Isolation and structure determination of a new analog of polycavernosides from marine Okeania sp. cyanobacterium.

Polycavernoside E (1), a new polycavernoside analog, was isolated from a marine Okeania sp. cyanobacterium. The relative configuration was elucidated primarily by analyzing the two dimensional nuclear magnetism resonance (2D NMR) data. The absolute configuration was clarified by comparing the electronic circular dichroism (ECD) data of 1 with those of known analogs. Polycavernoside E (1) exhibited moderate antitrypanosomal activity against Trypanosoma brucei rhodesiense. Furthermore, the isolation of polycavernoside E (1) from marine cyanobacteria provides additional evidence that marine cyanobacteria, and not red algae, are responsible for the biosynthesis of polycavernosides.

Kagimminols A and B, Cembrene-Type Diterpenes from an Okeania sp. Marine Cyanobacterium.

Kagimminols A (1) and B (2), new cembrene-type diterpenoids, were isolated from an Okeania sp. marine cyanobacterium. By combining DP4 analysis with an efficient NMR chemical shift calculation protocol, we clarified the relative configurations of 1 and 2 without consuming precious natural products. We determined the absolute configurations by a comparison of theoretical electronic circular dichroism (ECD) spectra with experimental spectra, and the absolute configuration of 1 was verified experimentally. Finally, we found that 1 and 2 showed selective growth-inhibitory activity against the causative agent of human African trypanosomiasis. This study exemplifies that computational chemistry is an efficient tool for clarifying the configurations of natural products possessing tautomers in equilibrium.

Isolation and Structure Determination of Akunolides, Macrolide Glycosides from a Marine Okeania sp. Cyanobacterium.

Akunolides A (1), B (2), C (3), and D (4), new macrolide glycosides, were isolated from a marine Okeania sp. cyanobacterium. Their structures were elucidated by spectroscopic analyses and derivatization reactions. Akunolides A-D (1-4) are classified as 16-membered macrolide glycosides, which are relatively rare structures for marine cyanobacterium-derived natural products. Akunolides A-D (1-4) showed moderate antitrypanosomal activities against Trypanosoma brucei rhodesiense, with IC50 values ranging from 11 to 14 µM. Furthermore, akunolides A (1) and C (3) exhibited no cytotoxicity against normal human WI-38 cells even at a concentration of 150 µM.

Glycerol biosynthetic pathway plays an essential role in proliferation and antioxidative defense in the human enteric protozoan parasite Entamoeba histolytica.

Amebiasis is caused by the protozoan parasite Entamoeba histolytica. Treatment options other than metronidazole and its derivatives are few, and their low efficacy against asymptomatic cyst carriers, and experimental evidence of resistance in vitro justify the discovery/repurposing campaign for new drugs against amebiasis. Global metabolic responses to oxidative stress and cysteine deprivation by E. histolytica revealed glycerol metabolism may represent a rational target for drug development. In this study using 14C-labelled glucose, only 11% of the total glucose taken up by E. histolytica trophozoites is incorporated to lipids. To better understand the role of glycerol metabolism in this parasite, we focused on characterizing two important enzymes, glycerol kinase (GK) and glycerol 3-phosphate dehydrogenase (G3PDH). Recombinant GK was biochemically characterized in detail, while G3PDH was not due to failure of protein expression and purification. GK revealed novel characteristics and unprecedented kinetic properties in reverse reaction. Gene silencing revealed that GK is essential for optimum growth, whereas G3PDH is not. Gene silencing of G3PDH caused upregulated GK expression, while that of GK resulted in upregulation of antioxidant enzymes as shown by RNA-seq analysis. Although the precise molecular link between GK and the upregulation of antioxidant enzymes was not demonstrated, the observed increase in antioxidant enzyme expression upon GK gene silencing suggests a potential connection between GK and the cellular response to oxidative stress. Together, these results provide the first direct evidence of the biological importance and coordinated regulation of the glycerol metabolic pathways for proliferation and antioxidative defense in E. histolytica, justifying the exploitation of these enzymes as future drug targets.

Molecular identification of phlebotomine sand flies and the harbored Leishmania spp. in Sokoto State, Nigeria.

