Src- and Abl-family kinases activate spleen tyrosine kinase to maximize phagocytosis and Leishmania infection.

Leishmania spp. are obligate intracellular parasites that must be internalized by phagocytic cells to evade immune responses and cause disease. The uptake of both Leishmania promastigotes (insect-stage parasites) and amastigotes (proliferative-stage parasites in humans and mice) by phagocytes is thought to be mainly host cell driven, not parasite driven. Our previous work indicates that host Src- and Abl-family kinases facilitate Leishmania entry into macrophages and pathogenesis in murine cutaneous leishmaniasis. Here, we demonstrate that host spleen tyrosine kinase (SYK) is required for efficient uptake of Leishmania promastigotes and amastigotes. A Src-family kinase-Abl-family kinase-SYK signaling cascade induces Leishmania amastigote internalization. Finally, lesion size and parasite burden during Leishmania infection is significantly decreased in mice lacking SYK in monocytes or by treatment with the SYK inhibitor entospletinib. In summary, SYK is required for maximal Leishmania uptake by macrophages and disease in mice. Our results suggest potential for treating leishmaniasis using host cell-directed agents.

A variant in sperm-specific glycolytic enzyme enolase 4 (ENO4) causes human male infertility.

BACKGROUND: Although defects in sperm morphology and physiology lead to male infertility, in many instances, the exact disruption of molecular pathways in a given patient is often unknown. The glycolytic pathway is an essential process to supply energy in sperm cell motility. Enolase 4 (ENO4) is crucial for the glycolytic process, which provides the energy for sperm cells in motility. ENO4 is located in the sperm principal piece and is essential for the motility and organization of the sperm flagellum. In the present study, we characterized a family with asthenozoospermia and abnormal sperm morphology as a result of a variant in the enolase 4 (ENO4) gene. METHODS: Computer-assisted semen analysis, papanicolaou smear staining and scanning electron microscopy were used to examine sperm motility and morphology for semen analysis in patients. For genetic analysis, whole-exome sequencing followed by Sanger sequencing was performed. RESULTS: Two brothers in a consanguineous family were being clinically investigated for sperm motility and morphology issues. Genetic analysis by whole-exome sequencing revealed a homozygous variant [c.293A>G, p.(Lys98Arg)] in the ENO4 gene that segregated with infertility in the family, shared by affected but not controls. CONCLUSIONS: In view of the association of asthenozoospermia and abnormal sperm morphology in Eno4 knockout mice, we consider this to be the first report describing the involvement of ENO4 gene in human male infertility. We also explore the possible involvement of another variant in explaining other phenotypic features in this family.

Clinical and genetic investigation of 14 families with various forms of short stature syndromes.

Skeletal dysplasias are a heterogeneous group of disorders presenting mild to lethal defects. Several factors, such as genetic, prenatal, and postnatal environmental may contribute to reduced growth. Fourteen families of Pakistani origin, presenting the syndromic form of short stature either in the autosomal recessive or autosomal dominant manner were clinically and genetically investigated to uncover the underlying genetic etiology. Homozygosity mapping, whole exome sequencing, and Sanger sequencing were used to search for the disease-causing gene variants. In total, we have identified 13 sequence variants in 10 different genes. The variants in the HSPG2 and XRCC4 genes were not reported previously in the Pakistani population. This study will expand the mutation spectrum of the identified genes and will help in improved diagnosis of the syndromic form of short stature in the local population.

Mutational analysis in different genes underlying severe combined immunodeficiency in seven consanguineous Pakistani families.

BACKGROUND: Severe Combined Immunodeficiency (SCID) is an autosomal recessive inborn error of immunity (IEI) characterized by recurrent chest and gastrointestinal (GI) infections and in some cases associated with life-threatening disorders. METHODOLOGY AND RESULTS: This current study aims to unwind the molecular etiology of SCID and also extended the patients' phenotype associated with identified particular variants. Herein, we present 06 disease-causing variants identified in 07 SCID-patients in three different SCID related genes. Whole Exome Sequencing (WES) followed by Sanger Sequencing was employed to explore genetic variations. The results included identification of two previously reported heterozygous variants in homozygous form for the first time in RAG1gene [(p.Arg410Gln);(p.Arg737His)], followed by a recurrent variant (p.Trp959*) in RAG1, a novel variant in IL2RG (p.Asp48Lfs*24), a recurrent variant in IL2RG (p.Gly271Glu) and a recurrent variant in DCLRE1C (p.Arg191*) gene. CONCLUSION: To conclude, the immune-profiling and WES revealed two novel, two as homozygous state for the first time, and two recurrent disease causing variants contributing valuably to our existing knowledge of SCID.

