Plasma metabolite profiling identifies non-diabetic CP patients with metabolic alterations progressing to prediabetes prior to HbA1c.

INTRODUCTION: Diabetes (T3cDM) secondary to chronic pancreatitis (CP) arises due to endocrine dysfunction and metabolic dysregulations. Currently, diagnostic tests are not available to identify patients who may progress from normoglycemia to hyperglycemia in CP. We conducted plasma metabolomic profiling to diagnose glycemic alterations early in the course of disease. METHODS: Liquid chromatography-tandem mass spectrometry was employed to generate untargeted, targeted plasma metabolomic profiles in CP patients, controls (n=445) following TRIPOD guidelines. Patients were stratified based on glucose tolerance tests following ADA guidelines. Multivariate analysis was performed using PLS-DA to assess discriminatory ability of metabolites among stratified groups. COMBIROC, logistic regression were employed to derive biomarker signatures. AI-ML tool(Rapidminer) was employed to verify these preliminary results. RESULTS: Ceramide, lysophosphatidylethanolamine, phosphatidylcholine, lysophosphatidic acid, phosphatidylethanolamine, carnitine and lysophosphatidylcholine discriminated T3cDM CP patients from healthy controls with AUC 93%(95%CI 0.81-0.98, p<0.0001), integration with pancreatic morphology improved AUC to 100%(95%CI 0.93-1.00, p<0.0001). Lysophosphatidic acid, phosphatidylinositol and ceramide discriminated non-diabetic CP with glycemic alterations (pre-diabetic CP);AUC 66% (95% CI 0.55-0.76, p=0.1),integration enhanced AUC to 74%,(95% CI 0.55-0.88,p=0.86). T3cDM was distinguished from pre-diabetic by lysophosphatidic acid, phosphatidylinositol and sphinganine(AUC 70%; 95%CI 0.54-0.83,p=0.08), integration improved AUC to 83% (95%CI 0.68-0.93,p=0.05). CombiROC cutoff identified 75% and 78% prediabetes in validation 1 and 2 cohorts. Random forest algorithm assessed performance of integrated panel demonstrating AUC of 72% in predicting glycemic alterations. DISCUSSION: We report for the first time that a panel of metabolites integrated with pancreatic morphology detects glycemia progression prior to HbA1c in CP patients.

Identification and characterization of impurities in an insecticide, commercial chlorantraniliprole using liquid chromatography-tandem mass spectrometry.

RATIONALE: Ensuring the global safety and effectiveness of agrochemicals has become imperative. An in-depth understanding of impurity profiles of products is crucial, especially for high-demand agrochemicals, where impurities may be more toxic and persistent than original agrochemicals. This study focuses on the detection and identification of impurities in a commercial chlorantraniliprole (CAP), an anthranilic diamide class broad-spectrum insecticide. METHODS: Commercial CAP was collected from an agrochemical supplier in India and was analyzed using a high-performance liquid chromatography-photodiode array (HPLC-PDA) (Agilent 1260; wavelength, 220 nm) with a Zorbax RP SB-C18 (250 × 4.6 mm, 5 µm) column and liquid chromatography-mass spectrometry (LC-MS) (Agilent 6545 quadrupole time of flight (Q-TOF)) techniques to identify the impurities. The impurities were isolated by preparative HPLC using a Zorbax-DB C18 (250 × 9.4 mm, 5 µm) column. liquid chromatography- tandem mass spectrometry (LC-MS/MS) experiments (Q-TOF) were performed on CAP and its impurities to obtain their structural data. RESULTS: HPLC-PDA analysis of CAP showed four major impurities (IM-1 to IM-4) ranging from 0.76% to 4.1%. The positive ion electrospray ionization (ESI) mass spectra of CAP and its impurities showed dominant [M + H]+ ions in addition to [M + Na]+ , [M + K]+ , and [2M + Na]+ ions. High-resolution mass spectrometry (HRMS) data provided the elemental composition of the compounds, and isotopic distribution patterns revealed the number of Cl and/or Br atoms present in them. The structures of impurities were proposed based on the LC-MS/MS) data and further confirmed by nuclear magnetic resonance (NMR) data on isolated impurities/synthesis. CONCLUSION: The quality and impurities of CAP, a popular insecticide, must be assessed and described for its efficacy and safety. In this study, four impurities of CAP were detected using HPLC and successfully characterized using LC-HRMS, LC-MS/MS, and NMR data. The method is useful for verifying the purity of CAP as well as helping in the identification of its possible impurities.

