Stable HIV decoy receptor expression after in vivo HSC transduction in mice and NHPs: Safety and efficacy in protection from SHIV.

We aim to develop an in vivo hematopoietic stem cell (HSC) gene therapy approach for persistent control/protection of HIV-1 infection based on the stable expression of a secreted decoy protein for HIV receptors CD4 and CCR5 (eCD4-Ig) from blood cells. HSCs in mice and a rhesus macaque were mobilized from the bone marrow and transduced by an intravenous injection of HSC-tropic, integrating HDAd5/35++ vectors expressing rhesus eCD4-Ig. In vivo HSC transduction/selection resulted in stable serum eCD4-Ig levels of ∼100 µg/mL (mice) and >20 µg/mL (rhesus) with half maximal inhibitory concentrations (IC50s) of 1 µg/mL measured by an HIV neutralization assay. After simian-human-immunodeficiency virus D (SHIV.D) challenge of rhesus macaques injected with HDAd-eCD4-Ig or a control HDAd5/35++ vector, peak plasma viral load levels were ∼50-fold lower in the eCD4-Ig-expressing animal. Furthermore, the viral load was lower in tissues with the highest eCD4-Ig expression, specifically the spleen and lymph nodes. SHIV.D challenge triggered a selective expansion of transduced CD4+CCR5+ cells, thereby increasing serum eCD4-Ig levels. The latter, however, broke immune tolerance and triggered anti-eCD4-Ig antibody responses, which could have contributed to the inability to eliminate SHIV.D. Our data will guide us in the improvement of the in vivo approach. Clearly, our conclusions need to be validated in larger animal cohorts.

Intravenous dexamethasone does not prolong the duration of pudendal nerve block in infants and children undergoing hypospadias surgery: A randomized clinical trial.

BACKGROUND: The administration of intravenous dexamethasone increases the duration of neuraxial block and improves the quality of analgesia. However, little is known about these effects of dexamethasone on peripheral nerve blocks in children. AIMS: In this study, we aimed to investigate the benefit of intravenous dexamethasone for enhancing the effect of pudendal block on postoperative analgesia in children who underwent hypospadias surgery. METHODS: In total, 46 children aged 6-36 months who underwent hypospadias surgery were randomly allocated to either a control group (normal saline, group C) or dexamethasone group (0.5 mg/kg, group D). Pudendal block was performed before the surgery using 0.3 mL/kg of 0.225% ropivacaine on both sides. Parents were instructed to press the patient-controlled analgesia bolus button when their children's pain score was >4 points. The primary outcome measure was the time at which the first patient-controlled analgesia by proxy bolus dose was administered. The secondary outcome measures were pain score, number of patient-controlled analgesia administration by proxy bolus attempts, number of rescue analgesics required, total amount of fentanyl administered, and overall parental satisfaction. RESULTS: The time of first patient-controlled analgesia bolus administration by proxy was not different between the control and dexamethasone groups (5.6 [5.2, 8.8] h versus 6.5 [5.4, 8.1] h, hazard ratio 0.8, 95% confidence intervals 0.43 to 1.47, p = .46). There were no statistically significant differences among the secondary outcomes. CONCLUSIONS: Administration of intravenous dexamethasone did not enhance the duration of pudendal nerve block in infants and children aged 6-36 months who underwent hypospadias surgery.

Design of a Self-Measuring Device Based on Bioelectrical Impedance Analysis for Regular Monitoring of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic disease, in which permanent joint deformation is largely preventable with the timely introduction of appropriate treatment strategies. However, there is no consensus for patients with RA to monitor their progress and communicate it to the rheumatologist till the condition progresses to remission. In response to this unmet need, we proposed the design of a self-measuring device based on bioelectrical impedance analysis (BIA) for regular monitoring of inflammation levels. Twenty joints of both hands were measured to monitor trends in inflammation levels. Three electrodes were used to measure two joints of each finger. A central electrode was used for two consecutive measurements. A suitable form factor for the device was proposed for the vertical placement of the hand. To ensure the stability of measurements, an air cushion was incorporated into the back of the hand, hand containers were designed on both sides, and a mobile application was designed. We conducted a convergence-assessment experiment with five air pressures to validate the consistency and convergence of bioimpedance measurements. A heuristic evaluation of the usability around the product and mobile application was conducted in parallel by six subject matter experts and validated the design. This study underscores the significance of considering patients' disease activity during intervals between hospital visits and introduces a novel approach to self-RA care.

