Sensitive Discrete Frequency Mid-Infrared Absorption Spectroscopy Using Digitally Referenced Detection.

Broadly tunable mid-infrared (IR) lasers, including quantum cascade lasers (QCL), are an asset for vibrational spectroscopy wherein high-intensity, coherent illumination can target specific spectral bands for rapid, direct chemical detection with microscopic localization. These emerging spectrometers are capable of high measurement throughputs with large detector signals from the high-intensity lasers and fast detection speeds as short as a single laser pulse, challenging the decades old benchmarks of Fourier transform infrared spectroscopy. However, noise in QCL emissions limits the feasible acquisition time for high signal-to-noise ratio (SNR) data. Here, we present an implementation that is broadly compatible with many laser-based spectrometer and microscope designs to address these limitations by leveraging high-speed digitizers and dual detectors to digitally reference each pulse individually. Digitally referenced detection (DRD) is shown to improve measurement sensitivity, with broad spectral indifference, regardless of imbalance due to dissimilarities among system designs or component manufacturers. We incorporated DRD into existing instruments and demonstrated its generalizability: a spectrometer with a 10-fold reduction in spectral noise, a microscope with reduced pixel dwell times to as low as 1 pulse while maintaining SNR normally achieved when operating 8-fold slower, and finally, a spectrometer to measure vibrational circular dichroism (VCD) with a ∼ 4-fold reduction in scan times. The approach not only proves versatile and effective but can also be tailored for specific applications with minimal hardware changes, positioning it as a simple and promising module for spectrometer designs using lasers.

ASC-J9 Blocks Cell Proliferation and Extracellular Matrix Production of Keloid Fibroblasts through Inhibiting STAT3 Signaling.

Keloids are a fibrotic skin disorder caused by abnormal wound healing and featuring the activation and expansion of fibroblasts beyond the original wound margin. Signal transducer and activator of transcription 3 (STAT3) has been found to mediate the biological functions of keloid fibroblasts (KFs). Therefore, we aimed to demonstrate whether ASC-J9, an inhibitor of STAT3 phosphorylation, can suppress the activation of KFs. Western blotting results showed that ASC-J9 inhibited the levels of COL1A1 and FN1 proteins, which were upregulated in KFs, by decreasing the expression of pSTAT3 and STAT3. RNA sequencing and in vitro studies further demonstrated that ASC-J9 treatment of KFs reduced cell division, inflammation, and ROS generation, as well as extracellular matrix (ECM) synthesis. ELISA assays verified that ASC-J9 treatment significantly mitigated IL-6 protein secretion in KFs. Transmission electron microscopy images revealed that ASC-J9 induced the formation of multilamellar bodies in KFs, which is associated with autophagy-related signaling. These results suggested that inhibiting a vicious cycle of the ROS/STAT3/IL-6 axis by ASC-J9 may represent a potential therapeutic approach to suppress cell proliferation and ECM production in KFs.

Rapid high resolution 3D imaging of expanded biological specimens with lattice light sheet microscopy.

Expansion microscopy was invented to surpass the optical diffraction limit by physically expanding biological specimens with swellable polymers. Due to the large sizes of expanded specimens, 3D imaging techniques that are capable to acquire large volumetric data rapidly at high spatial resolution are therefore required for expansion microscopy. Lattice light sheet microscopy (LLSM) was developed to image biological specimens rapidly at high 3D spatial resolution by using a thin lattice light sheet for sample illumination. However, due to the current limitations of LLSM mechanism and the optical design of LLS microscopes, it is challenging to image large expanded specimens at isotropic high spatial resolution using LLSM. To address the problem, we first optimized the sample preparation and expansion procedure for LLSM. Then, we implement a tiling lattice light sheet method to minimize sample translation during imaging and achieve much faster 3D imaging speed at high spatial resolution with more isotropic performance. Taken together, we report a general and improved 3D super-resolution imaging method for expanded samples.

Quantifying molecular- to cellular-level forces in living cells.

Mechanical cues have been suggested to play an important role in cell functions and cell fate determination, however, such physical quantities are challenging to directly measure in living cells with single molecule sensitivity and resolution. In this review, we focus on two main technologies that are promising in probing forces at the single molecule level. We review their theoretical fundamentals, recent technical advancements, and future directions, tailored specifically for interrogating mechanosensitive molecules in live cells.

