The Synthesis of Branched Oligonucleotides as Signal Amplification Multimers for Use in Nucleic Acid Assays

Abstract

Branched oligonucleotides have been synthesized using phosphoramidite derivatives with two protected hydroxyl functions. These molecules are employed for a label amplification strategy used in DNA probe diagnostics.     

Pyrimidine and Purine Analogues,Effects on Cell Cycle Regulation and the Role of Cell Cycle Inhibitors to Enhance Their Cytotoxicity

In anti-cancer treatment, deoxynucleoside analogues are widely used in combination chemotherapy. Improvement can be achieved by rational design of novel combinations with cell cycle inhibitors. These compounds inhibit protein kinases, preventing the cell cycle from continuing when affected by deoxynucleoside analogs. The efficacy is dependent on the site of cell cycle inhibition, whether multiple cyclin-dependent kinases are inhibited and whether the inhibitors should be given before or after the deoxynucleoside analogs. The action of cell cycle inhibition in vivo may be limited by unfavorable pharmacokinetics. Preclinical and clinical studies will be discussed, aiming to design improved future strategies.     

Multiple Complex Congenital Malformations in a Rabbit Kit (Oryctolagus cuniculi)

Congenital malformations may occur during early embryogenesis in cases of genetic abnormalities or various environmental factors. Affected subjects most often have only one or 2 abnormalities; subjects rarely have several unrelated congenital defects. Here we describe a case of a stillborn New Zealand white rabbit with multiple complex congenital malformations, including synophthalmia, holoprosencephaly, gastroschisis, and a supernumerary hindlimb, among other anomalies. There was no historical exposure to teratogens or other known environmental causes. Although not confirmed, this case was most likely a rare spontaneous genetic event.Congenital malformations occur when there is derangement of the embryologic developmental process. Neural development and organogenesis is a critical time of development that occurs during early embryogenesis.^2,37 Congenital malformations that manifest at this stage of development may occur in association with various genetic abnormalities, such as point mutations and chromosomal abnormalities.^25,29 In addition, environmental factors, including maternal health status, nutritional deficiencies, and exposure to teratogenic drugs or chemicals, may play a role in the development of congenital malformations.^12,25 However, in 65% to 75% of human cases, the cause is unknown, resulting from a complex set of events such as polygenic or multifactorial genetic disorders, spontaneous genetic errors, and synergistic interactions of teratogens.^3,25 Approximately 78% of human cases demonstrate only a single developmental malformation, with cardiovascular defects accounting for approximately 30% to 35% of organ defects. Cases of more than 2 or 3 malformations in a single person are extremely rare.²⁸Here we describe a New Zealand white rabbit that was stillborn with numerous complex developmental abnormalities, including synophthalmia, a supraoptic proboscis, holoprosencephaly with other associated craniofacial deformities, Chiari malformation type I, gastroschisis, a supernumerary hindlimb, a fused (horseshoe) kidney with a supernumerary kidney, and male pseudohermaphroditism.     

The glyceraldehyde-3-phosphate dehydrogenase promoter of the food yeast Candida utilis strain NRRL Y-660 is functional in Agrobacterium tumefaciens

The glyceraldehyde-3-phosphate dehydrogenase promoter of the food yeast Candida utilis strain NRRL Y-660 was cloned to create a novel integrative vector for Agrobacterium tumefaciens-mediated transformation. The new binary vector harbors β-glucuronidase activity as reporter and kanamicin/geneticin resistance as selection marker. Recombinant clones of A. tumefaciens show kanamycin resistance and high β-glucuronidase activity under the control of the C. utilis promoter. This finding can be explained by the presence of a prokaryotic core in the yeast promoter, predicted by in silico analysis of the sequence. This is the first report about functionality of a yeast promoter in A. tumefaciens.     

The central role of protein S12 in organizing the structure of the decoding site of the ribosome

The ribosome decodes mRNA by monitoring the geometry of codon–anticodon base-pairing using a set of universally conserved 16S rRNA nucleotides within the conformationally dynamic decoding site. By applying single-molecule FRET and X-ray crystallography, we have determined that conditional-lethal, streptomycin-dependence mutations in ribosomal protein S12 interfere with tRNA selection by allowing conformational distortions of the decoding site that impair GTPase activation of EF-Tu during the tRNA selection process. Distortions in the decoding site are reversed by streptomycin or by a second-site suppressor mutation in 16S rRNA. These observations encourage a refinement of the current model for decoding, wherein ribosomal protein S12 and the decoding site collaborate to optimize codon recognition and substrate discrimination during the early stages of the tRNA selection process.     

