Short tandem repeat polymorphism in the promoter region of cyclophilin 19B drives its transcriptional upregulation and contributes to drug resistance in the malaria parasite Plasmodium falciparum.

Resistance of the human malaria parasites, Plasmodium falciparum, to artemisinins is now fully established in Southeast Asia and is gradually emerging in Sub-Saharan Africa. Although nonsynonymous SNPs in the pfk13 Kelch-repeat propeller (KREP) domain are clearly associated with artemisinin resistance, their functional relevance requires cooperation with other genetic factors/alterations of the P. falciparum genome, collectively referred to as genetic background. Here we provide experimental evidence that P. falciparum cyclophilin 19B (PfCYP19B) may represent one putative factor in this genetic background, contributing to artemisinin resistance via its increased expression. We show that overexpression of PfCYP19B in vitro drives limited but significant resistance to not only artemisinin but also piperaquine, an important partner drug in artemisinin-based combination therapies. We showed that PfCYP19B acts as a negative regulator of the integrated stress response (ISR) pathway by modulating levels of phosphorylated eIF2α (eIF2α-P). Curiously, artemisinin and piperaquine affect eIF2α-P in an inverse direction that in both cases can be modulated by PfCYP19B towards resistance. Here we also provide evidence that the upregulation of PfCYP19B in the drug-resistant parasites appears to be maintained by a short tandem repeat (SRT) sequence polymorphism in the gene's promoter region. These results support a model that artemisinin (and other drugs) resistance mechanisms are complex genetic traits being contributed to by altered expression of multiple genes driven by genetic polymorphism at their promoter regions.

A linkage and exome study implicates rare variants of KANK4 and CAP2 in bipolar disorder in a multiplex family.

OBJECTIVES: Bipolar disorder (BD) is a neuropsychiatric disorder with a complex pattern of inheritance. Although many genetic studies have been conducted on BD, its genetic correlates remain uncertain. This study was aimed at  identifying the genetic underpinnings of the disorder in an Indian family, which has been under comprehensive clinical evaluation and follow-up for over 12 years. METHODS: We analysed a four-generation family with several of its members diagnosed for BD employing a combination of genetic linkage and exome analysis. RESULTS: We obtained suggestive LOD score for a chromosome 1 and a chromosome 6 marker (D1S410; LOD = 3.01, Ó¨ = 0; and D6S289; LOD = 1.58, Ó¨ = 0). Manual haplotyping of the regions encompassing these two markers helped delimit a critical genomic interval of 32.44 Mb (D1S2700-D1S435; chromosome 1p31.1-13.2) and another of 10.34 Mb (D6S470-D6S422; chromosome 6p22.3-22.2). We examined the exomic sequences corresponding to these two intervals and found rare variants, NM_181712.4: c.2461G>T (p.Asp821Tyr) in KANK4 at 1p31.1-13.2; and NM_006366:c.-93G>A, in the 5' UTR of CAP2 at 6p22.3-22.2. CONCLUSIONS: Our studysuggests involvement of KANK4 or CAP2 or both in BD in this family. Further analysis of these two genes in BD patients and functional evaluation of the allelic variants identified are suggested.

A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage samples.

BACKGROUND: Sequencing technology advancements opened new opportunities to use transcriptomics for studying malaria pathology and epidemiology. Even though in recent years the study of whole parasite transcriptome proved to be essential in understanding parasite biology there is no compiled up-to-date reference protocol for the efficient generation of transcriptome data from growing number of samples. Here, a comprehensive methodology on how to preserve, extract, amplify, and sequence full-length mRNA transcripts from Plasmodium-infected blood samples is presented that can be fully streamlined for high-throughput studies. RESULTS: The utility of various commercially available RNA-preserving reagents in a range of storage conditions was evaluated. Similarly, several RNA extraction protocols were compared and the one most suitable method for the extraction of high-quality total RNA from low-parasitaemia and low-volume blood samples was established. Furthermore, the criteria needed to evaluate the quality and integrity of Plasmodium RNA in the presence of human RNA was updated. Optimization of SMART-seq2 amplification method to better suit AT-rich Plasmodium falciparum RNA samples allowed us to generate high-quality transcriptomes from as little as 10 ng of total RNA and a lower parasitaemia limit of 0.05%. Finally, a modified method for depletion of unwanted human haemoglobin transcripts using in vitro CRISPR-Cas9 treatment was designed, thus improving parasite transcriptome coverage in low parasitaemia samples. To prove the functionality of the pipeline for both laboratory and field strains, the highest  2-hour resolution RNA-seq transcriptome for P. falciparum 3D7 intraerythrocytic life cycle available to  date was generated, and the entire protocol was applied to create the largest transcriptome data from Southeast Asian field isolates. CONCLUSIONS: Overall, the presented methodology is an inclusive pipeline for generation of good quality transcriptomic data from a diverse range of Plasmodium-infected blood samples with varying parasitaemia and RNA inputs. The flexibility of this pipeline to be adapted to robotic handling will facilitate both small and large-scale future transcriptomic studies in the field of malaria.

