Laser fiber degradation following holmium laser enucleation of the prostate utilizing Moses technology versus regular mode.

PURPOSE: We sought to objectively compare laser fiber degradation for holmium laser enucleation of the prostate (HoLEP) cases performed with 550 µm standard fibers versus 550 µm Moses 2.0 fiber in BPH mode on a macroscopic and microscopic level. METHODS: We prospectively collected outcomes for 50 standardized HoLEP cases using 550 µm Moses fiber with 2.0 BPH mode compared to our historical cohort of 50 patients using 550 µm standard fibers on regular mode. Macroscopic degradation length was the difference in length of exposed fiber at the start and end of each case. Five consecutive 550 µm standard fibers, five 550 µm Moses fibers and their respective controls underwent novel utilization of three objective corroborating imaging techniques: Brightfield high resolution microscopy, high resolution 3-D microCT and Confocal Reflection Surface Analysis. Mann-Whitney U, 2-tailed T tests and Chi-squared tests were used. RESULTS: Standard fibers demonstrated greater degradation than the Moses fibers with 2.0 BPH mode [2.9 cm (IQR 1.7-4.3 cm) vs 0.2 cm (IQR 0.1-0.4 cm), p < 0.01]. This difference remained significant when comparing degradation per energy used, per minute enucleation and per gram enucleated (all p < 0.05). None of the cases with Moses fiber and 2.0 BPH mode required intraoperative interruption to re-strip the fiber. Objective fiber degradation by three microscopic techniques confirmed more damage to the standard fibers with regular mode. CONCLUSION: Overall, use of the 550 µm Moses fiber with 2.0 BPH mode resulted in less fiber degradation compared to a standard 550 µm fiber with regular mode as confirmed using 4 corroborating macroscopic and microscopic techniques.

Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N')-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N'-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N'-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.

Neutrophil-selective deletion of Cxcr2 protects against CNS neurodegeneration in a mouse model of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a chronic debilitating immune-mediated disease of the central nervous system (CNS) driven by demyelination and gray matter neurodegeneration. We previously reported an experimental autoimmune encephalomyelitis (EAE) MS mouse model with elevated serum CXCL1 that developed severe and prolonged neuron damage. Our findings suggested that CXCR2 signaling may be important in neuronal damage, thus implicating neutrophils, which express CXCR2 in abundance, as a potential cell type involved. The goals of this study were to determine if CXCR2 signaling in neutrophils mediate neuronal damage and to identify potential mechanisms of damage. METHODS: EAE was induced in wild-type control and neutrophil-specific Cxcr2 knockout (Cxcr2 cKO) mice by repeated high-dose injections of heat-killed Mycobacterium tuberculosis and MOG35-55 peptide. Mice were examined daily for motor deficit. Serum CXCL1 level was determined at different time points throughout disease development. Neuronal morphology in Golgi-Cox stained lumbar spinal cord ventral horn was assessed using recently developed confocal reflection super-resolution technique. Immune cells from CNS and lymphoid organs were quantified by flow cytometry. CNS-derived neutrophils were co-cultured with neuronal crest cells and neuronal cell death was measured. Neutrophils isolated from lymphoid organs were examined for expression of reactive oxygen species (ROS) and ROS-related genes. Thioglycolate-activated neutrophils were isolated, treated with recombinant CXCL1, and measured for ROS production. RESULTS: Cxcr2 cKO mice had less severe disease symptoms at peak and late phase when compared to control mice with similar levels of CNS-infiltrating neutrophils and other immune cells despite high levels of circulating CXCL1. Additionally, Cxcr2 cKO mice had significantly reduced CNS neuronal damage in the ventral horn of the spinal cord. Neutrophils isolated from control EAE mice induced vast neuronal cell death in vitro when compared with neutrophils isolated from Cxcr2 cKO EAE mice. Neutrophils isolated from control EAE mice, but not Cxcr2 cKO mice, exhibited elevated ROS generation, in addition to heightened Ncf1 and Il1b transcription. Furthermore, recombinant CXCL1 was sufficient to significantly increase neutrophils ROS production. CONCLUSIONS: CXCR2 signal in neutrophils is critical in triggering CNS neuronal damage via ROS generation, which leads to prolonged EAE disease. These findings emphasize that CXCR2 signaling in neutrophils may be a viable target for therapeutic intervention against CNS neuronal damage.

CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci.

Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a method for tracking any locus of interest is desirable. Recently clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) or transcription activator-like effectors were adapted for imaging endogenous loci; however, they are mostly limited to visualization of repetitive regions. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions without the need for a pre-existing repetitive sequence. SHACKTeR requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by polymerase chain reaction, was successful at labeling 10 different loci in HCT116 cells. We also showed the feasibility of knock-in into lamina-associated, heterochromatin regions, demonstrating that these regions prefer non-homologous end joining for knock-in. Using SHACKTeR, we were able to observe DNA replication at a specific locus by long-term live cell imaging. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.

A Confocal Reflection Super-Resolution Technique to Image Golgi-Cox Stained Neurons.

Metal-based Golgi-Cox (GC) staining is an established method used to visualise neurons with great morphological detail. Although GC stained samples are imaged routinely under transmitted light microscopy, this method is unable to yield information on the three-dimensional structure of dendrites and neurons and thus help reveal the connective properties of the central nervous system. Although a few studies have attempted simultaneous visualisation of GC staining and antigen-specific fluorescent labelling under a confocal reflection technique, the resolution of both confocal reflection and fluorescence modalities used to acquire GC reflection and fluorescently stained antibody signals are still limited by the diffraction limit of light at about 220 nm. Here, we report a confocal reflection super-resolution technique (CRSR) to break this diffraction barrier, which is achieved by minimising the pinhole size from 1 airy unit (AU) to 0.1 AU. This is achieved by minimising or closing the confocal pinhole size and is possible in this reflection modality, unlike fluorescence, because it is not a photon limited technique. Utilising the lowest wavelength of light available in the system (405 nm), the CRSR technique results in ∼30% lateral and axial resolution improvement. We also show that the CRSR technique can be used in conjunction to visualise both GC and immunofluorescence targets to create precise and improved three-dimensional visualisation and analysis. In addition, using these superresolution confocal reflection data sets from GC in CRSR mode significantly reduced the data overestimation, improving the accuracy of statistical analysis of dendritic spine density and average spine dimensions. Combining the 0.1 AU setting with deconvolution routines, the signal-to-noise ratio and resolution could further be improved an additional ∼20-25%, yielding CRSR images with resolutions up to 2-fold over the diffraction limit both laterally and axially. The improved precision of both visualisation and quantification of subdiffraction limited dendritic spines using the CRSR technique may prove to be critical in investigations that concern changes in detailed neuron morphology under central nervous system disease conditions such as multiple sclerosis and Alzheimer's disease. LAY DESCRIPTION: For over a century, Golgi-Cox (GC) has been a leading staining technique in the field of neuroscience, used to visualise neurons with great morphological detail. GC stained brain or spinal cord samples are conventionally visualised under transmitted light techniques. This limits the view of Golgi-staining to a two-dimensional image. A recent report showed that Golgi staining can be visualised in three-dimensions using the reflection modality of the confocal microscope. This visualisation also allows for the simultaneous acquisition of immunofluorescence signals. However, the reported resolution of Golgi staining confocal reflection is limited by the diffraction limit of light, which is around 220 nm. Here, we report a superresolution confocal reflection technique (CRSR) that achieves superresolution by minimising the pinhole size used in confocal microscopy. The CRSR technique results in ∼30% lateral and axial resolution improvement. Adding a deconvolution step in the final processing could improve the SNR and resolution even further up to 2-fold improvement in resolution over the diffraction limit both laterally and axially. We hope that this improved visualisation will help in investigations that concern changes in detailed neuron morphology under central nervous system disease conditions such as multiple sclerosis and Alzheimer's disease.

Adaptive Evolution of Escherichia coli to Ciprofloxacin in Controlled Stress Environments: Contrasting Patterns of Resistance in Spatially Varying versus Uniformly Mixed Concentration Conditions.

A microfluidic gradient chamber (MGC) and a homogeneous batch culturing system were used to evaluate whether spatial concentration gradients of the antibiotic ciprofloxacin allow development of greater antibiotic resistance in Escherichia coli strain 307 (E. coli 307) compared to exclusively temporal concentration gradients, as indicated in an earlier study. A linear spatial gradient of ciprofloxacin and Luria-Bertani broth (LB) medium was established and maintained by diffusion over 5 days across a well array in the MGC, with relative concentrations along the gradient of 1.7-7.7× the original minimum inhibitory concentration (MICoriginal). The E. coli biomass increased in wells with lower ciprofloxacin concentrations, and only a low level of resistance to ciprofloxacin was detected in the recovered cells (∼2× MICoriginal). Homogeneous batch culture experiments were performed with the same temporal exposure history to ciprofloxacin concentration, the same and higher initial cell densities, and the same and higher nutrient (i.e., LB) concentrations as in the MGC. In all batch experiments, E. coli 307 developed higher ciprofloxacin resistance after exposure, ranging from 4 to 24× MICoriginal in all replicates. Hence, these results suggest that the presence of spatial gradients appears to reduce the driving force for E. coli 307 adaptation to ciprofloxacin, which suggests that results from batch experiments may over predict the development of antibiotic resistance in natural environments.

