Differential Non-Coding RNA Profiles for Lung Cancer Early Detection in African and White Americans.

Introduction: Lung cancer leads in cancer-related deaths. Disparities are observed in lung cancer rates, with African Americans (AAs) experiencing disproportionately higher incidence and mortality compared to other ethnic groups. Non-coding RNAs (ncRNAs) play crucial roles in lung tumorigenesis. Our objective was to identify ncRNA biomarkers associated with the racial disparity in lung cancer. Methods: Using droplet digital PCR, we examined 93 lung-cancer-associated ncRNAs in the plasma and sputum samples from AA and White American (WA) participants, which included 118 patients and 92 cancer-free smokers. Subsequently, we validated our results with a separate cohort comprising 56 cases and 72 controls. Results: In the AA population, plasma showed differential expression of ten ncRNAs, while sputum revealed four ncRNAs when comparing lung cancer patients to the control group. In the WA population, the plasma displayed eleven ncRNAs, and the sputum had five ncRNAs showing differential expression between the lung cancer patients and the control group. For AAs, we identified a three-ncRNA panel (plasma miRs-147b, 324-3p, 422a) diagnosing lung cancer in AAs with 86% sensitivity and 89% specificity. For WAs, a four-ncRNA panel was developed, comprising sputum miR-34a-5p and plasma miRs-103-3p, 126-3p, 205-5p, achieving 88% sensitivity and 87% specificity. These panels remained effective across different stages and histological types of lung tumors and were validated in the independent cohort. Conclusions: The ethnicity-related ncRNA signatures have promise as biomarkers to address the racial disparity in lung cancer.

Standoff Deep Ultraviolet Raman Spectrometer for Trace Detection.

We developed a state-of-the-art, high-sensitivity, low-stray-light standoff deep-ultraviolet (DUV) Raman spectrometer for the trace detection of resonance Raman-enhanced chemical species. As an excitation source for Raman measurements, we utilized our recently developed, second-generation, miniaturized, diode-pumped, solid-state neodymium-doped gadolinium orthovanadate (Nd:GdVO4) laser that generates quasi-continuous wave 228 nm light. This 228 nm excitation enhances the Raman intensities of vibrations of NOx groups in explosive molecules, aromatic groups in biological molecules, and various aromatic hydrocarbons. Our DUV Raman spectrograph utilizes a custom DUV f/8 Cassegrain telescope with an ∼200 mm diameter primary mirror, high-efficiency DUV transmission gratings, custom DUV mirrors, and a custom 228 nm Rayleigh rejection filter. We utilized our new standoff DUV Raman spectrometer to measure high signal-to-noise ratio spectra of ∼50 µg/cm2 drop-cast explosives: ammonium nitrate (AN), trinitrotoluene, pentaerythritol tetranitrate as well as aromatic biological molecules: lysozyme, tryptophan, tyrosine, deoxycytidine monophosphate, deoxyadenosine monophosphate at an ∼3 m distance within 10-30 s accumulation times. We roughly estimate the average ultraviolet resonance Raman (UVRR) detection limits for the relatively homogeneous drop-cast films of explosives and biological molecules to be ∼1 µg/cm2 when utilizing a continuous raster scanning that averages Raman signal over ∼1 cm2 sample area to avoid quick analyte depletion due to ultraviolet (UV) photolysis. We determined 3 m standoff UVRR detection limits for drop-cast AN films and identified factors impacting UVRR detection limits such as analyte photochemistry and analyte morphology. We found a detection limit of ∼0.5 µg/cm2 for drop-cast AN films on glass substrates when the Raman signal is averaged over ∼0.5 cm2 of sample surface using a continuous raster scan. For a step raster scan, when the probed sample area is limited to the laser spot size, the detection limit is approximately tenfold higher (∼5 µg/cm2) due to the impact of UV photochemistry.

Calibration of Raman Bandwidths on the Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals (SHERLOC) Deep Ultraviolet Raman and Fluorescence Instrument Aboard the Perseverance Rover.

