Genome wide survey, and expression analysis of Ornithine decarboxylase gene associated with alkaloid biosynthesis in plants.

Plant ODC (ornithine decarboxylase) plays a vital role in normalizing cell division in actively growing tissues. The ODC is a key precursor enzyme for nicotine and nornicotine biosynthesis in plants. ODCs are widely present in many plant families but have not been functionally validated and characterized at the molecular level. In the present study, 58 plant ODCs were identified and were found to contain two putative regulatory motifs, specifically PLP (Pyridoxal 5'-phosphate) and Orn/DAP/Arg decarboxylase family 2 pyridoxal-phosphate, that are highly conserved among diverse plant species. Further, the cis-regulatory elements and interacting partners of the gene revealed the importance of ODC in various metabolic pathways. The qRT-PCR revealed highest relative expression of ODC in floral meristem and roots. Our results suggest that ODC can be effectively used as an ideal candidate for engineering polyamine biosynthesis and would be crucial for developing ultra-low nicotine content tobacco lines via genome editing.

Overexpression of Glyoxalase III gene in transgenic sugarcane confers enhanced performance under salinity stress.

The glyoxalase pathway is a check point to monitor the elevation of methylglyoxal (MG) level in plants and is mediated by glyoxalase I (Gly I) and glyoxalase II (Gly II) enzymes in the presence of glutathione. Recent studies established the presence of unique DJ-1/PfpI domain containing protein named glyoxalase III (Gly III) in prokaryotes, involved in the detoxification of MG into D-lactic acid through a single step process. In the present study, eleven transgenic sugarcane events overexpressing EaGly III were assessed for salinity stress (100 mM and 200 mM NaCl) tolerance. Lipid peroxidation as well as cell membrane injury remained very minimal in all the transgenic events indicating reduced oxidative damage. Transgenic events exhibited significantly higher plant water status, gas exchange parameters, chlorophyll, carotenoid, and proline content, total soluble sugars, SOD and POD activity compared to wild type (WT) under salinity stress. Histological studies by taking the cross section showed a highly stable root system in transgenic events upon exposure to salinity stress. Results of the present study indicate that transgenic sugarcane events overexpressing EaGly III performed well and exhibited improved salinity stress tolerance.

Differential expression of flavonoid biosynthesis genes and biochemical composition in different tissues of pigmented and non-pigmented rice.

Ever since the flavonoids and other antioxidants in rice were demonstrated with immense health benefits, much interest has been diverted to study the native indigenous rice landraces. In the present investigation, three pigmented rare Indian rice landraces and two non-pigmented rice varieties were analyzed for their phytoconstituents like total phenolic content (TPC), total flavonoid content (TFC), total anthocyanin content and antioxidant potential using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. The phytochemical profile was compared between seed, seedling, stem, leaf and root tissues of the different varieties. Pigmented rice cultivars demonstrated higher levels of bioactive compounds as compared to the non-pigmented cultivars exhibiting a strong correlation between the antioxidant activity of the grain and their phenolic and flavonoid content. Among the five different rice studied, black navara was found to be superior in terms of its phytochemical composition. Further, the expression levels of flavonoid genes among pigmented and non-pigmented rice was studied. Higher gene expression profiles were observed in the 8 flavonoid genes studied in pigmented rice varieties as compared to non-pigmented varieties. The colorlessness of non-pigmented rice and its lower levels of phytoconstituents correlated with the low transcript levels of flavonoid genes recorded in them. This study provides a basis for understanding the health-promoting properties of pigmented rice over non-pigmented rice at the biochemical and molecular levels.

Ectopic expression of DJ-1/PfpI domain containing Erianthus arundinaceus Glyoxalase III (EaGly III) enhances drought tolerance in sugarcane.