Introduction: Female sand flies are hematophagous, feeding on animals and in the process serve as vectors for Leishmania, the parasites that cause leishmaniasis in humans. Leishmaniasis are a group of parasitic neglected tropical diseases in 98 countries including Nigeria and kills ~60,000 people/year. In Nigeria, Sokoto State is endemic to leishmaniasis but there is a knowledge gap on the identity of the prevalent sand flies and the Leishmania species they transmit. Hence, this cross-sectional study was designed to take inventory of the species of sand flies in Sokoto using genetic methods. Methods: 1,260 (310 females) sand flies were collected from three Local Government Areas (L.G.A) of Sokoto State- Wamakko, Sokoto South and Kware. Genomic DNA was extracted from each fly and DNA amplification by polymerase chain reaction (PCR) was carried out on the DNA samples using primers targeting the arthropods mitochondrial cytochrome oxidase subunit 1 (mt-coI) gene, and nested PCR with primers targeting the gene for Leishmania internal transcribed spacer-1 (its-1) of ribosomal RNA its-1rRNA. The PCR products were sequenced. Results: Gene sequence analysis revealed five species of sand flies belonging to the old-world genera namely Phlebotomus and Sergentomyia. The identified species were P. papatasi (6.45%), S. adleri (6.45%), S. affinis (9.7%), S. distincta (9.7%), S. schwetzi (67.7%). Within the sampling period, sand flies were most abundant in the rainy months of August (104/33.5%) and September (116/37.4%) with all the five identified species occurring. Sequence analysis of its-1 gene identified Leishmania infantum in two sand flies (2/310)- P. papatasi (from Sokoto South) and S. affinis (from Wamakko). BLAST search in NCBI and phylogenetic analysis revealed that the sand fly species are related to the species reported in different parts of Africa, while the L. infantum is identical to strain reported in Brazil (KY379083.1). Discussion: Phlebotomus papatasi and four species belonging to the genus Sergentomyia are the most prevalent sand flies in Sokoto State, Nigeria and they harbor L. infantum solely. The results shed light on why visceral leishmaniasis is the most predominant form of the disease. Therefore, we recommend that adequate care for dogs must be instituted as dogs are the major animal reservoir for L. infantum.

Spatial and meteorological controls of stable water isotope dynamics of precipitation in Kashmir Valley, Western Himalaya, India.

In the Himalayas, the lives and livelihoods of millions of people are sustained by water resources primarily depending on the moisture brought by Western Disturbances and Indian Summer Monsoon. In the present study, a network of 12 precipitation stations was established across the Kashmir Valley to understand the spatial and meteorological factors controlling precipitation isotopes. Temperature and relative humidity are dominant meteorological factors, whereas altitude, proximity to forest canopy, land use/land cover, windward and leeward sides of the mountains are the main physical factors influencing precipitation isotopes. The study suggests that the Mediterranean Sea and nearby water bodies along with continental recycling are the dominant sources of moisture from October to May, while the Arabian Sea, Bay of Bengal and continental recycling are the main sources of moisture from June to September. However, some precipitation events from October to May collect moisture from the Arabian Sea and some precipitation events from June to September collect moisture from the Mediterranean Sea. The occasional passage of Western Disturbances in summer merging with the Indian Summer Monsoon yields heavy to very heavy precipitation. The study provides a better understanding of complex spatial and meteorological phenomena controlling precipitation isotopes across the Western Himalayas.

Isolation of Hennaminal and Isolation and Total Synthesis of Hennamide, Pyrrolinone Compounds from the Marine Cyanobacterium Rivularia sp.

Two new natural products were isolated from the marine cyanobacterium Rivularia sp. collected in Japan. Hennaminal possesses a very rare functional group, ß,ß-diamino unsaturated ketone, which has only been found in bohemamine-type natural products so far. Hennamide possesses a reactive N-acyl pyrrolinone moiety, which induces self-dimerization. The isolation and structure determination supported by computational chemistry and total synthesis, as well as the antitrypanosomal activities of hennaminal and hennamide are described.

Attenuation of In Vitro and In Vivo Virulence Is Associated with Repression of Gene Expression of AIG1 Gene in Entamoeba histolytica.