In this work, we derive a simple method for calibrating Raman bandwidths for the Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals (SHERLOC) instrument onboard NASA's Perseverance rover. Raman bandwidths and shapes reported by an instrument contain contributions from both the intrinsic Raman band (IRB) and instrumental artifacts. To directly correlate bandwidth to sample properties and to compare bandwidths across instruments, the IRB width needs to be separated from instrumental effects. Here, we use the ubiquitous bandwidth calibration method of modeling the observed Raman bands as a convolution of a Lorentzian IRB and a Gaussian instrument slit function. Using calibration target data, we calculate that SHERLOC has a slit function width of 34.1 cm-1. With a measure of the instrument slit function, we can deconvolve the IRB from the observed band, providing the width of the Raman band unobscured by instrumental artifact. We present the correlation between observed Raman bandwidth and intrinsic Raman bandwidth in table form for the quick estimation of SHERLOC Raman intrinsic bandwidths. We discuss the limitations of using this model to calibrate Raman bandwidth and derive a quantitative method for calculating the errors associated with the calibration. We demonstrate the utility of this method of bandwidth calibration by examining the intrinsic bandwidths of SHERLOC sulfate spectra and by modeling the SHERLOC spectrum of olivine.

Diverse organic-mineral associations in Jezero crater, Mars.

The presence and distribution of preserved organic matter on the surface of Mars can provide key information about the Martian carbon cycle and the potential of the planet to host life throughout its history. Several types of organic molecules have been previously detected in Martian meteorites1 and at Gale crater, Mars2-4. Evaluating the diversity and detectability of organic matter elsewhere on Mars is important for understanding the extent and diversity of Martian surface processes and the potential availability of carbon sources1,5,6. Here we report the detection of Raman and fluorescence spectra consistent with several species of aromatic organic molecules in the Máaz and Séítah formations within the Crater Floor sequences of Jezero crater, Mars. We report specific fluorescence-mineral associations consistent with many classes of organic molecules occurring in different spatial patterns within these compositionally distinct formations, potentially indicating different fates of carbon across environments. Our findings suggest there may be a diversity of aromatic molecules prevalent on the Martian surface, and these materials persist despite exposure to surface conditions. These potential organic molecules are largely found within minerals linked to aqueous processes, indicating that these processes may have had a key role in organic synthesis, transport or preservation.

Utilizing MiSeq Sequencing to Detect Circulating microRNAs in Plasma for Improved Lung Cancer Diagnosis.

Non-small cell lung cancer (NSCLC) is a major contributor to cancer-related deaths, but early detection can reduce mortality. NSCLC comprises mainly adenocarcinoma (AC) and squamous cell carcinoma (SCC). Circulating microRNAs (miRNAs) in plasma have emerged as promising biomarkers for NSCLC. However, existing techniques for analyzing miRNAs have limitations, such as restricted target detection and time-consuming procedures. The MiSeqDx System has been shown to overcome these limitations, making it a promising tool for routine clinical settings. We investigated whether the MiSeqDx could profile cell-free circulating miRNAs in plasma and diagnose NSCLC. We sequenced RNA from the plasma of patients with AC and SCC and from cancer-free smokers using the MiSeqDx to profile and compare miRNA expressions. The MiSeqDx exhibits high speed and accuracy when globally analyzing plasma miRNAs. The entire workflow, encompassing RNA to data analysis, was completed in under three days. We also identified panels of plasma miRNA biomarkers that can diagnose NSCLC with 67% sensitivity and 68% specificity, and detect SCC with 90% sensitivity and 94% specificity, respectively. This study is the first to demonstrate that rapid profiling of plasma miRNAs using the MiSeqDx has the potential to offer a straightforward and effective method for the early detection and classification of NSCLC.

Streptococcus pneumoniae promotes lung cancer development and progression.

