Triticum aestivum L.) rusts are a worldwide production problem. Plant breeders have used genetic resistance to combat these fungi. However, single‐gene resistance is rapidly overcome as a result of frequent occurrence of new virulent fungal strains. Thus, a supply of new resistance sources is continually needed, and new resistance sources are limited within hexaploid wheat genetic stocks. Wild relatives are able to be a resource for new resistance genes but are hindered because of chromosome incapability with domesticated wheats. Twenty‐eight double‐haploid hexaploid wheat/Amblyopyrum muticum (Boiss.) Eig introgression lines, with introgressions covering the majority of the T genome, were evaluated for resistance to Puccinia triticina Erikss., P. graminis Pers.:Pers. f.sp. tritici Erikss. & E. Henning, and P. striiformis Westend. f.sp. tritici Erikss.. At the seedling level, four lines were resistant to races of P. triticina, six lines were resistant to P. graminis, and 15 lines were resistant to P. striiformis. At the adult stage, 16 lines were resistant to P. triticina. Line 355 had resistance to all three rusts and line 161 had resistance to all tested races of P. triticina. Some of these lines will require further work to reduce the size of the introgressed segment; however, lines 92 and 355 have very small fragments and can be used directly as new resistance donors.Wheat ( Each can be controlled with chemical fungicide applications. However, in many areas, environmental constraints limit production resulting in low profit margins, hindering the use of fungicides.Wheat rusts are the most formidable group of pathogens influencing production. Three species, Lr34/Yr18/Sr57, providing various levels of resistance to all three rusts . Puccinia triticina is found in all world regions of wheat production and >70 different races are found each year in North America alone  Thell.] , Leymus mollis (Trin.) Pilg, L. racemosus (Lam.) Tzvelev, and Thinopyrum junceiforme (Á. Löve & D. Löve) Á. Löve  van Slageren] with Th. intermedium (Host) Barkworth & D. R. Dewey, Th. bessarabicum (Savul. & Rayss) Á. Löve, Th. elongatum (Host) D. R. Dewey, Th. ponticum (Podp.) Barkworth & D. R. Dewey, Anthosachne rectiseta (Nees) Barkworth & S. W. L. Jacobs, Ae. caudata L., and Ae. speltoides Tausch  is a wild relative of wheat originating from the Middle East and Armenia. Am. muticum possesses a Ph1 suppressor gene that facilitates recombination between Am. muticum chromosomes and the homoeologous chromosomes of wheat  and initially crossed to either ‘Chinese Spring’ (CI6223) or ‘Pavon 76’ (PI519847), The F1 products were crossed to either ‘Paragon’ , and 97AZ103 were suspended in 2 ml of Soltrol 170 parafin oil (Phillips Petroleum) and sprayed onto the seedlings with an atomizer  at 40 psi. Seedlings were transferred to a Percival humidity chamber at 100% relative humidity with wall settings of 5 °C and water basin of 40 °C for 16 h. Plants were transferred back into the greenhouse. At 14 d post inoculation (dpi), infection types were rated on a scale of 0–4 . Five seeds of the hexaploid parents Paragon and Pavon 76 along with a susceptible check ‘Thatcher’ were also included and placed throughout the pan. At the two‐ to three‐leaf seedling stage, 25 mg of P. striiformis tritici isolates collected in Kansas in 2012 and 2014 . Plants were inoculated as above but humidity chamber conditions were wall settings of 0 °C and water basin of 29 °C for 24 h. At 14 dpi, plants were scored using a scale of 1 (resistant) to 9 (susceptible) , as above except humidity chamber conditions were with wall settings of 6 °C and water basin of 42 °C for 16 h. At 14 dpi, infection types were scored on a scale of 0, ;, 1–4  as described in Hundie et al.  markers. Fifty to 100 ng of DNA was dried in a 384‐well plate and resuspended using 3 μl of KASP assay master mix containing primer, 2× KASP buffer and enzyme (Keygene), and MgCl2. For Lr46, the primer used was Lr46‐Yr29_JF2‐2‐KASP , and for Lr34, two separate primers Lr34Exon11‐KASP  were used to check for the Lr34 gene and also confirm there was not a false‐positive gene. For Lr34exon11 and Lr46, the thermal cycler conditions used were as follows: 94 °C for 15 min, 97 °C 15 sec, 68 °C for 1 min at −0.8 °C per cycle for 10 cycles, 97 °C for 20 sec, 60 °C for 1 min for 30 cycles, 59 °C for 30 sec at −1.0 °C per cycle for 34 cycles, and a final cool down of 10 °C for 5 min. For Lr34JagExon22, the thermal cycler conditions were as follows: 94 °C for 15 min, 94 °C for 20 sec, 60 °C for 1 min for 30 cycles, and a cool down for 5 min at 10 °C. Reactions were run on a GeneAmp Systems 9700 (Applied Biosystems) and results were processed using Klustercaller (LGC Biosearch Technologies).Twenty‐one lines were tested for the presence of 3P. triticina were used to screen seedlings of the introgression lines. Race 1 BBBD, the most avirulent race available with virulence only to Lr14a, Lr14b, Lr20, and Lr50, was used to identify any resistance contained in the lines. Only lines 28, 161, and 355 were resistant with infection type (IT) ; (fleck) meaning a very small, hypersensitive‐like point on the leaf with no pustule formation  wheat. Lines 161 and 355 were resistant to 97AZ103A  having reduced pustule sizes. Line 161 was scored resistant with very small pustules surrounded by chlorosis (IT‐1C)  and even pustule distribution across the flag leaf. Five of the introgression lines showed this reaction  (G) was heterozygous (G/C) in all lines except 196 and 202, which were homozygous (C/C), that is, Lr46 absent.A P. graminis f. sp. tritici isolates commonly found in the U.S. Great Plains. RKQQC and GFCFC have a combined virulence to the following Sr genes: 5, 6, 7b, 8a, 9a, 9b, 9d, 9g, 10, 17, 21, 36, Tmp, and McN  and were therefore expected to contain the same segments. Indeed, the molecular and cytogenetic characterization appeared to confirm this; however, some of the lines tested containing the same segments did not give the same resistance results. The most notable of these are lines 124 and 355 and line 202 compared with lines 191, 192, 195, 196, and 198. Line 124 was susceptible to all races tested while, as outlined above, line 355 shows useful resistance to leaf and stem rust. Lines 191, 192, 195, 196, and 198 all show adult resistance to P. triticina, while line 202 was susceptible. There are a number of possible explanations for these apparently conflicting results. Firstly, neither the molecular nor cytogenetic characterization would have revealed small differences in the size of the segments. The markers used for the characterization of the lines were part of the Axiom Wheat Wild Relative Array  (King et al., Am. muticum‐specific SNPs, 137 KASP markers have been derived that span the genome (Grewal et al., In this work we have evaluated a group of J. Fellers conceived and conducted the screening experiments, provided funding, and co‐wrote the manuscript. A. Matthews and A.K. Fritz provided marker analysis and co‐wrote the manuscript. M. Rouse conducted the Ug99 screening and revised the manuscript. S. Grewal, J. King, and I.P. King provided the lines, verified the introgressions with GISH and markers and co‐wrote the manuscript.The authors declare that there are no conflicts of interest.Am. muticum fragment, size, wheat chromosome integration site, and KASP marker to follow trait.Supplemental Table S1. Genetic background of each introgression line tested for rusts, including source of Click here for additional data file.


P < 0.001). With regard to the response to immunosuppressive therapy, the overall response rate (ORR) in SAA/VSAA treated with rabbit anti-thymocyte globulin with/without cyclosporin A (rATG ± CsA) were significantly superior to those treated with CsA alone, or anabolic steroids . The 2-year OS in SAA/VSAA treated with rATG ± CsA, CsA, and anabolic steroids were 54.8%, 54.5%, and 37.6% (P = 0.037), respectively. The incidence of adult AA in Thailand is higher than those in Western countries, and the peak incidence is in the elderly. rATG ± CsA provided a better response than anabolic steroids, translating to the superior survival in SAA/VSAA treated with rATG ± CsA.The incidence and outcomes of aplastic anemia (AA) in Asia remain limited. This study aimed to explore the incidence and outcomes of patients with adult AA across the country of Thailand. This is a prospective multi-center nationwide population-based observational study of AA patients aged at least 15 years old, diagnosed from August 2014 to July 2016, with a longitudinal follow-up period over 2 years. There were 348 newly diagnosed adult AA patients during the enrollment period, giving an annual incidence of 4.6 per million. The incidence of severe (SAA) and very severe aplastic anemia (VSAA) (3.8 per million) was higher than non-severe AA . The peak incidence was observed in the patients aged from 80 to 89 years old (14.4 per million). The 2-year overall survival (OS) in NSAA, SAA, and VSAA were 65.5%, 49.3%, and 20.1%, respectively (The online version contains supplementary material available at 10.1007/s00277-021-04566-0. The incidence of aplastic anemia (AA) varied worldwide. The estimated annual incidence in Western countries was 1.5–2.3 per million –9. The e. Although horse ATG (hATG) gives better responses over rabbit ATG (rATG) as first-line therapy [Immunosuppressive therapy (IST) is recommended for the treatment of SAA patients who are not eligible for hematopoietic stem cell transplantation (HSCT) , 13. Alt therapy –16. Anab9/L, or platelet count ≤ 50 × 109/L. Patients with myelodysplastic syndrome (MDS) or classic paroxysmal nocturnal hemoglobinuria and chemotherapy-induced bone marrow suppression were excluded. Patients with chromosomal abnormalities were also excluded. Those with nationalities other than Thai were excluded. All enrolled patients were reassessed for definite diagnosis by the central review committee. Treatment of choice was given at the discretion of the treating physicians, depending on the patient’s eligibility and accessibility to treatment. The research proposal and all subsequent amendments were approved by the local Ethics Committee at each study site. The study was conducted in accordance with ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. All patients provided written informed consent.This study was a multi-center prospective observational study. Newly diagnosed adult AA patients from 30 referral centers nationwide (aged at least 15 years) were enrolled during the period from August 1, 2014, to July 31, 2016. Criteria for diagnosis of AA included bone marrow hypocellularity with at least two of the following: anemia with corrected reticulocyte count ≤ 1%, absolute neutrophil count (ANC) ≤ 1.5 × 109/L, platelet count ≤ 20 × 109/L, and hypocellular bone marrow with ≤ 25% cellularity. VSAA fulfills the definition of SAA but with ANC ≤ 0.2 × 109/L. Those who do not meet the criteria for SAA/VSAA are NSAA.SAA has at least 2 of the following: reticulocyte count ≤ 1%, ANC ≤ 0.5 × 109/L and platelet ≥ 150 × 109/L; partial response (PR): transfusion independence and no longer met the criteria for severe disease, and no response (NR): not achieving CR/PR.Responses to treatment were evaluated at each visit every 3 months. In those who switched to subsequent therapy, the responses were reassessed to evaluate the efficacy of the second-line treatment. Criteria for