Reply to Deora et al. Multiplexing for Plasmodium spp.? Think Again! Comment on "Bhowmick et al. Dry Post Wintertime Mass Surveillance Unearths a Huge Burden of P. vivax, and Mixed Infection with P. vivaxP. falciparum, a Threat to Malaria Elimination, in Dhalai, Tripura, India. Pathogens 2021, 10, 1259".

We thank Deora et al. [...].

Validation of a Mobile Health Technology Platform (FeverTracker) for Malaria Surveillance in India: Development and Usability Study.

BACKGROUND: A surveillance system is the foundation for disease prevention and control. Malaria surveillance is crucial for tracking regional and temporal patterns in disease incidence, assisting in recorded details, timely reporting, and frequency of analysis. OBJECTIVE: In this study, we aim to develop an integrated surveillance graphical app called FeverTracker, which has been designed to assist the community and health care workers in digital surveillance and thereby contribute toward malaria control and elimination. METHODS: FeverTracker uses a geographic information system and is linked to a web app with automated data digitization, SMS text messaging, and advisory instructions, thereby allowing immediate notification of individual cases to district and state health authorities in real time. RESULTS: The use of FeverTracker for malaria surveillance is evident, given the archaic paper-based surveillance tools used currently. The use of the app in 19 tribal villages of the Dhalai district in Tripura, India, assisted in the surveillance of 1880 suspected malaria patients and confirmed malaria infection in 93.4% (114/122; Plasmodium falciparum), 4.9% (6/122; P vivax), and 1.6% (2/122; P falciparum/P vivax mixed infection) of cases. Digital tools such as FeverTracker will be critical in integrating disease surveillance, and they offer instant data digitization for downstream processing. CONCLUSIONS: The use of this technology in health care and research will strengthen the ongoing efforts to eliminate malaria. Moreover, FeverTracker provides a modifiable template for deployment in other disease systems.

Dry Post Wintertime Mass Surveillance Unearths a Huge Burden of P. vivax, and Mixed Infection with P. vivax P. falciparum, a Threat to Malaria Elimination, in Dhalai, Tripura, India.

With India aiming to achieve malaria elimination by 2030, several strategies have been put in place. With that aim, mass surveillance is now being conducted in some malaria-endemic pockets. As dry season mass surveillance has been shown to have its importance in targeting the reservoir, a study was undertaken to assess the parasite load by a sensitive molecular method during one of the mass surveys conducted in the dry winter period. It was executed in two malaria-endemic villages of Dhalai District, Tripura, in northeast India, also reported as P. falciparum predominated area. The present study found an enormous burden of Rapid Diagnostic Test negative malaria cases with P. vivax along with P. vivax and P. falciparum mixed infections during the mass surveillance from febrile and afebrile cases in dry winter months (February 2021-March 2021). Of the total 150 samples tested, 72 (48%) were positive and 78 (52%) negative for malaria by PCR. Out of the 72 positives, 6 (8.33%) were P. falciparum, 40 (55.55%) P. vivax, and 26 (36.11%) mixed infections. Out of 78 malaria negative samples, 6 (7.7%) were with symptoms, while among the total malaria positive, 72 cases 7 (9.8%) were with symptoms, and 65 (90.2%) were asymptomatic. Out of 114 samples tested by both microscopy and PCR, 42 samples turned out to be submicroscopic with 4 P. falciparum, 23 P. vivax, and 15 mixed infections. Although all P. vivax submicroscopic infections were asymptomatic, three P. falciparum cases were found to be febrile. Evidence of malaria transmission was also found in the vectors in the winter month. The study ascertained the use of molecular diagnostic techniques in detecting the actual burden of malaria, especially of P. vivax, in mass surveys. As Jhum cultivators in Tripura are at high risk, screening for the malarial reservoirs in pre-Jhum months can help with malaria control and elimination.

Mitigation of X-ray induced DNA damages and expression of DNA-repair genes by antioxidative Potentilla fulgens root extract and its ethyl-acetate fraction in mammalian cells.