Entamoeba histolytica virulence results from complex host-parasite interactions implicating multiple amoebic components (e.g., Gal/GalNAc lectin, cysteine proteinases, and amoebapores) and host factors (microbiota and immune response). UG10 is a strain derived from E. histolytica virulent HM-1:IMSS strain that has lost its virulence in vitro and in vivo as determined by a decrease of hemolytic, cytopathic, and cytotoxic activities, increased susceptibility to human complement, and its inability to form liver abscesses in hamsters. We compared the transcriptome of nonvirulent UG10 and its parental HM-1:IMSS strain. No differences in gene expression of the classical virulence factors were observed. Genes downregulated in the UG10 trophozoites encode for proteins that belong to small GTPases, such as Rab and AIG1. Several protein-coding genes, including iron-sulfur flavoproteins and heat shock protein 70, were also upregulated in UG10. Overexpression of the EhAIG1 gene (EHI_180390) in nonvirulent UG10 trophozoites resulted in augmented virulence in vitro and in vivo. Cocultivation of HM-1:IMSS with E. coli O55 bacteria cells reduced virulence in vitro, and the EhAIG1 gene expression was downregulated. In contrast, virulence was increased in the monoxenic strain UG10, and the EhAIG1 gene expression was upregulated. Therefore, the EhAIG1 gene (EHI_180390) represents a novel virulence determinant in E. histolytica.

Isolation and Total Synthesis of Beru'amide, an Antitrypanosomal Polyketide from a Marine Cyanobacterium Okeania sp.

A 68 µg amount of an acyclic polyketide, named beru'amide, was isolated from a marine cyanobacterium Okeania sp. Beru'amide contains six unique moieties in its relatively small skeleton. By applying several cutting-edge techniques, including DFT-based chemical shift calculations, we achieved the structure determination and the total synthesis of this highly functionalized scarce natural product. Furthermore, beru'amide was shown to have strong antitrypanosomal activity.

PTEN differentially regulates endocytosis, migration, and proliferation in the enteric protozoan parasite Entamoeba histolytica.

PTEN is a lipid phosphatase that is highly conserved and involved in a broad range of biological processes including cytoskeletal reorganization, endocytosis, signal transduction, and cell migration in all eukaryotes. Although regulation of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] signaling via PTEN has been well established in model organisms and mammals, it remains elusive in the parasitic protist E. histolytica, which heavily relies on PtdIns phosphate(s)-dependent membrane traffic, migration, and phago- and trogocytosis for its pathogenesis. In this study, we characterized the major PTEN from E. histolytica, EhPTEN1, which shows the highest expression at the transcript level in the trophozoite stage among 6 possible PTENs, to understand the significance of PtdIns(3,4,5)P3 signaling in this parasite. Live imaging of GFP-EhPTEN1 expressing amebic trophozoites showed localization mainly in the cytosol with a higher concentration at pseudopods and the extending edge of the phago- and trogocytic cups. Furthermore, quantitative analysis of phago- and trogocytosis using a confocal image cytometer showed that overexpression of EhPTEN1 caused reduction in trogo- and phagocytosis while transcriptional gene silencing of EhPTEN1 gene caused opposite phenotypes. These data suggest that EhPTEN1 has an inhibitory role in these biological processes. Conversely, EhPTEN1 acts as a positive regulator for fluid-phase and receptor-mediated endocytosis in E. histolytica trophozoites. Moreover, we showed that EhPTEN1 was required for optimal growth and migration of this parasite. Finally, the phosphatase activity of EhPTEN1 towards PtdIns(3,4,5)P3 was demonstrated, suggesting that the biological roles of EhPTEN1 are likely linked to its catalytic function. Taken together, these results indicate that EhPTEN1 differentially regulates multiple cellular activities essential for proliferation and pathogenesis of the organism, via PtdIns(3,4,5)P3 signaling. Elucidation of biological roles of PTEN and PtdIns(3,4,5)P3 signaling at the molecular levels promotes our understanding of the pathogenesis of this parasite.

Identifying and estimating the sources of river flow in the cold arid desert environment of Upper Indus River Basin (UIRB), western Himalayas.

A reliable water supply in different Himalayan River basins is increasingly important for domestic, agriculture, and hydropower generation. These water resources are under serious threat due to climate change, with the potential to alter the economic stability of 237 million people living in the Indus River Basin alone. In the present study, we used new stable water isotope data set to identify and estimate the different sources of streamflow and their controlling factors in the Upper Indus River Basin (UIRB), India. The data set presented wide spatial and temporal variability without the distinct isotopic signature of various sources of river flow. However, variable but distinct signatures of sources of river/stream flow exist at the sub-basin or catchment scale. These variabilities are ascribed to changing physiographical, meteorological, and local climatic conditions. Further, the distinct microclimatic conditions including altitudinal variability, aspect slope, etc. govern the spatio-temporal variability of sources and streamflow, hence different lapse rates at sub-basin/catchment scale. The study suggested that the contribution of snowmelt and glacier melt to river flow varies spatially and temporally. The Bayesian mixing model results suggested that snowmelt contribution is higher in Indus (63 ± 1.2%) and Shyok (58 ± 1.7%) while as, glacier melt contribution is higher in Nubra 64 ± 2.3% and Suru 60 ± 2.7% sub-basins/catchments. The groundwater contribution (baseflow) sustains and regulates the flow in rivers/streams during winter and spring, which is very vital for the local water supply. The study suggests that the spatially diverse rugged topography and microclimate in UIRB dominantly control the differential contribution from various sources of river flow. The warming climate, which has resulted in a decrease in solid precipitation, continuous glacier mass loss, early melting of snow cover, etc., would have an inconsistent impact on the perennial flow of rivers with the potential to alter the economic and political stability in the region.