Thiourea derivatives inhibit key diabetes-associated enzymes and advanced glycation end-product formation as a treatment for diabetes mellitus.

This study was designed to screen novel thiourea derivatives against different enzymes, such as α-amylase, α-glucosidase, protein tyrosine phosphatase 1 B, and advanced glycated end product (AGEs). A cytotoxicity analysis was performed using rat L6 myotubes and molecular docking analysis was performed to map the binding interactions between the active compounds and α-amylase and α-glucosidase. The data revealed the potency of five compounds, including E (1-(2,4-difluorophenyl)-3-(3,4-dimethyl phenyl) thiourea), AG (1-(2-methoxy-5-(trifluoromethyl) phenyl)-3-(3-methoxy phenyl) thiourea), AF (1-(2,4-dichlorophenyl)-3-(4-ethylphenyl) thiourea), AD (1-(2,4-dichlorophenyl)-3-(4-ethylphenyl) thiourea), and AH (1-(2,4-difluorophenyl)-3-(2-iodophenyl) thiourea), showed activity against α-amylase. The corresponding percentage inhibitions were found to be 85 ± 1.9, 82 ± 0.7, 75 ± 1.2, 72 ± 0.4, and 65 ± 1.1%, respectively. These compounds were then screened using in vitro assays. Among them, AH showed the highest activity against α-glucosidase, AGEs, and PTP1B, with percentage inhibitions of 86 ± 0.4% (IC50  = 47.9 µM), 85 ± 0.7% (IC50  = 49.51 µM), and 85 ± 0.5% (IC50  = 79.74 µM), respectively. Compound AH showed an increased glucose uptake at a concentration of 100 µM. Finally, an in vivo study was conducted using a streptozotocin-induced diabetic mouse model and PTP1B expression was assessed using real-time PCR. Additionally, we examined the hypoglycemic effect of compound AH in diabetic rats compared to the standard drug glibenclamide.

Sequence variants in different genes underlying Bardet-Biedl syndrome in four consanguineous families.

BACKGROUND: Bardet-Biedl Syndrome (BBS) is a rare (1:13,500-1-160,000) heterogeneous congenital disorder, characterized by postaxial polydactyly, obesity, hypogonadism, rod-cone dystrophy, cognitive impairment, and renal abnormalities (renal cystic dysplasia, anatomical malformation). To date about twenty-five genes have been identified to cause BBS, which accounts for about 80% of BBS diagnosis. METHODS: In the current study, we have performed mutational screening of four Pakistani consanguineous families (A-D) with clinical manifestation of BBS by microsatellite-based genotyping and whole exome sequencing. RESULTS: Analysis of the data revealed four variants, including a novel/unique inheritance pattern of compound heterozygous variants, p.(Ser40*) and p.(Thr259Leufs*21), in MKKS gene, novel homozygous variant, p.(Gly251Val)] in BBS7 gene and two previously reported p.(Thr259Leufs*21) in MKKS and p.(Met1Lys) in BBS5 gene. The variants were found segregated with the disorder within the families. CONCLUSION: The study not only expanded mutations spectrum in the BBS genes, but this will facilitate diagnosis and genetic counselling of families carrying BBS related phenotypes in Pakistani population.

Novel variants in the LRP4 underlying Cenani-Lenz Syndactyly syndrome.