Characterization of isomeric acetyl amino acids and di-acetyl amino acids by LC/MS/MS.

Acetylation of amino acids is important in the molecular biology and biochemistry because they are part of several metabolic pathways. N-acetyl amino acids can form through degradation of N-acetyl proteins or direct acetylation of amino acids by specific enzymes. Acetylation of α-amino acids can be either on the alpha -NH2 or on the side-chain functional group, where both the acetyl products are isomeric and can show different biological roles. Theoretically, all proteinogenic α-amino acids are expected to undergo acetylation and they can be a part of metabolome. Thus, it is essential to detect and identify all the possible acetylated products of α-amino acids for untargeted metabolomics studies. In this study, it is aimed to synthesize and characterize all acetylated products of natural α-amino acids. A total of 20 Nα -acetyl amino acids (1-20), six side-chain acetyl amino acids (21-26), and six diacetyl amino acids (27-32) were synthesized and characterized by liquid chromatography-electrospray ionizationtandem mass spectrometry (LC-ESI-MS/MS). The [M + H]+ ions of all the acetyl amino acids were subjected to MS/MS experiments to obtain their structural information. Apart from the expected loss of (H2 O + CO) (immonium ions), most of the acetyl amino acids specifically showed loss of H2 O and loss of a ketene (C2 H2 O) from [M+H]+ ions. The side-chain acetyl amino acids showed a clear-cut structure specific fragment ions that enabled easy differentiation from their isomeric Nα -acetyl amino acids. The other isomeric/isobaric acetyl amino acids could also be easily distinguished by their MS/MS spectra. The MS/MS of immonium ions of the acetyl amino acids were also studied, and they included characteristic products reflecting the structures of parent Nα -acetyl and side-chain acetyl amino acids.

LC-MS/MS Analysis of Reaction Products of Arginine/Methylarginines with Methylglyoxal/Glyoxal.

Methylglyoxal (MGO) and glyoxal (GO) are toxic α-dicarbonyl compounds that undergo reactions with amine containing molecules such as proteins and amino acids and result in the formation of advanced glycation end products (AGEs). This study aimed at investigating the reactivity of arginine (Arg) or dimethylarginine (SDMA or ADMA) with MGO or GO. The solutions of arginine and MGO or GO were prepared in PBS buffer (pH 7.4) and incubated at 37 °C. Direct electrospray ionization-high-resolution mass spectrometry (ESI-HRMS) analysis of the reaction mixture of Arg and MGO revealed the formation of Arg-MGO (1:1) and Arg-2MGO (1:2) products and their corresponding dehydrated products. Further liquid chromatography (LC)-MS analyses revealed the presence of isomeric products in each 1:1 and 1:2 product. The [M + H]+ of each isomeric product was subjected to MS/MS experiments for structural elucidation. The MS/MS spectra of some of the products showed a distinct structure indicative fragment ions, while others showed similar data. The types of products formed by the arginines with GO were also found to be similar to that of MGO. The importance of the guanidine group in the formation of the AGEs was reflected in similar incubation experiments with ADMA and SDMA. The structures of the products were proposed based on the comparison of the retention times and HRMS and MS/MS data interpretation, and some of them were confirmed by drawing analogy to the data reported in the literature.

Evaluation of amino acids and other related metabolites levels in end-stage renal disease (ESRD) patients on hemodialysis by LC/MS/MS and GC/MS.