Physiological roles and metabolism of γ-aminobutyric acid (GABA) in parasitic protozoa.

γ-aminobutyric acid (GABA) is a nonstructural amino acid that serves diverse functions in unicellular and multicellular organisms. Besides its widely established role in mammals as an inhibitory neurotransmitter, the diverse biological roles and metabolism of GABA in protozoan parasites have begun to be unveiled. GABA acts as either the intracellular signal or cell-to-cell messenger to mediate a variety of cellular responses that protect the parasites from environmental and host-derived stress. Moreover, GABA metabolism was found to be tightly regulated, involving protein machinery confined to the protozoa lineage. Meanwhile, host-parasite GABAergic interaction plays a role in the pathogenesis and disease manifestation of protozoan infections. Therefore, the GABAergic system apparently is broadly involved in essential biological and pathophysiological processes and is well conserved in parasitic and free-living protozoa.

Impact of Indian summer monsoon in westerly dominated water resources of western Himalayas.

We used stable water isotopes of oxygen and hydrogen to identify and estimate the seasonal contribution of precipitation to the regional hydrology of Sindh and Rambiara catchments of western Himalayas. The different source waters exhibit significant spatio-temporal variations that correspond to the change in seasonal meteorology, precipitation form and moisture sources. The two-component hydrograph separation based on d-excess suggests that the western disturbances (WD) contribute dominantly (76 ± 4 %) to the regional hydrology, compared to Indian summer monsoon (ISM) rainfall (24 ± 4 %). A comparison of d-excess values of WD and ISM indicates the groundwater consists of 90 ± 3 % WD sources and 10 ± 2 % ISM sources, signifying distinct seasonal variations in groundwater recharge sources. The sine wave model results showed that the annual mean residence time (MRT) of groundwater for the Sindh catchment (5.8 ± 0.6 months) is greater than the Rambiara groundwater (3.6 ± 0.5 months). The lower isotope values observed in the river water than in the precipitation suggest its origin from the snowmelt. This study provides valuable insights into the hydrological processes operating in the high altitude Himalayan catchments to facilitate the improved understanding of runoff generation mechanisms and water resource management in future climate change scenarios.

First Total Synthesis and Structure-Activity Relationship of Iheyamide A, an Antitrypanosomal Linear Peptide Isolated from a Dapis sp. Marine Cyanobacterium.

Iheyamide A (1) is an antitrypanosomal linear peptide isolated from a Dapis sp. marine cyanobacterium by our group in 2020, and based on structure-activity relationships of its natural analogues, the C-terminal pyrrolinone moiety has been identified as the phamacophore for its antiparasitic activity. Further, we isolated this pyrrolinone moiety by itself as a new natural product from the marine cyanobacterium and named it iheyanone (2). As expected, iheyanone (2) showed antitrypanosomal activity, but its potency was weaker than iheyamide A (1). To clarify more detailed structure-activity relationships, we completed a total synthesis of iheyamide A (1) along with iheyanone (2) and evaluated the antitrypanosomal activities of several synthetic intermediates. As a result, we found that the longer the peptide chain, the stronger the antitrypanosomal activity. As iheyamide A (1) showed selective toxicity against Trypanosoma brucei rhodesiense, these findings can provide design guidelines for antitrypanosomal drugs.

Isolation and Total Synthesis of Bromoiesol sulfates, Antitrypanosomal arylethers from a Salileptolyngbya sp. Marine Cyanobacterium.

