Transgenic mouse models support a protective role of type I IFN response in SARS-CoV-2 infection-related lung immunopathology and neuroinvasion.

Type I interferon (IFN-I) response is the first line of host defense against invading viruses. In the absence of definite mouse models, the role of IFN-I in SARS-CoV-2 infection remains perplexing. Here, we develop two mouse models, one with constitutively high IFN-I response (hACE2; Irgm1-/-) and the other with dampened IFN-I response (hACE2; Ifnar1-/-), to comprehend the role of IFN-I response. We report that hACE2; Irgm1-/- mice are resistant to lethal SARS-CoV-2 infection. In contrast, a severe SARS-CoV-2 infection along with immune cell infiltration, cytokine storm, and enhanced pathology is observed in the lungs and brain of hACE2; Ifnar1-/- mice. The hACE2; Irgm1-/-Ifnar1-/- double-knockout mice display loss of the protective phenotype observed in hACE2; Irgm1-/- mice, suggesting that heightened IFN-I response accounts for the observed immunity. Taking the results together, we demonstrate that IFN-I protects from lethal SARS-CoV-2 infection, and Irgm1 (IRGM) could be an excellent therapeutic target against SARS-CoV-2.

A proteomic perspective and involvement of cytokines in SARS-CoV-2 infection.

Infection with the SARS-CoV-2 virus results in manifestation of several clinical observations from asymptomatic to multi-organ failure. Biochemically, the serious effects are due to what is described as cytokine storm. The initial infection region for COVID-19 is the nasopharyngeal/oropharyngeal region which is the site where samples are taken to examine the presence of virus. We have now carried out detailed proteomic analysis of the nasopharyngeal/oropharyngeal swab samples collected from normal individuals and those tested positive for SARS-CoV-2, in India, during the early days of the pandemic in 2020, by RTPCR, involving high throughput quantitative proteomics analysis. Several proteins like annexins, cytokines and histones were found differentially regulated in the host human cells following SARS-CoV-2 infection. Genes for these proteins were also observed to be differentially regulated when their expression was analyzed. Majority of the cytokine proteins were found to be up regulated in the infected individuals. Cell to Cell signaling interaction, Immune cell trafficking and inflammatory response pathways were found associated with the differentially regulated proteins based on network pathway analysis.

Down regulation of defensin genes during SARS-CoV-2 infection.

Defensins, crucial components of the innate immune system, play a vital role against infection as part of frontline immunity. Association of SARS-CoV-2 infection with defensins has not been investigated. In this study, we have investigated the expression of defensin genes in the buccal cavity from patients with COVID-19 infection along with negative control samples. Nasopharyngeal/oropharyngeal swab samples collected for screening SARS-CoV-2 infection in early 2020 from Hyderabad, India, were analyzed for the expression of major defensin genes by the quantitative real-time reverse transcription polymerase chain reaction, qRT-PCR. Forty SARS-CoV-2 infected positive and 40 negative swab samples were selected for this study. Based on the qRT-PCR analysis involving gene specific primers for defensin genes, 9 defensin genes were found to be expressed in the nasopharyngeal/oropharyngeal cavity. Four defensin genes were found to be significantly down regulated in SARS-CoV-2 infected patients in comparison with the control samples based on differential expression analysis. The significantly down regulated genes were defensin beta 4A/B, 106B, 107B, and 103A. Down regulation of human beta defensin 2, 3, 6 and 7 suggests that antiviral innate immune response provided by defensins may be compromised in SARS-CoV-2 infection resulting in progression of the disease. Correction of the down regulation process through appropriate defensin peptide-based therapy could be an attractive method of treatment. Keywords: host defense; defensins; COVID-19; gene regulation; SARS-CoV-2.

Gramicidin S and melittin: potential anti-viral therapeutic peptides to treat SARS-CoV-2 infection.