Reversal of dual epigenetic repression of non-canonical Wnt-5a normalises diabetic corneal epithelial wound healing and stem cells.

AIMS/HYPOTHESIS: Diabetes is associated with epigenetic modifications including DNA methylation and miRNA changes. Diabetic complications in the cornea can cause persistent epithelial defects and impaired wound healing due to limbal epithelial stem cell (LESC) dysfunction. In this study, we aimed to uncover epigenetic alterations in diabetic vs non-diabetic human limbal epithelial cells (LEC) enriched in LESC and identify new diabetic markers that can be targeted for therapy to normalise corneal epithelial wound healing and stem cell expression. METHODS: Human LEC were isolated, or organ-cultured corneas were obtained, from autopsy eyes from non-diabetic (59.87±20.89 years) and diabetic (71.93±9.29 years) donors. The groups were not statistically different in age. DNA was extracted from LEC for methylation analysis using Illumina Infinium 850K MethylationEPIC BeadChip and protein was extracted for Wnt phospho array analysis. Wound healing was studied using a scratch assay in LEC or 1-heptanol wounds in organ-cultured corneas. Organ-cultured corneas and LEC were transfected with WNT5A siRNA, miR-203a mimic or miR-203a inhibitor or were treated with recombinant Wnt-5a (200 ng/ml), DNA methylation inhibitor zebularine (1-20 µmol/l) or biodegradable nanobioconjugates (NBCs) based on polymalic acid scaffold containing antisense oligonucleotide (AON) to miR-203a or a control scrambled AON (15-20 µmol/l). RESULTS: There was significant differential DNA methylation between diabetic and non-diabetic LEC. WNT5A promoter was hypermethylated in diabetic LEC accompanied with markedly decreased Wnt-5a protein. Treatment of diabetic LEC and organ-cultured corneas with exogenous Wnt-5a accelerated wound healing by 1.4-fold (p<0.05) and 37% (p<0.05), respectively, and increased LESC and diabetic marker expression. Wnt-5a treatment in diabetic LEC increased the phosphorylation of members of the Ca2+-dependent non-canonical pathway (phospholipase Cγ1 and protein kinase Cß; by 1.15-fold [p<0.05] and 1.36-fold [p<0.05], respectively). In diabetic LEC, zebularine treatment increased the levels of Wnt-5a by 1.37-fold (p<0.01)and stimulated wound healing in a dose-dependent manner with a 1.6-fold (p<0.01) increase by 24 h. Moreover, zebularine also improved wound healing by 30% (p<0.01) in diabetic organ-cultured corneas and increased LESC and diabetic marker expression. Transfection of these cells with WNT5A siRNA abrogated wound healing stimulation by zebularine, suggesting that its effect was primarily due to inhibition of WNT5A hypermethylation. Treatment of diabetic LEC and organ-cultured corneas with NBC enhanced wound healing by 1.4-fold (p<0.01) and 23.3% (p<0.05), respectively, with increased expression of LESC and diabetic markers. CONCLUSIONS/INTERPRETATION: We provide the first account of epigenetic changes in diabetic corneas including dual inhibition of WNT5A by DNA methylation and miRNA action. Overall, Wnt-5a is a new corneal epithelial wound healing stimulator that can be targeted to improve wound healing and stem cells in the diabetic cornea. DATA AVAILABILITY: The DNA methylation dataset is available from the public GEO repository under accession no. GSE229328 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229328 ).

