Periampullary tumors in a patient with pancreatic divisum and neurofibromatosis type 1: a case report.

INTRODUCTION: We present a case of a male patient with neurofibromatosis type 1 diagnosed with pancreatic divisum and several gastrointestinal tumors. A 55-year-old man was admitted to the hospital with recurrent chronic pancreatitis, indicating a large mass in the ampulla. In addition, genetic testing revealed two unique germline mutations in the neurofibromin (NF1) gene, and their potential interaction in promoting cancer was further investigated. CONCLUSION: The first similar case was reported in 2020. The current case was distinct from other cases since an additional two NF1 mutations were found in the patient. In conjunction with prior case reports, our findings imply that genetic testing in patients diagnosed with neurofibromatosis type 1 could be helpful in the development of effective treatments.

Centrin-deficient Leishmania mexicana confers protection against Old World visceral leishmaniasis.

Leishmaniasis is one of the top neglected tropical diseases with significant morbidity and mortality in low and middle-income countries (LMIC). However, this disease is also spreading in the developed world. Currently, there is a lack of effective strategies to control this disease. Vaccination can be an effective measure to control leishmaniasis and has the potential to achieve disease elimination. Recently, we have generated centrin gene-deleted new world L. mexicana (LmexCen-/-) parasites using CRISPR/Cas9 and showed that they protect mice against a homologous L. mexicana infection that causes cutaneous disease. In this study, we tested whether LmexCen-/- parasites can also protect against visceral leishmaniasis caused by L. donovani in a hamster model. We showed that immunization with LmexCen-/- parasites is safe and does not cause lesions. Furthermore, such immunization conferred protection against visceral leishmaniasis caused by a needle-initiated L. donovani challenge, as indicated by a significant reduction in the parasite burdens in the spleen and liver as well as reduced mortality. Similar control of parasite burden was also observed against a sand fly mediated L. donovani challenge. Importantly, immunization with LmexCen-/- down-regulated the disease promoting cytokines IL-10 and IL-4 and increased pro-inflammatory cytokine IFN-γ resulting in higher IFN-γ/IL-10 and IFN-γ/IL4 ratios compared to non-immunized animals. LmexCen-/- immunization also resulted in long-lasting protection against L. donovani infection. Taken together, our study demonstrates that immunization with LmexCen-/- parasites is safe and efficacious against the Old World visceral leishmaniasis.

Investigating the Leishmania donovani sacp Gene and Its Role in Macrophage Infection and Survival in Mice.

The protozoan parasite Leishmania donovani is a causative agent of the neglected tropical disease known as visceral leishmaniasis, which can be lethal when untreated. Studying Leishmania viru-lence factors is crucial in determining how the parasite causes disease and identifying new targets for treatment. One potential virulence factor is L. donovani's abundantly secreted protein: secreted acid phosphatase (SAcP). Whole-genome analysis revealed that the sacp gene was present in three copies in wild type L. donovani. Using CRISPR-Cas9 gene editing; we generated a sacp gene knockout termed LdΔSAcP, which demonstrated a loss of both the SAcP protein and an associated reduction in secreted acid phosphatase activity. Genome sequencing confirmed the precise dele-tion of the sacp gene in LdΔSAcP and identified several changes in the genome. LdΔSAcP demonstrated no significant changes in promastigote proliferation or its ability to infect and survive in macrophages compared to the wildtype strain. LdΔSAcP also demonstrated no change in murine liver infection; however, survival was impaired in the spleen. Taken together these results show that SAcP is not necessary for the survival of promastigotes in culture but may support long-term survival in the spleen. These observations also show that the use of CRISPR gene editing and WGS together are effective to investigate the function and phenotype of complex potential drug targets such as multicopy genes.

Reconstitution of Mycobacterium marinum Nonhomologous DNA End Joining Pathway in Leishmania.