The COVID19 pandemic has led to multipronged approaches for treatment of the disease. Since de novo discovery of drugs is time consuming, repurposing of molecules is now considered as one of the alternative strategies to treat COVID19. Antibacterial peptides are being recognized as attractive candidates for repurposing to treat viral infections. In this study, we describe the anti-SARS-CoV-2 activity of the well-studied antibacterial peptides gramicidin S and melittin obtained from Bacillus brevis and bee venom respectively. The EC50 values for gramicidin S and melittin were 1.571 µg and 0.656 µg respectively based on in vitro antiviral assay. Significant decrease in the viral load as compared to the untreated group with no/very less cytotoxicity was observed. Both the peptides treated to the SARS-CoV-2 infected Vero cells showed viral clearance from 12 h onwards with a maximal viral clearance after 24 h post infection. Proteomics analysis indicated that more than 250 proteins were differentially regulated in the gramicidin S and melittin treated SARS-CoV-2 infected Vero cells against control SARS-CoV-2 infected Vero cells after 24 and 48 h post infection. The identified proteins were found to be associated in the metabolic and mRNA processing of the Vero cells post-treatment and infection. Both these peptides could be attractive candidates for repurposing to treat SARS-CoV-2 infection.

Transcriptomic and proteomic insights into patulin mycotoxin-induced cancer-like phenotypes in normal intestinal epithelial cells.

Patulin (PAT) is a natural contaminant of fruits (primarily apples) and their products. Significantly, high levels of contamination have been found in fruit juices all over the world. Several in vitro studies have demonstrated PAT's ability to alter intestinal structure and function. However, in real life, the probability of low dose long-term exposure to PAT to humans is significantly higher through contaminated food items. Thus, in the present study, we have exposed normal intestinal cells to non-toxic levels of PAT for 16 weeks and observed that PAT had the ability to cause cancer-like properties in normal intestinal epithelial cells after chronic exposure. Here, our results showed that chronic exposure to low doses of PAT caused enhanced proliferation, migration and invasion ability, and the capability to grow in soft agar (anchorage independence). Moreover, an in vivo study showed the appearance of colonic aberrant crypt foci (ACFs) in PAT-exposed Wistar rats, which are well, establish markers for early colon cancer. Furthermore, as these neoplastic changes are consequences of alterations at the molecular level, here, we combined next-generation RNA sequencing with liquid chromatography mass spectrometry-based proteomic analysis to investigate the possible underlying mechanisms involved in PAT-induced neoplastic changes.

Understanding the complexity of epimorphic regeneration in zebrafish caudal fin tissue: A transcriptomic and proteomic approach.

The complex epimorphic regeneration of zebrafish caudal fin tissue is hasty and absolute. This study was executed to understand the role of various genes/proteins involved in the regeneration of zebrafish caudal fin tissue through differential transcriptomics and proteomics analysis. Based on our study 1408 genes and 661 proteins were found differentially regulated in the regenerating caudal fin tissue for having at least 1-log fold change. Interleukin, Solute carrier, Protein arginine methyltransferase, Homeobox, Neurotransmitter and several novel genes were found to be associated with regeneration for its differential regulation during the mechanism. Based on the network and pathway analysis the differentially regulated genes and proteins were found allied with activation of cell proliferation, cell viability, cell survival & cell movement and inactivation of organismal death, morbidity, necrosis, death of embryo & cell death. This study has mapped a detailed insight of the genes/proteins expression associated with the epimorphic regeneration more profoundly.

Proteome profile of telencephalon associates attenuated neurogenesis with chronic stress induced mood disorder phenotypes in zebrafish model.

