Owing to its wide band gap, extreme mechanical hardness and high thermal conductivity, GaN has found widespread applications in optoelectronic devices and high-power/-frequency devices. However, the ...growth of high-quality and large-size GaN crystal substrates is still a great challenge, which hinders the development of power and radio frequency (RF) devices. The Na-flux method can emerge as an effective strategy to address these challenges. Nevertheless, the growth quality of GaN crystals is influenced by several factors during the growth process. This study focused on investigating the growth rate and quality of GaN in relation to the growth temperature and pressure. It also explains the difference in the solubility between N
3−
and GaN as a function of temperature in liquid phase melts. The intricacy of the Na-flux method and the opacity of the growth process present significant obstacles to the growth of GaN crystals. In order to accurately determine and optimise the growth conditions, the temperature distribution and material transport during the growth process are predicted by simulation. A series of validation experiments were conducted to investigate the influence of temperature and pressure on GaN crystallisation. Under optimised growth conditions, high-quality GaN crystals with a full width at half maximum of 433 arcsec (002) were obtained. This work provides an effective strategy for the liquid-phase growth of high-quality GaN crystals, facilitating the development of high-performance blue-green lasers, RF and power devices.
GaN single crystals (15 × 15 mm) were epitaxially grown using the Na-flux method, and substrate self-peeling was achieved. The effect of temperature and pressure on the growth rate and the quality of GaN single crystals are investigated.
Abstract As the representative of substrate material, gallium nitride (GaN) has excellent mechanical properties and high thermal stability. Achieving high surface flatness is critical for subsequent ...epitaxial growth and device fabrication processes. Chemical mechanical polishing (CMP) technique of GaN is commonly one of the most effective ways to achieve atomically smooth surfaces. However, the current process is difficult to meet the needs of industrial development due to the characteristics of low material removal rate. Assisted enhanced CMP technique is deemed to possess significant potential due to its improved processing efficiency and surface topography quality. Herein, a variety of auxiliary enhanced CMP systems are designed and studied. In this review, recent advances both in conventional and assisted enhanced CMP of GaN are comprehensive presented, with a focus on their potential applications in various fields. The mechanism and design strategy of the process are discussed and summarized. The key issues in machining atomically flattened surface are outlined, and future strategies for sustainable development are also proposed. This review provides a novel perspective on GaN processing and offers more inspiration for future research to realize its development and commercial application.
Mutants of the Cucumber mosaic virus (CMV) movement protein (MP) were generated and analyzed for their effects on virus movement and pathogenicity in vivo. Similar to the wild-type MP, mutants M1, ...M2, and M3, promoted virus movement in eight plant species. Mutant M3 showed some differences in pathogenicity in one host species. Mutant M8 showed some host-specific alterations in movement in two hypersensitive hosts of CMV. Mutant M9 showed altered pathogenicity on three hosts and was temperature sensitive for long-distance movement, demonstrating that cell-to-cell and long-distance movement are distinct movement functions for CMV. Four mutants (M4, M5, M6, and M7) were debilitated from movement in all hosts tested. Mutants M4, M5, and M6 could be complemented in trans by the wild-type MP expressed transgenically, although not by each other or by mutant M9 (at the restrictive temperature). Mutant M7 showed an inability to be complemented in trans. From these mutants, different aspects of the CMV movement process could be defined and specific roles for particular sequence domains assigned. The broader implications of these functions are discussed.