In mammalian cells, DNA double-strand breaks (DSBs) are mainly repaired by nonhomologous end joining (NHEJ) pathway. Ku (a heterodimer formed by Ku70 and Ku80 proteins) and DNA ligase IV are the core NHEJ factors. Ku could also be involved in other cellular processes, including telomere length regulation, DNA replication, transcription, and translation control. Leishmania, an early branching eukaryote and the causative agent of leishmaniasis, has no functional NHEJ pathway due to its lack of DNA ligase IV and other NHEJ factors but retains Ku70 and Ku80 proteins. In this study, we generated Leishmania donovani Ku70 disruption mutants and Ku70 and Ku80 double gene (Ku70/80) disruption mutants. We found that Leishmania Ku is still involved in DSB repair, possibly through its binding to DNA ends to block and slowdown 5' end resections and Ku-Ku or other protein interactions. Depending on location of a DSB between the direct repeat genomic sequences, Leishmania Ku could have an inhibiting effect, no effect or a promoting effect on the DSB repair mediated by single strand annealing (SSA), the most frequently used DSB repair pathway in Leishmania. Ku70/80 proteins are also required for the healthy proliferation of Leishmania cells. Interestingly, unlike in Trypanosoma brucei and L. mexicana, Ku70/80 proteins are dispensable for maintaining the normal lengths of telomeres in L. donovani. We also show it is possible to reconstitute the two components (Ku and Ligase D) NHEJ pathway derived from Mycobacterium marinum in Leishmania. This improved DSB repair fidelity and efficiency in Leishmania and sets up an example that the bacterial NHEJ pathway can be successfully reconstructed in an NHEJ-deficient eukaryotic parasite. IMPORTANCE Nonhomologous end joining (NHEJ) is the most efficient double-stranded DNA break (DSB) repair pathway in mammalian cells. In contrast, the protozoan parasite Leishmania has no functional NHEJ pathway but retains the core NHEJ factors of Ku70 and Ku80 proteins. In this study, we found that Leishmania Ku heterodimers are still participating in DSB repair possibly through blocking 5' end resections and Ku-Ku protein interactions. Depending on the DSB location, Ku could have an inhibiting or promoting effect on DSB repair mediated by the single-strand annealing repair pathway. Ku is also required for the normal growth of the parasite but surprisingly dispensable for maintaining the telomere lengths. Further, we show it is possible to introduce Mycobacterium marinum NHEJ pathway into Leishmania. Understanding DSB repair mechanisms of Leishmania may improve the CRISPR gene targeting specificity and efficiency and help identify new drug targets for this important human parasite.

Centrin-deficient Leishmania mexicana confers protection against New World cutaneous leishmaniasis.

Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally with no approved vaccines for human use. New World cutaneous leishmaniasis (CL) caused by L. mexicana is characterized by the development of chronic non-healing skin lesions. Using the CRISPR/Cas9 technique, we have generated live attenuated centrin knockout L. mexicana (LmexCen-/-) parasites. Centrin is a cytoskeletal protein important for cellular division in eukaryotes and, in Leishmania, is required only for intracellular amastigote replication. We have investigated the safety and immunogenicity characteristics of LmexCen-/- parasites by evaluating their survival and the cytokine production in bone-marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) in vitro. Our data shows that LmexCen-/- amastigotes present a growth defect, which results in significantly lower parasitic burdens and increased protective cytokine production in infected BMDMs and BMDCs, compared to the wild type (WT) parasites. We have also determined the safety and efficacy of LmexCen-/- in vivo using experimental murine models of L. mexicana. We demonstrate that LmexCen-/- parasites are safe and do not cause lesions in susceptible mouse models. Immunization with LmexCen-/- is also efficacious against challenge with WT L. mexicana parasites in genetically different BALB/c and C57BL/6 mouse models. Vaccinated mice did not develop cutaneous lesions, displayed protective immunity, and showed significantly lower parasitic burdens at the infection site and draining lymph nodes compared to the control group. Overall, we demonstrate that LmexCen-/- parasites are safe and efficacious against New World cutaneous leishmaniasis in pre-clinical models.

Preclinical validation of a live attenuated dermotropic Leishmania vaccine against vector transmitted fatal visceral leishmaniasis.

Visceral Leishmaniasis (VL), a potentially fatal disease is caused by Leishmania donovani parasites with no vaccine available. Here we produced a dermotropic live attenuated centrin gene deleted Leishmania major (LmCen-/-) vaccine under Good Laboratory Practices and demonstrated that a single intradermal injection confers robust and durable protection against lethal VL transmitted naturally via bites of L. donovani-infected sand flies and prevents mortality. Surprisingly, immunogenicity characteristics of LmCen-/- parasites revealed activation of common immune pathways like L. major wild type parasites. Spleen cells from LmCen-/- immunized and L. donovani challenged hamsters produced significantly higher Th1-associated cytokines including IFN-γ, TNF-α, and reduced expression of the anti-inflammatory cytokines like IL-10, IL-21, compared to non-immunized challenged animals. PBMCs, isolated from healthy people from non-endemic region, upon LmCen-/- infection also induced more IFN-γ compared to IL-10, consistent with our immunogenicity data in LmCen-/- immunized hamsters. This study demonstrates that the LmCen-/- parasites are safe and efficacious against VL and is a strong candidate vaccine to be tested in a human clinical trial.

