Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system.

BACKGROUND: Pregnenolone and progesterone are the life-important steroid hormones regulating essential vital functions in mammals, and widely used in different fields of medicine. Microbiological production of these compounds from sterols is based on the use of recombinant strains expressing the enzyme system cholesterol hydroxylase/C20-C22 lyase (CH/L) of mammalian steroidogenesis. However, the efficiency of the known recombinant strains is still low. New recombinant strains and combination approaches are now needed to produce these steroid hormones. RESULTS: Based on Mycolicibacterium smegmatis, a recombinant strain was created that expresses the steroidogenesis system (CYP11A1, adrenodoxin reductase, adrenodoxin) of the bovine adrenal cortex. The recombinant strain transformed cholesterol and phytosterol to form progesterone among the metabolites. When 3-methoxymethyl ethers of sterols were applied as bioconversion substrates, the corresponding 3-ethers of pregnenolone and dehydroepiandrosterone (DHEA) were identified as major metabolites. Under optimized conditions, the recombinant strain produced 85.2 ± 4.7 mol % 3-methoxymethyl-pregnenolone within 48 h, while production of 3-substituted DHEA was not detected. After the 3-methoxymethyl function was deprotected by acid hydrolysis, crystalline pregnenolone was isolated in high purity (over 98%, w/w). The structures of steroids were confirmed using TLC, HPLC, MS and 1H- and 13C-NMR analyses. CONCLUSION: The use of mycolicybacteria as a microbial platform for the expression of systems at the initial stage of mammalian steroidogenesis ensures the production of valuable steroid hormones-progesterone and pregnenolone from cholesterol. Selective production of pregnenolone from cholesterol is ensured by the use of 3-substituted cholesterol as a substrate and optimization of the conditions for its bioconversion. The results open the prospects for the generation of the new microbial biocatalysts capable of effectively producing value-added steroid hormones.

Antimicrobial Activity and Phytochemical Characterization of Baccharis concava Pers., a Native Plant of the Central Chilean Coast.

Few sclerophyllous plants from the central coast of Chile have been systematically studied. This work describes the phytochemical composition and antimicrobial properties of Baccharis concava Pers. (sin. B. macraei), a shrub found in the first line and near the Pacific coast. B. concava has been traditionally used by indigenous inhabitants of today's central Chile for its medicinal properties. Few reports exist regarding the phytochemistry characterization and biological activities of B. concava. A hydroalcoholic extract of B. concava was prepared from leaves and small branches. Qualitative phytochemical characterization indicated the presence of alkaloids, steroids, terpenoids, flavonoids, phenolic, and tannin compounds. The antimicrobial activity of this extract was assessed in a panel of microorganisms including Gram-positive bacteria, Gram-negative bacteria, and pathogenic yeasts. The extract displayed an important antimicrobial effect against Gram-positive bacteria, Candida albicans, and Cryptococcus neoformans but not against Gram-negatives, for which an intact Lipopolysaccharide is apparently the determinant of resistance to B. concava extracts. The hydroalcoholic extract was then fractionated through a Sephadex LH-20/methanol-ethyl acetate column. Afterward, the fractions were pooled according to a similar pattern visualized by TLC/UV analysis. Fractions obtained by this criterion were assessed for their antimicrobial activity against Staphylococcus aureus. The fraction presenting the most antimicrobial activity was HPLC-ESI-MS/MS, obtaining molecules related to caffeoylquinic acid, dicaffeoylquinic acid, and quercetin, among others. In conclusion, the extracts of B. concava showed strong antimicrobial activity, probably due to the presence of metabolites derived from phenolic acids, such as caffeoylquinic acid, and flavonoids, such as quercetin, which in turn could be responsible for helping with wound healing. In addition, the development of antimicrobial therapies based on the molecules found in B. concava could help to combat infection caused by pathogenic yeasts and Gram-positive bacteria, without affecting the Gram-negative microbiota.

An indicator color chart for quick detection of Pigment Red 53 in cosmetic products in Indonesia.

