Fabrication and Characterization of Thinner Solid-State Nanopores

Solid State nanopores that are fabricated by the ion beam sculpting process and electron beam drilling have shown great promise as a sensing device for DNA and protein molecules. Even though biological pores such as the alpha-Haemolysin have been in use for quite some time, the use of solid state Nanopores in single biomolecule detection has been on the rise since the mid 1990s. Solid State nanopores have an advantage over biological pores in that they are more robust, stable, and can be sculpted to any desired size for use in translocation experiments. One of the major challenges in Nanopore fabrication by ion beam sculpting has been limited by the user\u27s ability to control the closure rate of pores in the fabrication process. Another challenge in nanopore sensing is the resolution limitation due to the thickness of the pore. This is because most of the nanopores fabricated by the ion beam sculpting method are often thicker than they should. This thesis will focus on the modification of nanopore fabrication using the ion beam sculpting system at the University of Arkansas by first baking the samples in vacuum under specified temperature conditions. Baking the samples will give the user better control over pore closure. This Thesis will also focus on thinning the sculpted pores by Reactive Ion Etching in an attempt to increase its resolution for single biomecule translocation experiments

Next-generation sequencing reveals large connected networks of intra-host HCV variants

Background: Next-generation sequencing (NGS) allows for sampling numerous viral variants from infected patients. This provides a novel opportunity to represent and study the mutational landscape of Hepatitis C Virus (HCV) within a single host. Results: Intra-host variants of the HCV E1/E2 region were extensively sampled from 58 chronically infected patients. After NGS error correction, the average number of reads and variants obtained from each sample were 3202 and 464, respectively. The distance between each pair of variants was calculated and networks were created for each patient, where each node is a variant and two nodes are connected by a link if the nucleotide distance between them is 1. The work focused on large components having > 5% of all reads, which in average account for 93.7% of all reads found in a patient. The distance between any two variants calculated over the component correlated strongly with nucleotide distances (r = 0.9499; p = 0.0001), a better correlation than the one obtained with Neighbour-Joining trees (r = 0.7624; p = 0.0001). In each patient, components were well separated, with the average distance between (6.53%) being 10 times greater than within each component (0.68%). The ratio of nonsynonymous to synonymous changes was calculated and some patients (6.9%) showed a mixture of networks under strong negative and positive selection. All components were robust to in silico stochastic sampling; even after randomly removing 85% of all reads, the largest connected component in the new subsample still involved 82.4% of remaining nodes. In vitro sampling showed that 93.02% of components present in the original sample were also found in experimental replicas, with 81.6% of reads found in both. When syringe-sharing transmission events were simulated, 91.2% of all simulated transmission events seeded all components present in the source. Conclusions: Most intra-host variants are organized into distinct single-mutation components that are: well separated from each other, represent genetic distances between viral variants, robust to sampling, reproducible and likely seeded during transmission events. Facilitated by NGS, large components offer a novel evolutionary framework for genetic analysis of intra-host viral populations and understanding transmission, immune escape and drug resistance

Assessing Staff Nurse Knowledge of Skills to Address Nurse Burnout

The cases of burnout among nurses are increasing. The problem that has been identified for this DNP project relates to the lack of hospital nursing staff knowledge regarding the skills to address nurse burnout. Many staff nurses continue to work as usual despite showing severe symptoms of nurse burnout. The purpose of the DNP project was to fill the identified practice gap, a lack of knowledge and skills among nurses on strategies of preventing and controlling burnout, by implementing an educational program. The focus for this DNP project was to develop an evidence-based nursing staff education program on nurse burnout to help increase nurses’ knowledge regarding burnout. The DNP project was informed by the conservation of resource theory and the Neumann systems model. Evidence from project participants was collected to establish the level of knowledge on burnout. An educational program was presented to 23 staff nurses via Zoom. A pre- and posttest was used to measure their change in knowledge. Ninety-eight percent of the participants attributed workload as the main cause of nurse burnout. Pre- and posttest data were analyzed using descriptive statistics, 22 participants completed both the pre- and posttest. Initial knowledge about nurse burnout was indicated by a 56.5% pretest score. After the educational presentation, participants scored an average of 98% posttest indicating that the nurses had acquired knowledge about nurse burnout. Implementing burnout reduction mechanisms through the training programs supports social change by helping nurses improve coordination, teamwork, productivity, and performance in inpatient care