responses were based on the British Committee for Standard Haematology (BCSH) guideline , 17. Bri9/L , and platelet ≥ 20 × 109/L .The response criteria for NSAA were similar to SAA for CR and NR, whereas the criteria for PR were transfusion independence (if previously dependent) or doubling or normalization of at least one lineage or increase of baseline Hb ≥ 3 g/dL, ANC ≥ 0.5 × 10http://bps.moph.go.th/new_bps/). Direct age standardization was presented as previously described [The incidence rate was calculated by the number of newly diagnosed AA patients divided by the number of inhabitants within the respective catchment areas. All eligible cases were recruited from almost all hospitals over the country. We excluded hospitals in which there were no hematologists. The catchment areas were defined to only the provinces with the hematology services Fig. . Only paescribed .Fig. 1I2, depending on variable types and data distribution. OS was analyzed using Kaplan–Meier plots. Cox regressions were used to estimate hazard ratios among given subgroup stratifications. Treatment responses to immunosuppressive therapy were pre-defined as previously described criteria [Comparisons of data were performed using t-test or Wilcoxon rank-sum test or χcriteria . The staA total of 348 patients were enrolled. Twenty-six patients were excluded for the incidence calculation because they had lived in the catchment area less than 6 months. Six patients were excluded from the treatment response analyses due to inadequate collected data.There were 322 patients with newly diagnosed adult AA during the 2-year periods, which yielded an overall annual incidence of 4.6 per million. The annual incidence of SAA/VSAA was 3.8 per million, higher than that of NSAA (0.8 per million with a ratio of 4.6:1). Forty-five patients had VSAA, giving the annual incidence of 0.6 per million. The incidence was high in older patients with the peak of 14.4 per million in patients aged from 80 to 89 years old Table .Table 1P = 0.005) and bleeding . Frequent environmental exposures were agriculture pesticides (n = 60), certain medications previously reported as possible cause of AA within 6 months (n = 32), ducks/geese, and animal fertilizer (n = 15).With regard to the severity, there were 45 patients (13.1%), 238 (69.5%), and 59 (17.2%) with VSAA, SAA, and NSAA, respectively Table . The majOf 283 SAA/VSAA patients, only 3 patients underwent HSCT, 143 received rATG ± CsA, 12 CsA only, 102 anabolic steroids as the frontline therapy, and supportive treatment in 23 patients (Table n = 58), inaccessibility to rATG (n = 35), concerns regarding medical facility (n = 17), and other reasons (n = 27).One hundred thirty-seven (137) patients with SAA/VSAA did not receive frontline rATG. The reasons included patient conditions not suitable for IST  received anabolic steroids as frontline therapy, while 8 received supportive treatment. The other 2 patients received CsA.In 59 NSAA patients, the majority of them (P = 0.002) (Table n = 153) at 3, 6, 12, and 24 months were 17.6% , 30.1% , 34.0% , and 37.9% , respectively (Table P = 0.04)  Table . The ORR4) Table .Table 3P = 0.02) while patients with VSAA had a trend of a poorer response than those with SAA  (Table P = 0.03) , acute renal failure (n = 2), and cardiac arrest (n = 1)  Table . At 24 m1) Table .Fig. 2On = 3). The ORR of anabolic steroids at 3, 6, 12, and 24 months were 28.6% , 34.7% , 40.8% , and 34.7% , respectively  responded to treatment with a CR rate of 6.1% (ly Table .P < 0.001) , respectively   with a 2-year OS of 48.1% . When stratified by the disease severity, the 2-year OS for NSAA, SAA, and VSAA were 65.5% , 49.3% , and 20.1% , respectively (01) Fig. . The 2-yely Fig. . There w24) Fig. .Fig. 3KP < 0.001), respectively  and non-specific treatment . From multivariate analysis, age > 60 years , VSAA , and non-specific treatment  were independently associated with inferior OS among patients with SAA/VSAA (Table P < 0.001) , 54.5% , 37.6% , and 0%  Fig. .Fig. 4KIn this nationwide prospective study of adult AA patients diagnosed from 2014 to 2016, the annual incidence of adult AA in Thailand was 4.6 per million, which considerably is more or less similar to 3.0–5.0 per million in the previous population-based study in Thailand conducted 20 years ago , 20. NevThe current study demonstrated a remarkable age-related distribution with steeply increased incidence after the age of 50 (6.2 per million) with a peak incidence at the age over 80 years old (14.3 per million). The pre-existing clonal hematopoiesis, causing bone marrow failure with overlapping features of hypocellular MDS and AA, may lead to a higher incidence of AA among elderly patients , 24. DueHSCT is recommended treatment for younger patients of less than 40–50 years old. However, not all eligible patients could undergo this expensive procedure. Furthermore, the government supports only specific number of patients. Therefore, only small number of patients underwent HSCT in this cohort.rATG is the mainstay of treatment for SAA in Thailand as well as in many countries in Asia. For patients with SAA/VSAA in this cohort, the ORR was considerably low (44.4%), compared with previously published reports of rATG , 16. HowAnabolic steroids have long been used to treat AA. Its mechanisms include increasing telomerase (TERT) gene expression in hematopoietic cells , stimulaThe outcomes of this study represented the real-world situation of patients with AA. The cases were collected from 30 hospitals, including university and public hospitals, located in all regions of the country; therefore, the data would reasonably reflect the actual AA incidence in Thailand. The further strengths of this study included the prospective design and centrally reviewed diagnosis. However, this study had some limitations. First, the incidence of NSAA may be underestimated especially non-transfusion dependent AA and asymptomatic cases. Second, there was a proportion of non-evaluable patients due to death and loss to follow-up. Third, this study was not randomized, and the comparison between treatment modalities should therefore be interpreted with caution as there would be a high selection bias for older and unfit patients to receive anabolic steroids or supportive care.In conclusion, the incidence of adult AA in Thailand from this nationwide population-based study is higher than in Western countries, and even higher among the elderly. The real-world outcome of patients with SAA, especially in those aged over 60 years, is substantially poor. The appropriate therapeutic options, as well as the accessibility to advanced treatment, are needed to ensure a better outcome of patients.Supplementary file1 (DOCX 49 KB)Below is the link to the electronic supplementary material.


Mycobacteroides abscessus, an emerging environmental opportunistic pathogen capable of causing chronic infections. M. abscessus disease is difficult to treat, and the current treatment recommendations include repurposed antibiotics, several of which are associated with undesirable side effects. In this study, we have evaluated the activity of omadacycline, a new tetracycline derivative, against M. abscessus using in vitro and in vivo approaches. Omadacycline exhibited an MIC90 of 0.5 µg/mL against a panel of 32 contemporary M. abscessus clinical isolates, several of which were resistant to antibiotics that are commonly used for treatment of M. abscessus disease. Omadacycline combined with clarithromycin, azithromycin, cefdinir, rifabutin, or linezolid also exhibited synergism against several M. abscessus strains and did not exhibit antagonism when combined with an additional nine antibiotics also commonly considered to treat M. abscessus disease. Concentration-dependent activity of omadacycline was observed in time-kill assessments. Efficacy of omadacycline was evaluated in a mouse model of lung infection against four M. abscessus strains. A dose equivalent to the 300-mg standard oral human dose was used. Compared to the untreated control group, within 4 weeks of treatment, 1 to 3 log10 fewer M. abscessus CFU were observed in the lungs of mice treated with omadacycline. Treatment outcome was biphasic, with bactericidal activity observed after the first 2 weeks of treatment against all four M. abscessus strains.The incidence of nontuberculous mycobacterial diseases in the United States is rising and has surpassed that of tuberculosis. Most notable among the nontuberculous mycobacteria is  Mycobacteroides abscessus (formerly Mycobacterium abscessus) (mycobacterium (NTM) and a causative pathogen of pulmonary and soft tissue infections, among others. This organism has been described as a “clinical and antibiotic nightmare”   is an enghtmare” , 3, as ightmare” , 5. As o experts –10. Cureexperts –, 12.M. abscessus is one of the most frequently recovered NTM from patients, causing ∼10% of all NTM pulmonary infections  that omadacycline is available in both intravenous and oral formulations, making administration simpler for patients and allowing for oral therapy, (ii) that omadacycline has an elevated and sustained concentration in epithelial lining fluid, alveolar cells, and plasma, as well as improved pulmonary pharmacokinetics (PK) compared to those of tigecycline  were isolated from cystic fibrosis and bronchiectasis patients  had omadacycline MIC values ≥2-fold higher in Middlebrook 7H9 broth compared to those in CAMHB. While a 2-fold variation is typically within the standard acceptable variability of an MIC assay, this trend should be reported. Further, 31% (10 of 32) of isolates tested indeed had omadacycline MIC values ≥4-fold higher in Middlebrook 7H9 broth compared to those in CAMHB, which is considered meaningful. MICs of some other antibiotics were even more strikingly different; for example, 100% (32 of 32) of azithromycin MIC values were ≥2-fold higher in Middlebrook 7H9 broth compared to those in CAMHB, and of these, 75% (24 of 32) were ≥4-fold higher and 66% (21 of 32) were ≥8-fold higher. On the other hand, the MIC values of imipenem consistently trended higher in CAMHB broth compared to those in Middlebrook 7H9 broth, where MIC values were ≥2-fold for 68% (19 of 32) of the M. abscessus strains (note that only 9% [3 of 32] differed by ≥4-fold). These data highlight the need for the utilization of consistent and appropriate media for susceptibility testing, and therefore CAMHB was utilized for other in vitro testing in this study in accordance with CLSI guidelines.The MICs of certain antibiotics against M. abscessus strains included in our study (n = 32) were randomly selected without prior knowledge of MIC values to omadacycline, and the MIC results showed that 100% were inhibited by ≤1 µg/mL of omadacycline when tested according to CLSI methodology, confirming its potent in vitro activity. The MIC of omadacycline was consistently within one dilution of its median MIC (0.25 µg/mL in CAMHB) against strains that displayed a high level of resistance to antibiotics that are frequently used to treat M. abscessus disease, such as amikacin, clarithromycin, azithromycin, cefoxitin, and imipenem. The MIC of omadacycline was also within one dilution of its median MIC against strains that were resistant to antibiotics less commonly used to treat M. abscessus disease, such as moxifloxacin, doripenem, and linezolid.Several strains included in this study were resistant to a range of