Debilitating mental illness like depression and related mood disorders is due to the disruption in circuitry that controls emotion, motivation, and reward, characterized by disparate phenotypes like decrease in socialization, motivation, threshold for threat apprehension, etc. Chronic stress is a major factor in the etiology of these disorders. Here, using a chronic unpredictable stress (CUS) paradigm the characterization of an array of mood disorder phenotypes in adult zebrafish, in comparison to normal control unstressed fish, was achieved using a battery of behavioral assays including novel ones comprising social interaction test, feed approach test, threat response test and novel tank test. For the predictive validity of the model for mood disorders, the mitigative role of a slow (imipramine) and fast (ketamine) acting antidepressant was assessed. The molecular changes associated with CUS-induced mood disorder phenotype was investigated utilizing a high throughput method called isobaric tag for relative and absolute quantification (iTRAQ) in telencephalon, the region critically associated with the processing of emotional information in the fish brain. Out of 222 proteins identified to be significantly altered, 58 were differentially expressed across the stress and antidepressant-treatment groups at more than one fold (in log2) change. Of these proteins, some were implicated in earlier studies on mood disorders such as CABP1, PER2, mTOR, etc. The enrichment of altered proteins by Ingenuity Pathway Analysis (IPA) led us to mTOR and opioid signaling pathways, the top canonical pathways affected in the fish telencephalon. Interestingly, most of the pathways affected converge at the one controlling cell proliferation thus indicating altered neurogenesis, which was validated using immunohistochemistry for cell proliferation markers BrdU, SOX2, and BLBP. The study concludes that molecules that regulate telencephalon neural progenitor cell proliferation or neurogenesis are crucially involved in chronic stress-induced mood disorders by affecting the circuitry that controls emotion and reward.

Transcriptomic and proteomic analysis of Hemidactylus frenatus during initial stages of tail regeneration.

Epimorphic regeneration of appendages is a complex and complete phenomenon found in selected animals. Hemidactylus frenatus, house gecko has the remarkable ability to regenerate the tail tissue upon autotomy involving epimorphic regeneration mechanism. This study has identified and evaluated the molecular changes at gene and protein level during the initial stages, i.e., during the wound healing and repair mechanism initiation stage of tail regeneration. Based on next generation transcriptomics and De novo analysis the transcriptome library of the gecko tail tissue was generated. A total of 254 genes and 128 proteins were found to be associated with the regeneration of gecko tail tissue upon amputation at 1, 2 and 5-day post amputation (dpa) against control, 0-dpa through differential transcriptomic and proteomic analysis. To authenticate the expression analysis, 50 genes were further validated involving RTPCR. 327 genes/proteins identified and mapped from the study showed association for Protein kinase A signaling, Telomerase BAG2 signaling, paxillin signaling, VEGF signaling network pathways based on network pathway analysis. This study empanelled list of transcriptome, proteome and the list of genes/proteins associated with the tail regeneration.

Functional role of annexins in zebrafish caudal fin regeneration - A gene knockdown approach in regenerating tissue.

Regeneration is an adaptive phenomenon with wide biological implications spread heterogeneously in almost all the organism including human beings. The ability of regeneration varies from species to species for its complexity. Epimorphic regeneration of zebrafish caudal fin tissue is the most widely studied regeneration mechanism for its discrete and rapid regenerative capability. Several genes and proteins were found to be associated with regenerative mechanisms of zebrafish caudal fin tissue. In this study we have evaluated the functional role of Annexin 2a and 2b genes during zebrafish caudal fin tissue regeneration using inventive electroporation techniques for targeting the gene involving CRISPR-Cas9 technology. The electroporation of the CRISPR was performed on the adult zebrafish caudal fin tissue post amputation. We report retarded growth during the regeneration of caudal fin tissue when Annexin 2a and 2b genes were knocked down, which was validated through gene expression & sequencing analysis and further supported by high-throughput quantitative proteomic analysis of the fin tissue. Annexin family genes such as ANXA13, ANXA1a, ANXA5b were also found to be repressed with their expression. Knocking down of ANXA2a and 2b in regenerating caudal fin tissue compromises regenerating capacity as these genes are involved in cell to cell communication and extracellular matrix growth. This study proves that ANXA2a and 2b plays a significant role in epimorphic regeneration of zebrafish caudal fin tissue.