End-stage renal disease (ESRD) is a rapidly increasing health problem, and every year, about 2 million ESRD cases are reported worldwide. Hemodialysis (HD) is the vital renal reinstatement therapy for ESRD, and HD patterns play a crucial role in patients' health. Plasma metabolomics is the potential approach to understanding the HD process, effectiveness, and patterns. The lack of protein vitality is a primary problem for HD patients, and the quantities of amino acids intracellularly and in the blood are considered to be a symbolic index of protein metabolism and nutrition conditions. In the current study, LC/MS/MS and GC/MS methods were developed for 29 targeted plasma metabolites and validated as per ICH bioanalytical method validation M10 guidelines. The 29 metabolites included 20 proteinogenic amino acids and nine other related metabolites. The methods were employed to measure the absolute quantities (µM) of the targeted metabolites in HD patients (n=60) before and after dialysis (PRE-HD and POST-HD), and compared with the healthy control (HC) group (n=60). Phenylacetylglutamine was found to be higher in both PRE-HD (72.88±14.5 µM) and POST-HD (26.62±7.9 µM), when compared to HC (1.61±0.6 µM). On the other hand, glutamic acid was lower in PRE-HD (14.90±6.5 µM), and POST-HD (13.6±6.1 µM) than that of HC (245.4±37.8 µM). The dialytic loss was found to be 52-45% for arginine, lysine, and histidine, while it was 38-26% for glycine, cysteine, proline, alanine, threonine, glutamine, valine, and methionine. The dialytic loss was low (≤12%) for aspartic acid, glutamic acid, asparagine, leucine, tyrosine, tryptophan, and isoleucine. Graphical abstract adapted from mass spectrometry templates by Biorender.com retrieved from https://app.biorender.com/biorender-templates .

Identification and Characterization of the Isomeric Impurity of the Fungicide "Cyazofamid".

Identification and characterization of biproducts/ impurities present in agrochemicals are critical in view of their efficacy and safety towards public health. We herein present our study on identification and characterization of an impurity, 5-chloro-2-cyano-N,N-dimethyl-4-p-tolylimidazole-1-sulfonamide (2) present in the fungicide, "cyazofamid". Intermittent HPLC analysis of the reaction of substituted imidazole (1) with N,N-dimethylsulfamoyl chloride suggested that 2 is formed during the reaction. Isolation by preparative HPLC and characterization by NMR, LC/HRMS, MS/MS and single crystal XRD analysis confirmed 2 as an isomer of cyazofamid, wherein the N,N-dimethyl sulfonamide group was positioned on the other nitrogen of imidazole in close proximity to chloride group. Computational studies further supported the formation of 2 and ruled out the other possible isomeric structures.

Assessment of uremic toxins in advanced chronic kidney disease patients on maintenance hemodialysis by LC-ESI-MS/MS.

INTRODUCTION: In the advanced stage of chronic kidney disease (CKD), electrolytes, fluids, and metabolic wastes including various uremic toxins, accumulate at high concentrations in the patients' blood. Hemodialysis (HD) is the conventional procedure used worldwide to remove metabolic wastes. The creatinine and urea levels have been routinely monitored to estimate kidney function and effectiveness of the HD process. This study, first from in Indian perspective, aimed at the identification and quantification of major uremic toxins in CKD patients on maintenance HD (PRE-HD), and compared with the healthy controls (HC) as well as after HD (POST-HD). OBJECTIVES: The study mainly focused on the identification of major uremic toxins in Indian perspective and the quantitative analysis of indoxyl sulfate and p-cresol sulfate (routinely targeted uremic toxins), and phenyl sulfate, catechol sulfate, and guaiacol sulfate (targeted for the first time), apart from creatinine and urea in PRE-HD, POST-HD, and HC groups. METHODS: Blood samples were collected from 90 HD patients (both PRE-HD and POST-HD), and 74 HCs. The plasma samples were subjected to direct ESI-HRMS and LC/HRMS for untargeted metabolomics and LC-MS/MS for quantitative analysis. RESULTS: Various known uremic toxins, and a few new and unknown peaks were detected in PRE-HD patients. The p-cresol sulfate and indoxyl sulfate were dominant in PRE-HD, the concentrations of phenyl sulfate, catechol sulfate, and guaiacol sulfate were about 50% of that of indoxyl sulfate. Statistical evaluation on the levels of targeted uremic toxins in PRE-HD, POST-HD, and HC groups showed a significant difference among the three groups. The dialytic clearance of indoxyl sulfate and p-cresol sulfate was found to be < 35%, while that of the other three sulfates was 50-58%. CONCLUSION: LC-MS/MS method was developed and validated to evaluate five major uremic toxins in CKD patients on HD. The levels of the targeted uremic toxins could be used to assess kidney function and the effectiveness of HD.

Alterations in the pH of pancreatic juice are associated with chymotrypsin C inactivation and lithostathine precipitation in chronic pancreatitis patients: a proteomic approach.