antibiotics based on CLSI breakpoints , according to CLSI interpretive criteria (Table S2). For example, the MIC of clarithromycin when assessed alone against isolate M9501 was 4.0 µg/mL  of the human 300-mg oral dose . The C3HeB/FeJ mouse strain PK parameters were determined because this mouse strain was utilized to evaluate omadacycline efficacy in an M. abscessus pulmonary infection model  were dose linear within the dose range of 7.5 to 30 mg/kg , and the Cmax is numerically less than dose proportional . For BALB/c mice, both AUC0–24 and Cmax were also dose linear , and the Cmax is numerically less than dose proportional . These data show that omadacycline AUC0–24 and Cmax, while similar in these two mouse strains at the doses tested, are not identical, with differences in PK parameters attributable to the different routes of administration. Specifically, intraperitoneal administration typically yields higher Cmax values than does subcutaneous injection (Omadacycline pharmacokinetic (PK) parameters were determined in C3HeB/FeJ mice by taking plasma samples at 0.5, 1, 2, 3, 6, 12, and 24 h after subcutaneous injection of omadacycline at 7.5, 15, or 30 mg/kg. Omadacycline PK parameters were also determined in BALB/c mice by taking plasma samples at 0.25, 0.5, 1, 2, 6, 12, and 24 h after intraperitoneal injection of omadacycline at 2.5, 7.5, 15, or 30 mg/kg. Plasma concentrations of omadacycline versus time were plotted  and B to30 mg/kg  and D. Te linear  and D. Tnjection .0–24 values were determined using an average mouse plasma protein binding of 33.9%, determined in BALB/c mice via equilibrium dialysis (data not shown). Linear regression analysis of the dose versus the C3HeB/FeJ mouse free AUC0–24 was performed and determined that an omadacycline subcutaneous dose of 15 mg/kg would result in a mouse free AUC0–24 of 9.2 h · µg/mL, which best represents the human free AUC0–24 of 8.92 h · µg/mL. It is of note that omadacycline lacks oral bioavailability in rodents  and thus extrapolation of the AUC obtained via subcutaneous administration in the mouse to the AUC obtained via oral administration in the human was required. Omadacycline concentrations in plasma from infected and uninfected C3HeB/FeJ mice were also measured. Plasma samples from infected and uninfected mice were compared and showed no significant difference in omadacycline concentrations, indicating that M. abscessus infection had no effect on omadacycline plasma levels in mice (data not shown).Free AUCM. abscessus isolates in a mouse model of pulmonary M. abscessus infection , clarithromycin (≤0.06 to 3 μg/mL), and imipenem (16 to 256 μg/mL) and were thus chosen to represent different phenotypes that may be encountered in the clinic  treatment at the same frequency as the test group, s burden . At the M. abscessus, the MICs against ATCC 19977 and M9501 recovered from lung homogenates of mice after 4 weeks of omadacycline treatment were determined along with those against the parent strains. Two colonies from each mouse, five mice per infection group, were randomly selected from 7H11 agar plates inoculated with lung homogenates. Overall, the MICs of omadacycline against the recovered isolates were identical or similar to the MIC value obtained against the parent strains (Table S5). Among the 10 colonies derived from ATCC 19977-infected mice, the MIC of omadacycline remained unchanged for eight isolates (MIC ≤ 0.5 µg/mL). For two isolates, the MIC was 0.75 µg/mL, which is within one dilution. The MICs of omadacycline against all 10 colonies derived from M9501-infected mice were identical to the MIC of omadacycline against the parent M9501 strain.To assess if prolonged exposure to omadacycline as a monotherapy in this model alters its MIC against M. abscessus disease, as demonstrated by the intrinsic resistance of this species to most antibiotics , it is possible that the omadacycline dose currently approved for treatment of community-acquired bacterial pneumonia (CABP) and acute bacterial skin and skin structure infections (ABSSSI) may also prove favorable for the treatment of M. abscessus infections. Indeed, the FDA-approved 300-mg oral dose is currently being evaluated in a phase 2, double-blind, randomized, parallel-group, placebo-controlled study to evaluate the efficacy, safety, and tolerability of oral omadacycline in adults with NTM pulmonary disease caused by M. abscessus complex . Importantly, we demonstrated that mice treated with 15 mg/kg, a dose that best represents the human equivalent exposure of the 300-mg oral dose, is efficacious in a murine model of pulmonary infection due to M. abscessus  and the inclusion of isolates that are resistant to several antibiotics, the actual proportion of clinical isolates against which the combination of omadacycline and clarithromycin may exhibit synergy may be slightly different. Additionally, although omadacycline in combination with rifabutin exhibited synergy against fewer isolates than in combination with clarithromycin, the FICI range of omadacycline plus rifabutin was the narrowest at 0.68 ± 0.20 (mean ± standard deviation [SD]). The FICI values for omadacycline plus clarithromycin showed a slightly wider range, with a mean ± standard deviation of 0.55 ± 0.30. An important finding was that antibiotics with which omadacycline exhibited synergy, including clarithromycin, azithromycin, cefdinir, linezolid, and rifabutin, are available in oral formulation. Therefore, there is potential, pending further evaluation in animal models of efficacy and clinical evaluation in humans, that a fully oral regimen containing omadacycline may be feasible. This type of regimen would reduce the logistical challenges associated with administering prolonged intravenous therapy in an outpatient setting, as is often required for treatment of M. abscessus lung disease.Treatment regimens for bination –8, 10, aM. abscessus pulmonary infections . In total, the data presented in our study provide additional support to this new treatment recommendation, as well as to the continued study of omadacycline in patients with pulmonary disease due to M. abscessus.The most recently published clinical practice guidelines for the treatment of NTM pulmonary disease were published in 2020 but failed to incorporate new antibiotics into these guidelines . A subsefections . The autregimens . In addiAnimal procedures used in the studies described here were performed in adherence to the Johns Hopkins University Animal Care and Use Committee and to the national guidelines.M. abscessus reference strain ATCC 19977  and imipenem . Mouse lung homogenates were cultured on Middlebrook 7H11 selective agar  supplemented with 10% albumin-dextrose-catalase enrichment, 50 µg/mL carbenicillin , and 50 µg/mL cycloheximide  as described previously  was calculated as described previously  containing omadacycline at 0.5×, 1×, 2×, 4×, 8×, and 16× MIC specific to each isolate in 4.8 mL CAMHB broth were prepared and inoculated with 200 µl of the M. abscessus suspension, or ∼105 CFU. A positive control for growth of M. abscessus without omadacycline was included in each assessment. The samples were incubated in an orbital shaker at 220 rpm and 37°C. At 0, 1, 3, and 7 days, a 100-µl aliquot was obtained from each sample, and appropriate 10-fold serial dilutions were prepared in CAMHB broth, inoculated onto CAMHB agar, and CFU enumerated after 3 days of incubation at 37°C. Similarly, to determine the time-kill activities of omadacycline in combination with another antibiotic, the five M. abscessus isolates were exposed to omadacycline and clarithromycin, azithromycin, cefdinir, linezolid, or rifabutin at 1×, 0.5×, and 0.25× MIC of each antibiotic specific to each strain, grown, and CFU determined. For each sample, CFU counts were converted to CFU/mL, and mean ± standard deviation data versus time were plotted.Activities of omadacycline alone and in combination with clarithromycin, azithromycin, cefdinir, linezolid, and rifabutin against the reference strain ATCC 19977 and four randomly selected clinical isolates, M9510, M9526, M9529, and M9530, were determined. Each g for 10 min to separate the plasma. Plasma was stored at −80°C until omadacycline concentration analysis.Uninfected C3HeB/FeJ mice  were injected subcutaneously with 7.5, 15, or 30 mg/kg omadacycline solution in 1× PBS. Blood samples were taken via terminal cardiac puncture at 0.5, 1, 2, 3, 6, 12, and 24 h postinjection, and at each time point, a single blood sample was collected from a single mouse. Five mice per dose and time point were utilized. Uninfected BALB/c mice  were injected intraperitoneally with 2.5, 7.5, 15, or 30 mg/kg omadacycline, and blood samples were taken via terminal cardiac puncture at 0.25, 0.5, 1, 2, 6, 12, and 24 h postinjection. Blood was transferred to lithium heparin vials and centrifuged at 1,500 × The mean (±SD) plasma concentration versus scheduled time points for uninfected BALB/c mice are shown in M. abscessus-infected C3HeB/FeJ mice, mice that had been infected were treated with 15 mg/kg omadacycline as described, and blood samples were taken via cardiac puncture from 5 mice at 24 h posttreatment at 0, 1, 2, and 4 weeks postinfection. Blood was transferred to lithium heparin vials and centrifuged at 1,500 × g for 10 min to separate the plasma. Plasma was stored at −80°C until omadacycline concentration analysis.To determine omadacycline concentrations in 18 high-performance liquid chromatography (HPLC) column  using a Sciex Exion LC AC system. Analyte molecules were detected using a Sciex 5500 mass spectrometer scanning in positive ion mode. Omadacycline tosylate drug substance (omadacycline) was used to prepare the stock standard and working standard omadacycline solutions. d9-Omadacycline was used as the internal standard for the analysis of omadacycline. The peak areas of omadacycline and its internal standard, d9-omadacycline, were acquired using Analyst 1.6.3 software . The calibration curves were obtained by fitting the peak area ratios of omadacycline/d9-omadacycline and the standard concentrations to a linear 1/×2 regression model using Analyst 1.6.3 software. The equations of the calibration curves were then used to interpolate the concentrations of omadacycline in the samples using their peak area ratios. The peak areas and peak area ratios used for the calculations were rounded to three precision points.Omadacycline plasma concentrations were determined by the Institute for Clinical Pharmacodynamics  using a qualified liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Samples were subjected to protein precipitation and chromatographically separated on an Ace 3 CM. abscessus infection  according to the manufacturer’s instructions . The infection cycle included preheating for 15 min, aerosol nebulization for 30 min, and cloud decay for 30 min, followed by surface decontamination for 15 min. This study was conducted in two phases. The first phase included evaluation of omadacycline efficacy in mice infected with ATCC 19977 and M9501. At the conclusion of this phase, two M. abscessus colonies were randomly selected from 7H11 agar plates containing lung homogenates of each mouse following completion of 4 weeks of daily omadacycline treatment. The MIC of omadacycline was determined against these 20 colonies to assess if daily exposure to omadacycline for 4 weeks altered its MIC against these strains. In the second phase, efficacy