Streptococcus pneumoniae (SP) is associated with lung cancer, yet its role in the tumorigenesis remains uncertain. Herein we find that SP attaches to lung cancer cells via binding pneumococcal surface protein C (PspC) to platelet-activating factor receptor (PAFR). Interaction between PspC and PAFR stimulates cell proliferation and activates PI3K/AKT and nuclear factor kB (NF-kB) signaling pathways, which trigger a pro-inflammatory response. Lung cancer cells infected with SP form larger tumors in BALB/C mice compared to untreated cells. Mice treated with tobacco carcinogen and SP develop more lung tumors and had shorter survival period than mice treated with the carcinogen alone. Mutating PspC or PAFR abolishes tumor-promoting effects of SP. Overabundance of SP is associated with the survival. SP may play a driving role in lung tumorigenesis by activating PI3K/AKT and NF-kB pathways via binding PspC to PAFR and provide a microbial target for diagnosis and treatment of the disease.

Aqueous alteration processes in Jezero crater, Mars-implications for organic geochemistry.

The Perseverance rover landed in Jezero crater, Mars, in February 2021. We used the Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals (SHERLOC) instrument to perform deep-ultraviolet Raman and fluorescence spectroscopy of three rocks within the crater. We identify evidence for two distinct ancient aqueous environments at different times. Reactions with liquid water formed carbonates in an olivine-rich igneous rock. A sulfate-perchlorate mixture is present in the rocks, which probably formed by later modifications of the rocks by brine. Fluorescence signatures consistent with aromatic organic compounds occur throughout these rocks and are preserved in minerals related to both aqueous environments.

Aptamer-functionalized 2D photonic crystal hydrogels for detection of adenosine.

Aptamer-functionalized two-dimensional photonic crystal (2DPC) hydrogels are reported for the detection of adenosine (AD). As a molecular recognition group, an AD-binding aptamer was covalently attached to 2DPC hydrogels. This aptamer selectively and sensitively binds AD, changing the conformation of the aptamer from a long single-stranded structure (AD-free conformation) to a short hairpin loop structure (AD-bound conformation). The AD-binding-induced changes of aptamer conformation reduced the volume of the 2DPC hydrogels and decreased the interparticle spacing of the 2DPC embedded in the hydrogel network. The particle spacing changes being dependent on AD concentration were determined by measuring 2DPC light diffraction using a simple laser pointer. The 2DPC hydrogel sensor showed a large particle spacing decrease of ~ 110 nm in response to 1 mM AD in phosphate-buffered saline (PBS). The linear range of determination of AD was 0.1 nM to 1 mM and the limit of detection was 0.09 nM. The hydrogel sensor response for real samples was then validated in diluted fetal bovine serum (FBS) and human urine. The average % difference in particle spacing changes measured between diluted FBS and pure PBS was only 3.99%. In diluted human urine, the recoveries for the detection of AD were 95-101% and the relative standard deviations were 4.9-7.8%. The results demonstrate the potential applicability of the hydrogel sensor for real samples. This sensing concept, using the aptamer-functionalized 2DPC hydrogels, allows for a simple, sensitive, selective, and reversible detection of AD. It may enable sensor development for a wide variety of analytes by simply changing the aptamer recognition group.

DNA-Crosslinked 2D Photonic Crystal Hydrogels for Detection of Adenosine Actuated by an Adenosine-Binding Aptamer.

There is a need to develop versatile sensing motifs that can be used to detect a variety of chemical targets in resource-limited settings, for example, at the point of care. While numerous sensing technologies have been developed toward this effort, these technologies can be overly complex and require a skilled technician, extensive sample preparation, or sophisticated instrumentation to use, limiting their generalizability and application in resource-limited settings. Here, we report a novel sensing motif that utilizes DNA-crosslinked two-dimensional photonic crystal (2DPC) hydrogels. These hydrogel sensors contain a DNA aptamer recognition group that binds a target analyte. As proof of concept, we fabricated 2DPC hydrogels using a well-studied adenosine-binding aptamer. This adenosine aptamer is duplexed with a partially complementary strand and forms responsive crosslinks in the hydrogel polymer network. When adenosine is introduced, aptamer-adenosine binding occurs, breaking the DNA crosslinks and causing the hydrogel to swell. This in turn increases the particle spacing of an embedded 2DPC array, shifting the 2DPC Bragg diffraction. Thus, adenosine concentration can be monitored through 2DPC Bragg diffraction measurements. A linear range of 20 µM to 2 mM was observed. The detection limits were calculated to be 13.9 µM in adenosine-binding buffer and 26.7 µM in fetal bovine serum. This reported sensing motif has a readout that is simple and rapid and requires minimal equipment. We hypothesize that this sensing motif is generalizable and that other sensors can be easily fabricated by simply exchanging the aptamer that serves as a molecular recognition group.

