Unraveling the role of cytochrome P450 enzymes in oleanane triterpenoid biosynthesis in arjuna tree.

Triterpenoids (C30-isoprenoids) represent a major group of natural products with various physiological functions in plants. Triterpenoids and their derivatives have medicinal uses owing to diverse bioactivities. Arjuna (Terminalia arjuna) tree bark accumulates highly oxygenated ß-amyrin-derived oleanane triterpenoids (e.g., arjunic acid, arjungenin, and arjunolic acid) with cardioprotective roles. However, biosynthetic routes and enzymes remain poorly understood. We mined the arjuna transcriptome and conducted cytochrome P450 monooxygenase (P450) assays using Saccharomyces cerevisiae and Nicotiana benthamiana to identify six P450s and two P450 reductases for oxidative modifications of oleanane triterpenoids. P450 assays using oleananes revealed a greater substrate promiscuity of C-2α and C-23 hydroxylases/oxidases than C-28 oxidases. CYP716A233 and CYP716A432 catalyzed ß-amyrin/erythrodiol C-28 oxidation to produce oleanolic acid. C-2α hydroxylases (CYP716C88 and CYP716C89) converted oleanolic acid and hederagenin to maslinic acid and arjunolic acid. CYP716C89 also hydroxylated erythrodiol and oleanolic aldehyde. However, CYP714E107a and CYP714E107b catalyzed oleanolic acid/maslinic acid/arjunic acid, C-23 hydroxylation to form hederagenin, arjunolic acid and arjungenin, and hederagenin C-23 oxidation to produce gypsogenic acid, but at a lower rate than oleanolic acid C-23 hydroxylation. Overall, P450 substrate selectivity suggested that C-28 oxidation is the first P450-catalyzed oxidative modification in the arjuna triterpenoid pathway. However, the pathway might branch thereafter through C-2α/C-23 hydroxylation of oleanolic acid. Taken together, these results provided new insights into substrate range of P450s and unraveled biosynthetic routes of triterpenoids in arjuna. Moreover, complete elucidation and reconstruction of arjunolic acid pathway in S. cerevisiae and N. benthamiana suggested the utility of arjuna P450s in heterologous production of cardioprotective compounds.

High-level biosynthesis of enantiopure germacrene D in yeast.

Germacrene D, a sesquiterpenoid compound found mainly in plant essential oils at a low level as (+) and/or (-) enantiomeric forms, is an ingredient for the fragrance industry, but a process for the sustainable supply of enantiopure germacrene D is not yet established. Here, we demonstrate metabolic engineering in yeast (Saccharomyces cerevisiae) achieving biosynthesis of enantiopure germacrene D at a high titer. To boost farnesyl pyrophosphate (FPP) flux for high-level germacrene D biosynthesis, a background yeast chassis (CENses5C) was developed by genomic integration of the expression cassettes for eight ergosterol pathway enzymes that sequentially converted acetyl-CoA to FPP and by replacing squalene synthase promoter with a copper-repressible promoter, which restricted FPP flux to the competing pathway. Galactose-induced expression of codon-optimized plant germacrene D synthases led to 13-30 fold higher titers of (+) or (-)-germacrene D in CENses5C than the parent strain CEN.PK2.1C. Furthermore, genomic integration of germacrene D synthases in GAL80, LPP1 and rDNA loci generated CENses8(+D) and CENses8(-D) strains, which produced 41.36 µg/ml and 728.87 µg/ml of (+) and (-)-germacrene D, respectively, without galactose supplementation. Moreover, coupling of mitochondrial citrate pool to the cytosolic acetyl-CoA, by expressing a codon-optimized ATP-citrate lyase of oleaginous yeast, resulted in 137.71 µg/ml and 815.81 µg/ml of (+) or (-)-germacrene D in CENses8(+D)* and CENses8(-D)* strains, which were 67-120 fold higher titers than in CEN.PK2.1C. In fed-batch fermentation, CENses8(+D)* and CENses8(-D)* produced 290.28 µg/ml and 2519.46 µg/ml (+) and (-)-germacrene D, respectively, the highest titers in shake-flask fermentation achieved so far. KEY POINTS: • Engineered S. cerevisiae produced enantiopure (+) and (-)-germacrene D at high titers • Engineered strain produced up to 120-fold higher germacrene D than the parental strain • Highest titers of enantiopure (+) and (-)-germacrene D achieved so far in shake-flask.