KEY MESSAGE: Sugarcane transgenic overexpressing EaGly III from Erianthus arundinaceus showed enhanced water deficit stress tolerance. Methylglyoxal (MG), an α-ketoaldehyde formed from either glycolysis or TCA cycle, is capable of causing total cellular damage via the generation of reactive oxygen species (ROS), advanced glycation end products (AGEs) and nucleic acid degradation. Glyoxalase pathway is a ubiquitous pathway known for detoxification of MG, involving key enzymes glyoxalase I (Gly I) and glyoxalase II (Gly II). Recently, a novel and an additional enzyme in glyoxalase pathway, viz., glyoxalase III (Gly III), has been discovered which possesses DJ-1/PfpI domain recognized for detoxifying MG in a single step process without requirement of any coenzyme. In the present study, a Gly III gene isolated from Erianthus arundinaceus, a wild relative of sugarcane, overexpressed in commercially cultivated sugarcane hybrid Co 86032 was assessed for drought tolerance. Morphometric observations revealed that transgenic sugarcane overexpressing EaGly III acquired drought tolerance trait. Oxidative damage caused by triggering generation of ROS has been determined to be low in transgenic plants as compared to wild type (WT). Transgenics resulted in higher relative water content, chlorophyll content, gas exchange parameters, photosynthetic efficiency, proline content and soluble sugars upon water deficit stress. In addition, higher and stable level of superoxide dismutase and peroxidase activities were observed along with minimal lipid peroxidation during drought stress signifying the tolerance mechanism exhibited by transgenic events. There was no significant structural change observed in the root anatomy of transgenic plants. Altogether, EaGly III gene could be considered as a potential candidate for conferring water deficit stress tolerance for sugarcane and other agricultural crops.

DNA barcoding detects contamination and substitution in North American herbal products.

BACKGROUND: Herbal products available to consumers in the marketplace may be contaminated or substituted with alternative plant species and fillers that are not listed on the labels. According to the World Health Organization, the adulteration of herbal products is a threat to consumer safety. Our research aimed to investigate herbal product integrity and authenticity with the goal of protecting consumers from health risks associated with product substitution and contamination. METHODS: We used DNA barcoding to conduct a blind test of the authenticity for (i) 44 herbal products representing 12 companies and 30 different species of herbs, and (ii) 50 leaf samples collected from 42 herbal species. Our laboratory also assembled the first standard reference material (SRM) herbal barcode library from 100 herbal species of known provenance that were used to identify the unknown herbal products and leaf samples. RESULTS: We recovered DNA barcodes from most herbal products (91%) and all leaf samples (100%), with 95% species resolution using a tiered approach (rbcL + ITS2). Most (59%) of the products tested contained DNA barcodes from plant species not listed on the labels. Although we were able to authenticate almost half (48%) of the products, one-third of these also contained contaminants and or fillers not listed on the label. Product substitution occurred in 30/44 of the products tested and only 2/12 companies had products without any substitution, contamination or fillers. Some of the contaminants we found pose serious health risks to consumers. CONCLUSIONS: Most of the herbal products tested were of poor quality, including considerable product substitution, contamination and use of fillers. These activities dilute the effectiveness of otherwise useful remedies, lowering the perceived value of all related products because of a lack of consumer confidence in them. We suggest that the herbal industry should embrace DNA barcoding for authenticating herbal products through testing of raw materials used in manufacturing products. The use of an SRM DNA herbal barcode library for testing bulk materials could provide a method for 'best practices? in the manufacturing of herbal products. This would provide consumers with safe, high quality herbal products.

Realising the value of plant molecular pharming to benefit the poor in developing countries and emerging economies.

Molecular Pharming, the production of recombinant pharmaceuticals through plant biotechnology, has the potential to transform the biologics sector of the pharmaceutical industry. More fascinating however, is how it might be used to improve access to modern medicines, and improve health of the poor in developing countries and emerging economies. Although improving global health through molecular pharming has been discussed for at least two decades, little progress has actually been made. In this manuscript, a four point plan is described to maximise the opportunity for molecular pharming to provide solutions. These are (i) to identify and prioritise important drug targets that are relevant to the poor; (ii) to support research and development partners in low to middle income countries to develop local expertise, transfer technology and build capacity; (iii) to increase collaboration between regulatory bodies to enable national regulatory frameworks to be developed in low to middle income countries; and (iv) to promote intellectual property management approaches that include socially responsible licensing. An existing case study is described to illustrate how this might be achieved.