Surface figure correction with roughness reduction using carbon-doped platinum film for high-precision X-ray mirror fabrication.

To obtain the surface shape of an X-ray mirror with high precision, a differential deposition method was used instead of a direct removal method. To modify the mirror surface shape using the differential deposition method, it is necessary to coat it with a thick film, and the co-deposition method is used to suppress the increase in surface roughness. The addition of C to the Pt thin film, which is often used as an X-ray optical thin film, resulted in lower surface roughness compared with that with the Pt coating alone, and the stress change according to the thin film thickness was evaluated. Differential deposition controls the speed of the substrate during coating based on continuous motion. The stage was controlled by calculating the dwell time through deconvolution calculations based on the accurate measurement of the unit coating distribution and target shape. We successfully fabricated an X-ray mirror with high precision. This study indicated that an X-ray mirror surface could be manufactured by modifying the surface shape at a micrometer level through the coating. Changing the shape of existing mirrors can not only result in the manufacture of high-precision X-ray mirrors but also improve their performance.

High-performance resistive random access memory using two-dimensional electron gas electrode and its switching mechanism analysis.

In this study, a two-dimensional electron gas (2DEG), which is a conductive layer formed at the interface of Al2O3and TiO2, was used as an electrode for resistive random access memory (RRAM) and implemented in a cell size down to 30 nm. For an RRAM device comprising W/2DEG/TiO2/W, we confirmed that the dominant switching mechanism changed from interfacial to filamentary as the cell size decreased from 500 nm to 30 nm. Through analyses of changes in forming characteristics and conduction mechanisms in the low resistive state depending on the cell size, it was identified that the 2DEG acted as an oxygen-scavenging layer of TiO2during the resistive switching process. By comparing the switching characteristics of RRAM devices with and without 2DEG for a 30 nm cell size, we confirmed that a high-performance 2DEG RRAM was realized, with highly uniform current-voltage characteristics, a low operating voltage (∼1 V), and a high on/off ratio (>102). Finally, the applicability of the proposed device to a crossbar array was validated by evaluating 1S1R operation with an NbO2-based selector. Considering the improved switching uniformity, the 2DEG RRAM shows promise for high-density memory applications.

Enhancement of NbO2-based oscillator neuron device performance via cryogenic operation.

The Niobium Dioxide (NbO2) oscillator neuron has garnered significant interest because of its simple structure compared to conventional CMOS-based circuits. However, the limited on/off resistance ratio narrows the range of series resistances that satisfy the self-oscillation conditions and limits its use in large-scale synaptic arrays. In this study, we report the possibility of improving the performance of NbO2-based oscillator neuron devices through cryogenic operation. The study emphasizes two crucial parameters: the on/off resistance ratio and the oscillation amplitude, both of which are essential for accurate weighted sum classification. The data suggest that these parameters can be effectively enhanced under cryogenic conditions. In addition, we revealed that 120 K is the optimal temperature for cryogenic operation, as it represents the temperature where the on/off resistance ratio ceases to increase. As a result, we revealed that the series resistance range satisfying the self-oscillation condition in a single oscillator increases from 20 to 126 kΩ. The research also probes the maximum possible array size at each temperature. At 300 K, representation is only possible for a 5 × 5 array, but at 120 K, a 30 × 30 array can be represented as a frequency. The evidence implies that the 120 K conditions not only broaden the range of series resistors that can be connected to a single oscillator but also increases the array size, thereby representing different weighted sum currents as frequencies. The research indicates that using carefully optimized cryogenic operation could be a viable method to enhance the necessary NbO2properties for an oscillator neuron device.

Discovery of Leishmania donovani topoisomerase IB selective inhibitors by targeting protein-protein interactions between the large and small subunits.