Hitchhiking on chromosomes: A persistence strategy shared by diverse selfish DNA elements.

An underlying theme in the segregation of low-copy bacterial plasmids is the assembly of a 'segrosome' by DNA-protein and protein-protein interactions, followed by energy-driven directed movement. Analogous partitioning mechanisms drive the segregation of host chromosomes as well. Eukaryotic extra-chromosomal elements, exemplified by budding yeast plasmids and episomes of certain mammalian viruses, harbor partitioning systems that promote their physical association with chromosomes. In doing so, they indirectly take advantage of the spindle force that directs chromosome movement to opposite cell poles. Molecular-genetic, biochemical and cell biological studies have revealed several unsuspected aspects of 'chromosome hitchhiking' by the yeast 2-micron plasmid, including the ability of plasmid sisters to associate symmetrically with sister chromatids. As a result, the plasmid overcomes the 'mother bias' experienced by plasmids lacking a partitioning system, and elevates itself to near chromosome status in equal segregation. Chromosome association for stable propagation, without direct energy expenditure, may also be utilized by a small minority of bacterial plasmids-at least one case has been reported. Given the near perfect accuracy of chromosome segregation, it is not surprising that elements residing in evolutionarily distant host organisms have converged upon the common strategy of gaining passage to daughter cells as passengers on chromosomes.

Testis-specific Arf promoter expression in a transposase-aided BAC transgenic mouse model.

CDKN2A is an evolutionarily conserved gene encoding proteins implicated in tumor suppression, ocular development, aging, and metabolic diseases. Like the human form, mouse Cdkn2a encodes two distinct proteins-p16Ink4a, which blocks cyclin-dependent kinase activity, and p19Arf, which is best known as a positive regulator of the p53 tumor suppressor-and their functions have been well-studied in genetically engineered mouse models. Relatively little is known about how expression of the two transcripts is controlled in normal development and in certain disease states. To better understand their coordinate and transcript-specific expression in situ, we used a transposase-aided approach to generate a new BAC transgenic mouse model in which the first exons encoding Arf and Ink4a are replaced by fluorescent reporters. We show that mouse embryo fibroblasts generated from the transgenic lines faithfully display induction of each transgenic reporter in cell culture models, and we demonstrate the expected expression of the Arf reporter in the normal testis, one of the few places where that promoter is normally expressed. Interestingly, the TGFß-2-dependent induction of the Arf reporter in the eye-a process essential for normal eye development-does not occur. Our findings illustrate the value of BAC transgenesis in mapping key regulatory elements in the mouse by revealing the genomic DNA required for Cdkn2a induction in cultured cells and the developing testis, and the apparent lack of elements driving expression in the developing eye.

Substitutions of strontium in bioactive calcium silicate bone cements stimulate osteogenic differentiation in human mesenchymal stem cells.

Calcium silicate cements have been considered as alternative bone substitutes owing to its extraordinary bioactivity and osteogenicity. Unfortunately, the major disadvantage of the cements was the slow degradation rate which may limit the efficiency of bone regeneration. In this study, we proposed a facile method to synthesize degradable calcium silicate cements by incorporating strontium into the cements through solid-state sintering. The effects of Sr incorporation on physicochemical and biological properties of the cements were evaluated. Although, our findings revealed that the incorporation of strontium retarded the hardening reaction of the cements, the setting time of different cements (11-19 min) were in the acceptable range for clinical use. The presence of Sr in the CS cements would hampered the precipitation of calcium phosphate products on the surface after immersion in SBF, however, a layer of precipitated calcium phosphate products can be formed on the surface of the Sr-CS cement within 1 day immersion in SBF. More importantly, the degradation rate of the cements increased with increasing content of strontium, consequentially raised the levels of released strontium and silicon ions. The elevated dissolving products may contribute to the enhancement of the cytocompatibility, alkaline phosphatase activity, osteocalcin secretion, and mineralization of human Wharton's jelly mesenchymal stem cells. Together, it is concluded that the strontium-incorporated calcium silicate cement might be a promising bone substitute that could accelerate the regeneration of irregularly shaped bone defects.

Replication-dependent and independent mechanisms for the chromosome-coupled persistence of a selfish genome.