BACKGROUND: Pigment Red 53 is a dangerous synthetic dye that is often added to cosmetics, even though its use in cosmetic products has been prohibited because of possible impacts on health. Faster and more sensitive detection of Pigment Red 53 is needed for onsite analysis to protect the community from illegal cosmetics that contain the dye. Indicator color charts are a kind of analytical method that can be used to detect Pigment Red 53 in cosmetic products, including lipstick, rouge, and eyeshadow. Such charts are practical, fast, and can be used for onsite analysis. METHODS: In this study, an indicator for Pigment Red 53 detection was obtained through a reagent reaction that caused a specific color change. An indicator color chart was then produced by setting out in paper form the series of colors which resulted from the reaction of specific chemical reagents and Pigment Red 53 solutions at concentrations of 10, 20, 40, 60, 80, and 100 ppm. RESULTS: The testing results showed that the indicator color chart may be used as an initial screening method for the detection of Pigment Red 53 in cosmetic products with a detectable minimum concentration of 10 ppm. Out of nine samples, only one (Eyeshadow 3) tested positive for Pigment Red 53. Further analysis was carried out on the indicator color chart and the results showed good agreement with TLC and UV-Vis spectrophotometry methods. CONCLUSION: The results reported in this paper demonstrate that the indicator color chart is a good prospective method for onsite analysis to detect Pigment Red 53 in cosmetic samples, with a lower detection limit compared to polymer-based indicators.

Factors associated with non-completion of palliative radiotherapy for spinal metastasis in patients with terminal cancer: a retrospective study.

BACKGROUND: Predictors of non-completion of radiotherapy (RT) should be identified to determine the optimal RT dose. Therefore, this study aimed to explore factors associated with non-completion of palliative RT in patients with terminal cancer. METHODS: In this retrospective study, patients with terminal cancer who received RT (not including single-fraction RT) for relief of pain caused by spinal metastasis were categorized into complete and incomplete groups. Baseline characteristics, hematologic test data [e.g., total lymphocyte count (TLC)], performance status, palliative performance scale (PPS) score, psoas muscle index (PMI), Charlson comorbidity index, and age-adjusted Charlson comorbidity index of the patients were compared between the two groups. RESULTS: The complete group comprised 58 patients (median age: 68 years; female/male: 17/41; number of irradiation fractions: ≥2 to <10, 20 patients; 10, 34 patients; and >10, 4 patients), and the incomplete group comprised 9 patients (median age: 68 years; female/male: 3/6; number of irradiation fractions: ≥2 to <10, 2 patients; 10, 7 patients; and >10, 0 patient). The proportion of patient death within 1 week or 1 month was higher in the incomplete group than in the complete group. Compared with that in the incomplete group, TLC measured 1 week before RT (pre-TLC) and PMI recorded before RT were significantly higher in the complete group (P=0.013 and P=0.012, respectively). In multivariable analyses, pre-TLC was significantly associated with the incomplete group (P=0.048). Compared with the complete group, the incomplete group included several patients whose PPS scores rapidly decreased. CONCLUSIONS: Pre-TLC can predict non-completion of palliative RT in patients with terminal cancer.

Association of Monocyte Count With Lung Function and Exercise Capacity Among Hospitalized COVID-19 Survivors: A 2-Year Cohort Study.

BACKGROUND: Abnormal changes of monocytes have been observed in acute COVID-19, whereas associations of monocyte count with long COVID were not sufficiently elucidated. METHODS: A cohort study was conducted among COVID-19 survivors discharged from hospital. The primary outcomes were core symptoms of long COVID, distance walked in 6 min, and lung function, and the secondary outcomes were health-related quality of life and healthcare use after discharge. Latent variable mixture modeling was used to classify individuals into groups with similar trajectory of monocyte count from discharge to 2-year after symptom onset. Multivariable adjusted generalized linear regression models and logistic regression models were used to estimate the associations of monocyte count trajectories and monocyte count at discharge with outcomes. RESULTS: In total, 1389 study participants were included in this study. Two monocyte count trajectories including high to normal high and normal trajectory were identified. After multivariable adjustment, participants in high to normal high trajectory group had an odds ratio (OR) of 2.52 (95% CI, 1.44-4.42) for smell disorder, 2.27 (1.27-4.04) for 6-min walking distance less than lower limit of normal range, 2.45 (1.08-5.57) for total lung capacity (TLC) < 80% of predicted, 3.37 (1.16-9.76) for personal care problem, and 1.70 (1.12-2.58) for rehospitalization after discharge at 2-year follow-up compared with those in normal trajectory group. Monocyte count at discharge showed similar results, which was associated with smell disorder, TLC < 80% of predicted, diffusion impairment, and rehospitalization. CONCLUSIONS: Monocyte count may serve as an easily accessible marker for long-term management of people recovering from COVID-19.