of omadacycline was evaluated in mice infected with M9529 and M9530. An identical protocol was used in both phases, but the studies were separated in time. Five mice per infecting strain group were sacrificed at 24 h postinfection (designated week −1), and lungs were homogenized, inoculated onto Middlebrook 7H11 selective plates described above, incubated at 37°C for 5 days, and CFU recorded to determine the initial M. abscessus burden in the lungs of mice. Similarly, five mice were sacrificed at 1 week postinfection (week zero) and CFU enumerated. CFU counts from each mouse lung were converted into CFU per lung, comprising the average of three consecutive steps of a 10-fold dilution series of a given lung sample. Mean CFU ± standard deviation of lung M. abscessus burden in five mice per group per time point was plotted determine the growth of each isolate under conditions tested.C3HeB/FeJ mice  were procured from Jackson Laboratories . As described in the protocol for a mouse model of pulmonary nfection , mice weM. abscessus lung burden was determined as described above.At 1 week postinfection (week zero), mice infected with each strain were further divided into three groups of 15 mice per group. All antibiotics and control treatments were administered via subcutaneous injection in the dorsal flank of hind limbs. Mice in the negative-control group were treated once daily with 200 µl sterile 1× PBS. Mice in the positive-control group were administered imipenem, and mice in the test group were administered omadacycline. Powdered omadacycline and imipenem were resuspended in sterile 1× PBS  for administration of 15 mg/kg/every 25 h (q24h) and 200 mg/kg/q12h of omadacycline and imipenem, respectively, using a 200-µl bolus. An average body weight of 25 g per mouse was considered based on our past experience with this mouse strain, age, and sex . Therefoin vitro and in vivo studies were analyzed to determine mean plus or minus standard deviation for each time point in each experimental group and graphed using GraphPad Prism v8.4.3.CFU data from


An analogous pattern was also observed for the other investigated temperatures. The quantitated TMZ solubility values were regressed using Apelblat and Van’t Hoff models and showed overall deviances of 0.96% and 1.33%, respectively. Apparent thermodynamic analysis indicated endothermic, spontaneous, and entropy-driven dissolution of TMZ in all solvents. TMZ solubility data may help to formulate dosage forms, recrystallize, purify, and extract/separate TMZ.The solubility parameters, and solution thermodynamics of temozolomide (TMZ) in 10 frequently used solvents were examined at five different temperatures. The maximum mole fraction solubility of TMZ was ascertained in dimethyl sulfoxide (1.35 × 10 The studied temperature range from “T = 298.2 K to 323.15 K” and interval of 5.0 K or 10.0 K were selected randomly in such a way that the maximum evaluated temperature, i.e., “T = 323.2 K,” should not exceed the melting temperature of TMZ nor the boiling points of the studied solvents. The melting temperature of TMZ was determined to be 474.8 K using thermal analysis. The maximum investigated temperature, “T = 323.2 K, was much lower than the melting temperature of TMZ and the boiling temperatures of the studied solvents. As a result, the above temperature range was selected in this study. Solubility data were further utilized for apparent thermodynamic calculation using the equations described by Van’t Hoff and Krug et al. [Thus, the objective of the current study was to determine the solubility of TMZ in 10 commonly used solvents  were pumped at a flow rate of 1 mL/min as the mobile phase. A “Nucleodur® ” HPLC column was used as the stationary phase and was maintained at room temperature [TMZ analysis was performed using a “HPLC system fitted with an SPD 20A UV/VIS detector ” set at 330 nm. Methanol and 0.5% acetic acid  in the mole fraction in each solvent was assessed using Equation (1) [m1 and m2 are the masses of pure TMZ and the solvent (g) and M1 and M2 are the molar masses of TMZ and the solvent (g mol−1), respectively.The static equilibrium method was utilized for the assessment of TMZ mole fraction solubility in commonly used solvents, namely, “H323.2 K” . In the 323.2 K” ,14. Then323.2 K” . Next, ttion (1) :(1) xe=mTfus) of 474.8 K and 475.7 K, respectively. A fusion enthalpy (∆Hfus) of −107.05 kJ mol−1 and a ∆Hfus value of −110.85 kJ mol−1 were observed for the pure and equilibrated TMZ samples, respectively , > PEG-400 (3.32 × 10−3), > TC (2.89 × 10−3), > EG (1.64 × 10−3), > PG (1.47 × 10−3), > H2O (7.70 × 10−4), > EA (5.44 × 10−4), > EtOH (1.80 × 10−4), > IPA (1.32 × 10−4), > 1-BuOH (1.07 × 10−4) at T = 323.2 K  of TMZ (30.30 MPa1/2) was closer to the δ value of PG (29.20 MPa1/2) than to the Hansen solubility parameter of IPA (22.30 MPa1/2). As a result, the xe values of TMZ were much higher in PG than in IPA. The xe values of TMZ were higher in H2O than in EtOH owing to the lower polarity of ethanol [2O, EA, and EtOH; and poorly soluble in IPA and 1-BuOH.The mole fraction solubility of TMZ was noticeably more prominent in DMSO, PEG-400, TC, EG, PG, Hd, δp, and δh symbolize dispersion, polarity, and hydrogen-bonding parameters for Hansen solubility, respectively.The assessment of the Hansen solubility parameter (δ) for TMZ and the other investigated solvents was calculated utilizing Equation (2) ,24,25: (1) for several solvents has been described [1 is shown in 2O, EtOH, EG, PG, TC, IPA, 1-BuOH, and EA”, were found to be considerably near the stated δ1 values. However, the calculated δ values for PEG-400 and DMSO differed to some extent from the described δ1 values [1/2, indicating that TMZ possesses moderate polarity  and PG , indicating the maximum solubility of TMZ in these solvents, as per the Hansen solubility parameter. “HSPiP ” was utilized to determine the values of these parameters  . The TMZescribed . The com1 values . The δ vpolarity . The δ vxe values were correlated using Apelblat and Van’t Hoff models. Equation (3) was employed to calculate the Apelblat model solubility (xApl) values of TMZ [A, B, and C were assessed using nonlinear multivariate regression analysis of the xe data of TMZ presented in The TMZ s of TMZ ,28:(3)lnxe values were interconnected with the TMZ xApl values and evaluated in terms of root mean square deviations (RMSD) as well as R2 values. The RMSD for TMZ was estimated using Equation (4) [N is the number of experimental temperature points. The graphical correlation between the logarithm xe (ln xe) and ln xApl values of TMZ in each solvent as a function of 1/T is shown in xe and ln xApl TMZ values in every examined solvent.The TMZ tion (4) :(4)RMSD=RMSD value for TMZ was observed for 1-BuOH (1.38%) and the lowest was for ethanol (0.22%)  . Further3–0.9999 . The RMSxVan’t) of TMZ [a and b are the model parameters of the Van’t Hoff model that are described by the schematization of the values of ln ex of TMZ as a function of 1/T. The xe values of TMZ were interrelated using the xVan’t values of TMZ in terms of RMSD and R2 values, and the relation between the ln xe and ln xVan’t values of TMZ in each solvent was calculated as a function of 1/T (Equation (5) was employed to calculate the Van’t Hoff model solubility  of TMZ .(5)ln xn of 1/T .RMSD value for TMZ was observed for IPA (1.90%) and the lowest was for EtOH (0.48%). The R2 values for TMZ in the examined solvents were in the range of 0.9957–0.9996. The assessed RMSD and R2 values indicated a good correlation for the xe values of TMZ by the Van’t Hoff model.The data of Van’t Hoff correlation are shown in solH0 , ΔsolG0 (the apparent standard Gibbs free energy), and ΔsolS0 (the apparent standard dissolution entropy) for TMZ dissolution, were evaluated using the Van’t Hoff and Krug et al. analysis methods [solH0 values for TMZ dissolution in the examined solvents were projected at the mean harmonic temperature (Thm) of 308.96 K by implementing Van’t Hoff analysis as described in Equation (6) [The dissolution feature of TMZ in the examined solvents was evaluated using the experimental solubility data of TMZ in apparent thermodynamic analysis. For this analysis, different apparent standard thermodynamic parameters, for instance, Δ methods . The Δsotion (6) ,32:(6) [The Krug et al. analysis was performed at tion (7) .(7)ΔsolThe intercept value for TMZ in each examined solvent was ascertained from the Van’t Hoff plots displayed in solS0, which reveal the dissolution characteristics of TMZ [Furthermore, the Van’t Hoff and Krug et al. analytical approaches (Equation (8)) were utilized to calculate the values of Δs of TMZ ,31,32., followed by that in 1-BuOH, EtOH, DMSO, EA, EG, TC, H2O, and PEG-400, and the minimum was observed in PG (20.55 kJ mol−1).The data of apparent thermodynamic analysis for TMZ dissolution are presented in solG0 values for TMZ dissolution in the examined solvents were also found to be positive values and within the range from 12.44 to 24.97 kJ mol−1. The maximum ΔsolG0 value for TMZ dissolution was detected in 1-BuOH (24.97 kJ mol−1) followed by that in IPA, EtOH, EA, H2O, PG, EG, TC, and PEG-400, and the minimum was observed in DMSO (12.44 kJ mol−1). The minimum ΔsolG0 value in DMSO could be on account of the maximum solubility of TMZ in DMSO. The lowermost value of ΔsolG0 in DMSO indicated that little energy is needed for the solubilization and dissolution of TMZ in DMSO. The results of the ΔsolG0 analysis for TMZ dissolution were found to be in good correspondence with the TMZ solubility values. The positive values of ΔsolH0 and ΔsolG0 suggest that TMZ showed endothermic and spontaneous dissolution characteristics in all the solvents examined in the current study [Further, the Δnt study ,33.solS0 values for the TMZ dissolution characteristic in all examined solvents were also detected as positive values and within the range from 8.74 J mol−1 K−1 to 64.03 J mol−1 K−1. The positive ΔsolS0 values suggest an entropy-driven dissolution of TMZ in all solvents. In general, the TMZ dissolution was regarded as endothermic and entropy-driven in all solvents examined in the current study [The Δnt study ,14,25. Tnt study .2O, EA, EtOH, IPA, and 1-BuOH”. In conclusion, TMZ is considered to be highly soluble in DMSO; soluble in PEG-400, TC, EG, and PG; slightly soluble in H2O, EA, and EtOH, and poorly soluble in IPA and 1-BuOH.In the present study, the experimental solubility and solution thermodynamics of TMZ, a classic DNA methylating anticancer drug, were examined in 10 frequently used solvents at five different temperatures. The solubility of TMZ in all the examined solvents was considerably augmented with an increase in temperature. The maximal mole fraction solubility of TMZ was detected in DMSO, followed by that in “PEG-400, TC, EG, PG, H