BACKGROUND: The progression of chronic pancreatitis (CP), an inflammatory disease of the pancreas, causes pancreatic stones to form within the pancreatic ductal lumen/parenchyma, which occurs via protein plug formation. Pain is the most common symptom that necessitates clinical attention, and pain relief is the therapeutic goal for these patients. Endoscopic therapy and surgery are complimentary forms of therapy for pain relief. This study was envisaged to clarify the mechanism by which protein plug/soft stones form in pancreatic ducts prior to undergoing calcification. METHODS: Protein plugs were obtained from twenty CP patients undergoing therapeutic ERCP for stone removal. Pancreatic juice was obtained from five CP patients without stones. Proteins were isolated by TCA/acetone precipitation, SDS PAGE and 2-D gel electrophoresis to determine the protein profile. Protein spots from the 2-D gel were excised and subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) for identification. The effect of altered pH and elevated concentrations of trypsin on pancreatic juice protein was assessed by SDS‒PAGE to determine the protein profile. Differentially expressed protein bands were excised and subjected to MALDI-TOF. In silico analysis was performed by docking lithostathine with the calcite molecule using AutoDock Vina and PyMOL to clarify their interaction during stone formation. RESULTS: Twenty-three and twenty-nine spots from 2D gels of protein plugs and pancreatic juice, respectively, revealed that lithostathine (Reg1A) was the only protein in the protein plugs, whereas digestive enzymes and lithostathine were identified in pancreatic juice. Altered pH levels and increased trypsin concentrations in the pancreatic juice caused a protein to degrade via an unknown mechanism, and this protein was identified as chymotrypsin C (CTRC) by MALDI-TOF. Docking studies showed that the binding affinity of calcite was higher with the cleaved lithostathine, explaining the deposition of calcium that was observed around the protein plugs after calcified stones were formed through precipitation. CONCLUSION: Our results suggest that chymotrypsin C (CTRC) is degraded in an acidic environment, leading to the precipitation of lithostathine in the ductal lumen.

Integration of metabolites from meta-analysis with transcriptome reveals enhanced SPHK1 in PDAC with a background of pancreatitis.

BACKGROUND: Pathophysiology of transformation of inflammatory lesions in chronic pancreatitis (CP) to pancreatic ductal adenocarcinoma (PDAC) is not clear. METHODS: We conducted a systematic review, meta-analysis of circulating metabolites, integrated this data with transcriptome analysis of human pancreatic tissues and validated using immunohistochemistry. Our aim was to establish biomarker signatures for early malignant transformation in patients with underlying CP and identify therapeutic targets. RESULTS: Analysis of 19 studies revealed AUC of 0.86 (95% CI 0.81-0.91, P < 0.0001) for all the altered metabolites (n = 88). Among them, lipids showed higher differentiating efficacy between PDAC and CP; P-value (< 0.0001). Pathway enrichment analysis identified sphingomyelin metabolism (impact value-0.29, FDR of 0.45) and TCA cycle (impact value-0.18, FDR of 0.06) to be prominent pathways in differentiating PDAC from CP. Mapping circulating metabolites to corresponding genes revealed 517 altered genes. Integration of these genes with transcriptome data of CP and PDAC with a background of CP (PDAC-CP) identified three upregulated genes; PIGC, PPIB, PKM and three downregulated genes; AZGP1, EGLN1, GNMT. Comparison of CP to PDAC-CP and PDAC-CP to PDAC identified upregulation of SPHK1, a known oncogene. CONCLUSIONS: Our analysis suggests plausible role for SPHK1 in development of pancreatic adenocarcinoma in long standing CP patients. SPHK1 could be further explored as diagnostic and potential therapeutic target.

Lutein and ß-carotene biosynthesis in Scenedesmus sp. SVMIICT1 through differential light intensities.