Evidence that a naturally occurring single nucleotide polymorphism in the RagC gene of Leishmania donovani contributes to reduced virulence.

Leishmaniasis is a widespread neglected tropical disease transmitted by infected sand flies resulting in either benign cutaneous infection or fatal visceral disease. Leishmania donovani is the principal species responsible for visceral leishmaniasis, yet an atypical L. donovani has become attenuated in several countries including Sri Lanka and causes cutaneous leishmaniasis. Previous studies have identified 91 genes altered in the atypical cutaneous L. donovani compared to typical visceral disease associated L. donovani including mutations in the RagC and Raptor genes that are part of the eukaryotic conserved TOR pathway and its upstream sensing pathway. In the present study, we investigate whether the RagC R231C mutation present in atypical cutaneous L. donovani introduced into the virulent L. donovani 1S2D chromosome by CRISPR gene editing could affect virulence for survival in visceral organs. Through bioinformatic analysis, we further investigated the presence of sensing pathway components upstream of TOR in L. donovani including RagC complexing proteins, RagA and Raptor. L. donovani 1S2D edited to express mutant RagC R231C were viable in promastigote but had reduced visceral parasitemia in infected BALB/c mice. The RagC R231C mutant retained the ability to interact with RagA and gene knockout experiments revealed that although the RagA gene was essential, the RagC gene was not essential under promastigote culture conditions but was essential for survival in the liver of experimentally infected mice. These results provide evidence that the TOR associated sensing pathway plays a prominent role in L. donovani visceral disease and the RagC R231C mutation contributed to the atypical pathology of cutaneous L. donovani in Sri Lanka.

Systematical Engineering of Synthetic Yeast for Enhanced Production of Lycopene.

Synthetic biology allows the re-engineering of biological systems and promotes the development of bioengineering to a whole new level, showing great potential in biomanufacturing. Here, in order to make the heterologous lycopene biosynthesis pathway compatible with the host strain YSy 200, we evolved YSy200 using a unique Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system that is built in the Sc2.0 synthetic yeast. By inducing SCRaMbLE, we successfully identified a host strain YSy201 that can be served as a suitable host to maintain the heterologous lycopene biosynthesis pathway. Then, we optimized the lycopene biosynthesis pathway and further integrated into the rDNA arrays of YSy201 to increase its copy number. In combination with culturing condition optimization, we successfully screened out the final yeast strain YSy222, which showed a 129.5-fold increase of lycopene yield in comparison with its parental strain. Our work shows that, the strategy of combining the engineering efforts on both the lycopene biosynthesis pathway and the host strain can improve the compatibility between the heterologous pathway and the host strain, which can further effectively increase the yield of the target product.

The Phosphoenolpyruvate Carboxykinase Is a Key Metabolic Enzyme and Critical Virulence Factor of Leishmania major.

There is currently no effective vaccine against leishmaniasis because of the lack of sufficient knowledge about the Ags that stimulate host-protective and long-lasting T cell-mediated immunity. We previously identified Leishmania phosphoenolpyruvate carboxykinase (PEPCK, a gluconeogenic enzyme) as an immunodominant Ag that is expressed by both the insect (promastigote) and mammalian (amastigote) stages of the parasite. In this study, we investigated the role of PEPCK in metabolism, virulence, and immunopathogenicity of Leishmania major We show that targeted loss of PEPCK results in impaired proliferation of L. major in axenic culture and bone marrow-derived macrophages. Furthermore, the deficiency of PEPCK results in highly attenuated pathology in vivo. BALB/c mice infected with PEPCK-deficient parasites failed to develop any cutaneous lesions despite harboring parasites at the cutaneous site of infection. This was associated with a dramatic reduction in the frequency of cytokine (IFN-γ, IL-4, and IL-10)-producing CD4+ T cells in spleens and lymph nodes draining the infection site. Cells from mice infected with PEPCK-deficient parasites also produced significantly low levels of these cytokines into the culture supernatant following in vitro restimulation with soluble Leishmania Ag. PEPCK-deficient parasites exhibited significantly greater extracellular acidification rate, increased proton leak, and decreased ATP-coupling efficiency and oxygen consumption rates in comparison with their wild-type and addback counterparts. Taken together, these results show that PEPCK is a critical metabolic enzyme for Leishmania, and its deletion results in altered metabolic activity and attenuation of virulence.

