Intermolecular Gene Conversion for the Equalization of Genome Copies in the Polyploid Haloarchaeon Haloferax volcanii: Identification of Important Proteins.

The model haloarchaeon Haloferax volcanii is polyploid with about 20 copies of its major chromosome. Recently it has been described that highly efficient intermolecular gene conversion operates in H. volcanii to equalize the chromosomal copies. In the current study, 24 genes were selected that encode proteins with orthologs involved in gene conversion or homologous recombination in archaea, bacteria, or eukaryotes. Single gene deletion strains of 22 genes and a control gene were constructed in two parent strains for a gene conversion assay; only radA and radB were shown to be essential. Protoplast fusions were used to generate strains that were heterozygous for the gene HVO_2528, encoding an enzyme for carotinoid biosynthesis. It was revealed that a lack of six of the proteins did not influence the efficiency of gene conversion, while sixteen mutants had severe gene conversion defects. Notably, lack of paralogous proteins of gene families had very different effects, e.g., mutant Δrad25b had no phenotype, while mutants Δrad25a, Δrad25c, and Δrad25d were highly compromised. Generation of a quadruple rad25 and a triple sph deletion strain also indicated that the paralogs have different functions, in contrast to sph2 and sph4, which cannot be deleted simultaneously. There was no correlation between the severity of the phenotypes and the respective transcript levels under non-stressed conditions, indicating that gene expression has to be induced at the onset of gene conversion. Phylogenetic trees of the protein families Rad3/25, MutL/S, and Sph/SMC/Rad50 were generated to unravel the history of the paralogous proteins of H. volcanii. Taken together, unselected intermolecular gene conversion in H. volcanii involves at least 16 different proteins, the molecular roles of which can be studied in detail in future projects.

One Advantage of Being Polyploid: Prokaryotes of Various Phylogenetic Groups Can Grow in the Absence of an Environmental Phosphate Source at the Expense of Their High Genome Copy Numbers.

Genomic DNA has high phosphate content; therefore, monoploid prokaryotes need an external phosphate source or an internal phosphate storage polymer for replication and cell division. For two polyploid prokaryotic species, the halophilic archaeon Haloferax volcanii and the cyanobacterium Synechocystis PCC 6803, it has been reported that they can grow in the absence of an external phosphate source by reducing the genome copy number per cell. To unravel whether this feature might be widespread in and typical for polyploid prokaryotes, three additional polyploid prokaryotic species were analyzed in the present study, i.e., the alphaproteobacterium Zymomonas mobilis, the gammaproteobacterium Azotobacter vinelandii, and the haloarchaeon Halobacterium salinarum. Polyploid cultures were incubated in the presence and in the absence of external phosphate, growth was recorded, and genome copy numbers per cell were quantified. Limited growth in the absence of phosphate was observed for all three species. Phosphate was added to phosphate-starved cultures to verify that the cells were still viable and growth-competent. Remarkably, stationary-phase cells grown in the absence or presence of phosphate did not become monoploid but stayed oligoploid with about five genome copies per cell. As a negative control, it was shown that monoploid Escherichia coli cultures did not exhibit any growth in the absence of phosphate. Taken together, all five polyploid prokaryotic species that have been characterized until now can grow in the absence of environmental phosphate by reducing their genome copy numbers, indicating that cell proliferation outperforms other evolutionary advantages of polyploidy.

Ploidy in Vibrio natriegens: Very Dynamic and Rapidly Changing Copy Numbers of Both Chromosomes.

Vibrio natriegens is the fastest-growing bacterium, with a doubling time of approximately 12-14 min. It has a high potential for basic research and biotechnological applications, e.g., it can be used for the cell-free production of (labeled) heterologous proteins, for synthetic biological applications, and for the production of various compounds. However, the ploidy level in V. natriegens remains unknown. At nine time points throughout the growth curve, we analyzed the numbers of origins and termini of both chromosomes with qPCR and the relative abundances of all genomic sites with marker frequency analyses. During the lag phase until early exponential growth, the origin copy number and origin/terminus ratio of chromosome 1 increased severalfold, but the increase was lower for chromosome 2. This increase was paralleled by an increase in cell volume. During the exponential phase, the origin/terminus ratio and cell volume decreased again. This highly dynamic and fast regulation has not yet been described for any other species. In this study, the gene dosage increase in origin-adjacent genes during the lag phase is discussed together with the nonrandom distribution of genes on the chromosomes of V. natriegens. Taken together, the results of this study provide the first comprehensive overview of the chromosome dynamics in V. natriegens and will guide the optimization of molecular biological characterization and biotechnological applications.