Preparation, Characterization, and Radiolabeling of Anti-HER2 scFv With Technetium Tricarbonyl and Stability Studies.

Breast cancer is the most common diagnosed cancer, and the second cause of cancer death among women, worldwide. HER2 overexpression occurred in approximately 15% to 20% of breast cancers. Invasive biopsy method has been used for detection of HER2 overexpression. HER2-targeted imaging via an appropriate radionuclide is a promising method for sensitive and accurate identification of HER2+ primary and metastatic lesions. 99m Tc-anti-HER2 scFv can specifically target malignancies and be used for diagnosis of the cancer type and metastasis as well as treatment of breast cancer. We radiolabeled anti-HER2 scFv that was expressed in Escherichia coli and purified through Ni-NTA resin under native condition with 99m Tc-tricarbonyl formed from boranocarbonate. HER2-based ELISA, BCA, TLC, and HPLC were used in this study. In the current study, anti-HER2 scFv was lyophilized before radiolabeling. It was found that freeze-drying did not change the binding activity of anti-HER2 scFv to HER2. Results demonstrated direct anti-HER2 scFv radiolabeling by 99m Tc-tricarbonyl to hexahistidine sequence (His-tag) without any changes in biological activity and radiochemical purity of around 98%. Stability analysis revealed that 99m Tc-anti-HER2 scFv is stable for at least 24 h in PBS buffer, normal saline, human plasma proteins, and histidine solution.

Lower Arginine Bioavailability, Increased FeNO Levels, and Airway Resistance on Impulse Oscillometry Are Characteristics of Asthma in Children and Young Adults with Sickle Cell Disease.

Background and Objectives: Data on characteristics of asthma in children with sickle cell disease (SCD) is conflicting. Recently, the L-arginine pathway has gained attention in the pathogenesis of asthma and SCD. This study aimed to determine the distinctive clinical and laboratory features and the role of arginine metabolism in asthmatic children with SCD. Materials and Methods: A total of 52 children and adolescents with SCD, including 24 with asthma (SCD-A) and 28 without asthma (SCD-NA), and 40 healthy controls were included. A questionnaire, atopy tests, fractional exhaled nitric oxide (FeNO), and lung function tests were employed. Serum metabolites of the arginine pathway were measured. The results of the three groups were compared. Results: The demographic characteristics and atopy markers of the three groups were similar. FEV1%, FEV1/FVC, MMEF%, and total lung capacity (TLC%) values of SCD-A patients were not significantly different from the SCD-NA group, but they were significantly lower than the values measured in the controls. FeNO values greater than 35 ppb were present only in the SCD-A group. In impulse oscillometry, median resistance values at 5 Hz (R5)% were higher in both SCD subgroups than in healthy controls (p = 0.001). The (R5-20/R5)% values were higher in the SCD-A group (p = 0.028). Serum arginine levels and arginine bioavailability indices were significantly lower in the SCD-A group than in the SCD-NA group and healthy controls (p = 0.003 and p < 0.001). Conclusions: Asthma in children with SCD was not associated with atopy or low FEV1/FVC levels. However, lower arginine bioavailability and higher FeNO levels differentiated asthma in patients with SCD. High R5% and (R5-20/R5)% values indicated increased airway resistance in SCD, with a predominance of small airway disease in asthmatics.

Dengue Cases Presenting to the Emergency Department of a Tertiary Care Hospital in Late 2021: A Cross-Sectional Study in Karachi.

Objectives: Dengue Fever (DF) is an arboviral disease caused by the Dengue virus (DENV). This study aims to assess the association of dengue prevalence with patients' residential areas and hematological laboratory findings (Total platelet count, immunoglobulins, and dengue antigens) during COVID-19 pandemic. Methods: A retrospective study was conducted at the Emergency department of Jinnah Postgraduate Medical Center, Karachi from October to December 2021. All the patients irrespective of their ages presenting to the ED with either of the following complaints: fever; GIT problems; vomiting; body ache; bleeding were included in our study. Results: Our study comprised 189 patients in total in which the females (n = 172) outnumbered the males (n = 17). Out of all, 84.7% of the patients were febrile having a low-grade fever on average. Korangi district had the most dengue cases, while Keamari had the fewest. There was no significant association reported between mean platelet count, mean TLC, and mean hemoglobin levels with dengue positivity. Conclusion: There is a concerning rise in DF cases in Karachi, particularly in the Korangi district. Despite the COVID-19 pandemic, DF demands urgent attention.