The yeast 2-micron plasmid epitomizes the evolutionary optimization of selfish extra-chromosomal genomes for stable persistence without jeopardizing their hosts' fitness. Analyses of fluorescence-tagged single-copy reporter plasmids and/or the plasmid partitioning proteins in native and non-native hosts reveal chromosome-hitchhiking as the likely means for plasmid segregation. The contribution of the partitioning system to equal segregation is bipartite- replication-independent and replication-dependent. The former nearly eliminates 'mother bias' (preferential plasmid retention in the mother cell) according to binomial distribution, thus limiting equal segregation of a plasmid pair to 50%. The latter enhances equal segregation of plasmid sisters beyond this level, elevating the plasmid close to chromosome status. Host factors involved in plasmid partitioning can be functionally separated by their participation in the replication-independent and/or replication-dependent steps. In the hitchhiking model, random tethering of a pair of plasmids to chromosomes signifies the replication-independent component of segregation; the symmetric tethering of plasmid sisters to sister chromatids embodies the replication-dependent component. The 2-micron circle broadly resembles the episomes of certain mammalian viruses in its chromosome-associated propagation. This unifying feature among otherwise widely differing selfish genomes suggests their evolutionary convergence to the common logic of exploiting, albeit via distinct molecular mechanisms, host chromosome segregation machineries for self-preservation.

Complexity Analysis of Beat-to-Beat Skin-Surface Laser-Doppler Flowmetry Signals in Stroke Patients.

OBJECTIVES: This study performed skin-surface LDF measurements and SampEn analysis with the aims of (i) capturing the temporal complexity of cerebral hemodynamics in stroke patients and (ii) discriminating stroke patients from healthy control subjects. We also investigated the response induced by AS in beat-to-beat SampEn indexes of LDF signals. METHODS: LDF signals were obtained at bilateral TaiYang acupoints in 52 stroke patients. Each assessment involved a 20-minute baseline recording, a 20-minute AS, and a subsequent 20-minute recording. The FDT, FRT, and PW were calculated for each pulse of the LDF signals, and then their SampEn values were calculated. RESULTS: The SampEn values of FRT were significantly larger in the stroke group (1.064 ± 0.052 and p = 0.013 at the stroke side; 1.059 ± 0.055 and p = 0.017 at the controlateral side) than in the control group (0.975 ± 0.120). On the stroke side, the SampEn of value of FRT was significantly decreased following AS (1.064 ± 0.052 to 1.008 ± 0.060; p = 0.027). CONCLUSION: Larger SampEn values of FRT can be partly attributed to the local regulatory activities that are present in the stroke subjects when facing the induced abnormal vascular conditions and blood flow perfusion resistance. The present findings could aid the development of a noninvasive monitoring technique that will enable discrimination of the different microcirculatory responses in stroke patients.

Co-segregation of yeast plasmid sisters under monopolin-directed mitosis suggests association of plasmid sisters with sister chromatids.

The 2-micron plasmid, a high copy extrachromosomal element in Saccharomyces cerevisiae, propagates itself with nearly the same stability as the chromosomes of its host. Plasmid stability is conferred by a partitioning system consisting of the plasmid-coded proteins Rep1 and Rep2 and a cis-acting locus STB. Circumstantial evidence suggests that the partitioning system couples plasmid segregation to chromosome segregation during mitosis. However, the coupling mechanism has not been elucidated. In order to probe into this question more incisively, we have characterized the segregation of a single-copy STB reporter plasmid by manipulating mitosis to force sister chromatids to co-segregate either without mother-daughter bias or with a finite daughter bias. We find that the STB plasmid sisters are tightly correlated to sister chromatids in the extents of co-segregation as well as the bias in co-segregation under these conditions. Furthermore, this correlation is abolished by delaying spindle organization or preventing cohesin assembly during a cell cycle. Normal segregation of the 2-micron plasmid has been shown to require spindle integrity and the cohesin complex. Our results are accommodated by a model in which spindle- and cohesin-dependent association of plasmid sisters with sister chromatids promotes their segregation by a hitchhiking mechanism.

Gender- and age-related differences in foveal pit morphology.