Application of an advanced maximum likelihood estimation restoration method for enhanced-resolution and contrast in second-harmonic generation microscopy.

Second-harmonic generation (SHG) microscopy has gained popularity because of its ability to perform submicron, label-free imaging of noncentrosymmetric biological structures, such as fibrillar collagen in the extracellular matrix environment of various organs with high contrast and specificity. Because SHG is a two-photon coherent scattering process, it is difficult to define a point spread function (PSF) for this modality. Hence, compared to incoherent two-photon processes like two-photon fluorescence, it is challenging to apply the various PSF-engineering methods to improve the spatial resolution to be close to the diffraction limit. Using a synthetic PSF and application of an advanced maximum likelihood estimation (AdvMLE) deconvolution algorithm, we demonstrate restoration of the spatial resolution in SHG images to that closer to the theoretical diffraction limit. The AdvMLE algorithm adaptively and iteratively develops a PSF for the supplied image and succeeds in improving the signal to noise ratio (SNR) for images where the SHG signals are derived from various sources such as collagen in tendon and myosin in heart sarcomere. Approximately 3.5 times improvement in SNR is observed for tissue images at depths of up to ∼480 nm, which helps in revealing the underlying helical structures in collagen fibres with an ∼26% improvement in the amplitude contrast in a fibre pitch. Our approach could be adapted to noisy and low resolution modalities such as micro-nano CT and MRI, impacting precision of diagnosis and treatment of human diseases.

Light sheet microscopy reveals more gradual light attenuation in light-green versus dark-green soybean leaves.

Light wavelengths preferentially absorbed by chlorophyll (chl) often display steep absorption gradients. This over-saturates photosynthesis in upper chloroplasts and deprives lower chloroplasts of blue and red light. Reducing chl content could create a more even leaf light distribution and thereby increase leaf light-use efficiency and overall canopy photosynthesis. This was tested on soybean cultivar 'Clark' (WT) and a near-isogenic chl b deficient mutant, Y11y11, grown in controlled environment chambers and in the field. Light attenuation was quantified using a novel approach involving light sheet microscopy. Leaf adaxial and abaxial surfaces were illuminated separately with blue, red, and green wavelengths, and chl fluorescence was detected orthogonally to the illumination plane. Relative fluorescence was significantly greater in deeper layers of the Y11y11 mesophyll than in WT, with the greatest differences in blue, then red, and finally green light when illuminated from the adaxial surface. Modeled relative photosynthesis based on chlorophyll profiles and Beer's Law predicted less steep gradients in mutant relative photosynthesis rates compared to WT. Although photosynthetic light-use efficiency was greater in the field-grown mutant with ~50% lower chl, light-use efficiency was lower in the mutant when grown in chambers where chl was ~80% reduced. This difference is probably due to pleiotropic effects of the mutation that accompany very severe reductions in chlorophyll and may warrant further testing in other low-chl lines.

Imaging horse tendons using multimodal 2-photon microscopy.

Injuries and damage to tendons plague both human and equine athletes. At the site of injuries, various cells congregate to repair and re-structure the collagen. Treatments for collagen injury range from simple procedures such as icing and pharmaceutical treatments to more complex surgeries and the implantation of stem cells. Regardless of the treatment, the level of mechanical stimulation incurred by the recovering tendon is crucial. However, for a given tendon injury, it is not known precisely how much of a load should be applied for an effective recovery. Both too much and too little loading of the tendon could be detrimental during recovery. A mapping of the complex local environment imparted to any cell present at the site of a tendon injury may however, convey fundamental insights related to their decision making as a function of applied load. Therefore, fundamentally knowing how cells translate mechanical cues from their external environment into signals regulating their functions during repair is crucial to more effectively treat these types of injuries. In this paper, we studied systems of tendons with a variety of 2-photon-based imaging techniques to examine the local mechanical environment of cells in both normal and injured tendons. These tendons were chemically treated to instigate various extents of injury and in some cases, were injected with stem cells. The results related by each imaging technique distinguish with high contrast and resolution multiple morphologies of the cells' nuclei and the alignment of the collagen during injury. The incorporation of 2-photon FLIM into this study probed new features in the local environment of the nuclei that were not apparent with steady-state imaging. Overall, this paper focuses on horse tendon injury pattern and analysis with different 2-photon confocal modalities useful for wide variety of application in damaged tissues.