Cobalt-Catalyzed Tandem Transformation of 2-Aminobenzonitriles to Quinazolinones Using Hydration and Dehydrogenative Coupling Strategy.

A tandem synthesis of quinazolinones from 2-aminobenzonitriles is demonstrated here by using an aliphatic alcohol-water system. For this transformation, a cheap and easily available cobalt salt and P(CH2CH2PPh2)3 (PP3) ligand were employed. The substrate scope, scalability, and synthesis of natural products exhibited the vitality of this protocol.

Microtubule-associated defects caused by EFHC1 mutations in juvenile myoclonic epilepsy.

Juvenile myoclonic epilepsy (JME) is a common form of epilepsy with a substantial genetic basis to its etiology. While earlier studies have identified EFHC1 as a causative gene for JME, subsequent studies have suggested that ethnicity may play a role in determining expression of the JME phenotype among individuals carrying EFHC1 mutations. Here, we report on our studies on EFHC1 in JME patients from India. We examined the complete structure of the EFHC1 transcript from 480 JME patients and 700 control chromosomes by direct sequencing. Functional correlates of mutations were studied by immunolocalization experiments in cultured mammalian cells and protein homology modeling by in silico methods. Thirteen mutations, of which 11 were previously not known, were identified in 28 JME patients. These mutations accounted for about 6% of the patients examined. Functional studies suggest that these EFHC1 mutations result in microtubule-related abnormalities during cell division. In silico analysis for a subset of mutations suggests that they may affect EFHC1 protein domains, compromising its ability to interact with other proteins. Our observations strengthen the evidence supporting a role for EFHC1 in JME in a population ethnically and geographically distinct from the one in which the gene was initially identified, and broaden the extent of allelic heterogeneity in the gene.

The artemisinin-induced dormant stages of Plasmodium falciparum exhibit hallmarks of cellular quiescence/senescence and drug resilience.

Recrudescent infections with the human malaria parasite, Plasmodium falciparum, presented traditionally the major setback of artemisinin-based monotherapies. Although the introduction of artemisinin combination therapies (ACT) largely solved the problem, the ability of artemisinin to induce dormant parasites still poses an obstacle for current as well as future malaria chemotherapeutics. Here, we use a laboratory model for induction of dormant P. falciparum parasites and characterize their transcriptome, drug sensitivity profile, and cellular ultrastructure. We show that P. falciparum dormancy requires a ~ 5-day maturation process during which the genome-wide gene expression pattern gradually transitions from the ring-like state to a unique form. The transcriptome of the mature dormant stage carries hallmarks of both cellular quiescence and senescence, with downregulation of most cellular functions associated with growth and development and upregulation of selected metabolic functions and DNA repair. Moreover, the P. falciparum dormant stage is considerably more resistant to antimalaria drugs compared to the fast-growing asexual stages. Finally, the irregular cellular ultrastructure further suggests unique properties of this developmental stage of the P. falciparum life cycle that should be taken into consideration by malaria control strategies.

Whole genome resequencing and phenotyping of MAGIC population for high resolution mapping of drought tolerance in chickpea.