Toxicity of TiO2, SiO2, ZnO, CuO, Au and Ag engineered nanoparticles on hatching and early nauplii of Artemia sp.

The potential of environmental release enhances with increased commercial applications of the nanomaterials. In this work, a simple and efficient test to estimate the acute toxicity of nanoparticles is carried out on Artemia species and their hatching rates. We have tested six different engineered nanoparticles (silver, gold, copper oxide, zinc oxide, TiO2 and SiO2 nanoparticles) and three soluble salts (CuSO4, ZnSO4 and AgNO3) on Artemia sp. The physicochemical properties of the nanoparticles involved in this study were analyzed in normal water and marine water. Hydrated and bleached Artemia cysts were allowed to hatch in continuously aerated, filtered sterile salt water containing nanoparticles; hatching of viable nauplii and total hatchlings have been recorded. In parallel, standard Artemia toxicity test was conducted on the nauplii monitoring the viability. In hatching experiments, a reduction in hatching rate was observed along with mortality of newly hatched nauplii. The results of the hatching experiment and of the standard Artemia test showed a good correlation. The toxicity of the nanoparticles was compared and the order of toxicity was estimated as Ag>CuO>ZnO>Au>TiO2>SiO2. The study thus suggests that the hatching test itself is a reliable assay for determining the toxicity of nanomaterials.

Elemental analysis of scorpion venoms.

Scorpion venom is a rich source of biomolecules, which can perturb physiological activity of the host on envenomation and may also have a therapeutic potential. Scorpion venoms produced by the columnar cells of venom gland are complex mixture of mucopolysaccharides, neurotoxic peptides and other components. This study was aimed at cataloguing the elemental composition of venoms obtained from medically important scorpions found in the Arabian peninsula. The global elemental composition of the crude venom obtained from Androctonus bicolor, Androctonus crassicauda and Leiurus quinquestriatus scorpions were estimated using ICP-MS analyzer. The study catalogued several chemical elements present in the scorpion venom using ICP-MS total quant analysis and quantitation of nine elements exclusively using appropriate standards. Fifteen chemical elements including sodium, potassium and calcium were found abundantly in the scorpion venom at PPM concentrations. Thirty six chemical elements of different mass ranges were detected in the venom at PPB level. Quantitative analysis of the venoms revealed copper to be the most abundant element in Androctonus sp. venom but at lower level in Leiurus quinquestriatus venom; whereas zinc and manganese was found at higher levels in Leiurus sp. venom but at lower level in Androctonus sp. venom. These data and the concentrations of other different elements present in the various venoms are likely to increase our understanding of the mechanisms of venom activity and their pharmacological potentials.

Quantitative proteomic analysis of normal and degenerated human intervertebral disc.

BACKGROUND CONTEXT: Degenerative disc disease (DDD) is the most common disease of aging in humans. DDD is characterized by the gradual damage of the intervertebral discs. The disease is characterized by progressive dehydration of nucleus pulposus and disruption of annulus fibrosus of intervertebral disc. PURPOSE: Even though it is highly prevalent, there is no effective therapy to regenerate the degenerated disc, or decrease or halt the disease progression. Therefore, novel monitoring and diagnostic tests are essential to develop an alternative therapeutic strategies which can prevent further progression of disc degeneration. STUDY DESIGN: The study was designed to understand the proteome map of annulus fibrosus and nucleus pulposus tissues of intervertebral disc and its differential expression in patients with DDD. METHODS: The proteome map of the annulus fibrosus and nucleus pulposus tissues of intervertebral disc was cataloged involving one-dimensional gel electrophoresis-Fourier transform mass spectrometry/ion trap tandem mass spectrometry (FTMS/ITMSMS) analysis. The altered proteome patterns of annulus fibrosus and nucleus pulposus tissues for DDD were identified using Isobaric tag for relative and absolute quantification (iTRAQ)-based quantitative proteomics coupled with FTMS/ITMSMS and network pathway analysis. RESULTS: The study identified a total of 759 and 692 proteins from the annulus fibrosus and the nucleus pulposus tissues of the disc based on FTMS/ITMSMS analysis, which includes 118 proteins commonly identified between the two tissues. Vibrant changes were observed between the normal and the degenerating annulus fibrosus and nucleus pulposus tissues. A total of 73 and 54 proteins were identified as differentially regulated in the annulus and the nucleus tissues, respectively, between the normal and the degenerated tissues independently. Network pathway analysis mapped the differentially expressed proteins to cell adhesion, cell migration, and interleukin13 signaling pathways. CONCLUSIONS: Altogether, the current study provides a novel vision in the biomechanism of human disc degeneration and a certain number of proteins with the potential biomarker value for the preliminary diagnosis and scenario of DDD.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine induced Parkinson's disease in zebrafish.