Targeted expression of SbMATE in the root distal transition zone is responsible for sorghum aluminum resistance.

Aluminum (Al) toxicity is one of the major limiting factors for crop production on acid soils that comprise significant portions of the world's lands. Aluminum resistance in the cereal crop Sorghum bicolor is mainly achieved by Al-activated root apical citrate exudation, which is mediated by the plasma membrane localized citrate efflux transporter encoded by SbMATE. Here we precisely localize tissue- and cell-specific Al toxicity responses as well as SbMATE gene and protein expression in root tips of an Al-resistant near-isogenic line (NIL). We found that Al induced the greatest cell damage and generation of reactive oxygen species specifically in the root distal transition zone (DTZ), a region 1-3 mm behind the root tip where transition from cell division to cell elongation occurs. These findings indicate that the root DTZ is the primary region of root Al stress. Furthermore, Al-induced SbMATE gene and protein expression were specifically localized to the epidermal and outer cortical cell layers of the DTZ in the Al-resistant NIL, and the process was precisely coincident with the time course of Al induction of SbMATE expression and the onset of the recovery of roots from Al-induced damage. These findings show that SbMATE gene and protein expression are induced when and where the root cells experience the greatest Al stress. Hence, Al-resistant sorghum plants have evolved an effective strategy to precisely localize root citrate exudation to the specific site of greatest Al-induced root damage, which minimizes plant carbon loss while maximizing protection of the root cells most susceptible to Al damage.

Halomonas sulfidaeris-dominated microbial community inhabits a 1.8 km-deep subsurface Cambrian Sandstone reservoir.

A low-diversity microbial community, dominated by the γ-proteobacterium Halomonas sulfidaeris, was detected in samples of warm saline formation porewater collected from the Cambrian Mt. Simon Sandstone in the Illinois Basin of the North American Midcontinent (1.8 km/5872 ft burial depth, 50°C, pH 8, 181 bars pressure). These highly porous and permeable quartz arenite sandstones are directly analogous to reservoirs around the world targeted for large-scale hydrocarbon extraction, as well as subsurface gas and carbon storage. A new downhole low-contamination subsurface sampling probe was used to collect in situ formation water samples for microbial environmental metagenomic analyses. Multiple lines of evidence suggest that this H. sulfidaeris-dominated subsurface microbial community is indigenous and not derived from drilling mud microbial contamination. Data to support this includes V1-V3 pyrosequencing of formation water and drilling mud, as well as comparison with previously published microbial analyses of drilling muds in other sites. Metabolic pathway reconstruction, constrained by the geology, geochemistry and present-day environmental conditions of the Mt. Simon Sandstone, implies that H. sulfidaeris-dominated subsurface microbial community may utilize iron and nitrogen metabolisms and extensively recycle indigenous nutrients and substrates. The presence of aromatic compound metabolic pathways suggests this microbial community can readily adapt to and survive subsurface hydrocarbon migration.

Osteopontin stabilization and collagen containment slows amorphous calcium phosphate transformation during human aortic valve leaflet calcification.

Calcification of aortic valve leaflets is a growing mortality threat for the 18 million human lives claimed globally each year by heart disease. Extensive research has focused on the cellular and molecular pathophysiology associated with calcification, yet the detailed composition, structure, distribution and etiological history of mineral deposition remains unknown. Here transdisciplinary geology, biology and medicine (GeoBioMed) approaches prove that leaflet calcification is driven by amorphous calcium phosphate (ACP), ACP at the threshold of transformation toward hydroxyapatite (HAP) and cholesterol biomineralization. A paragenetic sequence of events is observed that includes: (1) original formation of unaltered leaflet tissues: (2) individual and coalescing 100's nm- to 1 µm-scale ACP spherules and cholesterol crystals biomineralizing collagen fibers and smooth muscle cell myofilaments; (3) osteopontin coatings that stabilize ACP and collagen containment of nodules preventing exposure to the solution chemistry and water content of pumping blood, which combine to slow transformation to HAP; (4) mm-scale nodule growth via ACP spherule coalescence, diagenetic incorporation of altered collagen and aggregation with other ACP nodules; and (5) leaflet diastole and systole flexure causing nodules to twist, fold their encasing collagen fibers and increase stiffness. These in vivo mechanisms combine to slow leaflet calcification and establish previously unexplored hypotheses for testing novel drug therapies and clinical interventions as viable alternatives to current reliance on surgical/percutaneous valve implants.