Background: The current pilot study was designed to examine the association between hippocampal γ-aminobutyric acid (GABA) concentration and episodic memory in older individuals, as well as the impact of two major risk factors for Alzheimer’s disease (AD)—female sex and Apolipoprotein ε4 (ApoE ε4) genotype—on this relationship.Methods: Twenty healthy, community-dwelling individuals aged 50–71 (11 women) took part in the study. Episodic memory was evaluated using a Directed Forgetting task, and GABA+ was measured in the right hippocampus using a Mescher-Garwood point-resolved magnetic resonance spectroscopy (MRS) sequence. Multiple linear regression models were used to quantify the relationship between episodic memory, GABA+, ApoE ɛ4, and sex, controlling for age and education.Results: While GABA+ did not interact with ApoE ɛ4 carrier status to influence episodic memory (p = 0.757), the relationship between GABA+ and episodic memory was moderated by sex: lower GABA+ predicted worse memory in women such that, for each standard deviation decrease in GABA+ concentration, memory scores were reduced by 11% (p = 0.001).Conclusions: This pilot study suggests that sex, but not ApoE ɛ4 genotype, moderates the relationship between hippocampal GABA+ and episodic memory, such that women with lower GABA+ concentration show worse memory performance. These findings, which must be interpreted with caution given the small sample size, may serve as a starting point for larger studies using multimodal neuroimaging to understand the contributions of GABA metabolism to age-related memory decline. A receptor-mediated inhibitory synaptic decline, preceded Aβ-related pathology, was accompanied by cognitive impairments in an episodic-like memory task, and could be reversed via administration of a GABAA receptor agonist . Levetiracetam also mitigates memory impairment in patients with amnestic mild cognitive impairment  is a progressive neurodegenerative disorder whose hallmark cognitive symptom is episodic memory loss  is the strongest common genetic risk factor for late-onset AD, being associated with both higher risk and a markedly earlier mean age of AD onset , and eight were ApoE ε4− . The median education level was 6 (range: 5–7), which corresponds to a bachelor’s degree or equivalent, according to the International Standard Classification of Education (ISCE) classification. Written informed consent, as approved by the Institutional Review Board of the Columbia University Medical Center, was obtained prior to study participation.Healthy older adults were recruited for the study from two participant cohorts maintained by the Cognitive Neuroscience Division at Columbia University, the Cognitive Reserve Study, and the Reference Ability Neural Network Study. Participants were recruited to these studies by mail-market procedures targeting individuals within 10 miles of the Columbia University Medical Center. Participants were required to be right-handed, native English speakers with at least a fourth-grade reading level. As part of these cohort studies, participants were genotyped for ApoE ε4 and screened for neurological diagnoses and medication use, as detailed elsewhere  = 2,700 ms, repetition time (TR) = 7,156 ms, inversion time (TI) = 450 ms, 176 slices, 256 × 256 matrix size, slice thickness = 1 mm, flip angle (FA) = 12°). The second one was a two-dimensional (2D) axially-acquired structural T1-weighted fluid-attenuated inversion recovery (FLAIR) volume . The VOI with a size of 4 × 2 × 2 cm3 was centered in the right hippocampus  in one acquisition that lasted 768 s. A vendor-provided, semi-automatic shimming procedure was implemented prior to spectroscopic acquisition and was supplemented by interactive manual shimming, resulting in full-width at half-maximum (FWHM) water linewidths ranging from 9 to 22 Hz (mean line width = 13 ± 3.49 Hz).MRI data were acquired using a 32-channel head coil on a 3 Tesla MR scanner . Two anatomical images were acquired for the MRS volume of interest (VOI) placement; the first one was a three-dimensional (3D) brain volume (BRAVO) pocampus . 1H MRS The 3D T1-weighted images were analyzed using FreeSurfer (v5.1.0) an automated segmentation and cortical parcellation software package , which provides a direct measure of episodic memory, served as the outcome variable. Predictors of interest included MRS-measured GABA+ concentration from the right hippocampus, age, sex, education level , and ApoE ε4 genotype, coded as a binary variable (either positive or negative). There were no missing data pertaining to any of these variables.t-tests for continuous or categorical variables, respectively. Multiple linear regression models were used to evaluate the relationship between episodic memory and the predictors of interest. Memory served as the dependent variable, with GABA+, age, sex, education, and ApoE ε4 as independent variables. Independent variables were selected according to a priori hypotheses based on the previous literature or on univariate analysis results. To facilitate the interpretation of regression coefficients, GABA concentrations were standardized into z-scores. As our hypothesis involved ApoE ε4 and sex effects on GABA+ concentrations, interaction terms for ApoE ε4 × GABA+ and sex × GABA were included. Additionally, to avoid overfitting due to the large number of variables and small sample size, variables were selected via backward stepwise elimination according to the Akaike information criterion (AIC). Briefly, the AIC is a metric comparing the goodness of the fit of two models by selecting the one with the highest likelihood after penalizing for the number of parameters in the models. A lower AIC thus corresponds to better goodness of fit. The statistical assumptions  of the model obtained through variable selection were verified to confirm that they had been met.Univariate analyses were used to assess the association between GABA+, on the one hand, and age, sex, and ApoE ε4 variables, on the other hand, using Spearman’s rank correlation coefficients or Student’s a priori. In our case, we considered deciles from 10 to 90. This analysis allowed us to observe whether the correlation between GABA+ and memory remained constant across the distribution of TBR responses in men and women, giving robustness to our results. Pairwise comparisons of those models fitted at different τ were compared using Wald tests to assess whether the effect of GABA+ varied across the distribution of TBR responses.Overall accuracy on the task was high. To investigate as to whether the effect of GABA+ (the predictor variable) on memory was conditioned by the skewed distribution of TBR responses (the dependent variable), we implemented separate quantile regression models in men and women. In quantile regression, instead of fitting a model at the mean of the dependent variable, the effect of the independent variable is tested across the distribution of the dependent variable. Hence, coefficients are calculated at one or more quantiles of the distribution (expressed as τ), which are set All analyses were conducted using R software . For all analyses, α was set at 0.05.U = 56.5, p = 0.621), educational attainment  = 62.5, p = 0.318), or in its prevalence of the ApoE ε4 allele (p = 0.927). On the other hand, ApoE ε4 carriers were older [median (interquartile range) = 65.5 y  y] than non-carriers [57.5 y  y], but did not differ in educational attainment  = 64.5, p = 0.194).There was no significant sex-related difference in the sample’s age  or education level . Further, GABA+ was not associated with either age , or ApoE ɛ4 polymorphism  = 0.26, p = 0.802). However, overall, women had higher GABA+ concentration than did men  = −2.67, p = 0.016).The average proportion of correctly recognized TBR words was high, 0.9 ± 0.1. This score did not correlate with age  = −0.02 , p = 0.757], or a ApoE ε4 × GABA+ interaction [β = 0.00 , p = 0.990; see β = 0.00 , p = 0.935. As shown in Multiple linear regression models were used to analyze the relationship between episodic memory and the predictors of interest , the results of which are shown in τ10 = 0.10, t = 2.39, p = 0.040; τ20 = 0.20, t = 2.06, p = 0.070; τ30 = 0.30, t = 2.32, p = 0.045; τ40 = 0.40, t = 2.27, p = 0.049; τ50 = 0.50, t = 0.94, p = 0.370; τ60 = 0.60, t = 2.43, p = 0.038; τ70 = 0.70, t = 2.46, p = 0.036; τ80 = 0.80, t = 2.70, p = 0.025; τ90 = 0.90, t = 3.47, p = 0.007). As shown in The results of the quantile regression models revealed that GABA+ was not associated with memory performance in men in any portion of the TBR accuracy distribution. By contrast, in women, GABA+ was positively correlated with memory at all deciles except 20 and 50  performance on the memory task. On the other hand, hippocampal volume in our sample (μ ± σ = 3.84 ± 0.5 cm3) was on par with recently published normative data acquired from a large sample  of clinically healthy older adults , and these results were similar across sex. Specifically, the women in our sample had average hippocampal volumes of 3.64 ± 0.47 cm3, vs. 3.77 ± 0.37 cm3 in the normative sample. Men in our sample had average hippocampal volumes of 4.12 ± 0.35 cm3, vs. 3.97 ± 0.43 cm3 in the normative sample, which allays some concern over how representative the participants in our sample are  nor current levels or history of depression. These are important avenues of future inquiry, as they may provide insight into the mechanism driving the sex-specific effects found, and future studies should directly test the role of these factors in the relationship between GABA+ concentration and episodic memory in women. Third, while it is not possible to determine from the Data supporting the conclusions of this manuscript will be shared under petition of qualified researcher.The studies involving human participants were reviewed and approved by The Institutional Review Board of the Columbia University Medical Center. The patients/participants provided their written informed consent to participate in this study.JJ-B performed statistical analysis, created visualizations, and wrote the manuscript. AY wrote the manuscript. LK acquired behavioral and MRS data. KI and CL processed and analyzed the MRS imaging data. KB set up the MRS sequence and helped acquire the MRS data. JG provided technical support for the MRS analysis software. AI and AB edited the manuscript. TE conceived the study, acquired behavioral and MRS data, analyzed and interpreted data, wrote the manuscript, and acquired funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Tangwang Mingmu granule (TWMM), a traditional Chinese medicine, has been widely used in the treatment of diabetic retinopathy (DR), the most common microvascular complication in diabetes mellitus.To establish a method to select target compounds from herbs for a pharmacokinetic study using network pharmacology, which could be applied in clinical settings.in vivo were chosen. Then, Sprague-Dawley (SD) rats were administrated of TWMM at a 9.6 g/kg dose to investigating pharmacokinetic parameters using the UPLC-QQQ-MS.First, UPLC/Q Exactive Q-Orbitrap and GCMS 2010 were used to determine the non-volatile and volatile ingredients of TWMM. Based on the identified compounds, network pharmacology was used to screen the key compounds and targets of TWMM in the treatment of DR. Based on the compound-target-pathway network and identification of components emigrant into blood, the potential compound markers Ninety and forty-five compounds were identified by UPLC-MS and GC-MS, respectively. Based on the network pharmacology, nine compounds with a degree value above 15 were screened and implied that these compounds are the most active in DR treatment. Moreover, criteria of degree value greater than 7 were applied, and PTGS2, NOS2, AKT1, ESR1, TNF, and MAPK14 were inferred as the core targets in treating DR. After identification of components absorbed into blood, luteolin and formononetin were selected and used to investigate the pharmacokinetic parameters of TWMM after its