Automated Synthesis of [11C]PiB via [11CH3OTf]-as Methylating Agent for PET Imaging of ß-Amyloid.

AIM: Efficient synthesis of precursor from commercially available starting materials and automated radiosynthesis of [11C]PiB using commercially available dedicated [11C]- Chemistry module from the synthesized precursor. BACKGROUND: [11C]PiB is a promising radiotracer for PET imaging of ß-Amyloid, advancing Alzheimer's disease research. The availability of precursors and protocols for efficient radiolabelling foster the applications of any radiotracer. Efficient synthesis of PiB precursor was performed using anisidine and 4-nitrobenzoyl chloride as starting materials in 5 steps, having addition, substitutions, and cyclization chemical methodologies. This precursor was used for fully automated radiosynthesis of [11C]PiB in a commercially available synthesizer, MPS-100 (SHI, Japan). The synthesized [11C]PiB was purified via solid-phase methodology, and its quality control was by the quality and safety criteria required for clinical use. METHODS: The synthesis of desired precursors and standard authentic compounds started with commercially available materials with 70-80% yields. The standard analytical methods characterized all synthesized compounds. The fully automated [11C]-chemistry synthesizer (MPS-100) used for radiosynthesis of [11C]PiB with [11C]CH3OTf acts as a methylating agent. For radiolabelling, varied amounts of precursor and time of reaction were explored. The resulting crude product underwent purification through solid-phase cartridges. The synthesized radiotracer was analyzed using analytical tools such as radio TLC, HPLC, pH endo-toxicity, and half-life. RESULTS: The precursor for radiosynthesis of [11C]PiB was achieved in excellent yield using simple and feasible chemistry. A protocol for radiolabelling of precursor to synthesized [11C]PiB was developed using an automated synthesizer. The crude radiotracer was purified by solid-phase cartridge, with a decay-corrected radiochemical yield of 40±5% and radiochemical purity of more than 97% in approx 20 minutes (EOB). The specific activity was calculated and found in a 110-121 mCi/µmol range. CONCLUSION: A reliable methodology was developed for preparing precursor followed by fully automated radiolabeling using [11C]MeOTf as a methylating agent to synthesize [11C]PiB. The final HPLC-free purification yielded more than 97% radiochemical purity tracer within one radionuclide half-life. The method was reproducible and efficient for any clinical center.

Efficient Expression and Characterization of an Endo-Type Lyase HCLase_M28 and Its Gradual Scale-Up Fermentation for the Preparation of Chondroitin Sulfate Oligosaccharides.

Glycosaminoglycan (GAG) lyases have been critical in structural and functional studies of GAGs. HCLase_M28, a lyase identified from the genome of Microbacterium sp. M28 was heterologously expressed, enzymatically characterized, and prepared in large-scale fermentation for the production of chondroitin sulfate (CS) oligosaccharides. Results showed that the expression of HCLase_M28 in Escherichia coli BL21 (DE3)-pET24a-HCLase_M28opt1 and Bacillus subtilis W800-pSTOP1622-HCLase_M28opt2 were 108-fold and 25-fold that of wide strain. The optimal lytic reaction of HCLase_M28 happened in 20 mM Tris-HCl (pH 7.2) at 50 °C with a specific activity of 190.9 U/mg toward CS-A. The degrading activity was slightly simulated in presence of 1 mM Ca2+ and Mn2+ while severely inhibited by Hg+, Cu2+, Fe3+, and SDS. TLC and ESI-MS analysis proved HCLase_M28 was an endolytic lyase and degraded CS and hyaluronic acid into unsaturated disaccharides. Through a gradual scale-up of fermentation in 5 L, 100 L, and 1000 L, a highly efficient intracellular expression of HCLase_M28 with an activity of 3.88 × 105 U/L achieved within a 34 h of cultivation. Through ultrafiltration, CS oligosaccharides with DP of 2 to 8 as the main components could be controllably prepared. The successful large-scale fermentation made HCLase_M28 a promising enzyme for industrial production of CS oligosaccharides.