AIMS: To measure the foveal pit morphology parameters and evaluate their correlations with age and sex. SETTINGS AND DESIGN: A retrospective cross-sectional matched comparison study in a tertiary center. METHODS AND MATERIALS: Forty men and 40 age-matched women who had normal macular structures and foveal contours were enrolled. Foveal pit parameters including top width, base width, nasal width, temporal width, minimal thickness, nasal thickness, temporal thickness, nasal height, temporal height, nasal slope, and temporal slope were measured on horizontal B-scan macular optical coherence tomography and compared between men and women. STATISTICAL ANALYSIS USED: Paired t-tests and Pearson's correlation analysis. RESULTS: The average patient age was 51.4 ± 17.5 (21-84) years. Women had a wider base width (313.1 ± 68.0 µm vs 266.8 ± 70.9 µm, P = 0.006), wider temporal width (1043.1 ± 245.6 µm vs 968.9 ± 261.0 µm, P = 0.006), thinner nasal thickness (345.6 ± 36.2 µm vs 359.7 ± 35.8 µm, P = 0.048), and flatter temporal slope (11.60 ± 2.52° vs 12.98 ± 2.68°, P = 0.016) than men. With age, the base width (r = 0.35, P = 0.025) and temporal width (r = 0.54, P = 0.0003) tended to be wider and the temporal slope was flatter (r = -0.45, P = 0.003) in women but not men. The minimal thickness tended to be thinner in the elderly group (r = 0.038, P = 0.015). CONCLUSIONS: Women had a significantly wider base width, wider temporal width, thinner nasal thickness, and flatter temporal slope of the foveal pit than age-matched men. The base width and temporal width were wider and the temporal slope was flatter with age in women but not men.

Rapid and Label-Free Histopathology of Oral Lesions Using Deep Learning Applied to Optical and Infrared Spectroscopic Imaging Data.

Oral potentially malignant disorders (OPMDs) are precursors to over 80% of oral cancers. Hematoxylin and eosin (H&E) staining, followed by pathologist interpretation of tissue and cellular morphology, is the current gold standard for diagnosis. However, this method is qualitative, can result in errors during the multi-step diagnostic process, and results may have significant inter-observer variability. Chemical imaging (CI) offers a promising alternative, wherein label-free imaging is used to record both the morphology and the composition of tissue and artificial intelligence (AI) is used to objectively assign histologic information. Here, we employ quantum cascade laser (QCL)-based discrete frequency infrared (DFIR) chemical imaging to record data from oral tissues. In this proof-of-concept study, we focused on achieving tissue segmentation into three classes (connective tissue, dysplastic epithelium, and normal epithelium) using a convolutional neural network (CNN) applied to three bands of label-free DFIR data with paired darkfield visible imaging. Using pathologist-annotated H&E images as the ground truth, we demonstrate results that are 94.5% accurate with the ground truth using combined information from IR and darkfield microscopy in a deep learning framework. This chemical-imaging-based workflow for OPMD classification has the potential to enhance the efficiency and accuracy of clinical oral precancer diagnosis.

Topological similarity between the 2µm plasmid partitioning locus and the budding yeast centromere: evidence for a common evolutionary origin?

The partitioning locus STB of the selfish plasmid, the 2µm circle, of Saccharomyces cerevisiae is essential for the propagation of this multi-copy extra-chromosomal DNA element with nearly chromosome-like stability. The functional competence of STB requires the plasmid-coded partitioning proteins Rep1 and Rep2 as well as host-coded proteins. Host factors that associate with STB in a Rep1- and Rep2-dependent manner also interact with centromeres, and play important roles in chromosome segregation. They include the cohesin complex and the centromere-specific histone H3 variant Cse4. The genetically defined point centromere of S. cerevisiae differs starkly from the much more widespread epigenetically specified regional centromeres of eukaryotes. The particularly small size of the S. cerevisiae centromere and the association of chromosome segregation factors with STB raise the possibility of an evolutionary link between these two partitioning loci. The unusual positive supercoiling harboured by the S. cerevisiae centromere and STB in vivo in their functional states, unveiled by recent experiments, bolsters the notion of their potential descent from an ancestral plasmid partitioning locus.

The 2 micron plasmid of Saccharomyces cerevisiae: a miniaturized selfish genome with optimized functional competence.