Visceral leishmaniasis (VL) is a fatal infectious disease caused by viscerotropic parasitic species of Leishmania. Current treatment options are often ineffective and toxic, and more importantly, there are no clinically validated drug targets available to develop next generation therapeutics against VL. Topoisomerase IB (TopIB) is an essential enzyme for Leishmania survival. The enzyme is organized as a bi-subunit that is distinct from the monomeric topoisomerase I of human. Based on this unique feature, we synthesized peptides composed of partial amino acid sequences of small subunit of Leishmania donovani (Ld) TopIB to confirm a decrease in catalytic activity by interfering the interaction between the two subunits. One of the synthetic peptides, covering essential amino acids for catalytic activity of LdTopIB, interrupted the enzymatic activity. Next, we examined 151 compounds selected from virtual screening in a functional assay and identified three LRL-TP compounds with a significant decrease in LdTopIB activity (IC50 of LRL-TP-85: 1.3 µM; LRL-TP-94: 2.9 µM; and LRL-TP-101: 35.3 µM) and no effects on Homo sapiens (Hs) TopIB activity. Based on molecular docking, the protonated tertiary amine of inhibitors formed key interactions with S415 of the large subunit. The EC50 values of LRL-TP-85, LRL-TP-94, and LRL-TP-101 were respectively 4.9, 1.4, and 27.8 µM in extracellular promastigote assay and 34.0, 53.7, and 11.4 µM in intracellular amastigote assay. Overall, we validated the protein-protein interaction site of LdTopIB as a potential drug target and identified small molecule inhibitors with anti-leishmanial activity.

Tracheal intubation with or without a neuromuscular blocking agent for a short surgical procedure in children: Prospective, randomized, double-blind trial.

BACKGROUND: Although a neuromuscular blocking agent during induction of anesthesia is the standard of care in adults, some pediatric anesthesiologists remain concerned about their use for several reasons. Therefore, propofol and short-acting opioids with a moderate concentration of sevoflurane have been used as alternatives to a neuromuscular blocking agent. AIMS: This study compared propofol, alfentanil, and rocuronium to determine the optimal anesthetic agent for intubation conditions as well as emergence in a short pediatric procedure. METHODS: In this prospective, randomized, double-blind study, 114 pediatric patients, aged 1-9 years, were randomly assigned to one of three groups receiving either propofol 2 mg kg-1 (propofol group), alfentanil 14 mcg kg-1 (alfentanil group), or rocuronium 0.3 mg kg-1 (rocuronium group). The primary outcome was intubating conditions, which were evaluated 90 s after test drug administration. Vital signs were recorded during the intubation period. Complications during and after emergence, time to recovery, airway-related complications, and severity of emergence agitation were recorded. RESULTS: Compared with the propofol group (60%), significantly more excellent intubating conditions were observed in the alfentanil group (97%, percent difference -37, 95% confidence interval (CI) -54.4--21.0, p < .001) and the rocuronium group (87%, percent difference -27, 95% CI -46.5--8.2, p = .041). Hemodynamic responses were different between the rocuronium and alfentanil groups, although the incidence of adverse events was not different among the three groups. The emergence duration was only statistically different between the rocuronium group [9.9 ± 3.2 min] and the propofol group [11.7 ± 2.2 min] (difference 95% CI 0.667-3.583, p = .001), while that of the alfentanil group [10.9 ± 2.4 min] was comparable with the other groups. CONCLUSIONS: Both 0.3 mg kg-1 rocuronium and 14 µg kg-1 alfentanil are superior adjuncts for tracheal intubation in children undergoing frenulectomy in comparison with 2 mg kg-1 propofol. Hemodynamic adverse events and recovery profiles were comparable among the three groups.

Identification of inhibitors of Bcl-2 family protein-protein interaction by combining the BRET screening platform with virtual screening.