Abstract Background and Aims Patients with kidney disease, especially those on dialysis, are more susceptible to infections such as peritonitis, sepsis, pneumonia, and are at a higher risk for ...infection-related mortality. Pathogenic mechanisms underlying the secondary immunodeficiency in kidney disease include gut dysbiosis and barrier dysfunction, persistent inflammation, immune paralysis due to increased levels of immunoregulatory metabolites (e.g. uric acid) and proteins (e.g. FGF23) 1. Currently, it is unknown whether CKD-related hyperuricemia may contribute to the immune paralysis in this context. We hypothesized that hyperuricemia (HU) may aggravate the innate immune response to infection in CKD in mice. Method Mono- and polybacterial sepsis was induced by LPS injection or cecal ligation and puncture in Alb-creERT2;Glut9lox/lox mice with HU and/or CKD, and in Glut9lox/lox (healthy) control mice. After 24 hours, systemic inflammation was evaluated by measuring serum cytokine levels, immune cell activation and recruitment, and neutrophil extracellular trap release. Tissue injury in liver, heart, and kidney was assessed by performing RT-qPCR and immunohistochemistry. In addition, kidney function was determined by measuring the glomerular filtration rate (GFR). Additionally, mice were treated with urate-lowering therapy to reduce serum uric acid levels and the immune response and functional outcomes after sepsis is quantified. Results We found that both mono- and polybacterial sepsis caused systemic inflammation as indicated by an increased number of blood neutrophils (see Figure), neutrophil activation and pro-inflammatory cytokine levels as compared to the PBS-treated or sham-operated control groups in Glut9lox/lox healthy mice, respectively. Mice with sepsis also displayed a slightly worsened kidney function. Interestingly, HU significantly increased neutrophil numbers but decreased their activation status and the ability of neutrophils to form neutrophil extracellular traps in Alb-creERT2;Glut9lox/lox mice with sepsis as compared to the control groups, which was further aggravated in mice with CKD-related HU (CKD+HU). This impaired inflammatory response was partially reversible by lowering serum uric acid with febuxostat. Conclusion Our results identify hyperuricemia related or unrelated to kidney disease as immune regulator in bacterial infection by suppressing neutrophil functions in mice. Specifically targeting uric acid may help to overcome the secondary immunodeficiency related to kidney disease during infection but enhances sterile inflammation 2.
Tobacco plants were transformed with the cucumber mosaic cucumovirus (CMV) 3a gene and the
in planta-expressed 3a protein was detected immunologically. The 3a protein was predominantly localized in a ...subcellular fraction corresponding to the cytosol. Two frameshift and four deletion mutants were created within the 3a open reading frame of CMV RNA 3. Five of these mutants, containing an N-terminal, large central, or C-terminal 70-amino-acid deletion could not infect nontransformed tobacco plants, but could infect the 3a transgenic tobacco plants, and generally accumulated to wild-type levels. The sixth mutant, lacking the C-terminal 43 amino acids of the 3a protein, was able to infect nontransformed tobacco plants. A delay in accumulation of viral RNA in both the inoculated and the systemically infected leaves was demonstrated for one of the mutants. Thus, the CMV 3a protein is a virus movement protein, the functions of which can be complemented in a transgenic plant. The CMV 3a transgenic plants were able to complement the long-distance movement of a pseudorecombinant cucumovirus defective for this function in tobacco, as well as the cell-to-cell, but not the long-distance, movement of two other related viruses. However, these transgenic plants were unable to complement the long-distance movement of viruses from several other taxonomic groups that could move cell to cell but not long distance in tobacco.
GTP-binding protein/transglutaminases (tissue transglutaminases or TGases) have been implicated in a variety of cellular processes including retinoic acid (RA)-induced apoptosis. Recently, we have ...shown that RA activates TGases as reflected by stimulated GTP binding, increased membrane association, and stimulated phosphoinositide lipid turnover. This prompted us to search for cellular proteins that bind TGases in a RA-stimulated manner. In this report, we show that the eukaryotic initiation factor (eIF-5A), a protein that is essential for cell viability, perhaps through effects on protein synthesis and/or RNA export, associates with the TGasein vivo. The interaction between eIF-5A and TGase is specific for the GDP-bound form of the TGase and is not detected when the TGase is pre-loaded with GTPγS. The TGase-eIF-5A interaction also is promoted by Ca2+, Mg2+, and RA treatment of HeLa cells. In the presence of retinoic acid, millimolar levels of Ca2+ are no longer required for the TGase-eIF-5A interaction. Nocodazole treatment, which blocks the cell cycle at mitosis (M phase), strongly inhibits the interaction between eIF-5A and cytosolic TGase. The interaction between TGase and eIF-5A and its sensitivity to the nucleotide-occupied state of the TGase provides a potentially interesting connection between RA signaling and protein synthesis and/or RNA trafficking activities.