Pseudomonas aeruginosa pyocyanin causes airway goblet cell hyperplasia and metaplasia and mucus hypersecretion by inactivating the transcriptional factor FoxA2.

The redox-active exotoxin pyocyanin (PCN) can be recovered in 100 µM concentrations in the sputa of bronchiectasis patients chronically infected with Pseudomonas aeruginosa (PA). However, the importance of PCN within bronchiectatic airways colonized by PA remains unrecognized. Recently, we have shown that PCN is required for chronic PA lung infection in mice, and that chronic instillation of PCN induces goblet cell hyperplasia (GCH), pulmonary fibrosis, emphysema and influx of immune cells in mouse airways. Many of these pathological features are strikingly similar to the mouse airways devoid of functional FoxA2, a transcriptional repressor of GCH and mucus biosynthesis. In this study, we postulate that PCN causes and exacerbates GCH and mucus hypersecretion in bronchiectatic airways chronically infected by PA by inactivating FoxA2. We demonstrate that PCN represses the expression of FoxA2 in mouse airways and in bronchial epithelial cells cultured at an air-liquid interface or conventionally, resulting in GCH, increased MUC5B mucin gene expression and mucus hypersecretion. Immunohistochemical and inhibitor studies indicate that PCN upregulates the expression of Stat6 and EGFR, both of which in turn repress the expression of FoxA2. These studies demonstrate that PCN induces GCH and mucus hypersecretion by inactivating FoxA2.

Impact of mono(2-ethylhexyl) phthalate (MEHP) on the development of mouse embryo in vitro.

Mono(2-ethylhexyl) phthalate (MEHP) is the most studied metabolite of di(2-ethylhexyl) phthalate (DEHP), a phthalate found in cosmetics, flooring, paints, and plastics products, including toys and medical tubing. Humans are frequently exposed to this compound due to its ubiquitous presence in our environment. DEHP and MEHP are known to be endocrine-disrupting chemicals and exposure levels have been associated to decreased reproductive success. However, few studies have focused on the direct effects of MEHP on embryos. The present study investigated effects of MEHP (0.1, 1, 10, 100 and 1000 µM) on mice preimplantation embryonic development, evaluating percentage of blastocyst formation, hatching from zona pellucida, methylation-related genes, cell lineage commitment, micronucleation, and adherens junction marker at different stages of development during in vitro culture for 6 days. We show MEHP negatively impacts embryo competence by reducing blastocyst formation and hatching at 100 and 1000 µM. In addition, 100 µM MEHP increases the expression of Tet3 gene in blastocysts, which is related to a reduction of DNA methylation, an important mechanism regulating gene expression. Exposed embryos that completed the hatching process in groups 0.1, 1 and 10 µM MEHP had similar number of inner cell mass and trophectoderm cells compared to the control, while micronucleation occurrence and E-cadherin expression was not affected in exposed morulae by MEHP at 10 or 100 µM. Our results showed that high concentrations of MEHP can negatively impact embryo development. New studies unveiling the mechanism of toxicity involved and encompassing further developmental stages are warranted for further understanding.

CHCHD2 mediates glioblastoma cell proliferation, mitochondrial metabolism, hypoxia­induced invasion and therapeutic resistance.