oral administration.The reported strategy provides a method that combines ingredient profiling, network pharmacology, and pharmacokinetics to determine luteolin and formononetin as the pharmacokinetic markers of TWMM. This strategy provides a clinically relevant methodology that allows for the screening of pharmacokinetic markers in Chinese medicines. Clinical applications show that TWMM can significantly improve the vision of patients with DR, alleviate clinical symptoms such as asthenopia, and improve the state of blood vessels in the fundus  were purchased from Shanghai Yuanye Pharmaceutical Technology Co., Ltd and Sichuan Weikeqi Pharmaceutical Technology Co., Ltd. Acetonitrile and formic acid both in HPLC grade were obtained from Fisher Scientific . Ultrapure water was prepared using a Milli-Q water purification system . Other reagents were all analytical grade.The TWMM was provided by Beijing HanDian Pharmaceutical Co., Ltd. . Chlorogenic acid, linarin, luteolin, jaspolyside, tolbutamide, stachydrine, magnoflorine, calycosin-7-g for 10 min at 4 °C, followed by subsequent collection of the supernatants. All the solutions were stored at −20 °C before the experiment.Ten mg of each standard was dissolved in 10 mL of methanol solution. Next, an equal volume of stock solution was mixed in a 10 mL volumetric flask to acquire a mixed standard solution with a certain concentration. Samples of TWMM (20 mg) were added to 10 mL of 70% methanol for UPLC-MS and n-hexane for GC-MS analyses, respectively. The samples were exposed to ultrasound extraction for 15 min and centrifuged at 15,000 Analytical standards (10 mg) of oleuropein, chlorogenic acid, formononetin, verbascoside, linarin, luteolin, jaspolyside, specnuezhenide, and tolbutamide  were dissolved in a 10 mL volumetric flask with methanol. An appropriate volume of solution containing oleuropein, chlorogenic acid, formononetin, verbascoside, linarin, luteolin, jaspolyside, and specnuezhenide solution was diluted to obtain a stock solution. Subsequently, the mixture was serially diluted to prepare the reference working solutions and then tolbutamide was added to achieve an IS concentration of 5 μg/mL.v/v) acetic acid in water (A) and acetonitrile (B) under the following gradient conditions: 0–30 min, 8–20% B; 30–35 min, 20–25% B; 35–45 min, 25–80% B; 45–45.5 min, 80–8% B; 45.5–49 min, 8–8% B. The flow rate was set at 0.4 mL/min and the temperatures of the autosampler and analytical column were maintained at 35 °C and 4 °C, respectively. In addition, the HESI of the Q-Orbitrap mass spectrometer was set to both negative and positive ionisation modes. The ion source parameters were as follows: spray voltage, +2.9 kV/–2.8 kV; auxiliary gas rate, 10 L/h; sheath gas rate, 35 L/h; auxiliary gas temperature, 400 °C; capillary temperature, 350 °C; normalised collision energy, 10/20/40 V, and mass range, 150–1500 m/z.The identification of non-volatile components was determined using a UPLC/Q Exactive Q-Orbitrap system . Chromatographic separation was performed on a 1.8 µm HSS T3 analytical column (100 mm × 2.1 i.d.). The mobile phase was composed of 0.1% . Separation was conducted on a DB-17 column (30 m × 0.25 mm × 0.25 μm). The oven temperature program setting is shown in http://lsp.nwu.edu.cn/tcmspsearch.php), which provided the chemical compounds and their related target proteins. Related targets of DR were predicted and screened using the Comparative Toxicogenomics Database , Online Mendelian Inheritance in Man database , and DrugBank Database . The intersection targets were imported into Venny 2.1.0 software (https://bioinfogp.cnb.csic.es/tools/venny/index.html). The protein-protein interaction network (PPI) of intersection targets was created using the STRING Database platform  with medium confidence (0.4) to remove the isolated target proteins. Furthermore, the drug-disease crossover genes were annotated and visualised using the Ingenuity Pathway Analysis software . Lastly, the compound-target-pathway network was established using the Cytoscape interaction network visualisation software .All targets of all compounds constituting TWMM were screened by using the Traditional Chinese Medicine Systems Pharmacology Database  aqueous formic acid (A) and acetonitrile (B) under the following gradient conditions: 0–2 min, 8–13% B; 2–7 min, 13–50% B; 7–9 min, 50–95% B; 9–11 min, 95–95% B; 11–12.5 min, 95–8% B; 12.5–15 min, 8–8% B. The flow rate was set at 0.3 mL/min and the column and autosampler temperatures were maintained at 35 °C and 15 °C, respectively. ESI source was set to negative ionisation mode while the analysis was performed using multiple reaction monitorin. Ion spray voltage (3000 V), capillary temperature (450 °C), and the source parameters of the nine compounds are shown in Chromatographic separation was performed using an ACQUITY UPLC H-Class system equipped with a 1.8 μm HSS T3 analytical column . The mobile phase consisted of 0.1%  database. The pharmacokinetic data were processed through MassLynx 4.1, and DAS 2.0 software was used to calculate the pharmacokinetic parameters according to the compartment model.r). The lower limit of quantification (LLOQ) could fulfil the analytical requirements and achieved reliable accuracy and precision with a signal-to-noise ratio (S/N) ≥ 10. To determine intra- and inter-day precision and accuracy, six replicate quality control (QC) samples at three concentrations were prepared. Inter- and intra-day precision was evaluated by determining relative standard deviation (RSD) values, while accuracy was expressed in terms of the relative error (RE). The extraction recoveries were determined using three concentration levels and calculated by comparing the peak area of extracted samples with post-extracted spiked samples. Subsequently, matrix effects were assessed using the peak area of post-extracted spiked samples contrasted with QC samples at the three concentration levels. The stability test of QC samples at the three concentration levels was as follows: storage at 25 °C for 4 h, storage in an autosampler for 12 h, freeze-thaw cycle for three times, and storage at −80 °C for 7 d.Method specificity was assessed by comparing the chromatograms of blank plasma, blank plasma spiked with oleuropein, chlorogenic acid, formononetin, verbascoside, linarin, luteolin, jaspolyside, specnuezhenide, and IS, and plasma after oral administration of TWMM. The calibration curves were assessed at eight concentration levels using the correlation coefficient  were acquired from Beijing Vital-River Laboratory Animal Technology Co., Ltd. All rats were acclimatized to an environmentally controlled laboratory for a week. Following this period, the rats were exposed to fasting conditions for 12 h but were allowed free access to water before the experiment. The rats were orally administered TWMM at a dose of 9.6 g/kg. Blood samples (300 µL) were collected into heparinized centrifuge tubes from the fossa orbitalis at pre-dose and 0.03, 0.08, 0.17, 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 36 h intervals. The samples were immediately centrifuged at 6000 g for 10 min at 4 °C after being vortexed for 3 min. The obtained supernatants were transferred to clean 1.5 mL centrifuge tubes, followed by evaporation under a milt nitrogen stream. Next, the obtained residues were individually redissolved in 100 µL of methanol and centrifuged at 15,000 g for 10 min at 4 °C after vortexed for 3 min. Lastly, 10 µL of individual supernatant was injected for analysis.Each 100 µL plasma sample was mixed with 20 µL of methanol, 20 µL of IS (5 µg/mL), and 600 µL acetonitrile. The mixtures were centrifuged at 15,000 http://zinc.docking.org/). The conformation of the top 5 proteins screened by network pharmacology was collected from the Protein Data Bank (PDB) database: PTGS2 (PDB ID: 5IKR), NOS2 (PDB ID: 4NOS), AKT1 (PDB ID: 3CQW), ESR1 (PDB ID: 1XP9), and TNF (PDB ID: 2AZ5). The CDOCKER program of Discovery Studio 2019 was applied to investigating molecular docking parameters after molecule and protein preparing procedures. Then, PyMol 2.4.0 was used to visualize and verified the result of molecular docking.The 3D structures of luteolin and formononetin were obtained from the ZINC database  or formononetin  for 2 h followed by 30 mM high glucose (HG) for 24 h and incubated for 20 minutes with DCFH-DA (10 mM) at 37 °C. DCF fluorescence was assessed at F488/525 nm by using a bioassay multi-detection fluorescent plate reader .The generation of ROS was detected using dichloro-dihydro-fluorescein diacetate  as per manufacturer instructions. In this study, we seeded HUVEC cells in a 96-well plate at 7 × 106) were seeded into 6-well plates. When cell confluence reached about 70%, cells were co-transfected with pGL4.37, pGL4.75 following the manufacturer’s instructions . Luciferase assays were performed with the dual-luciferase reporter assay system  according to the manufacturer’s instructions. Luminescent signals were quantified by a luminometer , and each value from the firefly luciferase construct was normalized by Renilla luciferase assay.DH5a competent cells  chromatograms obtained in both positive and negative modes are shown in The volatile components of TWMM were detected by GC-MS 2010, and 45 compounds were identified using the NIST database. The GC-MS chromatogram of TIC traces is depicted in The 135 identified components were used as the foundation of network pharmacology. To acquire the formula-disease-related genes, the identified compounds were added to the TCMSP database to screen active ingredients and related targets. Following this, a total of 68 compounds and 287 targets were retrieved. Further, 646 genes related to DR were acquired from the CTD, OMIM, and DrugBank databases. Subsequently, after the intersection, 57 shared targets were obtained.The STRING database was used to form the intersection gene targets into a complex protein-protein network. The core gene targets are shown in The 27 key targets are analyzed by the "Core Analysis" function in IPA. As shown in A total of 72 functional entries related to DR were obtained by predicting the diseases and functions map of 27 core targets by IPA, namely organismal injury and abnormalities, cell death and survival, cellular development, cellular growth and proliferation, hematological system development, tissue development, organismal development, cellular movement, etc. As shown in As shown in Luteolin has been reported to inhibit proliferation and angiogenesis in human umbilical vein endothelial cells (HUVECs), human retinal microvascular endothelial cells (HRMECs), and retinal vascular endothelial rhesus (RF/6A) cells induced by VEGF  of all calibration curves was greater than 0.9906.The calibration curves of eight compounds were assessed by linear regression analysis with a weight factor of 1/The intra- and inter-day precision and accuracy at each investigated concentration level are shown in The extraction recoveries and matrix effects of QC samples are summarized in The stability tests were carried out under three freeze-thaw cycles, at 25 °C for 4 h, in an autosampler for 12 h, and at −80 °C for 7 d. The result shown in Tmax values of chlorogenic acid, formononetin, verbascoside, linarin, jaspolyside, and specnuezhenide were within 1 h, which suggests that these six compounds attained maximum plasma concentration rapidly. The t1/2 values show rapid elimination of