Unraveling the terpene synthase family and characterization of BsTPS2 contributing to (S)-( +)-linalool biosynthesis in Boswellia.

Boswellia tree bark exudes oleo-gum resin in response to wounding, which is rich in terpene volatiles. But, the molecular and biochemical basis of wound-induced formation of resin volatiles remains poorly understood. Here, we combined RNA-sequencing (RNA-seq) and metabolite analysis to unravel the terpene synthase (TPS) family contributing to wound-induced biosynthesis of resin volatiles in B. serrata, an economically-important Boswellia species. The analysis of large-scale RNA-seq data of bark and leaf samples representing more than 600 million sequencing reads led to the identification of 32 TPSs, which were classified based on phylogenetic relationship into various TPSs families found in angiosperm species such as TPS-a, b, c, e/f, and g. Moreover, RNA-seq analysis of bark samples collected at 0-24 h post-wounding shortlisted 14 BsTPSs that showed wound-induced transcriptional upregulation in bark, suggesting their important role in wound-induced biosynthesis of resin volatiles. Biochemical characterization of a bark preferentially-expressed and wound-inducible TPS (BsTPS2) in vitro and in planta assays revealed its involvement in resin terpene biosynthesis. Bacterially-expressed recombinant BsTPS2 catalyzed the conversion of GPP and FPP into (S)-( +)-linalool and (E)-(-)-nerolidol, respectively, in vitro assays. However, BsTPS2 expression in Nicotiana benthamiana found that BsTPS2 is a plastidial linalool synthase. In contrast, cytosolic expression of BsTPS2 did not form any product. Overall, the present work unraveled a suite of TPSs that potentially contributed to the biosynthesis of resin volatiles in Boswellia and biochemically characterized BsTPS2, which is involved in wound-induced biosynthesis of (S)-( +)-linalool, a monoterpene resin volatile with a known role in plant defense.

A Middle Eocene lowland humid subtropical "Shangri-La" ecosystem in central Tibet.

Tibet's ancient topography and its role in climatic and biotic evolution remain speculative due to a paucity of quantitative surface-height measurements through time and space, and sparse fossil records. However, newly discovered fossils from a present elevation of ∼4,850 m in central Tibet improve substantially our knowledge of the ancient Tibetan environment. The 70 plant fossil taxa so far recovered include the first occurrences of several modern Asian lineages and represent a Middle Eocene (∼47 Mya) humid subtropical ecosystem. The fossils not only record the diverse composition of the ancient Tibetan biota, but also allow us to constrain the Middle Eocene land surface height in central Tibet to ∼1,500 ± 900 m, and quantify the prevailing thermal and hydrological regime. This "Shangri-La"-like ecosystem experienced monsoon seasonality with a mean annual temperature of ∼19 °C, and frosts were rare. It contained few Gondwanan taxa, yet was compositionally similar to contemporaneous floras in both North America and Europe. Our discovery quantifies a key part of Tibetan Paleogene topography and climate, and highlights the importance of Tibet in regard to the origin of modern Asian plant species and the evolution of global biodiversity.

BAHD acetyltransferase contributes to wound-induced biosynthesis of oleo-gum resin triterpenes in Boswellia.

Triterpenes (30-carbon isoprene compounds) represent a large and highly diverse class of natural products that play various physiological functions in plants. The triterpene biosynthetic enzymes, particularly those catalyzing the late-stage regio-selective modifications are not well characterized. The bark of select Boswellia trees, e.g., B. serrata exudes specialized oleo-gum resin in response to wounding, which is enriched with boswellic acids (BAs), a unique class of C3α-epimeric pentacyclic triterpenes with medicinal properties. The bark possesses a network of resin secretory structures comprised of vertical and horizontal resin canals, and amount of BAs in bark increases considerably in response to wounding. To investigate BA biosynthetic enzymes, we conducted tissue-specific transcriptome profiling and identified a wound-responsive BAHD acetyltransferase (BsAT1) of B. serrata catalyzing the late-stage C3α-O-acetylation reactions in the BA biosynthetic pathway. BsAT1 catalyzed C3α-O-acetylation of αBA, ßBA, and 11-keto-ßBA in vitro and in planta assays to produce all the major C3α-O-acetyl-BAs (3-acetyl-αBA, 3-acetyl-ßBA, and 3-acetyl-11-keto-ßBA) found in B. serrata bark and oleo-gum resin. BsAT1 showed strict specificity for BA scaffold, whereas it did not acetylate the more common C3ß-epimeric pentacyclic triterpenes. The analysis of steady-state kinetics using various BAs revealed distinct substrate affinity and catalytic efficiency. BsAT1 transcript expression coincides with increased levels of C3α-O-acetyl-BAs in bark in response to wounding, suggesting a role of BsAT1 in wound-induced biosynthesis of C3α-O-acetyl-BAs. Overall, the results provide new insights into the biosynthesis of principal chemical constituents of Boswellia oleo-gum resin.