Glioblastoma (GBM) is the most common and malignant primary brain tumor affecting adults and remains incurable. The mitochondrial coiled­coil­helix­coiled­coil­helix domain­containing protein 2 (CHCHD2) has been demonstrated to mediate mitochondrial respiration, nuclear gene expression and cell migration; however, evidence of this in GBM is lacking. In the present study, it was hypothesized that CHCHD2 may play a functional role in U87 GBM cells expressing the constitutively active epidermal growth factor receptor variant III (EGFRvIII). The amplification of the CHCHD2 gene was found to be associated with a decreased patient overall and progression­free survival. The CHCHD2 mRNA levels were increased in high­vs. low­grade glioma, IDH­wt GBMs, and in tumor vs. non­tumor tissue. Additionally, CHCHD2 protein expression was greatest in invasive, EGFRvIII­expressing patient­derived samples. The CRISPR­Cas9­mediated knockout of CHCHD2 in EGFRvIII­expressing U87 cells resulted in an altered mitochondrial respiration and glutathione status, in decreased cell growth and invasion under both normoxic and hypoxic conditions, and in an enhanced sensitivity to cytotoxic agents. CHCHD2 was distributed in both the mitochondria and nuclei of U87 and U87vIII cells, and the U87vIII cells exhibited a greater nuclear expression of CHCHD2 compared to isogenic U87 cells. Incubation under hypoxic conditions, serum starvation and the reductive unfolding of CHCHD2 induced the nuclear accumulation of CHCHD2 in both cell lines. Collectively, the findings of the present study indicate that CHCHD2 mediates a variety of GBM characteristics, and highlights mitonuclear retrograde signaling as a pathway of interest in GBM cell biology.

Lysine acetylation is a widespread protein modification for diverse proteins in Arabidopsis.

Lysine acetylation (LysAc), a form of reversible protein posttranslational modification previously known only for histone regulation in plants, is shown to be widespread in Arabidopsis (Arabidopsis thaliana). Sixty-four Lys modification sites were identified on 57 proteins, which operate in a wide variety of pathways/processes and are located in various cellular compartments. A number of photosynthesis-related proteins are among this group of LysAc proteins, including photosystem II (PSII) subunits, light-harvesting chlorophyll a/b-binding proteins (LHCb), Rubisco large and small subunits, and chloroplastic ATP synthase (ß-subunit). Using two-dimensional native green/sodium dodecyl sulfate gels, the loosely PSII-bound LHCb was separated from the LHCb that is tightly bound to PSII and shown to have substantially higher level of LysAc, implying that LysAc may play a role in distributing the LHCb complexes. Several potential LysAc sites were identified on eukaryotic elongation factor-1A (eEF-1A) by liquid chromatography/mass spectrometry and using sequence- and modification-specific antibodies the acetylation of Lys-227 and Lys-306 was established. Lys-306 is contained within a predicted calmodulin-binding sequence and acetylation of Lys-306 strongly inhibited the interactions of eEF-1A synthetic peptides with calmodulin recombinant proteins in vitro. These results suggest that LysAc of eEF-1A may directly affect regulatory properties and localization of the protein within the cell. Overall, these findings reveal the possibility that reversible LysAc may be an important and previously unknown regulatory mechanism of a large number of nonhistone proteins affecting a wide range of pathways and processes in Arabidopsis and likely in all plants.

Compartmentalization of S-RNase and HT-B degradation in self-incompatible Nicotiana.

Pollen-pistil interactions are crucial for controlling plant mating. For example, S-RNase-based self-incompatibility prevents inbreeding in diverse angiosperm species. S-RNases are thought to function as specific cytotoxins that inhibit pollen that has an S-haplotype that matches one of those in the pistil. Thus, pollen and pistil factors interact to prevent mating between closely related individuals. Other pistil factors, such as HT-B, 4936-factor and the 120 kDa glycoprotein, are also required for pollen rejection but do not contribute to S-haplotype-specificity per se. Here we show that S-RNase is taken up and sorted to a vacuolar compartment in the pollen tubes. Antibodies to the 120 kDa glycoprotein label the compartment membrane. When the pistil does not express HT-B or 4936-factor, S-RNase remains sequestered, unable to cause rejection. Similarly, in wild-type pistils, compatible pollen tubes degrade HT-B and sequester S-RNase. We suggest that S-RNase trafficking and the stability of HT-B are central to S-specific pollen rejection.

GeoBioMed perspectives on kidney stone recurrence from the reactive surface area of SWL-derived particles.