Functional role of microRNA in the regulation of biotic and abiotic stress in agronomic plants.

The increasing demand for food is the result of an increasing population. It is crucial to enhance crop yield for sustainable production. Recently, microRNAs (miRNAs) have gained importance because of their involvement in crop productivity by regulating gene transcription in numerous biological processes, such as growth, development and abiotic and biotic stresses. miRNAs are small, non-coding RNA involved in numerous other biological functions in a plant that range from genomic integrity, metabolism, growth, and development to environmental stress response, which collectively influence the agronomic traits of the crop species. Additionally, miRNA families associated with various agronomic properties are conserved across diverse plant species. The miRNA adaptive responses enhance the plants to survive environmental stresses, such as drought, salinity, cold, and heat conditions, as well as biotic stresses, such as pathogens and insect pests. Thus, understanding the detailed mechanism of the potential response of miRNAs during stress response is necessary to promote the agronomic traits of crops. In this review, we updated the details of the functional aspects of miRNAs as potential regulators of various stress-related responses in agronomic plants.

Isolation of 5' regulatory region of COLD1 gene and its functional characterization through transient expression analysis in tobacco and sugarcane.

Chilling Tolerant Divergence 1 (COLD1) gene consists of Golgi pH Receptor (GPHR) as well as Abscisic Acid-linked G Protein-Coupled Receptor (ABA_GPCR), which are the major transmembrane proteins in plants. This gene expression has been found to be differentially regulated, under various stress conditions, in wild Saccharum-related genera, Erianthus arundinaceus, compared to commercial sugarcane variety. In this study, Rapid Amplification of Genomic Ends (RAGE) technique was employed to isolate the 5' upstream region of COLD1 gene to gain knowledge about the underlying stress regulatory mechanism. The current study established the cis-acting elements, main promoter regions, and Transcriptional Start Site (TSS) present within the isolated 5' upstream region (Cold1P) of COLD1, with the help of specific bioinformatics techniques. Phylogenetic analysis results revealed that the isolated Cold1P promoter is closely related to the species, Sorghum bicolor. Cold1P promoter-GUS gene construct was generated in pCAMBIA 1305.1 vector that displayed a constitutive expression of the GUS reporter gene in both monocot as well as dicot plants. The histochemical GUS assay outcomes confirmed that Cold1P can drive expression in both monocot as well as dicot plants. Cold1P's activities under several abiotic stresses such as cold, heat, salt, and drought, revealed its differential expression profile in commercial sugarcane variety. The highest activity of the GUS gene was found after 24 h of cold stress, driven by the isolated Cold1P promoter. The outcomes from GUS fluorimetric assay correlated with that of the GUS expression findings. This is the first report on Cold1P isolated from the species, E. arundinaceus. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03650-8.

Trends in the Epidemiology and Outcomes of Pneumocystis Pneumonia among Human Immunodeficiency Virus (HIV) Hospitalizations.

Introduction: Pneumocystis Pneumonia (PCP) is a common opportunistic infection among people living with the human immunodeficiency virus (HIV). This study's objective was to assess temporal trends in PCP epidemiology among hospitalized patients with HIV/AIDS in the US and to compare data for hospitalizations with HIV with PCP to those without PCP. Methods: The national inpatient sample (NIS) data were analyzed from 2002−2014. The discharge coding identified hospitalized patients with HIV or AIDS and with or without PCP. Results: We identified 3,011,725 hospitalizations with HIV/AIDS during the study period; PCP was present in 5% of the patients with a diagnosis of HIV. The rates of PCP progressively declined from 6.7% in 2002 to 3.5 % in 2014 (p < 0.001). Overall mortality in patients with HIV was 3.3% and was significantly higher in those with PCP than without PCP (9.9% vs. 2.9%; p < 0.001). After adjusting for demographics and other comorbidities, PCP had higher odds of hospital mortality 3.082 (OR 3.082; 95% CI, 3.007 to 3.159; p < 0.001). Conclusion: From 2002 to 2014, the rate of PCP in HIV patients has decreased significantly in the United States but is associated with substantially higher mortality.