Potentilla fulgens is a medicinal plant in North-East India whose root is reported to have anti-diabetic, anticarcinogenic and antioxidant properties. The potential of hydro-alcoholic extract of P. fulgens root (PRE) for providing protection to mammalian cells exposed to ionising radiation was investigated in this study. The methanolic extract of PRE shows an enhanced radical scavenging ability in a concentration dependent manner. PRE-pre-treatment to stimulated human blood lymphocytes (HBLs) reduced the frequency of deletion and exchange aberrations induced by X-irradiation. Similar protection of chromosome aberrations was also observed in mouse bone marrow cells (BMCs) where mice were given PRE extract (1 mg extract/day/mice) ad libitum in the drinking water for 45 days before whole-body X-irradiation. Of the various extracts prepared by partitioning of the methanol extract, the ethyl-acetate (EA) fraction was found to possess better antioxidant, radical scavenging and DNA-damage reduction activities. PRE-pre-treatment also reduced the radiation-induced cell-cycle delay effectively in HBL. In HEK-293 cells, PRE reduced radiation-induced G2-block in cell kinetics. Interestingly, PRE-treatment alone increased the concentration of endogenous glutathione (GSH) in mouse BMC and in stimulated HBL along with the elevated expression of γ-glutamyl-cysteine synthetase heavy/catalytic subunit, a key determinant of GSH synthesis. Studies on expression of two DNA-repair genes revealed that there was a marked increase in the expression of GADD45 and H2AX genes after X-irradiation in stimulated HBL, and such expression was reduced significantly if PRE-treatment was given prior to radiation. The present findings show the ability of PRE to reduce radiation-induced DNA damages probably by free radical scavenging whereas modulation of expression of DNA-repair genes' and endogenous GSH-increment emerge as effective strategies. The present study is the first report on the selected medicinal plant species that suggests it to be a potential natural radioprotector when used as root extract or its EA fraction for mitigating radiation toxicity.

Bioprospection of anti-inflammatory phytochemicals suggests rutaecarpine and quinine as promising 15-lipoxygenase inhibitors.

15-Lipoxygenase (15-LOX) belongs to the family of nonheme iron containing enzymes that catalyzes the peroxidation of polyunsaturated fatty acids (PUFAs) to generate eicosanoids that play an important role in signaling pathways. The role of 15-LOX has been demonstrated in atherosclerosis as well as other inflammatory diseases. In the present study, drug-like compounds were first screened from a set of anti-inflammatory phytochemicals based on Lipinski's rule of five (ROF) and in silico toxicity filters. Two lead compounds-quinine (QUIN) and rutaecarpine (RUT) were shortlisted by analyzing molecular interactions and binding energies of the filtered compounds with the target using molecular docking. Molecular dynamics simulation studies indicate stable trajectories of apo_15-LOX and docked complexes (15-LOX_QUIN and 15-LOX_RUT). In vitro 15-LOX inhibition studies shows that both QUIN and RUT have lower inhibitory concentration (IC50 ) value than the control (quercetin). Both QUIN and RUT exhibit moderate antioxidant activities. The cell viability study of these compounds suggests no significant toxicity in HEK-293 cell lines. Further, QUIN and RUT both did not show any inhibition against selected Gram-positive and Gram-negative bacterial species. Thus, based on our present findings, rutaecarpine and quinine may be suggested as promising 15-LOX inhibitor for the prevention of the atherosclerosis development.

Secondary metabolite profiling, cytotoxicity, anti-inflammatory potential and in vitro inhibitory activities of Nardostachys jatamansi on key enzymes linked to hyperglycemia, hypertension and cognitive disorders.