Cenani-Lenz syndrome (CLS) is a rare autosomal-recessive congenital disorder affecting development of distal limbs. It is characterized mainly by syndactyly and/or oligodactyly, renal anomalies, and characteristic facial features. Mutations in the LRP4 gene, located on human chromosome 11p11.2-q13.1, causes the CLS. The gene LRP4 encodes a low-density lipoprotein receptor-related protein-4, which mediates SOST-dependent inhibition of bone formation and Wnt signaling. In the study, presented here, three families of Pakistani origin, segregating CLS in the autosomal recessive manner were clinically and genetically characterized. In two families (A and B), microsatellite-based homozygosity mapping followed by Sanger sequencing identified a novel homozygous missense variant [NM_002334.3: c.295G>C; p.(Asp99His)] in the LRP4 gene. In the third family C, exome sequencing revealed a second novel homozygous missense variant [NM_002334.3: c.1633C>T; p.(Arg545Trp)] in the same gene. To determine the functional relevance of these variants, we tested their ability to inhibit canonical WNT signaling in a luciferase assay. Wild type LRP4 was able to inhibit LRP6-dependent WNT signaling robustly. The two mutants p.(Asp99His) and p.(Arg545Trp) inhibited WNT signaling less effectively, suggesting they reduced LRP4 function.

In Vivo Efficacy and Metabolism of the Antimalarial Cycleanine and Improved In Vitro Antiplasmodial Activity of Semisynthetic Analogues.

Bisbenzylisoquinoline (BBIQ) alkaloids are a diverse group of natural products that demonstrate a range of biological activities. In this study, the in vitro antiplasmodial activity of three BBIQ alkaloids (cycleanine [compound 1], isochondodendrine [compound 2], and 2'-norcocsuline [compound 3]) isolated from the Triclisia subcordata Oliv. medicinal plant traditionally used for the treatment of malaria in Nigeria are studied alongside two semisynthetic analogues (compounds 4 and 5) of cycleanine. The antiproliferative effects against a chloroquine-resistant Plasmodium falciparum strain were determined using a SYBR green 1 fluorescence assay. The in vivo antimalarial activity of cycleanine is then investigated in suppressive, prophylactic, and curative murine malaria models after infection with a chloroquine-sensitive Plasmodium berghei strain. BBIQ alkaloids (compounds 1 to 5) exerted in vitro antiplasmodial activities with 50% inhibitory concentration (IC50) at low micromolar concentrations and the two semisynthetic cycleanine analogues showed an improved potency and selectivity compared to those of cycleanine. At oral doses of 25 and 50 mg/kg body weight of infected mice, cycleanine suppressed the levels of parasitemia and increased mean survival times significantly compared to those of the control groups. The metabolites and metabolic pathways of cycleanine were also studied using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Twelve novel metabolites were detected in rats after intragastric administration of cycleanine. The metabolic pathways of cycleanine were demonstrated to involve hydroxylation, dehydrogenation, and demethylation. Overall, these in vitro and in vivo results provide a basis for the future evaluation of cycleanine and its analogues as leads for further development.

The relative rate of kill of the MMV Malaria Box compounds provides links to the mode of antimalarial action and highlights scaffolds of medicinal chemistry interest.

OBJECTIVES: Rapid rate-of-kill (RoK) is a key parameter in the target candidate profile 1 (TCP1) for the next-generation antimalarial drugs for uncomplicated malaria, termed Single Encounter Radical Cure and Prophylaxis (SERCaP). TCP1 aims to rapidly eliminate the initial parasite burden, ideally as fast as artesunate, but minimally as fast as chloroquine. Here we explore whether the relative RoK of the Medicine for Malaria Venture (MMV) Malaria Box compounds is linked to their mode of action (MoA) and identify scaffolds of medicinal chemistry interest. METHODS: We used a bioluminescence relative RoK (BRRoK) assay over 6 and 48 h, with exposure to equipotent IC50 concentrations, to compare the cytocidal effects of Malaria Box compounds with those of benchmark antimalarials. RESULTS: BRRoK assay data demonstrate the following relative RoKs, from fast to slow: inhibitors of PfATP4>parasite haemoglobin catabolism>dihydrofolate reductase-thymidylate synthase (DHFR-TS)>dihydroorotate dehydrogenase (DHODH)>bc1 complex. Core-scaffold clustering analyses revealed intrinsic rapid cytocidal action for diamino-glycerols and 2-(aminomethyl)phenol, but slow action for 2-phenylbenz-imidazoles, 8-hydroxyquinolines and triazolopyrimidines. CONCLUSIONS: This study provides proof of principle that a compound's RoK is related to its MoA and that the target's intrinsic RoK is also modified by factors affecting a drug's access to it. Our findings highlight that as we use medicinal chemistry to improve potency, we can also improve the RoK for some scaffolds. Our BRRoK assay provides the necessary throughput for drug discovery and a critical decision-making tool to support development campaigns. Finally, two scaffolds, diamino-glycerols and 2-phenylbenzimidazoles, exhibit fast cytocidal action, inviting medicinal chemistry improvements towards TCP1 candidates.