Green remediation potential of immobilized oxidoreductases to treat halo-organic pollutants persist in wastewater and soil matrices - A way forward.

The alarming presence of hazardous halo-organic pollutants in wastewater and soils generated by industrial growth, pharmaceutical and agricultural activities is a major environmental concern that has drawn the attention of scientists. Unfortunately, the application of conventional technologies within hazardous materials remediation processes has radically failed due to their high cost and ineffectiveness. Consequently, the design of innovative and sustainable techniques to remove halo-organic contaminants from wastewater and soils is crucial. Altogether, these aspects have led to the search for safe and efficient alternatives for the treatment of contaminated matrices. In fact, over the last decades, the efficacy of immobilized oxidoreductases has been explored to achieve the removal of halo-organic pollutants from diverse tainted media. Several reports have indicated that these enzymatic constructs possess unique properties, such as high removal rates, improved stability, and excellent reusability, making them promising candidates for green remediation processes. Hence, in this current review, we present an insight of green remediation approaches based on the use of immobilized constructs of phenoloxidases (e.g., laccase and tyrosinase) and peroxidases (e.g., horseradish peroxidase, chloroperoxidase, and manganese peroxidase) for sustainable decontamination of wastewater and soil matrices from halo-organic pollutants, including 2,4-dichlorophenol, 4-chlorophenol, diclofenac, 2-chlorophenol, 2,4,6-trichlorophenol, among others.

Analytical and regulatory considerations to mitigate highly hazardous toxins from environmental matrices.

The ubiquitous occurrence, toxicological influence, and bioaccumulation of toxic entities, e.g., pesticides and toxic elements in the environment, biota, and humans, directly or indirectly, are posing severe social, ecological, and human health concerns. Much attention has been given to the rising bioaccumulation of toxins and their adverse impact on various environmental matrices. For example, the inappropriate and exacerbated use of xenobiotics and related hazardous substances have caused the deterioration of the agricultural environment, e.g., fertile soils where plants are grown. Moreover, the harmful toxins have negatively impacted human health through the trophic chains. However, the analytical and regulatory considerations to effectively monitor and mitigate any or many pesticides and toxic elements from environmental matrices are still lacking in the existing literature. For decades, the scientific community has overseen the consequences caused by pollutants, however, the improvement of analytical detection methods and regulatory considerations are not yet fully covered. This review covers the notable literature gap by stressing the development and deployment of robust analytical and regulatory considerations for an efficient abatement of hazardous substances. Following detailed information on occurrence, toxicological influence, and bioaccumulation of pesticides and toxic elements, the most relevant analytical detection tools and regulatory measures are given herein, with suitable examples, to mitigate or reduce the damage caused by these pollutants.

Production of Genistein in Amaranthus tricolor var. tristis and Spinacia oleracea by Expression of Glycine max Isoflavone Synthase.