Abstract Background and Aims Nephron number highly varies between different species and within species, ranging in humans from 200 000 to 2 000 000 nephrons per kidney. Low nephron numbers can ...promote early onset of end-stage kidney disease. Nephron assessment is highly needed for better diagnosis and more targeted treatment. However, its assessment in vivo is not established in clinical practice yet. Pioneer studies suggest nephron number estimation in vivo through a combination of glomerular densities in kidney biopsies together with medical imaging. In contrast, post-mortem nephron assessment either using optical clearing or by exhaustive kidney sectioning is well studied but remains laborious. Here, we propose a new semi-automated nephron counting approach using serial PAS-stained whole slide kidney sections, deep learning segmentation and slide registration. Method Mice were sacrificed, kidneys harvested, measured, halved in the coronary plane and then opposingly embedded in paraffin. Subsequently, the entire kidney was sectioned with 10 µm thickness per slide. Tissue was stained with PAS and slides were digitalized using Aperio GT 450 DX scanner (Leica, Wetzlar, Germany) (Fig. a). Deep learning segmentation of glomeruli was performed in the first and every 10th (reference) section and subsequent (look-up) section using Tensorflow. Obtained segmentation results were manually quality controlled and adjusted, if necessary, in QuPath (Fig. b). Final segmentations of the look-up slides were registered to the reference slides in ImageJ using manual landmarks with the ImageJ BigWarp plugin (Fig. c). Glomeruli being present either in the look-up slide, or in the reference slide were counted as disector particles (Q-). Kidney areas on every examined slide were measured. Total number of glomeruli and total kidney area were determined as previously described (Fig. d). Results A kidney from a 12-week-old mouse suffering chronic kidney disease (mouse line Alb-creERT2 (ki/ki); Glut9 (fl/fl)) was used. Kidney weight was 162 mg, length 11 mm, width 6 mm, depth 4 mm and kidney volume 260 mm³. In total the kidney was sectioned in 95 slices. 20 reference sections and its respective look-up sections were stained and digitalized. In total 5981 glomeruli were segmented in 40 kidney slices using the deep learning algorithm. Glomerular segmentation quality was verified by two independent observers (mean dice score 0.79). Inaccuracies, if any, where manually corrected. For image registration in average 18.1 landmarks per slide were created within the ImageJ BigWarp plugin. Registration took in average 5 minutes per slide. Glomeruli without match (disector particles, Q-) were automatically counted. Number of disector particles (Q-) ranged from 32 to 244 per slide. Total nephron number was 11.085 and kidney area 210 mm³. The differences to the manual measured volume (260 mm³) might be explained by discard of small kidney fractions for the used sectioning technique. Conclusion We report, to our knowledge, the first deep learning assisted disector/fractionator approach to determine nephron number in whole slide kidney specimens. This method requires no physical disector setup, is scalable, cheap, time efficient, and unbiased. The method was so far validated in a mouse model of chronic kidney disease. The method will further be validated on different animal models. Additionally, occurrence of atubular glomeruli will be considered in nephron number determination. Furthermore, the integration of GFR measurements might allow the calculation of mean single nephron GFR and might together with automated glomerular area measurements unravel pathophysiologic processes in kidney ageing and disease.
Abstract
Background and Aims
Currently, it is unknown whether CKD patients with hyperuricemia (HU)-related uric acid (UA) crystalluria, also known as chronic UA nephropathy, would benefit from ...targeting HU, UA crystallization, and/or inflammation to slow down the progressive decline of kidney function. Although large multi-center RCTs with xanthine oxidase inhibitors (XOIs) such as allopurinol and two Mendelian randomization studies have disproven a causal link between HU and CKD progression, one cannot rule out that XOIs may still represent a viable therapeutic approach for patients with HU-related UA crystalluria in order to prevent CKD progression. This issue requires clarification. To address this, we used our well-established CKD mouse model of HU with UA crystalluria and tested numerous therapeutic approaches including XOI and anakinra.