Transdifferentiation of α-1,3-galactosyltransferase knockout pig bone marrow derived mesenchymal stem cells into pancreatic ß-like cells by microenvironment modulation.

OBJECTIVE: To evaluate the pancreatic differentiation potential of α-1,3-galactosyltransferase knockout (GalTKO) pig-derived bone marrow-derived mesenchymal stem cells (BM-MSCs) using epigenetic modifiers with different pancreatic induction media. METHODS: The BM-MSCs have been differentiated into pancreatic ß-like cells by inducing the overexpression of key transcription regulatory factors or by exposure to specific soluble inducers/small molecules. In this study, we evaluated the pancreatic differentiation of GalTKO pig-derived BM-MSCs using epigenetic modifiers, 5-azacytidine (5-Aza) and valproic acid (VPA), and two types of pancreatic induction media - advanced Dulbecco's modified Eagle's medium (ADMEM)-based and N2B27-based media. GalTKO BM-MSCs were treated with pancreatic induction media and the expression of pancreas-islets-specific markers was evaluated by real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. Morphological changes and changes in the 5'-C-phosphate-G-3' (CpG) island methylation patterns were also evaluated. RESULTS: The expression of the pluripotent marker (POU class 5 homeobox 1 [OCT4]) was upregulated upon exposure to 5-Aza and/or VPA. GalTKO BM-MSCs showed increased expression of neurogenic differentiation 1 in the ADMEM-based (5-Aza) media, while the expression of NK6 homeobox 1 was elevated in cells induced with the N2B27-based (5-Aza) media. Moreover, the morphological transition and formation of islets-like cellular clusters were also prominent in the cells induced with the N2B27-based media with 5-Aza. The higher insulin expression revealed the augmented trans-differentiation ability of GalTKO BM-MSCs into pancreatic ß-like cells in the N2B27-based media than in the ADMEM-based media. CONCLUSION: 5-Aza treated GalTKO BM-MSCs showed an enhanced demethylation pattern in the second CpG island of the OCT4 promoter region compared to that in the GalTKO BM-MSCs. The exposure of GalTKO pig-derived BM-MSCs to the N2B27-based microenvironment can significantly enhance their trans-differentiation ability into pancreatic ß-like cells.

Pancreatic endocrine-like cells differentiated from human umbilical cords Wharton's jelly mesenchymal stem cells using small molecules.

Following success of pancreatic islet transplantation in the treatment of Type I diabetes mellitus, there is a growing interest in using cell-based treatment approaches. However, severe shortage of donor islets-pancreas impeded the growth, and made researchers to search for an alternative treatment approaches. In this context, recently, stem cell-based therapy has gained more attention. The current study demonstrated that epigenetic modification improves the in vitro differentiation of Wharton's jelly mesenchymal stem cells (WJMSCs) into pancreatic endocrine-like cells. Here we used two histone deacetylase (HDAC) inhibitors namely trichostatin A (TSA) and TMP269. TSA inhibits both class I and II HDACs whereas TMP269 inhibits only class IIa HDACs. WJMSCs were differentiated using a multistep protocol in a serum-free condition with or without TSA pretreatment. A marginal improvement in differentiation was observed after TSA pretreatment though it was not significant. However, exposing endocrine precursor-like cells derived from WJMSCs to TMP269 alone has significantly improved the differentiation toward insulin-producing cells. Further, increase in the expression of paired box 4 (PAX4), insulin, somatostatin, glucose transporter 2 (GLUT2), MAF bZIP transcription factor A (MAFA), pancreatic duodenal homeobox 1 (PDX-1), and NKX6.1 was observed both at messenger RNA and protein levels. Nevertheless, TMP269-treated cells secreted higher insulin upon glucose challenge, and demonstrated increased dithizone staining. These findings suggest that TMP269 may improve the in vitro differentiation of WJMSCs into insulin-producing cells.