RGG-motif containing mRNA export factor Gbp2 acts as a translation repressor.

Complex cascades of RNA-binding proteins regulate the mRNA metabolism and influence gene expression. Several distinct proteins act at different stages of mRNA life cycle. SR family proteins in yeast are implicated in mRNA processing and nuclear export. In this report, we uncover the role of an SR/RGG-motif containing mRNA export factor Gbp2 in mRNA translation regulation. We demonstrate that Gbp2 localizes to cytoplasmic granules upon heat shock and oxidative stress. Our pull-down assays demonstrate that Gbp2 directly binds to the conserved translation factor eIF4G1 via its RGG motif. We further mapped the region on eIF4G1 to which Gbp2 binds and observed that the binding region overlaps with another translation repressor Sbp1. We found that the RGG-motif deletion mutant is defective in localizing to polysome fractions. Upon tethering Gbp2 to a GFP reporter mRNA in vivo, translation of GFP reporter decreased significantly indicating that Gbp2 acts as a translation repressor. Consistent with these results, we show that Gbp2 can directly repress mRNA translation in the in vitro translation systems in an RGG-motif dependent manner. Taken together, our results establish that the mRNA export factor Gbp2 has a vital role in repressing translation of mRNA. We propose that Gbp2 is a multifaceted RGG-motif protein responsible for translational repression without affecting mRNA levels.

Paleocene Ipomoea (Convolvulaceae) from India with implications for an East Gondwana origin of Convolvulaceae.

The morning glory family, Convolvulaceae, is globally important in medicine and food crops. The family has worldwide distribution in a variety of habitats; however, its fossil record is very poorly documented. The current fossil record suggests an origin in North America, which is in contrast to molecular data that indicate an East Gondwana origin. We report Ipomoea leaves from the late Paleocene (Thanetian; 58.7-55.8 million years ago) of India, which was a part of East Gondwana during this time. This is the earliest fossil record for both the family Convolvulaceae and the order Solanales. This suggests that the sister families Convolvulaceae and Solanaceae diverged before the Eocene in Gondwana-derived continents. The evidence presented here supports the conclusion from molecular phylogenetic analysis of an East Gondwana origin of Convolvulaceae.

Gastrointestinal bleeding in a chronic myeloid leukaemia patient precipitated by dasatinib-induced platelet dysfunction: Case report.

Bleeding in patients with chronic myeloid leukaemia (CML) receiving the second-line tyrosine kinase inhibitor (TKI) dasatinib is a well-documented side effect, occurring in up to 24% of patients. In most cases, it is attributed directly to a secondary grade 3 or 4 thrombocytopaenia. Platelet dysfunction precipitated by dasatinib has been demonstrated in multiple in vitro and in vivo studies; however, there is currently no correlative data that definitively associates this with clinically significant bleeding. In this case, we report a patient with chronic-phase CML receiving dasatinib who developed significant gastrointestinal bleeding secondary to angiodysplasia in the absence of a severe thrombocytopaenia or coagulopathy. Platelet function testing on the PFA-100 assay and formal platelet aggregometry demonstrated impaired platelet aggregation, however, upon cessation of dasatinib, platelet function normalised and the bleeding resolved without further intervention. This case demonstrates that dasatinib-induced platelet dysfunction can cause clinically significant bleeding and highlights the need for physicians to be aware of this adverse effect.

Primary Ewing sarcoma of the cavernous sinus.