TLC-Bioautography-Guided Isolation and Assessment of Antibacterial Compounds from Manuka (Leptospermum scoparium) Leaf and Branch Extracts.

A rapid procedure for the targeted isolation of antibacterial compounds from Manuka (Leptospermum scoparium) leaf and branch extracts was described in this paper. Antibacterial compounds from three different Manuka samples collected from New Zealand and China were compared. The active compounds were targeted by TLC-bioautography against S. aureus and were identified by HR-ESI-MS, and -MS/MS analysis in conjunction with Compound Discoverer 3.3. The major antibacterial component, grandiflorone, was identified, along with 20 ß-triketones, flavonoids, and phloroglucinol derivatives. To verify the software identification, grandiflorone underwent purification via column chromatography, and its structure was elucidated through NMR analysis, ultimately confirming its identity as grandiflorone. This study successfully demonstrated that the leaves and branches remaining after Manuka essential oil distillation serve as excellent source for extracting grandiflorone. Additionally, we proposed an improved TLC-bioautography protocol for evaluating the antibacterial efficacy on solid surfaces, which is suitable for both S. aureus and E. coli. The minimum effective dose (MED) of grandiflorone was observed to be 0.29-0.59 µg/cm2 against S. aureus and 2.34-4.68 µg/cm2 against E. coli, respectively. Furthermore, the synthetic plant growth retardant, paclobutrazol, was isolated from the samples obtained in China. It is hypothesized that this compound may disrupt the synthesis pathway of triketones, consequently diminishing the antibacterial efficacy of Chinese Manuka extract in comparison to that of New Zealand.

Green Chromatographic Assay of Toxic Impurities of Cyproheptadine: In Silico Toxicity Profiling.

Cyproheptadine (CYP) is an antihistaminic and anti-serotonin drug used to improve the clinical course of hospitalized patients severely affected by COVID-19 and requiring supplemental oxygen. Thin layer chromatography (TLC)-densitometry method was developed and validated for the estimation of CYP and its impurity, dibenzosuberone and its degradation product: 10, 11-dihydroxy-dibenzosuberone. The toxicity profiles of CYP impurity and degradation product were studied and the in silico data, using pre-ADMET software, proved their toxicity. A developing system of hexane: methanol: ethyl acetate: formic acid: triethylamine (8:1:1:0.05:0.1, by volume) was used with densitometric scanning at 245 nm. International Conference of Harmonization guidelines were obeyed for method validation. The proposed method was applied on CYP pharmaceutical formulation whose results showed no significant difference from results of the reported chromatographic method regarding accuracy and precision. Method's greenness was evaluated by analytical Eco scale, Green Analytical Procedure Index, and Analytical greenness approach tools. The developed method can be applied for safety check analysis of CYP containing formulations and detection and even quantification of its related toxic components in quality control laboratories at high sensitivity up to 20-fold than the reported chromatographic method.

COVID-19 after 2 Years from Hospital Discharge: A Pulmonary Function and Chest Computed Tomography Follow-Up Study.

INTRODUCTION: Serial follow-up with pulmonary function testing (PFT) and chest computed tomography (CT) after severe COVID-19 are recommended. As a result, many longitudinal studies have been published on COVID-19 of different grade of severity up to 1-year follow-up. Therefore, we aimed at a long-term observational study throughout 2 years after severe COVID-19. METHODS: Severe COVID-19 patients were consecutively recruited after hospital discharge between March and June 2020 and prospectively followed up for 24 months, with mMRC dyspnea scale and PFT at 6, 12, and 24 months. Chest CT was performed when clinically indicated. RESULTS: One hundred one patients enrolled completed the observational study. At 24 months, those with reduced total lung capacity (TLC) were 16%, associated with fibrotic ground glass opacity (GGO) and mMRC score >1, respectively, in 75% and 69% of them. At 24 months, those with a reduced diffusing capacity of the lung for CO were 41%, associated with fibrotic GGO and mMRC score >1, respectively, in 53% and 22% of them. CONCLUSION: Two years after hospitalization for severe COVID-19, a non-negligible number of patients still suffer from "long COVID" due to respiratory damage.

Personalized pulmonary rehabilitation program for patients with post-acute sequelae of COVID-19: A proof-of-concept retrospective study.

