HIV-2 derived lentiviral vectors: gene transfer in Parkinson's and Fabry disease models in vitro

Lentiviral vectors are prime candidate vectors for gene transfer into dividing and non-dividing cells, including neuronal cells and stem cells. For safety, HIV-2 lentiviral vectors may be better suited for gene transfer in humans than HIV-1 lentiviral vectors. HIV-2 vectors cross-packaged in HIV-1 cores may be even safer. Demonstration of the efficacy of these vectors in disease models will validate their usefulness. Parkinson's disease and Fabry disease provide excellent models for validation. Parkinson's disease is a focal degeneration of dopaminergic neurons in the brain with progressive loss of ability to produce the neurotransmitter dopamine. Current treatment entails administration of increasing doses of L-dopa, with attendant toxicity. We explore here the hypothesis that gene transfer of aromatic acid decarboxylase (AADC), a key enzyme in the pathway, will make neuronal cells more efficiently convert L-dopa into dopamine. Fabry disease on the other hand is a monogenic inherited disease, characterized by alpha-galactosidase A (AGA) deficiency, resulting in glycolipid accumulation in several cell types, including fibroblasts. Animal models for preclinical investigations of both of these diseases are available. We have designed monocistronic HIV-1 and HIV-2 vectors with the AADC transgene and monocistronic and bicistronic HIV-2 vectors with the AGA and puromycin resistance transgenes. They were packaged with either HIV-2 cores or HIV-1 cores (hybrid vectors). Gene transfer of AADC gene in neuronal cells imparted the ability on the transduced cells to efficiently convert L-dopa into dopamine. Similarly, the AGA vectors induced Fabry fibroblasts to produce high levels of AGA enzyme and caused rapid clearance of the glycolipids from the cells. Both monocistronic and bicistronic vectors were effective. Thus, the insertion of a second gene downstream in the bicistronic vector was not deleterious. In addition, both the self-packaged vectors and the cross-packaged hybrid vectors were effective in gene transfer.     

Reaktionen von α-Aminonitrilen mit Grignard-Verbindungen. III. Mitt. über Umsetzungen von α-Aminonitrilen. Einfluß von Verzweigungsgrad und Kettenlänge auf den Reaktionsverlauf

Reactions of α-Aminonitriles with Grignard-Reagents, III α-Aminonitriles are able to undergo different types of reactions with Grignard-reagents. The relationship between the course of reaction and the structure of reaction partners has been pointed out, using the reactions of 4 isomers of butylmagnesiumchloride with α-piperidinoacetonitriles as an example.     

Cloning and sequencing of cDNA of bovine N-acetylglucosamine (beta 1-4)galactosyltransferase.

Galactosyltransferases constitute a family of enzymes, each member of which transfers galactose from UDPgalactose to a specific acceptor molecule, generating a specific galactose-acceptor linkage. Two synthetic oligonucleotides, 27mer and 21mer, were synthesized, based on the amino acid sequences of two peptides derived from bovine milk N-acetylglucosaminide (beta 1-4)galactosyltransferase (EC 2.4.1.90), and used as hybridization probes to isolate cDNA clones for galactosyltransferase from a bovine mammary gland cDNA library. One of the plasmids, designated pLbGT-1, contains an insert of about 3.7 kilobases that hybridizes to both of the probes and encodes the amino acid sequences of five peptides obtained from bovine milk (beta 1-4)galactosyltransferase. A second plasmid, designated pLbGT-2, contains an insert of about 4.1 kilobases that hybridizes to only the 27mer and that encodes a polypeptide containing the sequence of the carboxyl-terminal 120 residues identical to the peptide encoded by pLbGT-1; the rest of the protein sequence, however, does not contain known sequences from bovine galactosyltransferase. The two cDNAs contain a 3'-untranslated region of about 2.7 kilobases that includes two copies of the Alu-equivalent sequences. pLbGT-1 and pLbGT-2 hybridize to mRNAs of various sizes obtained from the bovine and rat mammary gland and the human mammary tumor cell line MCF-7, with the longest mRNA from each species being around 4.5 kilobases. The results show that pLbGT-1 is a cDNA clone for bovine (beta 1-4)galactosyltransferase, and pLbGT-2 encodes a protein that is structurally and may be functionally related to transferases.     