Isoflavonoids, the diverse group of secondary metabolites derived from the phenylpropanoid pathway, are distributed predominantly in leguminous plants. It has received considerable attention in recent days due to its health promoting benefits and is known to prevent certain diseases in humans. These isoflavonoids are synthesized from flavonoid intermediates of phenylpropanoid pathway by the enzyme isoflavone synthase. Metabolic engineering of isoflavonoid biosynthesis in non-legume crop plants could offer the health benefits of these compounds in diverse plant species further contributing for crop improvement. The transient expression of heterologous genes in the host is considered as an alternative to stable expression, that can provide a rapid way of studying the pathway engineering for metabolite production and could also act as a production platform for nutraceuticals and biopharmaceuticals. In this study, isoflavone genistein was produced in Amaranthus tricolor var. tristis and Spinacia oleracea by transiently expressing Glycine max isoflavone synthase (GmIFS). The GmIFS gene was cloned in plant expression vector pEarleyGate 102 HA and pEAQ-HT-DEST 3 and transformed into plants by agroinfiltration. The presence of transgene in the agroinfiltrated leaves was confirmed by semiquantitative reverse-transcription polymerase chain reaction. The flavonoid substrate naringenin and isoflavonoid genistein were quantified using high performance liquid chromatography in both wild-type and infiltrated leaf samples of both the plants. The naringenin content varied in the range of 65.5-338.5 nM/g fresh weight, while the accumulation of genistein was observed with varying concentrations from 113 to 182.6 nM/g fresh weight in the agroinfiltrated leaf samples of both A. tricolor var. tristis and S. oleracea. These results indicate that the transient expression of GmIFS gene has led to the synthesis of isoflavonoid genistein in A. tricolor var. tristis and S. oleracea providing an insight that stable expression of this gene could enrich the nutraceutical content in the crop plants. To the best of our knowledge, this is the first report on transient expression of GmIFS gene for the production of genistein in A. tricolor var. tristis and S. oleracea.

Disseminated Nocardia infection-A rare presentation.

Nocardia infection is rare and requires early diagnosis, extended use of sulfonamides for good prognosis, and prevention of recurrence. It is crucial to suspect nocardiosis in cases of atypical pneumonias or unexplained visceral organ dysfunction.

A Unique Case of Spontaneous Pneumomediastinum in a Patient With COVID-19 and Influenza Coinfection.

Spontaneous pneumomediastinum is reported in patients with coronavirus disease-2019 (COVID-19) and influenza infection independently, usually associated with noninvasive and mechanical ventilation. We report a case of spontaneous pneumomediastinum in a patient with COVID-19 and influenza coinfection. A 58-year-old male admitted with shortness of breath, diagnosed with COVID-19 and influenza infection. A computed tomography angiogram showed pneumomediastinum. He was treated conservatively with 15 L of oxygen, remdesivir, convalescent plasma, and oseltamivir. The case is being reported for its uniqueness since this is the first documented case of spontaneous pneumomediastinum in COVID-19 and influenza coinfection.

Isolated Radial Vein Thrombosis: Upper Extremity Deep Vein Thrombosis in a Patient With COVID-19 Infection.

In general, upper extremity deep vein thrombosis (DVT) is less common than lower extremity DVT. Among upper extremity DVT cases, most of them are due to secondary causes like indwelling catheters, cancer, surgery, trauma or immobilization by plaster casts, pregnancy, oral contraceptives, and estrogen. Patients with coronavirus disease 2019 (COVID-19) infection are known to have coagulation dysfunction and a high incidence of DVT, mostly in the lower extremities; however, upper extremity DVT has been rarely reported. We present a rare case of upper extremity DVT in COVID-19 infection. A 56-year-old male with no significant past medical history was admitted with acute respiratory failure due to COVID-19 pneumonia. During hospitalization, he developed right upper extremity swelling, and an ultrasonogram showed right radial vein thrombosis. He was initially started on low molecular weight heparin (LMWH) and was discharged on apixaban. Patients with COVID-19 infection who develop DVT are recommended treatment with a direct oral anticoagulant (DOAC) for three months.

Clostridium Difficile and Noncirrhotic Hyperammonnemia in a Patient With COVID-19 Infection.

Clostridium difficile is a bacterial infection that usually presents with diarrhea and is mostly associated with previous antibiotics use. Patients with coronavirus disease 2019 (COVID-19) generally have respiratory symptoms but can also present with diarrhea. Noncirrhotic hyperammonemia is an infrequent presentation and is treated with lactulose. We report the case of a 40-year-old male who was admitted to our hospital with abdominal pain, diarrhea, shortness of breath, and confusion. During hospitalization, the patient tested positive for COVID-19 and C. difficile, and oral vancomycin was administered. His kidney functions improved, but he remained confused. His ammonia levels were elevated, and he was not treated with lactulose due to ongoing diarrhea secondary to C. difficile infection.