Bcl-2 family proteins play key roles in tumor initiation, progression, and resistance to therapy. Therefore, the protein-protein interactions (PPIs) between the pro-survival proteins, B-cell lymphoma (Bcl)-2 and Bcl-xL, and the pro-apoptotic proteins, Bax and Bak, could be attractive therapeutic targets for anti-cancer drug discovery. Here, we found new small molecules, BIP-A1001 and BIP-A2001 that modulated Bak/Bax and Bcl-xL interactions by combining the Nanoluc/YFP-based bioluminescence resonance energy transfer (BRET) assay with structure based virtual screening. In addition, we chose compounds with similar structures to BIP-A1001 and BIP-A2001 and tested their inhibitory effects using the BRET assay as a dose-response function. The results indicated that identifying compounds that inhibit interactions between Bak/Bax and Bcl-xL could be a promising approach to enhance cancer therapy.

Reactivation of γ-globin in adult ß-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing.

Disorders involving ß-globin gene mutations, primarily ß-thalassemia and sickle cell disease, represent a major target for hematopoietic stem/progenitor cell (HSPC) gene therapy. This includes CRISPR/Cas9-mediated genome editing approaches in adult CD34+ cells aimed toward the reactivation of fetal γ-globin expression in red blood cells. Because models involving erythroid differentiation of CD34+ cells have limitations in assessing γ-globin reactivation, we focused on human ß-globin locus-transgenic (ß-YAC) mice. We used a helper-dependent human CD46-targeting adenovirus vector expressing CRISPR/Cas9 (HDAd-HBG-CRISPR) to disrupt a repressor binding region within the γ-globin promoter. We transduced HSPCs from ß-YAC/human CD46-transgenic mice ex vivo and subsequently transplanted them into irradiated recipients. Furthermore, we used an in vivo HSPC transduction approach that involves HSPC mobilization and the intravenous injection of HDAd-HBG-CRISPR into ß-YAC/CD46-transgenic mice. In both models, we demonstrated efficient target site disruption, resulting in a pronounced switch from human ß- to γ-globin expression in red blood cells of adult mice that was maintained after secondary transplantation of HSPCs. In long-term follow-up studies, we did not detect hematological abnormalities, indicating that HBG promoter editing does not negatively affect hematopoiesis. This is the first study that shows successful in vivo HSPC genome editing by CRISPR/Cas9.

Production of heterozygous alpha 1,3-galactosyltransferase (GGTA1) knock-out transgenic miniature pigs expressing human CD39.

Production of transgenic pigs for use as xenotransplant donors is a solution to the severe shortage of human organs for transplantation. The first barrier to successful xenotransplantation is hyperacute rejection, a rapid, massive humoral immune response directed against the pig carbohydrate GGTA1 epitope. Platelet activation, adherence, and clumping, all major features of thrombotic microangiopathy, are inevitable results of immune-mediated transplant rejection. Human CD39 rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. In this study, we developed a vector-based strategy for ablation of GGTA1 function and concurrent expression of human CD39 (hCD39). An hCD39 expression cassette was constructed to target exon 4 of GGTA1. We established heterozygous GGTA1 knock-out cell lines expressing hCD39 from pig ear fibroblasts for somatic cell nuclear transfer (SCNT). We also described production of heterozygous GGTA1 knock-out piglets expressing hCD39 and analyzed expression and function of the transgene. Human CD39 was expressed in heart, kidney and aorta. Human CD39 knock-in heterozygous ear fibroblast from transgenic cloned pigs, but not in non-transgenic pig's cells. Expression of GGTA1 gene was lower in the knock-in heterozygous ear fibroblast from transgenic pigs compared to the non-transgenic pig's cell. The peripheral blood mononuclear cells (PBMC) from the transgenic pigs were more resistant to lysis by pooled complement-preserved normal human serum than that from wild type (WT) pig. Accordingly, GGTA1 mutated piglets expressing hCD39 will provide a new organ source for xenotransplantation research.

Nanoluciferase signal brightness using furimazine substrates opens bioluminescence resonance energy transfer to widefield microscopy.