The study evaluated the biosynthesis of lutein and ß-carotene by Scenedesmus sp. SVMIICT1 under five different light intensities (50, 250, 500, 750 and 1000 µE/m2/s). Liquid chromatography/mass spectrometry (LC/MS) was used to determine relative quantities of lutein and ß-carotene. Relatively, high lutein content of 1.43 ± 0.04 and 0.70 ± 0.02 mg/g was found with 50 and 500 µE/m2/s conditions respectively. ß-Carotene content was quantified as 0.15 ± 0.01, 0.1 ± 0.01 and 0.12 ± 0.02 mg/g with 50, 250 and 500 µE/m2/s conditions respectively. The light intensities altered photosystem II and photosystem I. At 50 µE intensity, high chlorophyll content and high photosystem II quantum efficiency (FV/FM) was observed. Low FV/FM ratio of around 0.3 was detected in high light intensities (750 µE and 1000 µE) due to photoinhibition. Lipid fraction increased with increasing light intensity and the fatty acid profiles were similar in all five conditions.

Identification, characterization and evaluation of novel antifungal cyclic peptides from Neobacillus drentensis.

Marine microbes secrete exopolymeric substances (EPS), which surrounds the biofilm and inhibits the fungal growth. Elucidation of the structure and function of the extracellular exopolymeric substances is of vital relevance therapeutically. The active compound responsible for bioactivity was purified and characterized using TLC, LC/MS/MS, GC/MS and FT-IR. Bioactivity of the characterized cyclic peptides (CLPs) against azole resistant and susceptible Candida strains were examined for growth and biofilm formation using scanning electron microscopy, flow cytometry, confocal microscopy. In the present study we identified bioactive cyclic peptides from marine isolated Neobacillus drentensis that exhibited promising tensio-active properties and antifungal efficacy against azole resistant and susceptible Candida albicans. The cluster is composed of five CLP isoforms which were sequenced and identified as new peptides with compositional and structural variations in the amino acid sequence and fatty acid chain. In vitro cytotoxic activity of CLPs was tested in human fibroblast normal cells. We have observed that the CLPs repressed the Candida albicans growth and multiplication by inhibiting the biofilm formation and disruption of branching filamentous hyphae. CLPs have been found to arrest the C. albicans cell cycle by a block at G1-S transition followed by apoptotic cell death. The current studies suggest these natural marine derived CLPs function as potential anti-biofilm agents against azole C. albicans resistant strains.

Identification and characterization of impurities in an insecticide, bifenthrin technical.

The HPLC-DAD and GC/MS methods were successfully used for the identification and characterization of the impurities in an agrochemical insecticide, bifenthrin technical. Three impurities ranging from 0.175%-0.541% were detected by the HPLC-DAD method. The LC/MS technique with ESI or APCI source failed to detect the impurities detected by HPLC-DAD, due to lack of ionization in ESI or APCI. The three impurities were enriched by prep-HPLC, and then their structures were elucidated based on the GC/EIMS and CIMS data. The EI mass spectra of bifenthrin and its impurities displayed molecular ion and provided structure indicative fragment ions; the CIMS data further confirmed their molecular weight. The identity of the impurity 1 was further confirmed by the synthesis of the authentic sample followed by NMR and GC/MS data.

Identification and characterization of forced degradation products of vortioxetine by LC/MS/MS and NMR.

Vortioxetine (VTX) is a novel multimodal antidepressant drug that affects the serotoninergic and noradrenergic systems. In this work, the forced degradation of VTX was studied according to (ICH) Q1A (R2) guidelines. The study revealed that VTX was stable under thermal stress conditions and hydrolytic stress conditions i.e., acidic, basic and neutral conditions. In contrast, six degradation products (DPs) were formed under photolytic and oxidative stress conditions. The DPs were identified and characterized by high-resolution LC/MS and LC/MS/MS. The structures of major DPs were further confirmed by the synthesis and characterization by 1H and 13C NMR data. A possible mechanism for the formation of the VTX DPs via photolytic/oxidative stress degradation pathway was proposed.

Plausible diagnostic value of urinary isomeric dimethylarginine ratio for diabetic nephropathy.

