microscopy after 24 h. The endpoint was scored as the last dilution at which a cytopathic effect (CPE) was observed.To prepare supernatants for cell cytotoxicity assays, P. sordellii cultures in HIS broth were diluted to an OD600 of 0.3 before a 1/100 dilution into 20 mL TY broth in glass bottles. The TY cultures were incubated for 22 h at 37 °C in an anaerobic environment. The OD600 of the cultures was recorded every two h from T = 1 to T = 11, with a final reading at T = 22.Mid-logarithmic phase P. sordellii RNA was obtained from 3 mL of TY broth culture at T = 10 of the TcsL release assay, equating to an early stationary growth phase. RNA was isolated and its concentration determined using a published method [tcsL in P. sordellii was determined by reverse-transcriptase digital droplet PCR (RT-ddPCR) based on a previous protocol [® III reverse transcriptase (Invitrogen). PCR samples were set up to contain 5 ng or 0.5 ng of cDNA with genespecific primers at 200 nM each, along with an equal volume of QX200TMddPCRTMEvaGreen Supermix . PCR reactions were converted to droplets in DG8TM cartridges (Bio-Rad) using the QX200TM droplet generator (Bio-Rad) and amplified in a C1000 TouchTM thermal cycler (Bio-Rad). Amplification events within individual droplets were measured by the QX200TM droplet reader (Bio-Rad), and the resulting data were analysed using QuantaSoftTM software (Bio-Rad). tcsL expression levels represent the number of transcripts per ng of cDNA, as a percentage of the transcripts of the housekeeping gene rpoA. Statistical analysis was carried out using the Mann–Whitney U test on four independent biological replicates.Total d method . The expprotocol . All reaP. sordellii secreted proteins, ice-cold 100% (w/v) tri-chloroacetic acid was added to 1 mL aliquots of filtered culture supernatant to achieve a final concentration of 10% (v/v). The supernatants were left on ice overnight, and precipitated proteins were then pelleted at 17,000× g for 30 min at 4 °C. The pellets were washed twice in 1 mL of −20 °C 90% (v/v) acetone by incubation on ice for 15 min followed by centrifugation for 30 min at 4°C. All remaining acetone was removed, and protein pellets were air-dried in a fume hood for 10 min and then stored at −80°C until needed.To concentrate Proteins were resuspended in 100 μL 1× LDS buffer (Thermo Fisher Scientific). The concentration of total protein (>10 kDa) in each sample was determined using densitometry. To do this, samples were separated by SDS-PAGE in a 4–15% gradient Stain-Free gel (Bio-Rad), and proteins in each lane were detected using the ChemiDoc Imaging system (Bio-Rad). Using Image Lab (Bio-Rad) software, densitometry against the BenchMark protein standard (Thermo Fisher Scientific) allowed protein concentrations to be determined. Samples were then diluted to 0.4 μg/ μL using 1× LDS buffer.g for 5 min, and the denser aqueous phase was collected and desalted using SDB-RPS StageTips [Then, 10 μg of protein from each sample  was simultaneously reduced and alkylated with 50 mM TCEP and 50 mM chloroacetamide, respectively, for 5 min at 95 °C. Proteins were then digested in-solution with 500 ng trypsin (Promega) and incubated overnight at 37 °C. Deoxycholate (DOC) was removed by phase transfer using an equal volume of ethyl acetate and acidified to 0.5% trifluoroacetic acid. The samples were then centrifuged at 14,000× tageTips . The elu−1 for 3 min. The trap-column was then switched in line with the analytical column . Peptide separation was performed at 250 nl min−1 using reverse phase chromatography  and Buffer B . The non-linear gradient conditions for Buffer B were as follows: 2.5–7.5% over 1 min, 7.5–37.5% over 90 min, 37.5–42.5% over 3 min, 42.5–99% over 5 min, 99% hold 6 min, 99-2.5% over 1 min, and 2.5% hold 17 min. Data were collected in positive mode using data-dependent acquisition. MS scan properties were as follows: scan range (m/z) 375–1575, Orbitrap resolution 120,000, AGC target 1E6, maximum injection time 54 ms, and dynamic exclusion 15 s. MS/MS scan properties were as follows: HCD activation, isolation window of 1.8 (m/z), collision energy 32%, Orbitrap resolution 30,000, AGC target 2E5, maximum injection time 54 ms.The peptides (4 μL) were analysed by nanoliquid chromatography–tandem mass spectrometry on a Dionex Ultimate 3000 UPLC coupled to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). Peptides were first loaded onto a trap column  at 15 μL minP. sordellii ATCC9714 coding sequences from the available genome sequence . Contaminant proteins from the media (yeast proteins (UniProt Proteome ID UP000002311) and casein subtypes ) and products encoded by the Group II Intron or pRPF185 vector (ErmB (UniprotKB P20173) and β-glucuronidase (UniprotKB P05804)) were also identified but removed from further comparative analysis. Spectra were searched with the following criteria: specific digestion mode; maximum 2 missed cleavages; fixed modification of carbamidomethylcysteine; variable modifications of methionine oxidation; and N-terminal acetylation. Label-free quantification (LFQ), and match between run options were enabled, and false discovery rates of 1% for proteins and peptides were applied, with 2 minimum peptides required for identification. The raw data from this analysis are provided in Raw files were analysed using the MaxQuant platform  version 2 transformation. Non-valid values (below the limit of detection) were inputted through random selection of values within ±0.2 standard deviations from the mean of the Gaussian distribution of the data set for their respective biological replicate, having first being shifted 1.9 standard deviations to the left. Statistical analysis of LFQ values for two strains for a particular protein was performed using a 2-tailed student’s t-test. For screening purposes, the statistical significance cut-off was considered a permutation-based false discovery ratio of 0.1 using 250 randomisations, while implementing an artificial within group’s variance of 0.1.Analysis of LFQ data was performed using Perseus version 1.5.6.0 . Identif