Calibration of the SHERLOC Deep Ultraviolet Fluorescence-Raman Spectrometer on the Perseverance Rover.

We describe the wavelength calibration of the spectrometer for the scanning of habitable environments with Raman and luminescence for organics and chemicals (SHERLOC) instrument onboard NASA's Perseverance Rover. SHERLOC utilizes deep ultraviolet Raman and fluorescence (DUV R/F) spectroscopy to enable analysis of samples from the Martian surface. SHERLOC employs a 248.6 nm deep ultraviolet laser to generate Raman-scattered photons and native fluorescence emission photons from near-surface material to detect and classify chemical and mineralogical compositions. The collected photons are focused on a charge-coupled device and the data are returned to Earth for analysis. The compact DUV R/F spectrometer has a spectral range from 249.9 nm to 353.6 nm (∼200 cm-1 to 12 000 cm-1) (with a spectral resolution of 0.296 nm (∼40 cm-1)). The compact spectrometer uses a custom design to project a high-resolution Raman spectrum and a low-resolution fluorescence spectrum on a single charge-coupled device. The natural spectral separation enabled by deep ultraviolet excitation enables wavelength separation of the Raman/fluorescence spectra. The SHERLOC spectrometer was designed to optimize the resolution of the Raman spectral region and the wavelength range of the fluorescence region. The resulting illumination on the charge-coupled device is curved, requiring a segmented, nonlinear wavelength calibration in order to understand the mineralogy and chemistry of Martian materials.

Rapid and Sensitive Detection of SARS-CoV-2 Using Clustered Regularly Interspaced Short Palindromic Repeats.

Rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for controlling the pandemic of coronavirus disease 2019. Polymerase chain reaction (PCR)-based technique is the standard test for detection of SARS-CoV-2, which, however, requires complicated sample manipulation (e.g., RNA extraction) and is time-consuming. We previously demonstrated that clustered regularly interspaced short palindromic repeats (CRISPR) could precisely detect Human papillomavirus and somatic mutations of Epidermal growth factor receptor gene and Kirsten rat sarcoma viral oncogene homolog gene in plasma. The objective of this study was to develop CRISPR as a rapid test for sensitive detection of SARS-CoV-2. We first combined reverse transcription-isothermal recombinase polymerase amplification and CRSIPR to detect SARS-CoV-2 in genomic RNA of cells infected with the virus. The CRISPR assay with guide RNA against the M gene of SARS-CoV-2 had a sensitivity of 0.1 copies per µL for detection of the virus. We then used the CRSIPR assay to directly analyze raw SARS-CoV-2 samples. The CRISPR assay could sensitively detect SARS-CoV-2 in one hour without RNA extraction. This assay can be performed at a single temperature and with minimal equipment. The results were immediately visualized either by a UV light illuminator or paper strips. The diagnostic value of the test was confirmed in nasopharyngeal swab specimens. Altogether, we have developed a rapid CRISPR test for sensitive detection of SARS-CoV-2.

Human Serum Phenylpyruvate Quantification Using Responsive 2D Photonic Crystal Hydrogels via Chemoselective Oxime Ligation: Progress toward Developing Phenylalanine-Sensing Elements.