Differentiation potential of different regions-derived same donor human Wharton's jelly mesenchymal stem cells into functional smooth muscle-like cells.

The present study evaluates the transdifferentiation potential of different region-derived same donor Wharton's jelly MSCs (WJMSCs) into functional smooth muscle-like cells (SMLCs). All regions showed baseline expression for early smooth muscle cell (SMC) markers (αSMA and SM22-α) whereas mid marker CALPONIN gradually reduced during in vitro culture expansion and late marker myosin heavy chain type-11 (MHY-11) was completely absent. Furthermore, WJMSCs were induced to SMLCs using DMEM containing 10% FBS supplemented with different concentrations/combinations of TGF-ß1 and PDGF-BB under normoxia (20% O2) condition. Three treatment groups namely group A: 2.5 ng/ml TGF-ß1, group B: 5 ng/ml PDGF-BB and group C: 2.5 ng/ml TGF-ß1 + 5 ng/ml PDGF-BB were used for the induction of WJMSCs into SMLCs. Cells were evaluated for SMC-specific marker expression at different time intervals. Finally, selection of the SMC-specific highly potent region along with the most suitable treatment group was done on the basis of highest outcome in terms of SMC-specific marker expression and functional competence of transdifferentiated cells. Among all regions, baby region-derived WJMSCs (B-WJMSCs) exhibited highest SMC marker expression and functional ability. To mimic the in vivo physiological conditions, hypoxic conditions (3% O2) were used to evaluate the effect of low oxygen on the SMLC differentiation potential of selected WJMSCs using previously used same parameters. Annexin-V assay was performed to check the effect of cytokines and different oxygen concentrations, which revealed no significant differences. It was concluded that different induction conditions have different but positive effects on the functional SMLC differentiation ability of WJMSCs.

A spatial fuzzy C-means algorithm for segmentation of brain MRI images.

Brain and its structure are extremely complex with deep levels of details. Applying image processing methods of brain image can be very useful in many practical domains. Magnetic Resonance Imaging (MRI) is widely used imaging technique and has particular advantage by possessing the capability of providing highly detailed images of brain soft tissues than any other imaging techniques. The real challenge at hand for researchers is to perform precise segmentation while overcoming the effects of noise and other imaging artifacts like intensity in homogeneity introduced in medical images during image acquisition process. In this research work, a directional weighted optimized Fuzzy C-Means (dwsFCM) method has been proposed for segmentation of brain MR images. This method works by incorporating the spatial information of the pixels of the images and assigning the directional weights to the neighborhood. In order to validate the proposed segmentation framework, a comprehensive set of experiments have been performed on publically available standard simulated as well as real datasets. The experimental results showed 95% of accuracy and the performance of the proposed segmentation framework is much better and the framework suppress the sufficient amount of noise especially rician noise and reproduce good segmentation by overcoming the effect of intensity in homogeneity.

Comparative analysis of human Wharton's jelly mesenchymal stem cells derived from different parts of the same umbilical cord.