Quantification of Human Immunodeficiency Virus -1 Viral Load using Nucleic Acid Sequence-based Amplification (NASBA) in North Central Nigeria

Viral load (VL) quantification is considered an integral part of the standard care in human immunodeficiency virus (HIV) infected individuals but in Nigeria as in most of sub-SaharanAfrica, this has not reached themajority of patients. We report the first field application of the NucliSens EasyQ HIV-1 platform for the real time quantification of HIV-1 VL combining NASBAamplification and real time detection with molecular beacons among HIV-1 infected individuals in north central Nigeria where the predominant HIV-1 subtypes are CRF02_AGandG.CD4 countswere enumerated using a fluorescence-activated cell sorter system. Of one hundred and forty nine (n=149) plasma sample from patients with mean age of 32 years andmade up of 77males and 72 females, fifty {n = 50 (37.9%); 28males and 22 females}hadVLs below the lower detection limit (LDL=25 IU/ml) set by the assay while eighty- two {n = 82 (62.1%); 39 males and 43 females}hadVLlevels above the LDL. Furthermore, 13 of 82 (15.9%) patientswith viral loads above the LDL had VLs between 26-1000 IU/ml while 69 (84.1%) had VLs of 1001-2400000 IU/ml. 17 (11.4%) of the samples could not be analyzed due to poor viral amplification. Among individuals with both CD4 and VL results (n=56), those with CD4 of 1-418 cell/μl presented with higher VL usually above 45,000 IU/ml when comparedwith thosewithCD4 of over 500 cell/μl. Our findings highlight the pattern, usefulness and feasibility of VL quantification by NucliSens EasyQinmonitoringHIV-1 patients inNigeria.Keywords: HIV-1,Viral load quantitation,Nigeri

Efficient error correction and haplotypes reconstruction for deep sequencing of hepatitis c amplicons

Секция 1. Защита информации и компьютерный анализ данныхWe present two new highly efficient pyrosequencing error correction algorithms: (i) k-mer – based error correction (KEC); and (ii) empirical frequency threshold (ET). Both were compared to the recently published clustering algorithm SHORAH to evaluate the relative performance using 24 experimental datasets obtained by 454-sequencing of amplicons with known sequences. We found that all three algorithms showed similar performance in terms of finding true haplotypes, but KEC and ET methods significantly outperformed SHORAH both in terms of their ability to remove false haplotypes and to estimate the frequency of true ones

Prevalence of Hepatitis B e Antigen in Chronic HBV Carriers in North-central Nigeria

Hepatitis B virus (HBV) is an important clinical problem due to its worldwide distribution and potential of adverse sequelae, including hepatocellular carcinoma (HCC). We studied the prevalence of hepatitis B virus e antigen (HBeAg) among individuals determined to be HBV surface antigen-positive (HBsAg+) and analyzed the gender/age category associated with more active HBV infection. A total of 572 HBsAg+ individuals, as determined by a double antibody sandwich ELISA method, participated in the study. They were tested for HbeAg, using a lateral flow chromatographic immunoassay. One hundred and ten individuals were found to be HBeAg-positive giving an overall prevalence of 19.2%. Of these 110 individuals, 20 (18.2%) were females, and 90 (81.8%) were males. Thus, the prevalence of HBeAg appears to be higher in males than in females (p<0.05). Our data also revealed that the prevalence of HBeAg was higher in patients between the age-group of 0-10 years and 11-20 years and appeared to decrease with increase in age. Taken together, our data show that approximately 1/5 of HBV-infected individuals are HBeAg+, suggesting that the virus is actively replicating and infecting liver-cells thereby ensuring an HBV-transmission pool within the Nigerian population. We suggest strengthening of the childhood HBV vaccination programmes, massive intervention activities, and treatment programmes, especially among young people to reverse the possible devastating effect of HBV infection. The success of these efforts will depend on our resolution to make the elimination of HBV infection a top priority on the public-health agenda as we start the second decade of this new century