Long-COVID patients present with a decline in physical fitness. The aim of this study is to reveal the impact of pulmonary rehabilitation (PR) on physical fitness, quality of life (QoL), and parameters of quantified thorax CT. Long-COVID patients enrolled in a 3-month PR program were retrospectively studied. PR included endurance and resistance training three times a week. Assessments pre- and post-rehabilitation included quantified chest CT, pulmonary function tests (PFT), six-minute walk test (6MWT), cardiopulmonary exercise test, and questionnaires: Hospital Anxiety and Depression Scale, post-COVID-19 Functional Status scale, Borg score, and EuroQol. Seventeen subjects (5M/12F), mean age 42 ± 13 years, were included. PR improved all questionnaires' results significantly. Only significant difference in PFT parameters was correlation between baseline total lung capacity (TLC) and difference in TLC pre- and post-rehabilitation (p = 0.002). 6MWT increased from 329 to 365 m (p < 0.001), VO2max changed from 21 to 24 mL/kg/min (p = 0.007), peak load increased from 116 to 141 Watt (p < 0.001). Blood volume in small pulmonary vessels of 1.25 to 5 mm2 in cross-sectional area (BV5%) was higher than observed in patients with acute COVID-19 infection. After rehabilitation, BV5% decreased from 65% to 62% (p = 0.020). These changes correlated directly with changes in TLC (p = 0.039). Quantified CT airway volume increased after rehabilitation (p = 0.013). After rehabilitation, TLC tended to normalize due to (re)opening of small airways, with decline in air trapping and recruitment of alveoli. Furthermore, this study revealed that pulmonary rehabilitation can improve QoL and physical fitness in long-COVID patients.

Fructus mori: An Updated Review on Botany, Phytonutrient, Detection, Bioactivity, Quality Marker, and Application.

BACKGROUND: Fructus mori (mulberry) is not only a delicious fruit with rich phytonutrients and health functions but also a medicinal plant with many clinical therapeutic values for tonifying kidneys and consolidating essence, making hair black and eyes bright. METHODS: The related references about F. mori in this review from 1996 to 2022 had been collected from both online and offline databases, including PubMed, Elsevier, SciFinder, Willy, SciHub, Scopus, Web of Science, ScienceDirect, SpringerLink, Google Scholar, Baidu Scholar, ACS publications, and CNKI. The other information was acquired from ancient books and classical works about F. mori. RESULTS: An updated summary of phytonutrients from F. mori was listed as fellows: flavonoids (1-20) (23.5%), phenolic acids (21-34) 16.5%), alkaloids (35-75) (48.2%), polysaccharides (76- 78) (3.5%), other compounds (79-85) (8.3%). The above chemical components were detected by TLC, UV-Vis, HPLC, GC-MS, and AAS methods for their quality standards. The various bioactivities (hepatoprotective, immunomodulatory, anti-oxidant, hypoglycemic, anti-cancer, and other activities) of mulberry are summarized and discussed in this review, which laid an important basis for analyzing their mechanisms and quality markers. This review summarized its applications for vinegar, wine, yogurt, drink, jelly, and sweetmeat in food fields, and the existing problems and future development directions are also discussed in this review. CONCLUSIONS: This review made a comprehensive description of F. mori, including botany, phytonutrient, detection, bioactivity, quality marker, and application. It will not only provide some important clues for further studying F. mori, but also provide some valuable suggestions for indepth research and development of F. mori.

In vitro and in vivo antitrypanosomal activity of the fresh leaves of Ranunculus Multifidus Forsk and its major compound anemonin against Trypanosoma congolense field isolate.