INTRODUCTION: Ewing sarcoma (ES) is a part of a larger family of round blue-cell tumors that occasionally manifest as osseous or extraosseous lesions adjacent to or within the central nervous system (CNS). Although a large body of literature exists on ES of bone, data are lacking on tumors with cranial or spinal components that affect the CNS. Moreover, primary intracranial ES has been uncommonly reported, and its location in the cavernous sinus is extremely rare, with only a few cases reported in literature. METHOD: We describe a case of a 15-year-old boy who presented initially with a seizure along with acute abducens, trochlear, and oculomotor nerve pareses. Magnetic resonance imaging (MRI) revealed a cavernous sinus mass. Given the rapid growth and aggressive nature of the mass, a biopsy of the lesion was performed. Microscopic examination of the specimen showed immunohistological features consistent with ES. The patient was treated with multi-agent chemotherapy and fractionated radiation therapy. RESULT: Clinical assessment 7 months after completion of chemotherapy and 4 months after completion of radiotherapy revealed that the patient's ocular motility and alignment had improved significantly, and MRI showed almost complete disappearance of the tumor. CONCLUSION: This is one of only six cases of extraosseous primary intracranial ES of the cavernous sinus reported in literature to date. The availability of multi-agent chemotherapy and fractionated radiation therapy in this patient resulted in his marked clinical and imaging improvement. It remains to be seen if this therapy will result in long-term control or cure of the lesion in our patient. In the meantime, increasing awareness and identification of children primary intracranial ES will hopefully allow a better understanding of the molecular biology of this tumor and the development of standardized treatment regimens.

Body Mass Index a Forecast of Sputum Culture Conversion Among Drug-Resistant Tuberculosis Patients.

Background Drug-resistant tuberculosis is a major health issue around the world. The time it takes to find a sputum-positive patient is a major risk factor for the spread of tuberculosis, and many things can indicate a longer time to culture conversion. Also, there is strong proof that poor nutrition is linked to infectious diseases. So, this study aimed to look into the link between a person's body mass index (BMI) and the change of a sputum culture within three months in people who have rifampicin-resistant (RR)/multidrug-resistant (MDR)-tuberculosis (TB) kept on a bedaquiline-based regimen. Materials and methods The Department of Respiratory Medicine at King George's Medical University, Lucknow, hosted an observational, analytical, prospective, single-center study from May 2020 to April 2021. The study included 105 people who had been identified with RR/MDR-TB and were on an optimized background regimen that included a bedaquiline-based regimen. The result we were interested in was sputum culture conversion within three months, and we looked at how BMI related to that outcome. Analytical analyses utilized Pearson's chi-square test for categorical variables and the t-test for continuous variables. Differences with a P-value of <0.05 were considered significant. SPSS software (version 18.0, IBM Corp., Armonk, NY, USA) was used for all analyses, with missing data not replaced or credited. Results A total of 105 people who met the inclusion and exclusion criteria were analyzed. The patients had a mean age of 33.34 years and were mostly male 61 (58%). Fifty-eight (58; 55%) patients lived in rural areas. Most patients had fever 77 (73%), cough 72 (69%), and weight loss 66 (63%). Sixty-nine (69; 66%) patients had a history of TB. Fifty-seven patients had a BMI of <18.5 kg/m2, and 48 patients had with BMI of ≥18.5. At the end of the study, 75/105 patients converted their sputum culture. Of the 105 patients, 57 (54%) had a low BMI (less than 18.5 kg/m2). Among the 57 patients with a BMI of <18.5 kg/m2, only 28 (46%) achieved sputum culture conversion after 3 months while 29 (60%) of 48 with BMI ≥18.5 achieved sputum culture conversion after 3 months. Among the patients with a BMI <18.5, 15/57 (26%) tested positive for sputum culture after three months. In patients with a BMI of ≥18.5, only 4/48 (8%) patients tested positive for sputum culture after three months. Conclusion In patients with drug-resistant tuberculosis, low BMI (<18.5 kg/m2) was an independent risk factor for failing to convert sputum cultures within three months.

Legumes from the Paleocene sediments of India and their ecological significance.

During the early Paleogene, greenhouse gases created warm global climates. These warm climates redistributed the habitat of marine and terrestrial biota globally. Understanding the ecology of biotas under extremely warm climates is important to decipher their behavior in future climate warming. Here we report two new legume fossils (Leguminocarpum meghalayensis Bhatia, Srivastava et Mehrotra sp. nov., and Parvileguminophyllum damalgiriensis Bhatia, Srivastava et Mehrotra sp. nov.) from the late Paleocene sediments of Tura Formation of Meghalaya, northeast India. Globally, the Paleocene legume fossil records indicate that legumes most likely immigrated to India from Africa via the Ladakh-Kohistan Arc during the early Paleogene. Moreover, previously reconstructed climate data from the Tura Formation indicate that legumes were well adapted to a warm seasonal climate with monsoon rains.