Easy isolation, lack of ethical issues, high proliferation, multi-lineage differentiation potential and immunomodulatory properties of umbilical cord (UC)-derived mesenchymal stem cells (MSCs) make them a valuable tool in stem cell research. Recently, Wharton's jelly (WJ) was proven as the best MSC source among various compartments of UC. However, it is still unclear whether or not Wharton's jelly-derived MSCs (WJMSCs) from different parts of the whole cord exhibit the same characteristics. There may be varied MSCs present in different parts of WJ throughout the length of the UC. For this purpose, using an explant attachment method, WJMSCs were isolated from three different parts of the UC, mainly present towards the placenta (mother part), the center of the whole cord (central part) and the part attached to the fetus (baby part). WJMSCs from all three parts were maintained in normal growth conditions (10% ADMEM) and analyzed for mesenchymal markers, pluripotent genes, proliferation rate and tri-lineage differentiation potential. All WJMSCs were highly proliferative, positively expressed CD90, CD105, CD73 and vimentin, while not expressing CD34, CD45, CD14, CD19 or HLA-DR, differentiated into adipocytes, osteocytes and chondrocytes and expressed pluripotency markers OCT-4, SOX-2 and NANOG at gene and protein levels. Furthermore, MSCs derived from all the parts were shown to have potency towards hepatocyte-like cell differentiation. Human bone marrow-derived MSCs were used as a positive control. Finally, we conclude that WJMSCs derived from all the parts are valuable sources and can be efficiently used in various fields of regenerative medicine.

Immune molecular profiling of whole blood drawn from a non-human primate cardiac xenograft model treated with anti-CD154 monoclonal antibodies.

Most studies of xenografts have been carried out with complex immunosuppressive regimens to prevent immune rejection; however, such treatments may be fatal owing to unknown causes. Here, we performed immune molecular profiling following anti-CD154 monoclonal antibody (mAb) treatment in heterotopic abdominal cardiac xenografts from α-1,3-galactosyltransferase-knockout pigs into cynomolgus monkeys to elucidate the mechanisms mediating the undesirable fatal side effects of immunosuppressive agents. Blood samples were collected from healthy monkeys as control and then at 2 days after xenograft transplantation and just before humane euthanasia; 94 genes related to the immune system were analyzed. The basic immunosuppressive regimen included cobra venom factor, anti-thymocyte globulin, and rituximab, with and without anti-CD154 mAbs. The maintenance therapy was followed with tacrolimus, MMF, and methylprednisolone. The number of upregulated genes was initially decreased on Day 2 (-/+ anti-CD154 mAb, 22/13) and then increased before euthanasia in recipients treated with anti-CD154 mAbs (-/+ anti-CD154 mAb, 30/37). The number of downregulated genes was not affected by anti-CD154 mAb treatment. Additionally, the number of upregulated genes increased over time for both groups. Interestingly, treatment with anti-CD154 mAbs upregulated coagulation inducers (CCL2/IL6) before euthanasia. In conclusion, immunosuppressive regimens used for cardiac xenografting affected upregulation of 6 inflammation genes (CXCL10, MPO, MYD88, NLRP3, TNFα, and TLR1) and downregulation of 8 genes (CCR4, CCR6, CD40, CXCR3, FOXP3, GATA3, STAT4, and TBX21).

The synthesis and evaluation of thymoquinone analogues as anti-ovarian cancer and antimalarial agents.

Thymoquinone (TQ), 2-isopropyl-5-methyl-1,4-benzoquinone, a natural product isolated from Nigella sativa L., has previously been demonstrated to exhibit antiproliferative activity in vitro against a range of cancers as well as the human malarial parasite Plasmodium falciparum. We describe here the synthesis of a series of analogues of TQ that explore the potential for nitrogen-substitution to this scaffold, or reduction to a hydroquinone scaffold, in increasing the potency of this antiproliferative activity against ovarian cancer cell lines and P. falciparum. In addition, alkyl or halogen-substituted analogues were commercially sourced and tested in parallel. Several TQ analogues with improved potency against ovarian cancer cells and P. falciparum were found, although this increase is suggested to be moderate. Key aspects of the structure activity relationship that could be further explored are highlighted.

Cholinergic Nerve Differentiation of Mesenchymal Stem Cells Derived from Long-Term Cryopreserved Human Dental Pulp In Vitro and Analysis of Their Motor Nerve Regeneration Potential In Vivo.