The 2 micron plasmid of Saccharomyces cerevisiae is a relatively small multi-copy selfish DNA element that resides in the yeast nucleus at a copy number of 40-60 per haploid cell. The plasmid is able to persist in host populations with almost chromosome-like stability with the help of a partitioning system and a copy number control system. The first part of this article describes the properties of the partitioning system comprising two plasmid coded proteins, Rep1 and Rep2, and a partitioning locus STB. Current evidence supports a model in which the Rep-STB system couples plasmid segregation to chromosome segregation by promoting the physical association of plasmid molecules with chromosomes. In the second part, the focus is on the Flp site-specific recombination system housed by the plasmid, which plays a critical role in maintaining steady state plasmid copy number. The Flp system corrects any decrease in plasmid population by promoting plasmid amplification via a recombination induced rolling circle replication mechanism. Appropriate plasmid amplification, without runaway increase in copy number, is ensured by positive and negative regulation of FLP gene expression by plasmid coded proteins and by the control of Flp level/activity through post-translational modification of Flp by the cellular sumoylation system. The Flp system has been successfully utilized to understand mechanisms of site-specific recombination and to bring about directed genetic alterations for addressing fundamental problems in biology and for accomplishing bio-engineering objectives. A particularly interesting, and perhaps less well known and underappreciated, application of Flp in revealing unique DNA topologies required to confer functional competence to DNA-protein machines is discussed.

Ossification of posterior atlantoaxial membrane causing spinal stenosis - A case report.

Background: Ossification of the posterior atlantoaxial membrane (PAAM) is a rare cause of spinal cord compression. Case presentation: A 46-year-old woman with rheumatoid arthritis (RA) and a 2-year history of slowly progressive gait disturbance underwent surgery for right knee stiffness and right lower limb mild weakness. A neurologic examination revealed brisk deep tendon reflexes (DTR) and spasticity in her four limbs. A computed tomography (CT) scan revealed spinal stenosis caused by ossification of the PAAM, a rare cause of spinal cord compression. The patient's lower limbs weakness and walking capability were ameliorated post-surgery. Conclusions: Although the exact mechanism of ossification of PAAM remains unclear, chronic mechanical stress as well as persistent atlantoaxial instability may promote the development of the ossification.

Methyltransferase Inhibition Enables Tgfß Driven Induction of CDKN2A and B in Cancer Cells.

CDKN2A/B deletion or silencing is common across human cancer, reinforcing the general importance of bypassing its tumor suppression in cancer formation or progression. In rhabdomyosarcoma (RMS) and neuroblastoma, two common childhood cancers, the three CDKN2A/B transcripts are independently expressed to varying degrees, but one, ARF, is uniformly silenced. Although TGFß induces certain CDKN2A/B transcripts in HeLa cells, it was unable to do so in five tested RMS lines unless the cells were pretreated with a broadly acting methyltransferase inhibitor, DZNep, or one targeting EZH2. CDKN2A/B induction by TGFß correlated with de novo appearance of three H3K27Ac peaks within a 20 kb cis element ∼150 kb proximal to CDKN2A/B. Deleting that segment prevented their induction by TGFß but not a basal increase driven by methyltransferase inhibition alone. Expression of two CDKN2A/B transcripts was enhanced by dCas9/CRISPR activation targeting either the relevant promoter or the 20 kb cis elements, and this "precise" manipulation diminished RMS cell propagation in vitro. Our findings show crosstalk between methyltransferase inhibition and TGFß-dependent activation of a remote enhancer to reverse CDKN2A/B silencing. Though focused on CDKN2A/B here, such crosstalk may apply to other TGFß-responsive genes and perhaps govern this signaling protein's complex effects promoting or blocking cancer.

Infrared spectroscopic laser scanning confocal microscopy for whole-slide chemical imaging.

Chemical imaging, especially mid-infrared spectroscopic microscopy, enables label-free biomedical analyses while achieving expansive molecular sensitivity. However, its slow speed and poor image quality impede widespread adoption. We present a microscope that provides high-throughput recording, low noise, and high spatial resolution where the bottom-up design of its optical train facilitates dual-axis galvo laser scanning of a diffraction-limited focal point over large areas using custom, compound, infinity-corrected refractive objectives. We demonstrate whole-slide, speckle-free imaging in ~3 min per discrete wavelength at 10× magnification (2 µm/pixel) and high-resolution capability with its 20× counterpart (1 µm/pixel), both offering spatial quality at theoretical limits while maintaining high signal-to-noise ratios (>100:1). The data quality enables applications of modern machine learning and capabilities not previously feasible - 3D reconstructions using serial sections, comprehensive assessments of whole model organisms, and histological assessments of disease in time comparable to clinical workflows. Distinct from conventional approaches that focus on morphological investigations or immunostaining techniques, this development makes label-free imaging of minimally processed tissue practical.