Altered circulatory asymmetric and symmetric dimethylarginines have been independently reported in patients with end-stage renal failure suggesting their potential role as mediators and early biomarkers of nephropathy. These alterations can also be reflected in urine. Herein, we aimed to evaluate urinary asymmetric to symmetric dimethylarginine ratio (ASR) for early prediction of diabetic nephropathy (DN). In this cross-sectional study, individuals with impaired glucose tolerance (IGT), newly diagnosed diabetes (NDD), diabetic microalbuminuria (MIC), macroalbuminuria (MAC), and normal glucose tolerance (NGT) were recruited from Dr. Mohans' Diabetes Specialties centre, India. Urinary ASR was measured using a validated high-throughput MALDI-MS/MS method. Significantly lower ASR was observed in MIC (0.909) and MAC (0.741) in comparison to the NGT and NDD groups. On regression models, ASR was associated with MIC [OR: 0.256; 95% CI: 0.158-0.491] and MAC [OR 0.146; 95% CI: 0.071-0.292] controlled for all the available confounding factors. ROC analysis revealed ASR cut-point of 0.95 had C-statistic of 0.691 (95% CI: 0.627-0.755) to discriminate MIC from NDD with 72% sensitivity. Whereas, an ASR cut-point of 0.82 had C-statistic of 0.846 (95% CI: 0.800 - 0.893) had 91% sensitivity for identifying MAC. Our results suggest ASR as a potential early diagnostic biomarker for DN among the Asian Indians.

Design of DNA-intercalators based copper(II) complexes, investigation of their potential anti-cancer activity and sub-chronic toxicity.

In the present paper, we synthesized and characterized four N-donor polypyridyl copper(II) complexes (C1-C4); [Cu(mono-CN-PIP)2]2+ (C1), [Cu(tri-OMe-PIP)2]2+ (C2), [Cu(di-CF3-PIP)2]2+ (C3) and [Cu(DPPZ)2]2+ (C4). The (Calf-Thymus) CT-DNA binding studies depicted that the complexes could interact with DNA via intercalative mode. All the complexes, particularly C3 and C4 attenuated the proliferation as well as migration of various cancer cells, indicating their anti-cancer and anti-metastatic activity. Additionally, chick embryo angiogenesis (CEA) assay exhibited the inhibition of vascular sprouting in presence of C3 and C4, suggesting their potential in inhibiting the blood vessel growth. Mechanistic studies revealed that the complexes induced the excessive production of cellular reactive oxygen species (ROS) leading to apoptosis through up regulation of p53 and downregulation of Bcl-xL, which might be the plausible mechanisms underlying their anti-cancer properties. To understand the feasibility of practical application of anti-cancer copper complexes C3 and C4, in vivo sub-chronic toxicity study (4 weeks) was performed in C57BL6 mice and the results exhibited almost non-toxic effects induced by these complexes in terms of haematology and serum biochemical analyses, suggesting their biocompatible nature. The current study provides the basis for future advancement of other novel biocompatible metal complexes that could be employed for the therapy of different cancers.

ESI-MS/MS analysis of protonated N-methyl amino acids and their immonium ions.

Methylation is one of the important posttranslational modifications of biological systems. At the metabolite level, the methylation process is expected to convert bioactive compounds such as amino acids, fatty acids, lipids, sugars, and other organic acids into their methylated forms. A few of the methylated amino acids are identified and have been proved as potential biomarkers for several metabolic disorders by using mass spectrometry-based metabolomics workstation. As it is possible to encounter all the N-methyl forms of the proteinogenic amino acids in plant/biological systems, it is essential to have analytical data of all N-methyl amino acids for their detection and identification. In earlier studies, we have reported the ESI-MS/MS data of all methylated proteinogenic amino acids, except that of mono-N-methyl amino acids. In this study, the N-methyl amino acids of all the amino acids (1-21; including one isomeric pair) were synthesized and characterized by ESI-MS/MS, LC/MS/MS, and HRMS. These data could be useful for detection and identification of N-methyl amino acids in biological systems for future metabolomics studies. The MS/MS spectra of [M + H]+ ions of most N-methyl amino acids showed respective immonium ions by the loss of (H2 O, CO). The other most common product ions detected were [MH-(NH2 CH3 ]+ , [MH-(RH)]+ (where R = side chain group) ions, and the selective structure indicative product ions due to side chain and N-methyl group. The isomeric/isobaric N-methyl amino acids could easily be differentiated by their distinct MS/MS spectra. Further, the MS/MS of immonium ions inferred side chain structure and methyl group on α-nitrogen of the N-methyl amino acids.

Changes in the photosynthetic apparatus and lipid droplet formation in Chlamydomonas reinhardtii under iron deficiency.