oleuropein, chlorogenic acid, verbascoside, and linarin. The areas under the concentration-time curve (AUC0–∞) of oleuropein, chlorogenic acid, formononetin, verbascoside, linarin, luteolin, jaspolyside, and specnuezhenide were 1.03 ± 0.75, 33.95 ± 4.55, 45.96 ± 7.89, 174.81 ± 40.83, 12.04 ± 4.72, 43.98 ± 4.19, 53.37 ± 32.17, and 57.19 ± 30.25 mg/mL/min, respectively.The plasma concentration-time curve after oral administration of TWMM is illustrated in in vivo revealed the potential ingredients and targets in the DR treatment of TWMM. To verified the bioactivity, the validated promoter or inhibitor binding sites of 5 targets were selected for docking with luteolin and formononetin. Then, PyMOL was used to verify and visualize the key hydrogen bonds of docking analysis (Supplementary Materials.The result of network pharmacology and identification of compounds analysis . The -CDin vivo markers having a high probability in the treatment of DR.Network pharmacology can be applied to screen compounds that have a high contribution in treating diseases, unveiling the target compounds to obtain better clinical adaptability that is similar to that of the parent herb or formulation. After systematic qualitative and network pharmacology analyses, apigenin, luteolin, daidzein, kaempferol, baicalein, rutin, chrysoeriol, glycitein, and formononetin were selected as potential target compounds based on degree and betweenness centrality. Furthermore, based on the qualitative analysis of plasma samples after oral administration of TWMM, luteolin and formononetin were found to immigrate into the blood. Therefore, luteolin and formononetin were selected as detectable Tmax and lower t1/2 than those of the extract. In addition, luteolin has been reported to have Tmax and t1/2 values ranging between 0.50–2.83 h and 3.63–12.10 h in different herbal extracts and prescriptions, respectively (Guan et al. Tmax and t1/2 values from the various herbs and prescriptions, the influence on target compounds is diverse. Network pharmacology is adept at analyzing compound mixtures and presenting degrees to which each compound can be selected as a target molecule for pharmacokinetic studies of any herbal mixture.Network pharmacology can identify pharmacokinetic target compounds with high efficiency and accuracy. However, the pharmacokinetic parameters of the same compound in different herbs or prescriptions are not consistent. Wang et al.  showed tin vivo was greatly affected by the external environment. Therefore, these results prompt us to carry out a pharmacokinetic study using a DR mouse model in the future.Network pharmacology can also identify pharmacokinetic target compounds in herbs or prescriptions based on their clinical application. The different pathological conditions also influence the ADME of drugs, resulting in different therapeutic outcomes. On comparing the absorption rate of formononetin and luteolin in diabetic and healthy rats, Wei et al.  observedUsing network pharmacology, we focussed on the qualitative components of TWMM obtained using UPLC-MS and GC-MS to screen the key molecules that share the same targets with DR. Luteolin and formononetin were determined as the target compounds in explaining the pharmacokinetic properties of TWMM. This study provides a suitable combination strategy to unveil pharmacokinetic markers based on clinical application with high efficiency and clinical focus.Click here for additional data file.


Results showed that 600 MPa/20 min processing was the most effective controlling mesophilic group, presenting 3.8 log cfu/g after 12 months of storage, and inactivating moulds and yeasts after HPP treatment. Colour was affected by storage, including a reduction in the L* parameter in all conditions to final values between 37.8 and 39.3, while the a* parameter increased during storage time at −12 °C and 4 °C to final values of around 12, and parameter b* decreased at storage temperature 20 °C to 5.3. Storage time affected the rheological behaviour of HPP-treated samples, increasing G′1Hz from the 2nd to 12th month of storage time to the final values between 1603 kPa and 2139 kPa. Moisture, aw and pH were not affected by HPP treatment nor storage time.The aim of this study was to evaluate the impact of high-pressure processing (HPP) on the long-term storage of chocolate fillings at room temperature, compared with conventional storage at lower temperatures. Dark chocolate fillings were treated at different pressure levels, holding times and stored at 20 °C for 12 months. Unprocessed batches were stored at 4 °C and at −12 °C. Moisture, water activity (aw), pH, colour, G′ Chocolate is one of most common confectionery products worldwide and has been studied for many years. Chocolate is a suspension of cocoa mass and sugar in the cocoa butter matrix, solid at room temperature that melts at oral temperature 37 °C) creating a smooth sensation . However7 °C creaw/w [Salmonella and Listeria spp. [Salmonella spp. [There is not a standard preparation for ganache; however, the percentage of cream in a recipe may range from 9 to 41% ria spp. . Additiolla spp. . This silla spp. , reductilla spp.  or creatlla spp. . Currentlla spp. ,11,12,13lla spp. .The aim of this study was to investigate the potential use of different HPP treatments to extend the shelf life of chocolate fillings, compared to standard long-term storage and considering physical, chemical, mechanical and microbiological parameters. The present work is of pertinence considering that there are very few studies covering the use of nonthermal processing technologies, such as HPP, to extend the shelf life of chocolate fillings.Chocolate fillings were produced in a local confectionery  using dark chocolate Apamate 70%  and cream (33% fat) in a 2:1 proportion. The preparation of chocolate filling started by heating up the cream to 90 °C using a heating plate with a magnetic stirrer. After reaching 90 °C, the chocolate was added using a blender to homogenize the mixture for 2 min . The mixture was allowed to cool down to 30 °C at room temperature. Then, approximately 50 g of filling was piped manually into polyethylene PET-MET/PE bags  and vacuum packaged at 800 mbar . A similar procedure was adopted for high pressure processed and unprocessed samples.HPP treatments were carried out in a 55 L high pressure equipment  at 400 MPa/10 min, 400 Mpa/20 min, 600 Mpa/10 min and 600 Mpa/20 min. HPP samples are identified in Unprocessed sample Control, NoHPP/−12 and NoHPP/4  were stoThe moisture content was determined according to the gravimetric method . The watEnterobacteriaceae on Violet Red Bile Glucose Agar (VRBG) CM0485 , incubated at 37 °C for 24 h, and moulds and yeasts on Rose-Bengal Chloramphenicol Agar Base CM0549 , incubated at 25 °C for 120 h. The limit of detection (LoD) was set to 1 log cfu/g. Microbiological analyses were conducted in duplicate.Samples of chocolate fillings (10 g) were diluted  in Ringer solution BR0052G  and homogenized for 2 min in a Stomacher 400 Circulator . A 1.0 mL aliquot of the homogenate was serially 10-fold diluted and 1 mL aliquots of appropriate dilutions were plated on specific media. Total aerobic mesophiles viable organisms were grown on Plate Count Agar (PCA) CM0325 , incubated at 30 °C for 72 h, The obtained results were subjected to statistical analysis where the significance level was set to 1% and samples having a size less than or equal to two were not considered. In order to use one-way analysis of variance (one-way ANOVA), assumption of normality was previously tested by using Shapiro–Wilk’s test ; furtherw/w, higher than other traditional formulations [p > 0.01), regardless of the storage temperature or pressure. The initial values of aw were, approximately, 0.92 ± 0.01 (p > 0.01). The presence of water in a ganache creates a pleasant, creamy smooth and light structure; however, it increases aw and considerably decreases the microbiological stability of chocolate fillings, limiting the shelf life of traditional filled chocolates. The observed values can be considered normal in such type of formulations and are a key factor for limiting shelf life [The evolution of physical and chemical parameters during storage are displayed in ulations ,4,10 due2 ± 0.01  higher tp > 0.01). Although an apparent maximum peak was observed at the 8th and 10th month of Control, the results are not conclusive.The initial pH value was 5.20 ± 0.01 , slightlp < 0.01), while neither the storage temperature nor HPP influenced L* (p > 0.01). Previous works on chocolate products also concluded that storage time for one year, at 18–20 °C, presented a significant impact on both the sensory and instrumental measurement of chocolate using colorimetry [p < 0.01) at lower temperatures, namely −12 °C and 4 °C. At a storage temperature of 20 °C, no significant differences were observed (p > 0.01), regardless of the HPP conditions. The initial value of colour parameter b* was 10.8 ± 0.5, decreasing during storage on all HPP-processed samples (p < 0.01). At the end of the storage time, processing NoHPP/4 presented no significant differences (p > 0.01) when compared with month 0, although lower values were observed at the 6th and 8th month. Processing NoHPP/−12 presented no significant differences during storage time (p > 0.01).The initial value of colour parameter L* was 44.3 ± 0.9, similarly to previous studies ,23, decrorimetry . The iniThe mechanical behaviour during storage of Control and 600/20 processing is presented in 1Hz was 6061 ± 153 kPa . At the end of storage time, the highest G′1Hz values were observed in HPP condition 400/10. Previous studies on milk chocolate [The initial value of G′ 153 kPa , followehocolate  and fillhocolate . Additiohocolate , which mThe average values of total mesophilic counts of untreated and HPP-treated chocolate fillings stored for 12 months are presented in p < 0.01), however, at different rates, as a consequence of HPP conditions and storage temperature. From month 2 to month 12, three distinct groups could be identified (p < 0.01): (i) NoHPP/−12 and 600/20; (ii) 400/20 and 600/10; and (iii) NoHPP/4, Control and 400/10 (data not shown). As expected, the Control sample presented the highest mesophilic counts during storage, wherein values around 5.4 ± 4.7 log cfu/g could be observed after 12 months of storage (p < 0.01), as a consequence of the high aw and high storage temperature [p < 0.01). Indeed, the impact of pressure on mesophilic growth during storage has been previously reported in several food products [During the first two months of storage time, all samples presented microbiological proliferation  were observed until the end of storage time. The exposure time also presented a considerable influence on the development of moulds, where samples 400/20 presented the first contaminations only after 8 months of storage, with 0.7 ± 0.8 log cfu/g counts, lower than 400/10 processing (P. roqueforti was only completely inactivated at 500 MPa [p < 0.01) due to a combined effect of storage at higher temperatures, high aw and neutral pH [After processing, mould counts were below detection level , alike pocessing . Such de 500 MPa . As expep < 0.01) where 2.7 ± 2.1 log cfu/g were observed at the end of storage time. Previous studies have demonstrated the possibility of previously injured cells by HPP recovering their metabolic functions during shelf life evaluation studies, and