Characterization of microRNAs from neem (Azadirachta indica) and their tissue-specific expression study in leaves and stem.

Neem (Azadirachta indica) is a very popular traditional medicinal plant used since ancient times to treat numerous ailments. MicroRNAs (miRNAs) are highly conserved, non-coding, short RNA molecules that play important regulatory roles in plant development and metabolism. In this study, deploying a high stringent genome-wide computational-based approach and following a set of strict filtering norms a total of 44 potential conserved neem miRNAs belonging to 21 families and their corresponding 48 potential target transcripts were identified. Important targets include Squamosa promoter binding protein-like proteins, NAC, Scarecrow proteins, Auxin response factor, and F-box proteins. A biological network has also been developed to understand the miRNA-mediated gene regulation using the minimum free energy (MFE) values of the miRNA-target interaction. Moreover, six selected miRNAs were reported to be involved in secondary metabolism in other plant species (miR156a, miR156l, miR160, miR164, miR171, miR395) were validated by qPCR and their tissue-specific differential expression pattern was observed in leaves and stem. Except for ain-miR395, all the other miRNAs were found overexpressed in the stem as compared to leaves. To the best of our knowledge, this is the first report of neem miRNAs and we believe the finding of the present study will be useful for the functional genomic study of medicinal plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02839-z.

Identification of microRNAs from Medicinal Plant Murraya koenigii by High-Throughput Sequencing and Their Functional Implications in Secondary Metabolite Biosynthesis.

MicroRNAs (miRNAs) are small noncoding RNA molecules that play crucial post-transcriptional regulatory roles in plants, including development and stress-response signaling. However, information about their involvement in secondary metabolism is still limited. Murraya koenigii is a popular medicinal plant, better known as curry leaves, that possesses pharmaceutically active secondary metabolites. The present study utilized high-throughput sequencing technology to investigate the miRNA profile of M. koenigii and their association with secondary metabolite biosynthesis. A total of 343,505 unique reads with lengths ranging from 16 to 40 nt were obtained from the sequencing data, among which 142 miRNAs were identified as conserved and 7 as novel miRNAs. Moreover, 6078 corresponding potential target genes of M. koenigii miRNAs were recognized in this study. Interestingly, several conserved and novel miRNAs of M. koenigii were found to target key enzymes of the terpenoid backbone and the flavonoid biosynthesis pathways. Furthermore, to validate the sequencing results, the relative expression of eight randomly selected miRNAs was determined by qPCR. To the best of our knowledge, this is the first report of the M. koenigii miRNA profile that may provide useful information for further elucidation of the involvement of miRNAs in secondary metabolism. These findings might be crucial in the future to generate artificial-miRNA-based, genetically engineered M. koenigii plants for the overproduction of medicinally highly valuable secondary metabolites.

The Effects of Warfarin and Direct Oral Anticoagulants on Systemic Vascular Calcification: A Review.

Warfarin has been utilized for decades as an effective anticoagulant in patients with a history of strong risk factors for venous thromboembolism (VTE). Established adverse effects include bleeding, skin necrosis, teratogenicity during pregnancy, cholesterol embolization, and nephropathy. One of the lesser-known long-term side effects of warfarin is an increase in systemic arterial calcification. This is significant due to the association between vascular calcification and cardiovascular morbidity and mortality. Direct oral anticoagulants (DOACs) have gained prominence in recent years, as they require less frequent monitoring and have a superior side effect profile to warfarin, specifically in relation to major bleeding. The cost and lack of data for DOACs in some disease processes have precluded universal use. Within the last four years, retrospective cohort studies, observational studies, and randomized trials have shown, through different imaging modalities, that multiple DOACs are associated with slower progression of vascular calcification than warfarin. This review highlights the pathophysiology and mechanisms behind vascular calcification due to warfarin and compares the effect of warfarin and DOACs on systemic vasculature.