Fluorescence and bioluminescence resonance energy transfer (FRET, BRET) techniques are powerful tools for studying protein-protein interactions in cellular assays. In contrast to fluorescent proteins, chemiluminescent proteins do not require excitation light, known to trigger autofluorescence, phototoxicity, and photobleaching. Regrettably, low signal intensity of luciferase systems restricts their usage as they require specialized microscopes equipped with ultra low-light imaging cameras. In this study, we report that bioluminescence quantification in living cells using a standard widefield automated microscope dedicated to screening and high content analysis is possible with the newer luciferase systems, Nanoluciferase (Nluc). With such equipment, we showed that robust intramolecular BRET can be measured using a combination of Nluc and yellow fluorescent protein (YFP). Using the human Superoxide Dismutase 1 (SOD1) dimer model, we next validated that intermolecular BRET could be quantified at a single cell level. The enhanced signal brightness of Nluc enabling BRET imaging to widefield microscopy shows strong potential to open up single cell protein-protein interactions studies to a wider audience. © 2016 International Society for Advancement of Cytometry.

Deposition of bioactive human epidermal growth factor in the egg white of transgenic hens using an oviduct-specific minisynthetic promoter.

Currently, transgenic animals have found a wide range of industrial applications and are invaluable in various fields of basic research. Notably, deposition of transgene-encoded proteins in the egg white (EW) of hens affords optimal production of genetically engineered biomaterials. In the present study, we developed a minisynthetic promoter modulating transgene transcription specifically in the hen's oviduct, and assayed the bioactivity of human epidermal growth factor (hEGF) driven by that promoter, after partial purification of epidermal growth factor (EGF) from transgenic hen eggs. Our minisynthetic promoter driving expression of chicken codon-optimized human epidermal growth factor (cEGF) features 2 consecutive estrogen response elements of the ovalbumin (OV) promoter, ligated with a 3.0 kb OV promoter region carrying OV regulatory elements, and a 5'-UTR. Subsequently, a 3'-UTR carrying the poly-A tail sequence of the OV gene was added after incorporation of the cEGF transgene. Finally, we partially purified cEGF from transgenic hen eggs and evaluated the biofunctional activities thereof in vitro and in vivo. In the in vitro assay, EW-derived hEGF exhibited a proliferative effect on HeLa cells similar to that of commercial hEGF. In the in vivo assay, compared to the nontreated control, transgenic hen egg-derived EGF afforded slightly higher levels of re-epithelialization (via fibroplasia) and neovascularization of wounded skin of miniature pigs than did the commercial material. In conclusion, transgenic hens may be used to produce genetically engineered bioactive biomaterials driven by an oviduct-specific minisynthetic promoter.

Soft hydrated sliding interfaces as complex fluids.

Hydrogel surfaces are biomimics for sensing and mobility systems in the body such as the eyes and large joints due to their important characteristics of flexibility, permeability, and integrated aqueous component. Recent studies have shown polymer concentration gradients resulting in a less dense region in the top micrometers of the surface. Under shear, this gradient is hypothesized to drive lubrication behavior due to its rheological similarity to a semi-dilute polymer solution. In this work we map 3 distinct lubricating regimes between a polyacrylamide surface and an aluminum annulus using stepped-velocity tribo-rheometry over 5 decades of sliding speed in increasing and decreasing steps. These regimes, characterized by weakly or strongly time-dependent response and thixotropy-like hysteresis, provide the skeleton of a lubrication curve for hydrogel-against-hard material interfaces and support hypotheses of polymer mechanics-driven lubrication. Tribo-rheometry is particularly suited to uncover the lubrication mechanisms of complex interfaces such as are formed with hydrated hydrogel surfaces and biological surfaces.

'People say that we are already dead much as we can still walk': a qualitative investigation of community and couples' understanding of HIV serodiscordance in rural Uganda.