Parkinson's disease (PD) is the most common age associated neurodegenerative disease, which has been extensively studied for its etiology and phenotype. PD has been widely studied in alternate model system such as rodents towards understanding the role of neurotoxin by inducing PD. This study is aimed to understand the biomechanism of PD in zebrafish model system induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The phenotype and role of various genes and proteins for Parkinsonism were tested and evaluated in this study using behavior, molecular and proteomic approaches. Zebrafish PD model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine showed a significant level of decrease in the movement with erratic swimming pattern and increased freezing bouts. CHCHD2, EEF2B, LRRK2, PARK7, PARK2, POLG, SNCGB and SYNB genes were differentially regulated at the transcript level in PD zebrafish. Similarly a total of 73 proteins were recognized as differentially expressed in the nervous system of zebrafish due to Parkinsonism based on quantitative proteomics approach. Proteins such as NEFL, MUNC13-1, NAV2 and GAPVD1 were down regulated in the zebrafish brain for the PD phenotype, which were associated with the neurological pathways. This zebrafish based PD model can be used as a potential model system for screening prospective drug molecules for PD.

Role of annexin gene and its regulation during zebrafish caudal fin regeneration.

The molecular mechanism of epimorphic regeneration is elusive due to its complexity and limitation in mammals. Epigenetic regulatory mechanisms play a crucial role in development and regeneration. This investigation attempted to reveal the role of epigenetic regulatory mechanisms, such as histone H3 and H4 lysine acetylation and methylation during zebrafish caudal fin regeneration. It was intriguing to observe that H3K9,14 acetylation, H4K20 trimethylation, H3K4 trimethylation and H3K9 dimethylation along with their respective regulatory genes, such as GCN5, SETd8b, SETD7/9, and SUV39h1, were differentially regulated in the regenerating fin at various time points of post-amputation. Annexin genes have been associated with regeneration; this study reveals the significant up-regulation of ANXA2a and ANXA2b transcripts and their protein products during the regeneration process. Chromatin immunoprecipitation and PCR analysis of the regulatory regions of the ANXA2a and ANXA2b genes demonstrated the ability to repress two histone methylations, H3K27me3 and H4K20me3, in transcriptional regulation during regeneration. It is hypothesized that this novel insight into the diverse epigenetic mechanisms that play a critical role during the regeneration process may help to strategize the translational efforts, in addition to identifying the molecules involved in vertebrate regeneration.

Ocular Fluid As a Replacement for Serum in Cell Cryopreservation Media.