Method
Alb-creERT2;Glut9lox/lox (male and female) mice were injected with tamoxifen and placed on an acidogenic diet with the purine inosine to induce HU and crystalluria-related CKD. After chronic UA nephropathy was established on day 14, mice were treated either 1) with the XOIs allopurinol or febuxostat, 2) with sodium bicarbonate supplementation to neutralize urinary pH to prevent UA crystallization, or 3) with anakinra to inhibit IL-1β-driven inflammation, or 4) a combination of the aforementioned therapies for 2 weeks. Saline was used as control. On day 0, 14, and 28, GFR (as primary endpoint) as well as serum UA levels and urinary UA crystals (as secondary endpoints) were determined. At the end of the study, we quantified kidney injury, immune cell infiltration, inflammation, and interstitial fibrosis using histological analysis, ELISA, RT-qPCR, and flow cytometry.
Results
Therapy with XOIs but not with sodium bicarbonate and anakinra reduced serum UA levels in mice with HU and UA crystalluria. Treating mice with febuxostat improved kidney function (increase in GFR) by preventing kidney injury, UA crystalluria, UA crystal granuloma formation, and interstitial fibrosis compared with the control group, while allopurinol worsened the outcomes of chronic UA nephropathy. On the other hand, sodium bicarbonate supplementation had no effects on serum UA levels, kidney inflammation, immune cell infiltration and interstitial fibrosis but improved kidney function due to less urinary UA crystal deposition (neutralization of urine pH) and granuloma formation as compared with saline-treated mice. As expected, anakinra did not slow down the progression of chronic UA nephropathy as noticed by a similar GFR decline compared with the control group. Although, anakinra reduced the number of infiltrating immune cells and the mRNA expression of inflammatory mediators in the kidney as well as the serum IL-1β concentrations, we observed more interstitial fibrosis. In combination, allopurinol with anakinra did not improve the outcomes of chronic UA nephropathy despite the inhibitory effect of anakinra on the inflammatory response because allopurinol triggered tubulointerstitial nephritis and vasodilation. Interestingly, both combination therapies febuxostat with anakinra and febuxostat with sodium bicarbonate were protective by improving kidney function and preventing UA crystal deposition, kidney injury, inflammation, interstitial fibrosis and UA crystal granuloma formation compared with the single therapies.
Conclusion
Our interventional study is the first pre-clinical study, which reveals that the XOIs allopurinol and febuxostat have differential effects on the outcomes of HU-related UA crystalluria. While allopurinol contributes to the progression of CKD by causing tubulointerstitial nephritis in mice, febuxostat rather seems to be renoprotective similar to urinary pH neutralization with sodium bicarbonate. We identified both combination therapies febuxostat with anakinra or sodium bicarbonate as most beneficial in order to prevent CKD progression in mice. Thus, febuxostat in combination with IL-1β inhibition or prevention of urinary UA crystallization may represent therapeutic approaches for patients with chronic UA nephropathy.
The cucumber mosaic virus (CMV) 3a movement protein (MP) was compared directly to the well-characterized tobacco mosaic virus (TMV) 30K MP by cloning the genes encoding these proteins intoEscherichia ...coli,isolating theE. coli-expressed MPs, and characterizing them with regard to RNA- and NTP-binding activities. The two MPs were shown to bind single-stranded RNA and DNA cooperatively, but with no sequence specificity. However, discrete lengths of CMV RNA 3 could be protected against RNase digestion by the CMV 3a protein, indicating that the RNA was not uniformly covered by the MP after cooperative binding. The TMV 30K:RNA complex was more stable in NaCl than the CMV 3a:RNA complex; about 50% of the corresponding complexes were stable in 0.6 and 0.4MNaCl, respectively. Both MPs could bind GTP strongly and UTP weakly, but not ATP or CTP. The CMV 3a protein expressed either inE. coliorin plantafrom RNA 3 of CMV was tagged at its C-terminus with six histidine residues, which facilitated its purification by affinity chromatography on a matrix containing Ni2+-nitrilotriacetate. The soluble, His-tagged 3a proteins, affinity-purified fromE. coliand zucchini squash, both were able bind CMV RNA 3in vitro.
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