A novel polysilicon field-enhanced nanowire thin-film transistor with the TiN-hafnia-nitride-vacuum-silicon (THNVAS) structure for nonvolatile memory applications.

A novel poly-Si field-enhanced nanowire (FEN) TFT memory with the TiN-hafnia-nitride-vacuum-silicon (THNVAS) structure fabricated simply via a sidewall spacer formation has been presented. The THNVAS devices with superior memory performance were demonstrated by introducing the hafnia as blocking oxide and the vacuum, the lowest-k in nature, as tunneling layer. According to the simulation results, the memory device with oxide/nitride/vacuum gate dielectric exhibited a higher local electric-field of 4.72 x 10(7) V/cm as compared to 2.55 x 10(7) V/cm for the conventional oxide/nitride/oxide counterpart. In addition, the electric-field of tunneling layer could be further increased to 7.06 x 10(7) V/cm while the blocking oxide was substituted for hafnia. The experimental data showed that THNVAS possessed a greater threshold voltage shift of 3.75 V in 10 ms at V(GS) = 12 V, whereas the shift only 2.5 V for THNOS ones. These considerable improvements for THNVAS devices could be attributed to the evident field enhancement across the vacuum tunneling layer. Furthermore, owing to the empty feature of vacuum tunneling layer, the THNVAS demonstrated much-improved endurance performance and preferable data retention property. Hence, such excellent characteristics of THNVAS will be an attractive nonvolatile memory for future system-on-panel and 3-D Flash applications.

Revealing the nanometric structural changes in myocardial infarction models by time-lapse intravital imaging.

In the development of bioinspired nanomaterials for therapeutic applications, it is very important to validate the design of nanomaterials in the disease models. Therefore, it is desirable to visualize the change of the cells in the diseased site at the nanoscale. Heart diseases often start with structural, morphological, and functional alterations of cardiomyocyte components at the subcellular level. Here, we developed straightforward technique for long-term real-time intravital imaging of contracting hearts without the need of cardiac pacing and complex post processing images to understand the subcellular structural and dynamic changes in the myocardial infarction model. A two-photon microscope synchronized with electrocardiogram signals was used for long-term in vivo imaging of a contracting heart with subcellular resolution. We found that the structural and dynamic behaviors of organelles in cardiomyocytes closely correlated with heart function. In the myocardial infarction model, sarcomere shortening decreased from ∼15% (healthy) to ∼8% (diseased) as a result of impaired cardiac function, whereas the distances between sarcomeres increased by 100 nm (from 2.11 to 2.21 µm) in the diastolic state. In addition, T-tubule system regularity analysis revealed that T-tubule structures that were initially highly organized underwent significant remodeling. Morphological remodeling and changes in dynamic activity at the subcellular level are essential to maintain heart function after infarction in a heart disease model.

Optogenetic manipulation of cell migration with high spatiotemporal resolution using lattice lightsheet microscopy.

Lattice lightsheet microscopy (LLSM) featuring three-dimensional recording is improved to manipulate cellular behavior with subcellular resolution through optogenetic activation (optoLLSM). A position-controllable Bessel beam as a stimulation source is integrated into the LLSM to achieve spatiotemporal photoactivation by changing the spatial light modulator (SLM) patterns. Unlike the point-scanning in a confocal microscope, the lattice beams are capable of wide-field optical sectioning for optogenetic activation along the Bessel beam path.We show that the energy power required for optogenetic activations is lower than 1 nW (or 24 mWcm-2) for time-lapses of CRY2olig clustering proteins, and membrane ruffling can be induced at different locations within a cell with subcellular resolution through light-triggered recruitment of phosphoinositide 3-kinase. Moreover, with the epidermal growth factor receptor (EGFR) fused with CRY2olig, we are able to demonstrate guided cell migration using optogenetic stimulation for up to 6 h, where 463 imaging volumes are collected, without noticeable cellular damages.