BACKGROUND: Animal trypanosomiasis is a major livestock problem due to its socioeconomic impacts in tropical countries. Currently used trypanocides are toxic, expensive, and the parasites have developed resistance to the existing drugs, which calls for an urgent need of new effective and safe chemotherapeutic agents from alternative sources such as medicinal plants. In Ethiopian traditional medicine fresh leaves of Ranunculus multifidus Forsk, are used for the treatment of animal trypanosomiasis. The present study aimed to evaluate the antitrypanosomal activity of the fresh leaves of R. multifidus and its major compound anemonin against Trypanosoma congolense field isolate. METHODS: Fresh leaves of R. multifidus were extracted by maceration with 80% methanol and hydro-distillation to obtain the corresponding extracts. Anemonin was isolated from the hydro-distilled extract by preparative TLC. For the in vitro assay, 0.1, 0.4, 2 and 4 mg/ml of the test substances were incubated with parasites and cessation or drop in motility of the parasites was monitored for a total duration of 1 h. In the in vivo assay, the test substances were administered intraperitoneally daily for 7 days to mice infected with Trypanosoma congolense. Diminazene aceturate and 1% dimethylsulfoxide (DMSO) were used as positive and negative controls, respectively. RESULTS: Both extracts showed antitrypanosomal activity although the hydro-distilled extract demonstrated superior activity compared to the hydroalcoholic extract. At a concentration of 4 mg/ml, the hydro-distilled extract drastically reduced motility of trypanosomes within 20 min. Similarly, anemonin at the same concentration completely immobilized trypanosomes within 5 min of incubation, while diminazene aceturate (28.00 mg/kg/day) immobilized the parasites within 10 min. In the in vivo antitrypanosomal assay, anemonin eliminates parasites at all the tested doses (8.75, 17.00 and 35.00 mg/kg/day) and prevented relapse, while in diminazene aceturate-treated mice the parasites reappeared on days 12 to 14. CONCLUSIONS: The current study demonstrated that the fresh leaves of R. multifidus possess genuine antitrypanosomal activity supporting the use of the plant for the treatment of animal trypanosomiasis in traditional medicine. Furthermore, anemonin appears to be responsible for the activity suggesting its potential as a scaffold for the development of safe and cost effective antitrypanosomal agent.

Characterization of pigment produced by high carotenoid yielding bacteria Paracoccus marcusii RSPO1 and evaluation of its anti-diabetic, anti-microbial and antioxidant properties.

High pigment producing bacteria was isolated and identified as Paracoccus marcusii RSPO1 using biochemical and 16s rRNA identification. Bacterial pigment production was optimised using parameters like inoculum size, nitrogen source, pH, temperature, and agitation speed. Carotenoids production was 7,240 ± 41 µg L-1 after optimization. The silica column purified pigment was characterized using UV-visible spectroscopy, TLC, FTIR, LC-ESI-MS and NMR, which revealed its composition as astaxanthin, zeaxanthin, ζ-carotene and ß-zeacarotene. The inhibition assays against α-amylase and α-glucosidase showed IC50 values as 226 µg ml-1 and 0.7548 µg ml-1 respectively. The MIC of 1000 µg ml-1 of carotenoid was found to be effective against Escherichia coli and Enterobacter aerogenes when tested for antibacterial activity. Moreover, antioxidant activity of carotenoid sample was also determined where antioxidant potential of extracted carotenoid for DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) inhibition were 65 ± 0.06% and 42 ± 07% respectively at 20 µg ml-1 concentration.

Identification and characterization of major bioactive compounds from Andrographis paniculata (Burm. f.) extracts showed multi-biomedical applications.

The Andrographis paniculata recognized as most valuable medicinal plant in folk medicine. Hence, this research was designed to evaluate antibacterial potential of petroleum ether (PE) and methanol (ME) extracts of A. paniculata against skin infection causing bacterial pathogens such as Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae, Enterobacter aerogenes, Proteus vulgaris, and Propionibacterium acnes. Also assessed the antidiabetic (α-glucosidase and α-amylase inhibition assay), antioxidant, and photoprotective potential of PE and ME extract analyses. The major bioactive compounds were identified and characterized through UV, FTIR, 1H-NMR and 13C-NMR spectra analyses. The ME extract contain more number of phytochemicals (alkaloids, flavonoids, saponins, terpenoids, glycoside, protein, and phytosterol) than PE extract. The antibacterial activity result also revealed that the ME (as dose dependent) extract showed better activity at 250 mg mL-1 as in the following order: P. acnes (6-29 mm) > K. pneumoniae (3-28 mm) > S. aureus (3-27 mm) > P. vulgaris (3-26 mm) > S. pyogenes (2-25 mm) > E. aerogenes (1-23 mm). PE: E. aerogenes (3-20 mm) > P. vulgaris (2-19 mm) > P. acnes (3-18 mm) > K. pneumoniae (3-17 mm) > S. aureus (2-16 mm) > S. pyogenes (0-11 mm). The MIC value of ME extract was found as 100-150 mg mL-1 and it was better than PE extract. Similarly, the ME also possesses dose based α-glucosidase inhibition activity as up to 85% at 250 mg mL-1 concentration. The fluorescence spectra analysis method also stated that the ME extract possess photoprotective bioactive agent. The ME fractions (F01 and F02) obtained from TLC and column chromatogram were identified as 3-O-ß-d-glucosyl-14- deoxyandrographiside and 14-deoxyandrographolide respectively through UV, FTIR, 1H-NMR and 13C-NMR spectra analyses. Such compounds may be responsible for significant antibacterial activity against pathogenic bacteria causing skin infections, excellent antidiabetic activity, as well as photoprotective potential.