Genome Copy Number Quantification Revealed That the Ethanologenic Alpha-Proteobacterium Zymomonas mobilis Is Polyploid.

The alpha-proteobacterium Zymomonas mobilis is a promising biofuel producer, based on its native metabolism that efficiently converts sugars to ethanol. Therefore, it has a high potential for industrial-scale biofuel production. Two previous studies suggested that Z. mobilis strain Zm4 might not be monoploid. However, a systematic analysis of the genome copy number is still missing, in spite of the high potential importance of Z. mobilis. To get a deep insight into the ploidy level of Z. mobilis and its regulation, the genome copy numbers of three strains were quantified. The analyses revealed that, during anaerobic growth, the lab strain Zm6, the Zm6 type strain obtained from DSMZ (German Collection of Microorganisms), and the lab strain Zm4, have copy numbers of 18.9, 22.3 and 16.2, respectively, of an origin-adjacent region. The copy numbers of a terminus-adjacent region were somewhat lower with 9.3, 15.8, and 12.9, respectively. The values were similar throughout the growth curves, and they were only slightly downregulated in late stationary phase. During aerobic growth, the copy numbers of the lab strain Zm6 were much higher with around 40 origin-adjacent copies and 17 terminus-adjacent copies. However, the cells were larger during aerobic growth, and the copy numbers per µm3 cell volume were rather similar. Taken together, this first systematic analysis revealed that Z. mobilis is polyploid under regular laboratory growth conditions. The copy number is constant during growth, in contrast to many other polyploid bacteria. This knowledge should be considered in further engineering of the strain for industrial applications.

Characterization of Non-selected Intermolecular Gene Conversion in the Polyploid Haloarchaeon Haloferax volcanii.

Gene conversion is defined as the non-reciprocal transfer of genetic information from one site to a homologous, but not identical site of the genome. In prokaryotes, gene conversion can increase the variance of sequences, like in antigenic variation, but can also lead to a homogenization of sequences, like in the concerted evolution of multigene families. In contrast to these intramolecular mechanisms, the intermolecular gene conversion in polyploid prokaryotes, which leads to the equalization of the multiple genome copies, has hardly been studied. We have previously shown the intermolecular gene conversion in halophilic and methanogenic archaea is so efficient that it can be studied without selecting for conversion events. Here, we have established an approach to characterize unselected intermolecular gene conversion in Haloferax volcanii making use of two genes that encode enzymes involved in carotenoid biosynthesis. Heterozygous strains were generated by protoplast fusion, and gene conversion was quantified by phenotype analysis or/and PCR. It was verified that unselected gene conversion is extremely efficient and it was shown that gene conversion tracts are much longer than in antigenic variation or concerted evolution in bacteria. Two sites were nearly always co-converted when they were 600 bp apart, and more than 30% co-conversion even occurred when two sites were 5 kbp apart. The gene conversion frequency was independent from the extent of genome differences, and even a one nucleotide difference triggered conversion.

Exposure to Closed Loop Barriers Using Virtual Reality.

BACKGROUND: Closed loop (CL) automated insulin delivery systems are demonstrated to be safe and effective in regulating glucose levels and reducing cognitive burden in people with type 1 diabetes (T1D). However, given the limited market options and the do-it-yourself nature of most systems, it can be difficult for potential users to shape their expectations fitting them into daily lives and management routines. As such, we examined the potential feasibility of a virtual reality (VR) intervention. METHODS: A four-part VR intervention was created to expose adults with T1D to expected CL system barriers: body image, perceived hassles of using CL, deskilling fears, and unwanted social attention. Goals of the pilot were to assess feasibility and expose patients to CL. Surveys were conducted pre- and postparticipating in the VR experience. RESULTS: A total of 20 adults with T1D completed the pilot. Average time to complete the experience was 14.1 minutes (8.8-39.9). Reported VR sickness was low. Willingness to use VR was maintained in 90% (n = 18) and did not change expectations of CL in 95% (n = 19). Virtual reality changed perceived hassles of CL in 25% (n = 5) with four concerned over alarms and one connectivity issues: positive diabetes technology attitudes, confidence in managing hypoglycemia, overall perceptions of appearance, and positive affect maintained after the VR intervention. Negative affect significantly decreased after exposure and perceptions of being overweight trended toward significance. CONCLUSION: This pilot VR intervention demonstrated high potential in addressing expected barriers to uptake and usage of CL systems without decreasing enthusiasm or changing expectations of CL.