Cryostorage is of immense interest in biomedical research, especially for stem cell-based therapies and fertility preservation. Several protocols have been developed for efficient cryopreservation of cells and tissues, and a combination of dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS) is commonly used. However, there is a need for an alternative to FBS because of ethical reasons, high cost, and risk of contamination with blood-borne diseases. The objective of the present study was to examine the possibility of using buffalo (Bubalus bubalis) ocular fluid (BuOF) to replace FBS in cryomedia. Frozen-thawed cells, which were cryopreserved in a cryomedia with BuOF, were assessed for viability, early and late apoptosis, and proliferation. Three cell lines (CHO, HEK, and C18-4), mouse embryonic stem (mES) cells, and primary cells, such as mouse embryonic fibroblast (MEF) cells, human peripheral blood mononuclear cells (hPBMCs), and mouse bone marrow cells (mBMCs), were cryopreserved in cryomedia containing 10% DMSO (D10) with 20% FBS (D10S20) or D10 with 20% BuOF (D10O20). For all three cell lines and mES cells cryopreserved in either D10S20 or D10O20, thawed cells showed no difference in cell viability or cell recovery. Western blot analysis of frozen-thawed-cultured cells revealed that the expression of Annexin V and proliferating cell nuclear antigen (PCNA) proteins, and the ratio of BAX/BCL2 proteins were similar in all three cell lines, mES cells, and hPBMCs cryopreserved in D10S20 and D10O20. However, initial cell viability, cell recovery after culture, and PCNA expression were significantly lower in MEF cells, and the BAX/BCL2 protein ratio was elevated in mBMCs cryopreserved in D10O20. Biochemical and proteomic analysis of BuOF showed the presence of several components that may have roles in imparting the cryoprotective property of BuOF. These results encourage further research to develop an efficient serum-free cryomedia for several cell types using BuOF.

Proteomic and gene expression analysis of zebrafish brain undergoing continuous light/dark stress.

Several organisms irrespective of their complexity in structure and function have an inbuilt circadian rhythm. Zebrafish could be used as an alternate model animal in sleep research as it exhibits similar sleep-wake dynamics as mammals and Drosophila. In this study, we have analysed the adult zebrafish brain for its differential proteome and gene expression during perturbed light/dark cycle. A total of 53 and 25 proteins including sncb, peroxiredoxins and TCR alpha were identified based on two-dimensional gel electrophoresis Fourier transform mass spectrometer/ion trap tandem mass spectrometer and differential in-gel electrophoresis MALDI TOF MS/MS analysis, respectively, with at least 1.5-fold changes between the control and experimental brains. Real time-polymerase chain reaction revealed that many circadian pathway-associated genes, such as per1b, bmal1b, cry1b, bmal2 and nr1d2, were differentially regulated during continuous light/dark exposures. It is hypothesized that the differential regulation of these genes might lead to the discovery of potential diagnostic markers for gaining insight into the light/dark-associated stress in humans.

OncomiRdbB: a comprehensive database of microRNAs and their targets in breast cancer.

BACKGROUND: Given the estimate that 30% of our genes are controlled by microRNAs, it is essential that we understand the precise relationship between microRNAs and their targets. OncomiRs are microRNAs (miRNAs) that have been frequently shown to be deregulated in cancer. However, although several oncomiRs have been identified and characterized, there is as yet no comprehensive compilation of this data which has rendered it underutilized by cancer biologists. There is therefore an unmet need in generating bioinformatic platforms to speed the identification of novel therapeutic targets. DESCRIPTION: We describe here OncomiRdbB, a comprehensive database of oncomiRs mined from different existing databases for mouse and humans along with novel oncomiRs that we have validated in human breast cancer samples. The database also lists their respective predicted targets, identified using miRanda, along with their IDs, sequences, chromosome location and detailed description. This database facilitates querying by search strings including microRNA name, sequence, accession number, target genes and organisms. The microRNA networks and their hubs with respective targets at 3'UTR, 5'UTR and exons of different pathway genes were also deciphered using the 'R' algorithm. CONCLUSION: OncomiRdbB is a comprehensive and integrated database of oncomiRs and their targets in breast cancer with multiple query options which will help enhance both understanding of the biology of breast cancer and the development of new and innovative microRNA based diagnostic tools and targets of therapeutic significance. OncomiRdbB is freely available for download through the URL link http://tdb.ccmb.res.in/OncomiRdbB/index.htm.