ETHNOPHARMACOLOGICAL RELEVANCE: Nardostachys jatamansi (D. Don) DC., 'Spikenard' or 'Jatamansi', a highly valued, aromatic herb from alpine Himalayas has a long history of use as ethnomedicine and dietary supplements in Ayurveda, Unani and Chinese system of medicine since Vedic ages (1000-800 BC). In Ayurveda and traditional system of medicine, the species is used as stimulant, sedative, brain tonic or mind rejuvenator, antidiabetic, cardio tonic, and in the treatment of various neurological disorders such as insomnia, epilepsy, hysteria, anxiety and depression. It is considered as Sattvic herb in Ayurveda and is now commercially marketed either as single or poly-herbal formulations by many companies in national and international markets. AIM OF THE STUDY: The species has become threatened in its natural habitats due to over exploitation and illegal trade of its rhizomes for drug preparation in herbal and pharmaceutical industries. Considering the increasing demand and tremendous medicinal importance of this threatened plant species, a detailed study was undertaken to evaluate its antioxidant potential, secondary metabolite profiling, cytotoxicity, anti-inflammatory potential and in vitro enzyme inhibitory activities on key enzymes linked to hyperglycemia, hypertension and cognitive disorders in different plant parts of wild and in vitro-raised plants with respect to different solvent systems for its sustainable utilization. MATERIALS AND METHODS: Anti-cholinesterase activity of leaves and rhizome of wild and cultured plant extracts was investigated against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes. In vitro anti-hyperglycemic (α-amylase and PTP1B), anti-hypertensive (angiotensin-converting enzyme), anti-tyrosinase and anti-inflammatory potential (5-lipoxygenase and hyaluronidase) of different plant parts of wild and in vitro-raised plants with respect to different solvent systems were also evaluated. In vitro cytotoxic effect of rootstock extracts of wild and in vitro-derived plants were against cancer (HCT-116, MCF-7 and OE33) and two normal (HEK and MEF) cell lines. Secondary metabolite profiling of rhizome segments of wild and in vitro-derived plants was carried out by quantitative gas chromatography-mass spectrometry (GC-MS). RESULTS: In vitro-raised plantlets showed comparative higher yield of various secondary metabolites with a significantly high antioxidant activity as compared to the wild plants. Methanolic rootstock extracts of both wild and in vitro-derived plants of N. jatamansi exhibited significant AChE (IC50 36.46 ±â€¯2.1 and 31.18 ±â€¯2.6 µg/ml, respectively) and BuChE (IC50 64.6 ±â€¯3.5 and 60.12 ±â€¯3.6 µg/ml, respectively) inhibitory potential as compared to standard inhibitor galanthamine (IC50 0.94 ±â€¯0.03 and 4.45 ±â€¯0.5 µg/ml). Methanolic rootstock extract of in vitro-derived plants showed significant α-amylase (IC50 90.69 ±â€¯2.1 µg/ml), PTP1B (IC50 24.56 ±â€¯0.8 µg/ml), angiotensin-converting enzyme (IC50 42.5 ±â€¯3.6 µg/ml) and tyrosinase (IC50 168.12 ±â€¯3.6 µg/ml) inhibitory potential as compared to standard acarbose (IC50 52.36 ±â€¯3.1 µg/ml), ursolic acid (IC50 5.24 ±â€¯0.8 µg/ml), captopril (IC50 32.36 ±â€¯2.5 µg/ml) and kojic acid (IC50 = 54.44 ±â€¯2.3 µg/ml). Both the methanolic rootstock and leaf extracts of tissue culture-derived plants exhibited promising anti-5-LOX and anti-hyaluronidase activities against the known inhibitor of 5-LOX and hyaluronidase. Furthermore, methanolic rootstock extracts of both wild and in vitro-derived plants exhibited promising cytotoxic effects to HCT-116, MCF-7 and OE33 cell lines as compared to the normal HEK and MEF after 12 h of treatment. Secondary metabolite profiling of wild and in vitro-derived plants by quantitative GC-MS analysis revealed the presence of different classes of terpenoids and phenolic acids might be responsible for its effective biological activities. CONCLUSION: In vitro-derived plants revealed a substantial anti-cholinesterases, anti-hyperglycemic anti-inflammatory, anti-hypertensive and anti-tyrosinase potential with higher yield of various bioactive metabolites and significantly higher antioxidant activity which substantially explain medicinal importance of N. jatamansi in traditional medicine, used for centuries in different Ayurvedic formulations. The present findings suggest that cultured plants could be a promising alternative for the production of bioactive metabolites with comparative biological activities to the wild plants.

The N-terminal-truncated recombinant fibrin(ogen)olytic serine protease improves its functional property, demonstrates in vivo anticoagulant and plasma defibrinogenation activity as well as pre-clinical safety in rodent model.

An N-terminal truncated fibrino(geno)lytic serine protease gene encoding a ~42kDa protein from Bacillus cereus strain AB01 was produced by error prone PCR, cloned into pET19b vector, and expressed in E5 coli BL21 DE3 cells. The deletion of 24 amino acid residues from N-terminal of wild-type Bacifrinase improves the catalytic activity of [Bacifrinase (ΔN24)]. The anticoagulant potency of [Bacifrinase (ΔN24)] was comparable to Nattokinase and Warfarin and results showed that its anticoagulant action is contributed by progressive defibrinogenation and antiplatelet activities. Nonetheless, at the tested concentration of 2.0µM [Bacifrinase (ΔN24)] did not show in vitro cytotoxicity or chromosomal aberrations on human embryonic kidney cells-293 (HEK-293) and human peripheral blood lymphocytes (HPBL) cells. [Bacifrinase (ΔN24)], at a dose of 2mg/kg, did not show toxicity, adverse pharmacological effects, tissue necrosis or hemorrhagic effect after 72h of its administration in Swiss albino mice. However, at the tested doses of 0.125 to 0.5mg/kg, it demonstrated significant in anticoagulant effect as well as defibrinogenation after 6h of administration in mice. We propose that [Bacifrinase (ΔN24)] may serve as prototype for the development of potent drug to prevent hyperfibrinogenemia related disorders.