Touching The Human Neuron: User-Centric Augmented Reality Viewing and Interaction of in-vivo Cellular Confocal Laser Scanning Microscopy (CLSM) Utilizing High Resolution zStack Data Sets for Applications in Medical Education and Clinical Medicine Using GLASS and Motion Tracking Technology.

This is the first report of a prototype that allows for real-time interaction with high-resolution cellular modules using GLASS© technology. The prototype was developed* using zStack data sets which allow for real-time interaction of low polygon and direct surface models exported from primary source imaging. This paper also discusses potential educational and clinical applications of a wearable, interactive, user-centric, augmented reality visualization of cellular structures. * Prototype was developed using IMARIS© , ImageJ© , MAYA® , 3DCOAT© , UNITY© , ZBrush© , OcculusRift® , LeapMotion© , and output for multiplatform function with Google GLASS© technology as its viewing modality.

A unique foot-worn device for patients with degenerative meniscal tear.

PURPOSE: The purpose of the current study was to assess the effects of a new foot-worn device on the gait, physical function and pain in patients suffering from knee osteoarthritis (OA) who had a low-impact injury to the medial meniscus causing a degenerative meniscal tear. METHODS: A retrospective analysis of 34 patients with knee OA and a degenerative medial meniscal tear was performed. Patients underwent a gait evaluation, using an electronic walkway mat, and completed the SF-36 health survey and the WOMAC questionnaire at baseline and after 3 and 12 months of therapy. AposTherapy is a functional, biomechanical, non-invasive rehabilitation therapy consisting of a foot-worn device that is individually calibrated to each patient and is used during activities of daily living. Repeated-measures analyses were performed to compare gait parameters and self-evaluation questionnaires between baseline, and 3 and 12 months. RESULTS: Significant improvements were found in gait velocity, step length and single-limb support of the involved knee following 12 weeks of therapy (all p < 0.01), alongside an improvement in limb symmetry. These results were maintained at the 12-month follow-up examination. Significant improvements were also found in all three domains of the WOMAC index (pain, stiffness and physical function) and in the SF-36 Physical Health Scale and the SF-36 Mental Health Scale (all p < 0.01). CONCLUSIONS: Patients with knee OA and a degenerative medial meniscal tear using a biomechanical foot-worn device for a year showed improvement in gait, physical function and pain. Based on the findings of this study, it can be postulated that this biomechanical device might have a positive effect on this population. LEVEL OF EVIDENCE: Therapeutic study, Level IV.

The question of ethnic variability and the Darwinian significance of physiological neonatal jaundice in East Asian populations.

Recent work in Darwinian medicine has suggested that physiological neonatal jaundice (PNJ) might serve an adaptive function in scavenging reactive oxygen species that in later life are removed by the mature antioxidant enzyme system in the liver. This treatise examines this hypothesis in light of novel epidemiological and genetic findings which suggest that the incidence of PNJ is significantly increased in East Asian populations. Though found across all ethnic groups, it has been established that neonates of East Asian origin are at a significantly greater risk of developing PNJ, with more than one studying finding the incidence to be near double. For any Darwinian explanation of physiological neonatal jaundice to be considered in clinical circles, it is essential that the elevated incidence of PNJ in this population be explained both mechanistically and in terms of adaptation. Recent work has linked PNJ to a specific enzyme polymorphism, a variation of the UGT1A1 gene, in the glucoronidation pathway which is essential for bilirubin metabolism and is strongly correlated with ethnic origin. In this paper it is hypothesized that the elevated incidence of PNJ in East Asian populations is not random or due to a flaw in the system but rather due to an evolved mechanism. Two potential pressures which might have selected for an elevated neonatal bilirubin in East Asian populations versus other ethnic groups are a diminished ability to reduce harmful oxidant radicals due to variations the P450 liver metabolic pathway and the endemic nature of Hepatitis B in the Asia-Pacific region. This is the first work to attempt to explain PNJ through a Darwinian yet clinically relevant lens while suggesting a specific proximate mechanism that is correlated with a pre-existing evolutionary environment and can be associated with differential reproductive success.