this may be the case for samples processed at 400/10 and 400/20, as well as 600/10 [After processing, yeasts presented a 3.1 ± 2.5 log cfu/g count. In fact, chocolate fillings are considered “intermediate moisture food” (IMF) products ,35, usuas 600/10 . Processs 600/10 .Enterobacteriaceae group were observed after processing . This group is responsible for the production of bacteriocins with antimicrobial activity, which may explain the reduction in Enterobacteriaceae during storage [Initial counts around 2.5 ± 1.4 log in the ocessing . The appondition . Usually storage . The pre storage .1Hz during storage, possibly as a result of a change in the crystallinity of the cocoa butter with time. However, fillings submitted to HPP at 600 MPa for 20 min presented a weaker structure after 12 months of storage time. The microbiological evaluation was also influenced by HPP conditions, especially for yeasts and moulds. HPP at 600 MPa for 20 min showed the highest reduction in mesophiles, yeasts and moulds, compared with Control, and was the only HPP condition to fulfil the recommended food safety criteria after storage time, although storage at −12 °C also presented lower mesophile counts. However, when considering energy costs and energy efficiency during processing and storage, HPP may present a higher sustainability than the compression-vapour refrigeration cycle.The HPP is a non-thermal treatment used successfully in different food products, especially in high quality products, allowing longer storage time periods. The present study was designed to evaluate the impact of HPP on the long-term storage of chocolate fillings, based on physical, chemical and microbiological parameters. The obtained results concluded on the viability of HPP to improve the shelf life of chocolate fillings, however, depending on applied conditions, namely pressure and time. Moisture and aw did not present significant changes during storage, concluding that polyethylene PET-MET/PE material is an effective barrier against water transfer. Additionally, pH did not present significant changes during storage time or due to HPP conditions. However, physical properties of chocolate fillings were affected during storage. Colour parameter L* decreased in all conditions, parameter a* was affected by lower storage temperatures and parameter b* was affected by higher storage temperatures. Rheological behaviour was also affected, as all samples increased G′


Arisaematis, such as ScienceDirect, Wiley Online Library, Springer Link, Web of Science, CNKI, and WanFang Database. In this paper, the traditional uses, botany, phytochemistry, pharmacology, and toxicology of AR were reviewed. Finally, the existing problems and research directions of the research on AR are discussed.  This review is a collection of all possible studies on AR, published in scientific journals, papers, and books. Using the papers related to  Ninety-eight chemical constituents were isolated from AR. AR has a wide range of pharmacological effects, such as the effects on the central nervous system and cardiovascular system. It also has anti-tumor, sedative, analgesic, anticonvulsant, anti-inflammatory, expectorant, antiarrhythmic, anticoagulant, and other effects. It is also considered an effective drug for in vitro and in vivo validation.  AR is an excellent traditional medicinal plant in China. Pharmacological studies support the traditional use of AR and may verify the folk use of AR in the treatment of different diseases. The anti-tumor effect of AR has been widely concerned by scholars at home and abroad. It has become a hot spot in recent years and has made great contributions to the survival and development of human beings. Although it has a high value of comprehensive utilization, its development and utilization are far from enough. Therefore, the comprehensive development of AR is worthy of further analysis. Arisaematis rhizoma (hereafter referred to as AR) is a tuber of the Arisaema plant of the Araceae family . Th. ThA. erA. amurense Maxim. (东北天南星) is about 35–60 cm (see Arisaema amurensis), and they are obovate or broadly obovate, with a length of 11–15 cm and a width of 6–8 cm, all of which have irregular teeth. The inflorescence is 20–40 cm long, and the spathes are green or purple, with a total length of 11–14 cm. The inflorescence has rod-shaped appendages at the top. It is in berry red, flowers from July to August, and grows in a shady and humid forest on a shady slope.The height of 0 cm see , and theA. Heterophyllum Blume (异叶天南星) is a perennial herb  are oblate  are oblate and spherical   Table 5. Among ttosterol , ethyl gtosterol –29, galltosterol  . A variety of lectins have been found in AR, and blood agglutinin, lymph agglutinin, and sperm agglutinin have been found through cell research of humans and animals . Wu Hao toxicity , 35. Fentoxicity .It is rich in amino acids in AR. Wang Xing found 14 kinds of amino acids were obtained from the qualitative and quantitative analysis of AR  Table 8.Yi Jinghai eight nucleoside compounds were found in AR  Table 9. NucleosIn addition, Fu Ming obtained 8 compounds from 80% ethanol extract of rhizome of AR  Table 1. Zheng XAR has been reported to have a wide range of pharmacological activities, including the effects on the central nervous system and cardiovascular system; it is also used in the clinic for anti-tumor, sedation, analgesia, anti-convulsant, anti-inflammatory, expectorant, anti-arrhythmic, and anti-coagulant. In this section, we mainly introduce and analyze the pharmacological effects of AR .AR is a common traditional Chinese medicine for reducing phlegm. It has the effect of dryness and dampness reducing phlegm. The experiment was carried out by phenol red excretion method in mice showed AR aqueous solution (19 g/kg) had an expectorant effect . Nie RonArisaema (30°C10 g/kg) could significantly inhibit the convulsion rate of strychnine and reduce the convulsion mortality , and the inhibition rate of high dose group was 33.3%. It was found that all the treatment groups could inhibit the growth of S180 tumor, and the inhibition rate of the high-dose group was 33.3% [−1 [50 of 133.5 g/mL [50 of 0.24 mg/mL, 0.78 mg/mL, and 82.17 mg/mL [With the research on chemical constituents of AR in recent years, the study of its anti-tumor effect has become a hot spot in recent years. Zhang Zhilin found that 2.5 g/L and 1.25 g/L ethanol extracts of AR had significant effects on the proliferation of mouse spleen cells . Li Ling in mice . Jiang Sas 33.3% . Yang Zoas 33.3% . Zhao Ch3.3% [−1 . Liang F3.5 g/mL , 67. In 3.5 g/mL . Qiu Lim3.5 g/mL . Tang Hu3.5 g/mL . Li Feng3.5 g/mL . Qi Xiao3.5 g/mL . Qian Ji3.5 g/mL . Dong We3.5 g/mL . Tang Ji17 mg/mL –77.Oncomelania hupensis [Oncomelania hupensis and the examination of the scanning electron microscope has a cough-relieving effect [In addition to the pharmacological effects described above, AR also has some other pharmacological effects. Rats were given 60% ethanol extract of AR (1.4 g/kg), which could not only delay the occurrence time of arrhythmia but also shorten the duration of arrhythmia . It was hupensis . Sensitihupensis . In addihupensis . In addig effect .Araceae. Thus, the toxicities of AR have been comprehensively recorded and investigated. The toxic or irritant components of Araceae plants may be produced by special biological metabolites contained in the plants. It may include calcium oxalate needle crystal and its surface glycoprotein, and some trace polysaccharides [In China, AR is traditionally and commonly recognized as a toxic plant as other plants in charides , 81. Thecharides . The calcharides , resulticharides . The toxcharides , 86. Aft50 was 155.78 g/kg [50 of 21.58 g/kg [50 of the AR needle crystal group was 42.53 mg·kg−1, the LD50 of the raw AR powder group was 1062 mg·kg−1, and the LD50 of the processed product powder group was 2788 mg·kg−1 [Yang Zhonglin and Wu Lianying carried out an acute toxicity study and maximum tolerance test of AR , 88 Tab. Dong We.78 g/kg . In addi.58 g/kg . Tang Li mg·kg−1   = 149.106 ± 26.249 h·μg–1·mL–1, t1/2 = 2.300 ± 0.756 h, Cmax = 33.259 ± 3.192 μg/L, AUC (0⟶t) = 167.122 ± 95.766 h·μg–1·mL–1, AUC (0⟶∞) = 181.974 ± 108.915 h·μg–1·mL–1, t1/2 = 2.592 ± 1.175 h, and Cmax = 36.635 ± 19.53 μg/L. It can be seen from the blood concentration-time curve of the two flavonoids that the blood concentration of the two flavonoids rises obviously at 3 h, which may be related to the enterohepatic circulation, absorption, and distribution in vivo [So far, the pharmacokinetic studies on the extracts and compounds of this plant are very few. The pharmacokinetic studies of AR were mainly focused on flavonoid compounds, including schaftoside and isoschaftoside. Luo Fen studied the pharmacokinetic characteristics of schaftoside and isoschaftoside. In rats, the data are as follows: AUC (0⟶t) = 143.378 ± 26.249 h·To sum up, AR is a traditional Chinese medicine with a long history. It has more than 2,000 years of application history and continuous research of modern science. At present, many chemical constituents have been isolated and identified from this plant. There is no doubt that AR is an important traditional Chinese medicine, although it has certain toxicity. For this reason, in the past many years, many experts are constantly studying the south star and have made great contributions in many aspects. At the same time, in the process of continuous research, there are still some new problems and challenges, which need further research and exploration to meet the clinical needs.in vivo and in vitro verification to improve the safety of the clinical medication. So that AR can be better used better for the guidance of clinical medication to lay a certain foundation.First of all, as a traditional Chinese medicine, AR contains glycosides, sugars, flavonoids, alkaloids, saponins, and other chemical components with expectorant, anti-inflammatory, anti-convulsant, anti-tumor, and other pharmacological effects. There are many studies on it, and the anti-tumor effect of AR has also been widely concerned by scholars at home and abroad and has become a research hotspot in recent years. However, the material basis of other pharmacological effects is not clear, and how and what kind of effects some chemical components play are not clear. It is necessary to further study its monomer compounds and clarify the material basis through certain methods and experiments; AR has certain toxicity. Toxicity studies showed that most of them were irritant, and the long-term toxicity was not obvious. There are some problems in toxicity evaluation methods; for example, oral generated AR can stimulate the throat and make the throat smooth and hoarseness, and it is difficult to enlarge the throat so it is necessary to find a toxicity evaluation model to better evaluate the toxicity of AR. There are too few studies on the pharmacokinetics of AR, which should be paid more attention to In general, as a commonly used traditional Chinese medicine, AR still needs further study. This paper systematically and comprehensively introduces the research status of AR at home and abroad in recent years, including traditional application, phytochemistry, pharmacology, and toxicology. Although great progress has been made in its research, there are still some problems in all aspects. Therefore, we still need to make continuous efforts to develop and utilize AR.