BACKGROUND: Stable, co-habiting HIV serodiscordant couples are a key population in terms of heterosexual transmission in sub-Saharan Africa. Despite the wide availability of antiretroviral treatment and HIV educational programs, heterosexual transmission continues to drive the HIV epidemic in Africa. To investigate some of the factors involved in transmission or maintenance of serodiscordant status, we designed a study to examine participants' understanding of HIV serodiscordance and the implications this posed for their HIV prevention practices. METHODS: In-depth interviews were conducted with 28 serodiscordant couples enrolled in a treatment-as-prevention study in Jinja, Uganda. Participants were asked questions regarding sexual behaviour, beliefs in treatment and prevention, participants' and communities' understanding and context around HIV serodiscordance. Qualitative framework analysis capturing several main themes was carried out by a team of four members, and was cross-checked for consistency. RESULTS: It was found that most couples had difficulty explaining the phenomenon of serodiscordance and tended to be confused regarding prevention. Many individuals still held beliefs in pseudoscientific explanations for HIV susceptibility such as blood type and blood "strength". The participants' trust of treatment and medical services were well established. However, the communities' views of both serodiscordance and treatment were more pessimistic and wrought with mistrust. Stigmatization of serodiscordance and HIV-positive status were reported frequently. CONCLUSIONS: The results indicate that despite years of treatment and prevention methods being available, stigmatization and mistrust persist in the communities of HIV-affected individuals and may directly contribute to new cases and seroconversion. We suggest that to optimize the effects of HIV treatment and prevention, clear education and support of such methods are sorely needed in sub-Saharan African communities.

Analysis of Swine Leukocyte Antigen Haplotypes in Yucatan Miniature Pigs Used as Biomedical Model Animal.

The porcine major histocompatibility complex (MHC) is called swine leukocyte antigen (SLA), which controls immune responses and transplantation reactions. The SLA is mapped on pig chromosome 7 (SSC7) near the centromere. In this study, 3 class I (SLA-1, SLA-3, and SLA-2) and 3 class II (DRB1, DQB1, and DQA) genes were used for investigation of SLA haplotypes in Yucatan miniature pigs in Korea. This pig breed is a well-known model organism for biomedical research worldwide. The current study indicated that Korean Yucatan pig population had 3 Class I haplotypes (Lr-4.0, Lr-6.0, and Lr-25.0) and 3 class II haplotypes (Lr-0.5, Lr-0.7, and Lr-0.25). The combinations of SLA class I and II haplotype together, 2 homozygous (Lr-4.5/4.5 and Lr-6.7/6.7) and 3 heterozygous (Lr-4.5/6.7, Lr-4.5/25.25, and Lr-6.7/25.25) haplotypes were identified, including previously unidentified new heterozygous haplotypes (Lr-4.5/4.7). In addition, a new SLA allele typing method using Agilent 2100 bioanalyzer was developed that permitted more rapid identification of SLA haplotypes. These results will facilitate the breeding of SLA homozygous Yucatan pigs and will expedite the possible use of these pigs for the biomedical research, especially xenotransplantation research.

Dimerization, oligomerization, and aggregation of human amyotrophic lateral sclerosis copper/zinc superoxide dismutase 1 protein mutant forms in live cells.

More than 100 copper/zinc superoxide dismutase 1 (SOD1) genetic mutations have been characterized. These mutations lead to the death of motor neurons in ALS. In its native form, the SOD1 protein is expressed as a homodimer in the cytosol. In vitro studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and in vivo studies have shown that SOD1 forms aggregates in patients with familial forms of ALS. In this study, we analyzed WT SOD1 and 9 mutant (mt) forms of the protein by non-invasive fluorescence techniques. Using microscopic techniques such as fluorescence resonance energy transfer, fluorescence complementation, image-based quantification, and fluorescence correlation spectroscopy, we studied SOD1 dimerization, oligomerization, and aggregation. Our results indicate that SOD1 mutations lead to an impairment in SOD1 dimerization and, subsequently, affect protein aggregation. We also show that SOD1 WT and mt proteins can dimerize. However, aggregates are predominantly composed of SOD1 mt proteins.