Metastasis Suppressor microRNA-335 Targets the Formin Family of Actin Nucleators

MiRNAs can have pleiotropic effects by targeting multiple genes belonging to diverse signalling networks. Alternatively, miRNAs can enhance the potency of their cellular effects by targeting multiple genes within the same genetic pathway. Previously, we and others have demonstrated that miR-335 is a potent suppressor of tumour cell migration, invasion and metastasis, in part by targeting several genes involved in these cellular processes, including ROCK1, MAPK1, LRG1, SP1 and SOX4. Here, we demonstrate that direct targeting of multiple members of the formin family of actin nucleators contributes to the inhibitory effects of miR-335 in neuroblastoma cells. We demonstrate that miR-335 regulates the expression of at least five formin family members and validate three family members, FMNL3, FMN2 and DAAM2, as direct targets of miR-335. The contribution of the formin family genes to cancer progression and metastasis has recently begun to emerge and here we demonstrate for the first time the ability of FMN2 and DAAM2 to regulate tumour cell migration and invasion, using siRNA-mediated inhibition of each of these formin genes. Finally, we demonstrate that the formin genes, in particular FMNL3, are responsible for the protrusion of actin-rich filopodia structures that contribute to the enhanced migratory and invasive potential associated with reduced expression of miR-335. Thus, direct targeting of the formin family contributes to the metastasis suppressing abilities of miR-335 by providing a direct regulatory link to the actin assembly machinery of the cell. We conclude that miR-335 is a master regulator of tumour cell migration and invasion by directly targeting a plethora of genes that effectively control cell migratory processes.     

Chikungunya Virus Infection Results in Higher and Persistent Viral Replication in Aged Rhesus Macaques Due to Defects in Anti-Viral Immunity

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne Alphavirus that causes a clinical disease involving fever, myalgia, nausea and rash. The distinguishing feature of CHIKV infection is the severe debilitating poly-arthralgia that may persist for several months after viral clearance. Since its re-emergence in 2004, CHIKV has spread from the Indian Ocean region to new locations including metropolitan Europe, Japan, and even the United States. The risk of importing CHIKV to new areas of the world is increasing due to high levels of viremia in infected individuals as well as the recent adaptation of the virus to the mosquito species Aedes albopictus. CHIKV re-emergence is also associated with new clinical complications including severe morbidity and, for the first time, mortality. In this study, we characterized disease progression and host immune responses in adult and aged Rhesus macaques infected with either the recent CHIKV outbreak strain La Reunion (LR) or the West African strain 37997. Our results indicate that following intravenous infection and regardless of the virus used, Rhesus macaques become viremic between days 1–5 post infection. While adult animals are able to control viral infection, aged animals show persistent virus in the spleen. Virus-specific T cell responses in the aged animals were reduced compared to adult animals and the B cell responses were also delayed and reduced in aged animals. Interestingly, regardless of age, T cell and antibody responses were more robust in animals infected with LR compared to 37997 CHIKV strain. Taken together these data suggest that the reduced immune responses in the aged animals promotes long-term virus persistence in CHIKV-LR infected Rhesus monkeys.     

Syndromic Algorithms for Detection of Gambiense Human African Trypanosomiasis in South Sudan

Background

Active screening by mobile teams is considered the best method for detecting human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense but the current funding context in many post-conflict countries limits this approach. As an alternative, non-specialist health care workers (HCWs) in peripheral health facilities could be trained to identify potential cases who need testing based on their symptoms. We explored the predictive value of syndromic referral algorithms to identify symptomatic cases of HAT among a treatment-seeking population in Nimule, South Sudan.

Methodology/Principal Findings

Symptom data from 462 patients (27 cases) presenting for a HAT test via passive screening over a 7 month period were collected to construct and evaluate over 14,000 four item syndromic algorithms considered simple enough to be used by peripheral HCWs. For comparison, algorithms developed in other settings were also tested on our data, and a panel of expert HAT clinicians were asked to make referral decisions based on the symptom dataset. The best performing algorithms consisted of three core symptoms (sleep problems, neurological problems and weight loss), with or without a history of oedema, cervical adenopathy or proximity to livestock. They had a sensitivity of 88.9–92.6%, a negative predictive value of up to 98.8% and a positive predictive value in this context of 8.4–8.7%. In terms of sensitivity, these out-performed more complex algorithms identified in other studies, as well as the expert panel. The best-performing algorithm is predicted to identify about 9/10 treatment-seeking HAT cases, though only 1/10 patients referred would test positive.

Conclusions/Significance

In the absence of regular active screening, improving referrals of HAT patients through other means is essential. Systematic use of syndromic algorithms by peripheral HCWs has the potential to increase case detection and would increase their participation in HAT programmes. The algorithms proposed here, though promising, should be validated elsewhere.