Xanthan gum-based copper nano-magnetite doped carbon aerogel: A promising candidate for environmentally friendly catalytic dye degradation.

In this work, a novel copper nano-magnetite doped carbon aerogel (CXMCA) was created utilizing a simple graft co-polymerization approach with xanthan gum (XG) as a template to tackle the agglomeration problem caused by magnetic nanoparticle magnetism. The results indicated that the XG based CXMCA exhibited outstanding magnetic properties (Ms = 36.52 emu/g) as well as strong catalytic activity for the degradation of cationic and anionic dyes. Among all organic dyes, methylene blue and crystal violet (MB, CV) as cationic dyes, as well as congo red and methyl orange (CR, and MO) as anionic dyes, CXMCA demonstrated an exceptional dye degradation rate (8.06 × 10-3 s-1-1.12 × 10-2 s-1) and was highly competent for cationic dyes with degradation (90 %-98 %) as compared to its unsupported magnetic nanoparticles. The formation of CXMCA catalyst is clearly confirmed by the FTIR, XRD, XPS, VSM, SEM & TEM analyses. We report a very effective xanthan gum-based copper nano-magnetite doped carbon aerogel dye scavenger with application in percentage dye degradation and kinetic investigations, as well as a remarkable reusability assay up to 7 repetition cycles. The findings suggested that using biological macromolecules like xanthan gum as a foundation to generate magnetic aerogels might be a good choice for evaluating environmental aspects.

Acute pancreatitis-induced thrombotic thrombocytopenic purpura.

CONTEXT: Acute pancreatitis due to thrombotic thrombocytopenic purpura is a well recognized condition. Here, we are reporting a rare converse phenomenon, in which thrombocytopenic purpura occurred secondary to acute pancreatitis. CASE REPORT: A 19-year-old male referred to our intensive care unit with diagnosis of acute pancreatitis with multi-organ dysfunction. He had history of severe abdominal pain and recurrent vomiting about one month ago, requiring hospital admission. There, on diagnostic work-up at admission, abdominal ultrasonography was suggestive of pancreatitis. His serum amylase and lipase were 1,900 and 1,582 U/L, respectively. Other laboratory parameters were within normal limits. He was managed conservatively with intravenous fluids, antibiotics and analgesics; and discharged after about 2 weeks One week after discharge he was readmitted in same hospital with abdominal pain, multiple episodes of bilious vomiting and abdominal distention. Later on he was referred to our intensive care unit; having classical pentad of thrombocytopenic purpura, i.e., thrombocytopenia, micro-angiopathic hemolytic anemia, renal failure, encephalopathy, and fever. His condition improved with plasma exchange therapy and transferred out from our ICU to ward after 10 days of stay. CONCLUSION: Thrombocytopenic purpura may be precipitate by acute pancreatitis due to multiple mechanisms. A high clinical suspicion is required to make an early diagnosis and allow early initiation of plasma exchange therapy, resulting in a good prognosis.

CoviXNet: A novel and efficient deep learning model for detection of COVID-19 using chest X-Ray images.

The Coronavirus (COVID-19) pandemic has created havoc on humanity by causing millions of deaths and adverse physical and mental health effects. To prepare humankind for the fast and efficient detection of the virus and its variants shortly, COVID-19 detection using Artificial Intelligence and Computer-Aided Diagnosis has been the subject of several studies. To detect COVID-19, there are numerous publicly accessible datasets of Chest X-rays that the researchers have combined to solve the problem of inadequate data. The cause for concern here is that in combining two or more datasets, some of the images might be duplicates, so a curated dataset has been used in this study, taken from an author's paper. This dataset consists of 1281 COVID-19, 3270 Normal X-rays, and 1656 viral-pneumonia infected Chest X-ray images. Dataset has been pre-processed and divided carefully to ensure that there are no duplicate images. A comparative study on many traditional pre-trained models was performed, analyzing top-performing models. Fine-tuned InceptionV3, Modified EfficientNet B0&B1 produced an accuracy of 99.78% on binary classification, i.e., covid-19 infected and normal Chest X-ray image. ResNetV2 had a classification accuracy of 97.90% for 3-class classification i.e., covid-19 infected, normal, and pneumonia. Furthermore, a trailblazing custom CNN-based model, CoviXNet, has been proposed consisting of 15 layers that take efficiency into account. The proposed model CoviXNet exhibited a 10-fold accuracy of 99.47% on binary classification and 96.61% on 3-class. CoviXNet has shown phenomenal performance with exceptional accuracy and minimum computational cost. We anticipate that this comparative study, along with the proposed model CoviXNet, can assist medical centers with the efficient real-life detection of Coronavirus.