Sensing Host Arginine Is Essential for Leishmania Parasites' Intracellular Development.

Arginine homeostasis in lysosomes is critical for the growth and metabolism of mammalian cells. Phagolysosomes of macrophages are the niche where the parasitic protozoan Leishmania resides and causes human leishmaniasis. During infection, parasites encounter arginine deprivation, which is monitored by a sensor on the parasite cell surface. The sensor promptly activates a mitogen-activated protein kinase 2 (MAPK2)-mediated arginine deprivation response (ADR) pathway, resulting in upregulating the abundance and activity of the Leishmania arginine transporter (AAP3). Significantly, the ADR is also activated during macrophage infection, implying that arginine levels within the host phagolysosome are limiting for growth. We hypothesize that ADR-mediated upregulation of AAP3 activity is necessary to withstand arginine starvation, suggesting that the ADR is essential for parasite intracellular development. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport but lack the ability to respond to arginine starvation. While these mutants grow normally in culture, they were impaired in their ability to develop inside THP-1 macrophages and were ∼70 to 80% less infective in BALB/c mice. Hence, inside the host macrophage, Leishmania must overcome the arginine "hunger games" by upregulating the transport of arginine via the ADR. We show that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis.IMPORTANCE In this study, we report that the ability of the human pathogen Leishmania to sense and monitor the lack of arginine in the phagolysosome of the host macrophage is essential for disease development. Phagolysosomes of macrophages are the niche where Leishmania resides and causes human leishmaniasis. During infection, the arginine concentration in the phagolysosome decreases as part of the host innate immune response. An arginine sensor on the Leishmania cell surface activates an arginine deprivation response pathway that upregulates the expression of a parasite arginine transporter (AAP3). Here, we use CRISPR/Cas9-mediated disruption of the AAP3 locus to show that this response enables Leishmania parasites to successfully compete with the host macrophage in the "hunger games" for arginine.

A second generation leishmanization vaccine with a markerless attenuated Leishmania major strain using CRISPR gene editing.

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in occasional skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen-/-). Notably, LmCen-/- is a genetically engineered centrin gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with LmCen-/- have no visible lesions following challenge with L. major-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden. LmCen-/- immunization results in protection and an immune response comparable to leishmanization. LmCen-/- is safe since it is unable to cause disease in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.

Application of CRISPR/Cas9-Mediated Genome Editing in Leishmania.

CRISPR-Cas9 is an RNA guided endonuclease derived from the bacterium Streptococcus pyogenes. Due to its simplicity, versatility, and high efficiency, it has been widely used for genome editing in a variety of organisms including the protozoan parasite Leishmania, the causative agent of human leishmaniasis. Compared to the traditional homologous recombination gene targeting method, CRISPR-Cas9 has been shown to be a more efficient method to delete or disrupt Leishmania genes, generate point mutations, and add tags to endogenous genes. Notably, the stable CRISPR expression systems were shown to delete multicopy family Leishmania genes and genes present in multiploid chromosomes, identify essential Leishmania genes, and create specific chromosome translocations. In this chapter, we describe detailed procedures on using the stable CRISPR expression system for genome editing in Leishmania. These procedures include CRISPR targeting site selection, gRNA design, cloning single and double gRNA coding sequences into the Leishmania CRISPR vector pLdCN, oligonucleotide donor and drug resistance selection donor design, Leishmania cell transfection, screening, and isolation of CRISPR-edited mutants. As the principles of gene editing are generally similar, many of these procedures could also apply to the transient Leishmania CRISPR systems described by other labs.

Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in Leishmania.