The unicellular photosynthetic alga Chlamydomonas reinhardtii was propagated in iron deficiency medium and patterns of growth, photosynthetic efficiency, lipid accumulation, as well as the expression of lipid biosynthetic and photosynthesis-related proteins were analysed and compared with iron-sufficient growth conditions. As expected, the photosynthetic rate was reduced (maximally after 4 days of growth) as a result of increased non-photochemical quenching (NPQ). Surprisingly, the stress-response protein LHCSR3 was expressed in conditions of iron deficiency that cause NPQ induction. In addition, the protein contents of both the PSI and PSII reaction centres were gradually reduced during growth in iron deficiency medium. Interestingly, the two generations of Fe deficiency cells could be able to recover the photosynthesis but the second generation cells recovered much slower as these cells were severely in shock. Analysis by flow cytometry with fluorescence-activated cell sorting and thin layer chromatography showed that iron deficiency also induced the accumulation of triacylglycerides (TAG), which resulted in the formation of lipid droplets. This was most significant between 48 and 72 h of growth. Dramatic increases in DGAT2A and PDAT1 levels were caused by iron starvation, which indicated that the biosynthesis of TAG had been increased. Analysis using gas chromatography mass spectrometry showed that levels of 16:0, 18:0, 18:2 and 18:3Δ9,12,15 fatty acids were significantly elevated. The results of this study highlight the genes/enzymes of Chlamydomonas that affect lipid synthesis through their influence on photosynthesis, and these represent potential targets of metabolic engineering to develop strains for biofuel production.

Novel actinomycin group compound from newly isolated Streptomyces sp. RAB12: isolation, characterization, and evaluation of antimicrobial potential.

Streptomyces sp. RAB12 having potential to produce novel actinomycin group compounds was isolated from soil samples collected from CSIR-Indian Institute of Chemical Technology, Hyderabad, India, garden premises using International Streptomycetes Project (ISP) protocols. The 16S rRNA sequence of the strain RAB12 exhibited identity with Streptomyces sp. 13647M and the sequence was deposited in NCBI under the accession number KY 203650 while the strain RAB12 was deposited in The Microbial Type Culture Collection and Gene Bank (MTCC) with accession number MTCC 12747. Cell-free extract of this novel strain revealed two bioactive principles viz., RSP 01 and RSP 02. HR-MS analysis indicated a molecular mass of 1269.61 and 1270.63 m/z g/mol for RSP 01 and RSP 02, respectively. Proton 1H, 13C NMR, 2D NMR and mass spectroscopy analysis revealed a similar fingerprint to that actinomycin D except for a peak at δH3.59 J (1H NMR) and δ 208.88 (13C NMR) for RSP 01 compound suggesting the presence of keto carbonyl at 5-oxo position on the proline moiety which is absent in actinomycin D. Purified RSP 02 depicted a similarity with RSP 01 except a peak in the 1H proton NMR at δH 3.81 J. HR-ESI mass spectra confirmed the molecular formulae for RSP 01 and RSP 02 as C62H84N12O17 and C62H86N12O17, respectively. Antimicrobial activity profile revealed higher antimicrobial activity against bacterial strains (Pseudomonas aeruginosa, Micrococcus luteus, Staphylococcus aureus, Salmonella typhi, and Bacillus subtilis) and Candida albicans compared to standard actinomycin D. MIC and MBC for RSP 01 were observed to be 0.0039 and 0.0078 (µg/ml) against C. albicans, while for actinomycin D, it was found to be 0.031 and 0.62 (µg/ml), respectively indicating a tenfold higher potency. Thus, these RSP 01 and RSP 02 compounds from Streptomyces sp. RAB12 may be promising candidates for industrial and clinical applications.

Synthesis of 8',11'-dihydrospiro[cyclohexane-1,2'-oxepino[2,3-h] chromen]-4'(3'H)-ones with ring closing metathesis as a key step.

A series of novel hybrid molecular entities incorporating various spiro chromanone scaffolds onto the benzannulated oxepine core moiety were synthesised using allylation, Claisen rearrangement, Kabbe condensation and Ring Closing Metathesis (RCM) as a key step. During the synthesis we found that the nitrogen functionality in the substrate influences significantly the catalyst load due to electronic effects. Several iterations have been carried out to achieve complete conversion to products 6a-6e.