Ensemble of Deep Neural Networks based on Condorcet's Jury Theorem for screening Covid-19 and Pneumonia from radiograph images.

COVID-19 detection using Artificial Intelligence and Computer-Aided Diagnosis has been the subject of several studies. Deep Neural Networks with hundreds or even millions of parameters (weights) are referred to as "black boxes" because their behavior is difficult to comprehend, even when the model's structure and weights are visible. On the same dataset, different Deep Convolutional Neural Networks perform differently. So, we do not necessarily have to rely on just one model; instead, we can evaluate our final score by combining multiple models. While including multiple models in the voter pool, it is not always true that the accuracy will improve. So, In this regard, the authors proposed a novel approach to determine the voting ensemble score of individual classifiers based on Condorcet's Jury Theorem (CJT). The authors demonstrated that the theorem holds while ensembling the N number of classifiers in Neural Networks. With the help of CJT, the authors proved that a model's presence in the voter pool would improve the likelihood that the majority vote will be accurate if it is more accurate than the other models. Besides this, the authors also proposed a Domain Extended Transfer Learning (DETL) ensemble model as a soft voting ensemble method and compared it with CJT based ensemble method. Furthermore, as deep learning models typically fail in real-world testing, a novel dataset has been used with no duplicate images. Duplicates in the dataset are quite problematic since they might affect the training process. Therefore, having a dataset devoid of duplicate images is considered to prevent data leakage problems that might impede the thorough assessment of the trained models. The authors also employed an algorithm for faster training to save computational efforts. Our proposed method and experimental results outperformed the state-of-the-art with the DETL-based ensemble model showing an accuracy of 97.26%, COVID-19, sensitivity of 98.37%, and specificity of 100%. CJT-based ensemble model showed an accuracy of 98.22%, COVID-19, sensitivity of 98.37%, and specificity of 99.79%.

Autoproteolysis of Procerain and Procerain B mediated by structural changes.

Procerain (Pc) and Procerain B (PcB) are two latex proteases from Calotropis procera having potential applications in food and other industries. However, autolytic degradation of these proteases limits their potential use in industry. Nevertheless, basic mechanism underlying the autoproteolysis has not been detailed. In order to understand the same, we subjected the enzymes to various denaturing and activating conditions. The results showed that structural changes induced by different denaturing conditions trigger their autoproteolysis. We also observed differential response of Pc, PcB and other papain-like proteases towards autocatalysis in presence of reducing agent in-spite of sharing the same structural fold, including the number of disulfide bonds. The possible reason underlying this intriguing observation is also discussed. Further, present work establishes that structural changes in the proteases lead to autoproteolysis and the enzymes are stable unless they experience structural perturbation. These findings could thus be useful for their practical applications in industries.

Recombinant full-length Bacillus Anthracis protective antigen and its 63 kDa form elicits protective response in formulation with addavax.