CRISPR-Cas9 genome editing relies on an efficient double-strand DNA break (DSB) and repair. Contrary to mammalian cells, the protozoan parasite Leishmania lacks the most efficient nonhomologous end-joining pathway and uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair to repair DSBs. Here, we reveal that Leishmania predominantly uses single-strand annealing (SSA) (>90%) instead of MMEJ (<10%) for DSB repair (DSBR) following CRISPR targeting of the miltefosine transporter gene, resulting in 9-, 18-, 20-, and 29-kb sequence deletions and multiple gene codeletions. Strikingly, when targeting the Leishmania donovani LdBPK_241510 gene, SSA even occurred by using direct repeats 77 kb apart, resulting in the codeletion of 15 Leishmania genes, though with a reduced frequency. These data strongly indicate that DSBR is not efficient in Leishmania, which explains why more than half of DSBs led to cell death and why the CRISPR gene-targeting efficiency is low compared with that in other organisms. Since direct repeat sequences are widely distributed in the Leishmania genome, we predict that many DSBs created by CRISPR are repaired by SSA. It is also revealed that DNA polymerase theta is involved in both MMEJ and SSA in Leishmania Collectively, this study establishes that DSBR mechanisms and their competence in an organism play an important role in determining the outcome and efficacy of CRISPR gene targeting. These observations emphasize the use of donor DNA templates to improve gene editing specificity and efficiency in Leishmania In addition, we developed a novel Staphylococcus aureus Cas9 constitutive expression vector (pLdSaCN) for gene targeting in LeishmaniaIMPORTANCE Due to differences in double-strand DNA break (DSB) repair mechanisms, CRISPR-Cas9 gene editing efficiency can vary greatly in different organisms. In contrast to mammalian cells, the protozoan parasite Leishmania uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair (HDR) to repair DSBs but lacks the nonhomologous end-joining pathway. Here, we show that Leishmania predominantly uses single-strand annealing (SSA) instead of MMEJ for DSB repairs (DSBR), resulting in large deletions that can include multiple genes. This strongly indicates that the overall DSBR in Leishmania is inefficient and therefore can influence the outcome of CRISPR-Cas9 gene editing, highlighting the importance of using a donor DNA to improve gene editing fidelity and efficiency in Leishmania.

High quantum yield pure blue emission and fast proton conduction from an indium-metal-organic framework.

A multifunctional material, with two or more types of functionalities in a single material, might give new phenomena and novel applications. Metal-organic frameworks (MOFs) or porous coordination polymers (PCPs) can be utilized as a highly versatile and tunable platform for exploring multifunctional materials. In this study, an extra thermally stable indium-MOF (1) was synthesized via self-assembly of 1,1'-ethynebenzene-3,3',5,5'-tetracarboxylic acid (H4EBTC) with an indium salt under solvothermal conditions. MOF 1 crystallizes in a tetragonal space group P4/mmm and shows a three-dimensional (3D) anionic framework with one-dimensional (1D) channels along the c-axis direction, and charge-balancing [(CH3)2NH2]+ cations together with lattice DMF and water molecules are accommodated in channels. This multifunctional MOF emits pure blue luminescence under ambient conditions, arising from the π* →π electron transition in EBTC4-, with CIE coordinates of (0.16, 0.07), and shows the highest quantum yield of 61.4% among indium-MOFs. Alternatively, 1 features fast proton conductivity with σ = 3.49 × 10-3 S cm-1 at 25 °C and 99% RH, attributed to the efficiently synergistic proton transfer in the H-bond networks constructed by [NH2(CH3)2]+ cations, lattice water molecules in channels and carboxylate groups in the framework. The notable level of performance of 1, combined with good hydrolytic and extra thermal stability, offers great opportunities for its future applications, and it may behave as a kind of promising multifunctional MOF material.

Manganese-Catalyzed Ring-Opening Coupling Reactions of Cyclopropanols with Enones.

A manganese-catalyzed ring-opening coupling reaction of cyclopropanols with enones for the facile and efficient preparation of 1,6-diketones is described. A wide array of synthetically important 1,6-diketones bearing manifold functional groups are obtained with up to 93% yield. These reactions feature broad substrate scopes, environmentally benign conditions, inexpensive catalyst, and operational simplicity.

Rapid room temperature synthesis of red iridium(iii) complexes containing a four-membered Ir-S-C-S chelating ring for highly efficient OLEDs with EQE over 30.

Three red cyclometalated iridium(iii) complexes (4tfmpq)2Ir(dipdtc), (4tfmpq)2Ir(dpdtc) and (4tfmpq)2Ir(Czdtc) (4tfmpq = 4-(4-(trifluoromethyl)phenyl)quinazoline, dipdtc = N,N-diisopropyl dithiocarbamate, dpdtc = N,N-diphenyl dithiocarbamate, and Czdtc = N-carbazolyl dithiocarbamate) containing the unique four-membered Ir-S-C-S backbone ring were synthesized in five minutes at room temperature with good yields, and the Gibbs free energy calculation results indicate that all reactions are exothermic and thermodynamically favorable processes. The emission colors (λ peak = 641-611 nm), photoluminescence quantum efficiencies (Φ P = 58.3-93.0%) and bipolar properties can be effectively regulated by introducing different electron-donating substituents into the dithiocarbamate ancillary ligands. Employing these emitters, organic light emitting diodes (OLEDs) with double emissive layers exhibit excellent performances with a maximum brightness over 60 000 cd m-2, a maximum current efficiency of 40.68 cd A-1, a maximum external quantum efficiency (EQEmax) of 30.54%, and an EQE of 26.79% at the practical luminance of 1000 cd m-2. These results demonstrate that Ir(iii) complexes with sulfur-containing ligands can be rapidly synthesized at room temperature, which is key to the production of metal luminescent materials for large-scale application in highly efficient OLEDs.