TLC and HPTLC-APCI-MS for the rapid discrimination of plant resins frequently used for lacquers and varnishes by artists and conservators.

INTRODUCTION: Depending on their terpenoid and phenolic constituents plant resins can be classified as diterpenoid, triterpenoid or phenolic resins; thereby the profile of diterpenes and triterpenes is considered as genus- or even species-specific. OBJECTIVES: We aimed to develop a simple, rapid, inexpensive, sensitive and specific method for the identification of resin-specific triterpenoid and phenolic compounds in plant resins using (HP)TLC [(high-performance) thin-layer chromatography] combined with APCI-MS (atmospheric pressure chemical ionisation mass spectrometry) and post-chromatographic detection reactions. METHODS: Twenty resin samples from different plant species were analysed. Different extraction procedures, post-chromatographic detection reagents as well as various sorbents and solvents for planar chromatography were tested. To evaluate the potential of the optimised (HP)TLC-APCI-MS methods, parameter such as limit of detection (LOD) was determined for selected marker compounds. RESULTS: Our protocol enabled qualitative analyses of chemotaxonomic molecular markers in natural resins such as dammar, mastic, olibanum and benzoin. For the first time, the application of thionyl chloride-stannic chloride reagent for a specific post-chromatographic detection of triterpenes is reported, sometimes even allowing discrimination between isomers based on their characteristic colour sequences. For triterpene acids, triterpene alcohols and phenolic compounds, detection limits of 2-20 ng/TLC zone and a system precision with a relative standard deviation (RSD) in the range of 3.9%-7.0% were achieved by (HP)TLC-APCI-MS. The applicability of the method for the analysis of resin-based varnishes was successfully tested on a mastic-based varnish. Thus, the method we propose is a helpful tool for the discrimination of resins and resin-based varnishes with respect to their botanical origin.

Exercise capacity following SARS-CoV-2 infection is related to changes in cardiovascular and lung function in military personnel.

BACKGROUND: Since the COVID-19 pandemic, post-COVID syndrome (persistent symptoms/complications lasting >12 weeks) continues to pose medical and economic challenges. In military personnel, where optimal fitness is crucial, prolonged limitations affecting their ability to perform duties has occupational and psychological implications, impacting deployability and retention. Research investigating post-COVID syndrome exercise capacity and cardiopulmonary effects in military personnel is limited. METHODS: UK military personnel were recruited from the Defence Medical Services COVID-19 Recovery Service. Participants were separated into healthy controls without prior SARS-CoV-2 infection (group one), and participants with prolonged symptoms (>12 weeks) after mild-moderate (community-treated) and severe (hospitalised) COVID-19 illness (group 2 and 3, respectively). Participants underwent cardiac magnetic resonance imaging (CMR) and spectroscopy, echocardiography, pulmonary function testing and cardiopulmonary exercise testing (CPET). RESULTS: 113 participants were recruited. When compared in ordered groups (one to three), CPET showed stepwise decreases in peak work, work at VT1 and VO2 max (all p < 0.01). There were stepwise decreases in FVC (p = 0.002), FEV1 (p = 0.005), TLC (p = 0.002), VA (p < 0.001), and DLCO (p < 0.002), and a stepwise increase in A-a gradient (p < 0.001). CMR showed stepwise decreases in LV/RV volumes, stroke volumes and LV mass (LVEDVi/RVEDVi p < 0.001; LVSV p = 0.003; RVSV p = 0.001; LV mass index p = 0.049). CONCLUSION: In an active military population, post-COVID syndrome is linked to subclinical changes in maximal exercise capacity. Alongside disease specific changes, many of these findings share the phenotype of deconditioning following prolonged illness or bedrest. Partitioning of the relative contribution of pathological changes from COVID-19 and deconditioning is challenging in post-COVID syndrome recovery.