The reduction of choline acetyltransferase, caused by the loss of cholinergic neurons, leads to the absence of acetylcholine (Ach), which is related to motor nerve degeneration. The aims of the present study were to evaluate the in vitro cholinergic nerve differentiation potential of mesenchymal stem cells from cryopreserved human dental pulp (hDPSCs-cryo) and to analyze the scale of in vivo motor nerve regeneration. The hDPSCs-cryo were isolated and cultured from cryopreserved dental pulp tissues, and thereafter differentiated into cholinergic neurons using tricyclodecane-9-yl-xanthogenate (D609). Differentiated cholinergic neurons (DF-chN) were transplanted into rats to address sciatic nerve defects, and the scale of in vivo motor nerve regeneration was analyzed. During in vitro differentiation, the cells showed neuron-like morphological changes including axonal fibers and neuron body development, and revealed high expression of cholinergic neuron-specific markers at both the messenger RNA (mRNA) and protein levels. Importantly, DF-chN showed significant Ach secretion ability. At eight weeks after DF-chN transplantation in rats with sciatic nerve defects, notably increased behavioral activities were detected with an open-field test, with enhanced low-affinity nerve growth factor receptor (p75NGFR) expression detected using immunohistochemistry. These results demonstrate that stem cells from cryopreserved dental pulp can successfully differentiate into cholinergic neurons in vitro and enhance motor nerve regeneration when transplanted in vivo. Additionally, this study suggests that long-term preservation of dental pulp tissue is worthwhile for use as an autologous cell resource in the field of nerve regeneration, including cholinergic nerves.

Generation of a 3D Liver Model Comprising Human Extracellular Matrix in an Alginate/Gelatin-Based Bioink by Extrusion Bioprinting for Infection and Transduction Studies.

Bioprinting is a novel technology that may help to overcome limitations associated with two-dimensional (2D) cell cultures and animal experiments, as it allows the production of three-dimensional (3D) tissue models composed of human cells. The present study describes the optimization of a bioink composed of alginate, gelatin and human extracellular matrix (hECM) to print human HepaRG liver cells with a pneumatic extrusion printer. The resulting tissue model was tested for its suitability for the study of transduction by an adeno-associated virus (AAV) vector and infection with human adenovirus 5 (hAdV5). We found supplementation of the basic alginate/gelatin bioink with 0.5 and 1 mg/mL hECM provides desirable properties for the printing process, the stability of the printed constructs, and the viability and metabolic functions of the printed HepaRG cells. The tissue models were efficiently transduced by AAV vectors of serotype 6, which successfully silenced an endogenous target (cyclophilin B) by means of RNA interference. Furthermore, the printed 3D model supported efficient adenoviral replication making it suitable to study virus biology and develop new antiviral compounds. We consider the approach described here paradigmatic for the development of 3D tissue models for studies including viral vectors and infectious viruses.

A validated bioluminescence-based assay for the rapid determination of the initial rate of kill for discovery antimalarials.

Objectives: A future treatment for uncomplicated malaria will contain at least one component that exerts a rapid rate of kill. We describe here the validation and application of a simple, robust and rapid bioluminescence-based assay for the determination of the initial rate of kill in intra-erythrocytic asexual stages of Plasmodium falciparum . Methods: A modification to the concentration-response bioluminescence [here termed bioluminescence relative rate of kill (BRRoK)] assay, utilizing exposure to fold-IC 50 concentrations (0.33× to 9×), was used to monitor the immediate cytocidal effect of 372 open-source compounds for antimalarial drug discovery available through the Medicines for Malaria Venture Malaria Box. Results: Antimalarial drugs that exert a rapid cytocidal effect produce a concentration-dependent loss of bioluminescence signal that correlates with available in vitro and in vivo estimates of parasite clearance time and parasite reduction ratio. Following the measurement of IC 50 for the Malaria Box compounds in Dd2 luc , the BRRoK assay was used to identify and rank 372 compounds for their initial cytocidal activity. Fifty-three compounds in the Malaria Box show an initial relative rate of kill greater than that of chloroquine, with 17 of these having an initial relative rate of kill greater than that of dihydroartemisinin. Conclusions: The BRRoK assay provides a rapid assay format for the estimation of a key pharmacodynamic property of antimalarial drug action. The simplicity and robustness of the assay suggests it would be readily scalable for high-throughput screening and a critical decision-making tool for antimalarial drug development.