Correction: Rapid room temperature synthesis of red iridium(iii) complexes containing a four-membered Ir-S-C-S chelating ring for highly efficient OLEDs with EQE over 30.

[This corrects the article DOI: 10.1039/C8SC05605F.].

A complete Leishmania donovani reference genome identifies novel genetic variations associated with virulence.

Leishmania donovani is responsible for visceral leishmaniasis, a neglected and lethal parasitic disease with limited treatment options and no vaccine. The study of L. donovani has been hindered by the lack of a high-quality reference genome and this can impact experimental outcomes including the identification of virulence genes, drug targets and vaccine development. We therefore generated a complete genome assembly by deep sequencing using a combination of second generation (Illumina) and third generation (PacBio) sequencing technologies. Compared to the current L. donovani assembly, the genome assembly reported within resulted in the closure over 2,000 gaps, the extension of several chromosomes up to telomeric repeats and the re-annotation of close to 15% of protein coding genes and the annotation of hundreds of non-coding RNA genes. It was possible to correctly assemble the highly repetitive A2 and Amastin virulence gene clusters. A comparative sequence analysis using the improved reference genome confirmed 70 published and identified 15 novel genomic differences between closely related visceral and atypical cutaneous disease-causing L. donovani strains providing a more complete map of genes associated with virulence and visceral organ tropism. Bioinformatic tools including protein variation effect analyzer and basic local alignment search tool were used to prioritize a list of potential virulence genes based on mutation severity, gene conservation and function. This complete genome assembly and novel information on virulence factors will support the identification of new drug targets and the development of a vaccine for L. donovani.

Development of a sandwich ELISA to detect Leishmania 40S ribosomal protein S12 antigen from blood samples of visceral leishmaniasis patients.

BACKGROUND: Visceral leishmaniasis (VL), caused by Leishmania donovani complex parasites, is a neglected parasitic disease that is generally fatal if untreated. Despite decades of research to develop a sensitive VL diagnostic test, definitive diagnosis of VL still mainly relies on the visualization of the parasite in aspirates from the spleen, liver or bone marrow, an invasive and dangerous process with variable sensitivity. A sensitive assay that can detect Leishmania antigen from blood samples will help confirm cause, cure or recurrence of VL. METHODS: In this study, rabbit polyclonal antibodies were raised against eight recombinant Leishmania proteins that are highly abundant in Leishmania. The antibodies were purified and labeled with biotin for developing a prototype sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: The ELISA for the Leishmania 40S ribosomal protein S12 detected target antigen with the highest sensitivity and specificity and could detect 1 pg of purified protein or as few as 60 L. donovani parasites. The 40S ribosomal protein S12 sandwich ELISA could detect the target antigen from Peripheral Blood Mononuclear Cell (PBMC) samples in 68% of VL patients and post-kala-azar dermal leishmaniasis (PKDL) patients, providing an estimation of parasitemia ranging from 15 to 80 amastigotes per ml of blood. CONCLUSION: These results indicate that the 40S ribosomal protein S12 sandwich ELISA warrants further tests with more clinical samples of VL patients and other parasitic diseases. It is hopeful that this ELISA could become a useful tool for confirming VL diagnosis, monitoring treatment progress, disease recurrence and possibly detecting asymptomatic Leishmania infections with a high parasite load.

In(III)-TMSBr-Catalyzed Cascade Reaction of Diarylalkynes with Acrylates for the Synthesis of Aryldihydronaphthalene Derivatives.

A combined Lewis acid system comprising of two or more Lewis acids occasionally exhibits augmented catalytic activity in organic transformations which are otherwise unrealizable by either of the components exclusively. On the other hand, the efficient construction of multiple new C-C bonds and polycyclic structures in minimal steps remains a subject of great interest in both academia and industry. Herein we report an efficient method to assemble aryldihydronaphthalene derivatives via a cascade reaction of diarylalkynes with acrylates under the catalysis of a combined Lewis acid derived from In(III) salt and TMSBr.