Terminal drought is one of the major constraints to crop production in chickpea (Cicer arietinum L.). In order to map drought tolerance related traits at high resolution, we sequenced multi-parent advanced generation intercross (MAGIC) population using whole genome resequencing approach and phenotyped it under drought stress environments for two consecutive years (2013-14 and 2014-15). A total of 52.02 billion clean reads containing 4.67 TB clean data were generated on the 1136 MAGIC lines and eight parental lines. Alignment of clean data on to the reference genome enabled identification of a total, 932,172 of SNPs, 35,973 insertions, and 35,726 deletions among the parental lines. A high-density genetic map was constructed using 57,180 SNPs spanning a map distance of 1606.69 cM. Using compressed mixed linear model, genome-wide association study (GWAS) enabled us to identify 737 markers significantly associated with days to 50% flowering, days to maturity, plant height, 100 seed weight, biomass, and harvest index. In addition to the GWAS approach, an identity-by-descent (IBD)-based mixed model approach was used to map quantitative trait loci (QTLs). The IBD-based mixed model approach detected major QTLs that were comparable to those from the GWAS analysis as well as some exclusive QTLs with smaller effects. The candidate genes like FRIGIDA and CaTIFY4b can be used for enhancing drought tolerance in chickpea. The genomic resources, genetic map, marker-trait associations, and QTLs identified in the study are valuable resources for the chickpea community for developing climate resilient chickpeas.

Stereoselective synthesis of glycosyl azides from anomeric hydroxides via protecting group manipulations.

Herein, we report the direct conversion of anomeric hydroxides to glycosyl azides in one step using diphenylphosphoryl azide. Protecting group manipulations on the hexose sugars have enabled the stereoselective synthesis of either the α-glycosyl azides or the ß-anomeric azides in moderate to very good yields. The reaction has also been successfully used to enable the synthesis of ß-2-deoxy-2-aminoglucosyl azides.

Stochastic expression of invasion genes in Plasmodium falciparum schizonts.

Genetically identical cells are known to exhibit differential phenotypes in the same environmental conditions. These phenotypic variants are linked to transcriptional stochasticity and have been shown to contribute towards adaptive flexibility of a wide range of unicellular organisms. Here, we investigate transcriptional heterogeneity and stochastic gene expression in Plasmodium falciparum by performing the quasilinear multiple annealing and looping based amplification cycles (MALBAC) based amplification and single cell RNA sequencing of blood stage schizonts. Our data reveals significant transcriptional variations in the schizont stage with a distinct group of highly variable invasion gene transcripts being identified. Moreover, the data reflects several diversification processes including putative developmental "checkpoint"; transcriptomically distinct parasite sub-populations and transcriptional switches in variable gene families (var, rifin, phist). Most of these features of transcriptional variability are preserved in isogenic parasite cell populations (albeit with a lesser amplitude) suggesting a role of epigenetic factors in cell-to-cell transcriptional variations in human malaria parasites. Lastly, we apply quantitative RT-PCR and RNA-FISH approach and confirm stochastic expression of key invasion genes, such as, msp1, msp3, msp7, eba181 and ama1 which represent prime candidates for invasion-blocking vaccines.

Artemisinin resistance in the malaria parasite, Plasmodium falciparum, originates from its initial transcriptional response.

The emergence and spread of artemisinin-resistant Plasmodium falciparum, first in the Greater Mekong Subregion (GMS), and now in East Africa, is a major threat to global malaria elimination ambitions. To investigate the artemisinin resistance mechanism, transcriptome analysis was conducted of 577 P. falciparum isolates collected in the GMS between 2016-2018. A specific artemisinin resistance-associated transcriptional profile was identified that involves a broad but discrete set of biological functions related to proteotoxic stress, host cytoplasm remodelling, and REDOX metabolism. The artemisinin resistance-associated transcriptional profile evolved from initial transcriptional responses of susceptible parasites to artemisinin. The genetic basis for this adapted response is likely to be complex.

Short Synthesis of Molnupiravir (EIDD-2801) via a Thionated Uridine Intermediate.

Molnupiravir (MK-4482, EIDD-2801) is an experimental drug that has been demonstrated to be effective for the treatment of COVID-19 in human clinical trials. Herein, we report a concise synthesis of the drug via a novel thionated derivative that relies on one-pot methodologies, thus decreasing the number of purification steps required. This route provides the drug in an overall 62% yield and >99% purity and uses cheap and readily available bulk chemicals, thereby providing an affordable synthesis of the drug for cheaper and wider global accessibility.