Evaluation of Two Line Probe Assays for Rapid Detection of Mycobacterium tuberculosis,Tuberculosis (TB) Drug Resistance,and Non-TB Mycobacteria in HIV-Infected Individuals with Suspected TB

Limited performance data from line probe assays (LPAs), nucleic acid tests used for the rapid diagnosis of tuberculosis (TB), nontuberculosis mycobacteria (NTM), and Mycobacterium tuberculosis drug resistance are available for HIV-infected individuals, in whom paucibacillary TB is common. In this study, the strategy of testing sputum with GenoType MTBDRplus (MTBDR-Plus) and GenoType Direct LPA (Direct LPA) was compared to a gold standard of one mycobacterial growth indicator tube (MGIT) liquid culture. HIV-positive (HIV⁺) individuals with suspected TB from southern Africa and South America with <7 days of TB treatment had 1 sputum specimen tested with Direct LPA, MTBDR-Plus LPA, smear microscopy, MGIT, biochemical identification of mycobacterial species, and culture-based drug-susceptibility testing (DST). Of 639 participants, 59.3% were MGIT M. tuberculosis culture positive, of which 276 (72.8%) were acid-fast bacillus (AFB) smear positive. MTBDR-Plus had a sensitivity of 81.0% and a specificity of 100%, with sensitivities of 44.1% in AFB smear-negative versus 94.6% in AFB smear-positive specimens. For specimens that were positive for M. tuberculosis by MTBDR-Plus, the sensitivity and specificity for rifampin resistance were 91.7% and 96.6%, respectively, and for isoniazid (INH) they were 70.6% and 99.1%. The Direct LPA had a sensitivity of 88.4% and a specificity of 94.6% for M. tuberculosis detection, with a sensitivity of 72.5% in smear-negative specimens. Ten of 639 MGIT cultures grew Mycobacterium avium complex or Mycobacterium kansasii, half of which were detected by Direct LPA. Both LPA assays performed well in specimens from HIV-infected individuals, including in AFB smear-negative specimens, with 72.5% sensitivity for M. tuberculosis identification with the Direct LPA and 44.1% sensitivity with MTBDR-Plus. LPAs have a continued role for use in settings where rapid identification of INH resistance and clinically relevant NTM are priorities.     

Applications of glycosyltransferases in the site-specific conjugation of biomolecules and the development of a targeted drug delivery system and contrast agents for MRI

BACKGROUND: The delivery of drugs to the proposed site of action is a challenging task. Tissue and cell-specific guiding molecules are being used to carry a cargo of therapeutic molecules. The cargo molecules need to be conjugated in a site-specific manner to the therapeutic molecules such that the bioefficacy of these molecules is not compromised. METHODS: Using wild-type and mutant glycosyltransferases, the sugar moiety with a unique chemical handle is incorporated at a specific site in the cargo or therapeutic molecules, making it possible to conjugate these molecules through the chemical handle present on the modified glycan. RESULTS/CONCLUSIONS: The modified glycan residues introduced at specific sites on the cargo molecule make it possible to conjugate fluorophores for ELISA-based assays, radionuclides for imaging and immunotherapy applications, lipids for the assembly of immunoliposomes, cytotoxic drugs, cytokines, or toxins for antibody-based cancer therapy and the development of a targeted drug delivery system.     

Esophageal motility disorders—Symptomatic and manometric spectrum in Punjab,northern India

Background

Data on the spectrum of esophageal motility disorders in Indian population are scarce. We aimed to study the symptomatic and manometric profile of patients with suspected esophageal motility disorders.

Methods

Consecutive patients with esophageal symptoms who underwent esophageal high-resolution manometry (HRM) from January 2010 to December 2014 were included in this retrospective analysis of prospectively acquired data. HRM was performed with 22-channel water-perfusion system and patients classified using Chicago classification v3.0.

Results

Of the 401 patients studied [median age 43 (18–85) years; 61% males], 217 presented with dysphagia, 157 with predominant retrosternal discomfort and 27 with predominant regurgitation. Among patients with dysphagia, 43.8% had ineffective esophageal motility [IEM], 26.3% had achalasia cardia [AC], 6.9% had distal esophageal spasm [DES] and 19.4% had normal manometry [NM]. Among patients with retrosternal discomfort, 42.7% had IEM, 5.7% had AC, 4.5% had DES and 42% had NM. AC was significantly more common among patients presenting with dysphagia compared to those with retrosternal discomfort [p< 0.001] or regurgitation [p< 0.001]. NM was significantly more common among patients presenting with retrosternal symptoms compared to those with dysphagia [p< 0.001]. AC patients had longer duration of dysphagia, more frequent bolus obstruction and more weight loss compared to those with IEM or NM [p< 0.05].

Conclusion

Dysphagia was the commonest presenting symptom followed by retrosternal discomfort. Ineffective esophageal motility (not achalasia cardia) was the commonest manometric finding both among patients with dysphagia and retrosternal discomfort. This study highlights the high prevalence of IEM among patients with esophageal symptoms, which can present with dysphagia or retrosternal discomfort due to poor bolus transit.