Bromoiesol sulfates A (1) and B (2), new polyhalogenated aryl sulfates, were isolated from a Salileptolyngbya sp. marine cyanobacterium along with their hydrolyzed compounds, bromoiesols A (3) and B (4). To pick up the candidates of their structures, we used Small Molecule Accurate Recognition Technology (SMART), an artificial intelligence-based structure-prediction tool, and their structures were elucidated on the basis of single-crystal X-ray diffraction analysis of bromoiesols (3 and 4). In addition, to verify the structures, the total synthesis of bromoiesol A sulfate (1) and bromoiesol A (3) was achieved. The bromoiesol family, especially bromoiesols (3 and 4), selectively inhibited the growth of the bloodstream form of Trypanosoma brucei rhodesiense, the causative agent of human African sleeping sickness.

Isolation and Total Synthesis of Kinenzoline, an Antitrypanosomal Linear Depsipeptide Isolated from a Marine Salileptolyngbya sp. Cyanobacterium.

Kinenzoline (1), a new linear depsipeptide, was isolated from a marine Salileptolyngbya sp. cyanobacterium. Its structure was elucidated by spectroscopic analyses and degradation reactions. In addition, we achieved a total synthesis of 1 and confirmed its structure. Kinenzoline (1) showed highly selective antiproliferative activity against the causative organism of sleeping sickness, Trypanosoma brucei rhodesiense (IC50 4.5 µM), compared to normal human cells (WI-38, IC50 > 100 µM). Kinenzoline (1) is a promising lead compound for the development of new antitrypanosomal drugs.

Arsenic fate in upper Indus river basin (UIRB) aquifers: Controls of hydrochemical processes, provenances and water-aquifer matrix interaction.

Major river basins of the Himalayas contain a significant amount of arsenic (As) in the geological matrix, which tends to contaminate the groundwater at a local and regional scale. Although As enrichment in Quaternary deposits has been linked to primary provenances (Himalayan orogeny), limited studies have reported As enrichment in bedrock aquifers. In the present study, the hard rock and groundwater samples were collected across the upper Indus river basin (UIRB), Ladakh to assess the hydrogeochemical processes and environments responsible for As mobilization and release. The higher As concentrations were found in Khardung volcanics followed by Ophiolitic melange, Dras volcanics, Nindam sandstone, and Nindam Shale. The variability in As concentration among different rock samples is largely governed by the presence of felsic minerals and the type of magmatic setting. The groundwater is less mineralized, with moderate electrical conductivity (EC), and weakly acidic to alkaline in nature. The results indicated that mineral weathering, dissolution, and active cation exchange reactions have a strong influence on the major ion chemistry of the groundwater. Redox-sensitive processes are influencing the As mobilization and release under reducing environmental conditions. As in groundwater poses a serious threat to human health hence awareness is urgent towards achieving sustainable As mitigation globally. The study provided a significant dataset to better understand the processes and environmental conditions responsible for hydrogeochemical evolution, sources of solutes, and As mobilization and release in groundwater which will help in sustainable water resource management policies and ecosystem restoration across the Himalayas.

Motobamide, an Antitrypanosomal Cyclic Peptide from a Leptolyngbya sp. Marine Cyanobacterium.

Motobamide (1), a new cyclic peptide containing a C-prenylated cyclotryptophan residue, was isolated from a marine Leptolyngbya sp. cyanobacterium. Its planar structure was established by spectroscopic and MS/MS analyses. The absolute configuration was elucidated based on a combination of chemical degradations, chiral-phase HPLC analyses, spectroscopic analyses, and computational chemistry. Motobamide (1) moderately inhibited the growth of bloodstream forms of Trypanosoma brucei rhodesiense (IC50 2.3 µM). However, it exhibited a weaker cytotoxicity against normal human cells (IC50 55 µM).

Characterization of Plasmodium falciparum Pantothenate Kinase and Identification of Its Inhibitors From Natural Products.

Coenzyme A (CoA) is a well-known cofactor that plays an essential role in many metabolic reactions in all organisms. In Plasmodium falciparum, the most deadly among Plasmodium species that cause malaria, CoA and its biosynthetic pathway have been proven to be indispensable. The first and rate-limiting reaction in the CoA biosynthetic pathway is catalyzed by two putative pantothenate kinases (PfPanK1 and 2) in this parasite. Here we produced, purified, and biochemically characterized recombinant PfPanK1 for the first time. PfPanK1 showed activity using pantetheine besides pantothenate, as the primary substrate, indicating that CoA biosynthesis in the blood stage of P. falciparum can bypass pantothenate. We further developed a robust and reliable screening system to identify inhibitors using recombinant PfPanK1 and identified four PfPanK inhibitors from natural compounds.