Introduction: Bacillus anthracis is the causative agent for the lethal disease anthrax, primarily affecting animals and humans in close contact with an infected host. The pathogenicity of B. anthracis is attributed to the secreted exotoxins and their outer capsule. The host cell-binding exotoxin component "protective antigen" (PA) is reported to be a potent vaccine candidate. The aim of our study is to produce several PA constructs and analyze their vaccine potential. Methods: We have designed the various subunit, PA-based recombinant proteins, i.e., full-length Protective antigen (PA-FL), C-terminal 63 kDa fragment (PA63), Protective antigen domain 1-domain 4 chimeras (PA-D1-4) and protective antigen domain 4 (PA-D4) and analyzed their vaccine potential with different human-compatible adjuvants in the mouse model. We have optimized the process and successfully expressed our recombinant antigens as soluble proteins, except full-length PA. All the recombinant antigen formulations with three different adjuvants i.e., Addavax, Alhydrogel, and Montanide ISA 720, were immunized in different mouse groups. The vaccine efficacy of the formulations was analyzed by mouse serum antigen-specific antibody titer, toxin neutralization assay, and survival analysis of mouse groups challenged with a lethal dose of B. anthracis virulent spores. Results: We have demonstrated that the PA-FL addavax and PA63 addavax formulations were most effective in protecting spore-challenged mice and serum from the mice immunized with PAFL addavax, PA-FL alhydrogel, PA63 addavax, and PA63 alhydrogel formulations were equivalently efficient in neutralizing the anthrax lethal toxin. The higher levels of serum Th1, Th2, and Th17 cytokines in PA-FL addavax immunized mice correspond to the enhanced protection provided by the formulation in challenged mice. Discussion: We have demonstrated that the PA-FL addavax and PA63 addavax formulations exhibit equivalent efficiency as vaccine formulation both in a mouse model of anthrax and mammalian cell lines. However, PA63 is a smaller antigen than PA-FL and more importantly, PA63 is expressed as a soluble protein in E. coli, which imparts a translational advantage to PA63-based formulation. Thus, the outcome of our study has significant implications for the development of protective antigen-based vaccine formulations for human use against the lethal disease anthrax.

Leaf fossils of Sabalites (Arecaceae) from the Oligocene of northern Vietnam and their paleoclimatic implications.

Recent paleobotanical investigations in Vietnam provide a good opportunity to improve our understanding of the biodiversity and paleoclimatic conditions in the geological past of Southeast Asia. Palms (Arecaceae) are a diverse family of typical thermophilous plants with a relatively low tolerance for freezing. In this study, we describe well-preserved fossil palm leaves from the Oligocene Dong Ho Formation of Hoanh Bo Basin, northern Vietnam. Characters of the fossil leaves, such as a fan-shaped costapalmate lamina, an unarmed petiole, a costa slightly enlarged at the base that then tapers distally into the blade, and well-preserved amphistomatic leaves with cuticles, suggest that they represent a new fossil species, which we herein designate Sabalites colaniae A. Song, T. Su, T. V. Do et Z.K. Zhou sp. nov. Together with other paleontological and palaeoclimatic evidence, we conclude that a warm climate prevailed in northern Vietnam and nearby areas during the Oligocene.

Molecular and pathological screening of canine distemper virus in Asiatic lions, tigers, leopards, snow leopards, clouded leopards, leopard cats, jungle cats, civet cats, fishing cat, and jaguar of different states, India.

The present investigation was conducted to rule out canine distemper (CD) diseases in Indian wild felids (Asiatic lions, tigers, leopards, snow leopards, clouded leopards, leopard cats, jungle cats, civet cats, fishing cat, and jaguar). The collected samples were screened for CD virus (CDV) by histopathology (HP), immunohistochemistry (IHC) and reverse transcriptase-polymerase chain reaction (RT-PCR) targeting H gene and N gene. The HP and IHC of suspected samples portrayed that 22 [11 leopards, 6 lions, 3 tigers, 1 snow leopard and 1 civet cat] out of 129 (17.05%) wild felids were positive for CD. The major pathological consequences were observed in spleen, lung, kidney and brain. The syncytia and intranuclear as well as intracytoplasmic eosinophilic inclusion bodies were seen in CDV infected cells. Although the histopathological lesions in spleen were more specific and consistent, however, the severe demyelinated leukoencephalitis (usually expected in CD infected dog) was not observed in the brain of any Indian wild felids. Conversely, the CDV antigen has been portrayed via IHC in pancreatic islets of Langerhans of tiger species for the first time in this study. Moreover, the concurrent CD and babesiosis has also been observed in a lioness without a usual coffee-coloured urine. The N gene and H gene of CDV isolates were amplified, sequenced and subsequently constructed the phylogenetic tree. The phylogenetic analysis of H gene revealed that the CDV isolates from Indian lion formed separate clade with CDV isolates from Indian dog and Indian palm civet cat. Furthermore, two CDV isolates from Indian tigers formed clade with Onderstepoort vaccine strain and CDV isolates from dogs of Uttar Pradesh, USA and UK. Evidently, CDV is circulating in Indian wild felids and causing diseases in them.