Induction of Apoptosis and Reduction of Endogenous Glutathione Level by the Ethyl-Acetate Soluble Fraction of the Methanol Extract of the Roots of Potentilla fulgens in Cancer Cells.

Potentilla fulgens root traditionally used as a folk remedy in Meghalaya, India. However, systematic evaluation of its anticancer efficacy was limited. We investigated the anticancer potentials of the various extracts prepared by partitioning of the methanol extract of the root with the aim to discover major contributing factors from the most effective fractions. Methanol extract of P. fulgens roots (PRE) was prepared by maceration which was subsequently fractionated into hexane, ethyl-acetate (EA) and n-butanol soluble fractions. Various assays (clonogenic assay, Flow cytometry analysis, western blot, semiquantitative RT-PCR and the level of endogenous glutathione) were used to evaluate different parameters, such as Cell survivability, PARP-1 proteolysis, expression pattern of anti-apoptotic and γ-glutamyl-cysteine synthetase heavy subunit (GCSC) genes in both MCF-7 and U87 cancer cell lines. Since the EA-fraction showed most efficient growth inhibitory effect, it was further purified and a total of nine compounds and some monomeric and dimeric flavan-3-ols were identified and characterized. Three compounds viz., epicatechin (EC), gallic acid (GA) and ursolic acid (UA) were taken on the basis of their higher yield and 10 µg/ml of each was mixed together. The concentration used in this study for PRE, EA- and Hex-fraction was 100 µg/ml, which was higher than the IC50 value. Apoptotic cell death in the PRE, EA-fraction and EC+GA+UA treated cancer cell cultures was significantly greater than in normal cells due to suppression of anti-apoptotic protein Bcl2 following treatment. Depletion of glutathione by downregulating GCSC was also observed. Induction of apoptosis and lowering the level of glutathione are considered to be positive activity for an anticancer agent. Therefore, modulation of GSH concentration in tumor cells by PRE and its EA-fraction opened up the possibility of a new therapeutic approach because these plant products are not harmful to normal cells and may regulate the tumor cellular response to different anticancer treatments. Thus, it would be interesting to examine efficacy of these plant products or EA-fraction in human cancer treatment.

Bio-synthesis of silver nanoparticles using Potentilla fulgens Wall. ex Hook. and its therapeutic evaluation as anticancer and antimicrobial agent.

The present study aims to develop an easy and eco-friendly method for the synthesis of silver nanoparticles using extracts from the medicinal plant, Potentilla fulgens and evaluation of its anticancer and antimicrobial properties. The various parts of P. fulgens were screened and the root extract was found to have the highest potential for the synthesis of nanoparticles. The root extracts were able to quickly reduce Ag(+) to Ag(0) and stabilized the nanoparticles. The synthesis of nanoparticles was confirmed by UV-Visible spectrophotometry and further characterized using Zeta sizer, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscope (TEM) and X-ray diffraction (XRD). Electron microscopic study showed that the size of the nanoparticle was in the range of 10 to 15 nm and spherical in shape. The studies of phytochemical analysis of nanoparticles indicated that the adsorbed components on the surface of nanoparticles were mainly flavonoid in nature. Furthermore, nanoparticles were evaluated as cytotoxic against various cancer cell lines and 0.2 to 12 µg/mL nanoparticles showed good toxicity. The IC50 value of nanoparticles was found to be 4.91 and 8.23 µg/mL against MCF-7 and U-87 cell lines, respectively. Additionally, the apoptotic effect of synthesized nanoparticles on normal and cancer cells was studied using trypan blue assay and flow-cytometric analysis. The results indicate the synthesized nanoparticle ability to kill cancer cells compared to normal cells. The nanoparticles also exhibited comparable antimicrobial activity against both Gram-positive and Gram-negative bacteria.

Two new stereoisomeric antioxidant triterpenes from Potentilla fulgens.

The roots of Potentilla fulgens have been used for a long time as a folk remedy for many ailments without having information on its pharmacological action. Of the various extracts prepared by partitioning of the methanol extract, the ethyl acetate fraction was found to possess better antioxidant and cytotoxic activities. The degree of reduction in cloning efficiencies of MCF-7 cell lines was more with ethyl acetate than with hexane fraction of the root-extract. Hence, this fraction was further purified and nine compounds, including two new ursane type triterpenoids Fulgic acid A (4) and Fulgic acid B (5), were identified and characterized. Other compounds were identified as ursolic acid, euscaphic acid, corosolic acid, epicatechin, catechin, p-hydroxybenzaldehyde and gallic acid. Chemical structures were elucidated by spectroscopic methods, especially ESIHRMS and 2D NMR techniques. The new compounds showed good antioxidant activity and therefore this plant can be a source of natural antioxidants.