Shock wave lithotripsy (SWL) is an effective and commonly applied clinical treatment for human kidney stones. Yet the success of SWL is counterbalanced by the risk of retained fragments causing recurrent stone formation, which may require retreatment. This study has applied GeoBioMed experimental and analytical approaches to determine the size frequency distribution, fracture patterns, and reactive surface area of SWL-derived particles within the context of their original crystal growth structure (crystalline architecture) as revealed by confocal autofluorescence (CAF) and super-resolution autofluorescence (SRAF) microscopy. Multiple calcium oxalate (CaOx) stones were removed from a Mayo Clinic patient using standard percutaneous nephrolithotomy (PCNL) and shock pulse lithotripsy (SPL). This produced approximately 4-12 mm-diameter PCNL-derived fragments that were experimentally treated ex vivo with SWL to form hundreds of smaller particles. Fractures propagated through the crystalline architecture of PCNL-derived fragments in a variety of geometric orientations to form rectangular, pointed, concentrically spalled, and irregular SWL-derived particles. Size frequency distributions ranged from fine silt (4-8 µm) to very fine pebbles (2-4 mm), according to the Wentworth grain size scale, with a mean size of fine sand (125-250 µm). Importantly, these SWL-derived particles are smaller than the 3-4 mm-diameter detection limit of clinical computed tomography (CT) techniques and can be retained on internal kidney membrane surfaces. This creates clinically undetectable crystallization seed points with extremely high reactive surface areas, which dramatically enhance the multiple events of crystallization and dissolution (diagenetic phase transitions) that may lead to the high rates of CaOx kidney stone recurrence after SWL treatment.

Travertine crystal growth ripples record the hydraulic history of ancient Rome's Anio Novus aqueduct.

Travertine crystal growth ripples are used to reconstruct the early hydraulic history of the Anio Novus aqueduct of ancient Rome. These crystalline morphologies deposited within the aqueduct channel record the hydraulic history of gravity-driven turbulent flow at the time of Roman operation. The wavelength, amplitude, and steepness of these travertine crystal growth ripples indicate that large-scale sustained aqueduct flows scaled directly with the thickness of the aqueous viscous sublayer. Resulting critical shear Reynolds numbers are comparable with those reconstructed from heat/mass transfer crystalline ripples formed in other natural and engineered environments. This includes sediment transport in rivers, lakes, and oceans, chemical precipitation and dissolution in caves, and melting and freezing in ice. Where flow depth and perimeter could be reconstructed from the distribution and stratigraphy of the travertine within the Anio Novus aqueduct, flow velocity and rate have been quantified by deriving roughness-flow relationships that are independent of water temperature. More generally, under conditions of near-constant water temperature and kinematic viscosity within the Anio Novus aqueduct channel, the travertine crystal growth ripple wavelengths increased with decreasing flow velocity, indicating that systematic changes took place in flow rate during travertine deposition. This study establishes that travertine crystal growth ripples such as those preserved in the Anio Novus provide a sensitive record of past hydraulic conditions, which can be similarly reconstructed from travertine deposited in other ancient water conveyance and storage systems around the world.

Deficiency in the omega-3 fatty acid pathway results in failure of acrosome biogenesis in mice.

An omega-3 fatty acid, docosahexaenoic acid (DHA), is enriched in testicular membrane phospholipids, but its function is not well understood. The Fads2 gene encodes an enzyme required for the endogenous synthesis of DHA. Using Fads2-null mice (Fads2-/-), we found in our preceding studies that DHA deficiency caused the arrest of spermiogenesis and male infertility, both of which were reversed by dietary DHA. In this study, we investigated a cellular mechanism underlying the DHA essentiality in spermiogenesis. Periodic acid-Schiff staining and acrosin immunohistochemistry revealed the absence of acrosomes in Fads2-/- round spermatids. Acrosin, an acrosomal marker, was scattered throughout the cytoplasm of the Fads2-/- spermatids, and electron microscopy showed that proacrosomal granules were formed on the trans-face of the Golgi. However, excessive endoplasmic reticulum and vesicles were present on the cis-face of the Golgi in Fads2-/- spermatids. The presence of proacrosomal vesicles but lack of a developed acrosome in Fads2-/- spermatids suggested failed vesicle fusion. Syntaxin 2, a protein involved in vesicle fusion, colocalized with acrosin in the acrosome of wild-type mice. In contrast, syntaxin 2 remained scattered in reticular structures and showed no extensive colocalization with acrosin in the Fads2-/- spermatids, suggesting failed fusion with acrosin-containing vesicles or failed transport and release of syntaxin 2 vesicles from Golgi. Dietary supplementation of DHA in Fads2-/- mice restored an intact acrosome. In conclusion, acrosome biogenesis under DHA deficiency is halted after release of proacrosomal granules. Misplaced syntaxin 2 suggests an essential role of DHA in proper delivery of membrane proteins required for proacrosomal vesicle fusion.