Phenyl 1,2,3-triazole-thymidine ligands stabilize G-quadruplex DNA, inhibit DNA synthesis and potentially reduce tumor cell proliferation over 3'-azido deoxythymidine.

Triazoles are known for their non-toxicity, higher stability and therapeutic activity. Few nucleoside (L1, L2 and L3) and non-nucleoside 1,2,3-triazoles (L4-L14) were synthesised using click chemistry and they were screened for tumor cell cytotoxicity and proliferation. Among these triazole ligands studied, nucleoside ligands exhibited higher potential than non-nucleoside ligands. The nucleoside triazole analogues, 3'-Phenyl-1,2,3- triazole-thymidine (L2) and 3'-4-Chlorophenyl-1,2,3-triazole-thymidine (L3), demonstrated higher cytotoxicity in tumor cells than in normal cells. The IC50 value for L3 was lowest (50 µM) among the ligands studied. L3 terminated cell cycle at S, G2/M phases and enhanced sub-G1 populations, manifesting induction of apoptosis in tumor cells. Confocal studies indicated that nucleoside triazole ligands (L2/L3) cause higher DNA fragmentation than other ligands. Preclinical experiments with tumor-induced mice showed greater reduction in tumor size with L3. In vitro DNA synthesis reaction with L3 exhibited higher DNA synthesis inhibition with quadruplex forming DNA (QF DNA) than non quadruplex forming DNA (NQF DNA). T(m) of quadruplex DNA increased in the presence of L3, indicating its ability to enhance stability of quadruplex DNA at elevated temperature and the results indicate that it had higher affinity towards quadruplex DNA than the other forms of DNA (like dsDNA and ssDNA). From western blot experiment, it was noticed that telomerase expression levels in the tissues of tumor-induced mice were found to be reduced on L3 treatment. Microcalorimetry results emphasise that two nucleoside triazole ligands (L2/L3) interact with quadruplex DNA with significantly higher affinity (K(d)≈10⁻7 M). Interestingly the addition of an electronegative moiety to the phenyl group of L2 enhanced its anti-proliferative activity. Though IC50 values are not significantly low with L3, the studies on series of synthetic 1,2,3-triazole ligands are useful for improving and building potential pro-apoptotic ligands.

Evidence for dynamic and multiple roles for huntingtin in Ciona intestinalis.

Although mutations in the huntingtin gene (HTT) due to poly-Q expansion cause neuropathology in humans (Huntington's disease; HD), the normal function(s) of the gene and its protein (HTT) remain obscure. With new information from recently sequenced invertebrate genomes, the study of new animal models opens the possibility of a better understanding of HTT function and its evolution. To these ends, we studied huntingtin expression pattern and dynamics in the invertebrate chordate Ciona intestinalis. Ciona huntingtin (Ci-HTT) shows a biphasic expression pattern during larval development and prior to metamorphosis. A single form of huntingtin protein is present until the early larval stages, at which time two different mass proteins become evident in the metamorphically competent larva. An antibody against Ci-HTT labeled 50 cells in the trunk mesenchyme regions in pre-hatching and hatched larvae and probably represents the distribution of the light form of the protein. Dual labeling with anti-Ci-HTT and anti-aldoketoreductase confirmed the presence of Ci-HTT in mesenchyme cells. Suppression of Ci-HTT RNA by a morpholino oligonucleotide reduced the number and apparent mobility of Ci-HTT positive cells. In Ciona, HTT expression has a dynamic temporal and spatial expression pattern that in ontogeny precedes metamorphosis. Although our results may reflect a derived function for the protein in pre- and post-metamorphic events in Ciona, we also note that as in vertebrates, there is evidence for multiple differential temporal expression, indicating that this protein probably has multiple roles in ontogeny and cell migration.