There is a need to develop at-home phenylalanine (Phe) test kits, analogous to home glucose meters, for phenylketonuria patients who must measure their blood Phe levels frequently to adjust their diet. Unfortunately, such test kits are not available yet because of the lack of simple and inexpensive Phe-sensing elements. With the goal of developing a Phe-sensing element, we fabricated two-dimensional photonic crystal (2DPC) hydrogels that quantify human serum phenylpyruvate (PhPY), which is the product of the reaction between Phe and the enzyme phenylalanine dehydrogenase. The PhPY-sensing hydrogels have oxyamine recognition groups that link PhPY to the hydrogel polymer network via chemoselective oxime ligation. This structural modification induces the hydrogel to swell, which then increases interparticle spacings within the embedded 2DPC. The PhPY-induced particle spacing changes are measured from light diffraction and used to quantify the PhPY concentrations. The estimated limit of detection of PhPY in human serum for a detection time of 30 min is 19 µM, which is comparable to the minimum blood Phe concentrations of healthy people. Besides the potential application for developing Phe-sensing elements, this new hydrogel sensing approach via chemoselective oxime ligation is generalizable to the development of other chemical sensors working in complex biological environments.

Stimuli-Responsive Pure Protein Organogel Sensors and Biocatalytic Materials.

Utilizing protein chemistry in organic solvents has important biotechnology applications. Typically, organic solvents negatively impact protein structure and function. Immobilizing proteins via cross-links to a support matrix or to other proteins is a common strategy to preserve the native protein function. Recently, we developed methods to fabricate macroscopic responsive pure protein hydrogels by lightly cross-linking the proteins with glutaraldehyde for chemical sensing and enzymatic catalysis applications. The water in the resulting protein hydrogel can be exchanged for organic solvents. The resulting organogel contains pure organic solvents as their mobile phases. The organogel proteins retain much of their native protein function, i.e., protein-ligand binding and enzymatic activity. A stepwise ethylene glycol (EG) solvent exchange was performed to transform these hydrogels into organogels with a very low vapor pressure mobile phase. These responsive organogels are not limited by solvent/mobile phase evaporation. The solvent exchange to pure EG is accompanied by a volume phase transition (VPT) that decreases the organogel volume compared to that of the hydrogel. Our organogel sensor systems utilize shifts in the particle spacing of an attached two-dimensional photonic crystal (2DPC) to report on the volume changes induced by protein-ligand binding. Our 2DPC bovine serum albumin (BSA) organogels exhibit VPT that swell the organogels in response to the BSA binding of charged ligands like ibuprofen and fatty acids. To our knowledge, this is the first report of a pure protein organogel VPT induced by protein-ligand binding. Catalytic protein organogels were also fabricated that utilize the enzyme organophosphorus hydrolase (OPH) to hydrolyze toxic organophosphate (OP) nerve agents. Our OPH organogels retain significant enzymatic activity. The OPH organogel rate of OP hydrolysis is ∼160 times higher than that of un-cross-linked OPH monomers in a 1:1 ethylene glycol/water mixture.

Mechanisms by Which Organic Solvent Exchange Transforms Responsive Pure Protein Hydrogels into Responsive Organogels.

Responsive pure protein organogel sensors and catalysts are fabricated by replacing the aqueous mobile phase of protein hydrogels with pure ethylene glycol (EG). Exchanging water for EG causes irreversible volume phase transitions (VPT) in bovine serum albumin (BSA) polymers; however, BSA hydrogel and organogel sensors show similar volume responses to protein-ligand binding. This work elucidates the mechanisms involved in this enabling irreversible VPT by examining the protein secondary structure, hydration, and protein polymer morphology. Organogel proteins retain their native activity because their secondary structure and hydration shell are relatively unperturbed by the EG exchange. Conversely, the decreasing solvent quality initiates polymer phase separation to minimize the BSA polymer surface area exposed to EG, thus decreasing distances between BSA polymer strands. These protein polymer morphology changes promote interprotein interactions between BSA polymer strands, which increase the effective polymer cross-link density and prevent organogel swelling as the mobile phase is exchanged back to water.

Physical Frailty: ICFSR International Clinical Practice Guidelines for Identification and Management.

OBJECTIVE: The task force of the International Conference of Frailty and Sarcopenia Research (ICFSR) developed these clinical practice guidelines to overview the current evidence-base and to provide recommendations for the identification and management of frailty in older adults. METHODS: These recommendations were formed using the GRADE approach, which ranked the strength and certainty (quality) of the supporting evidence behind each recommendation. Where the evidence-base was limited or of low quality, Consensus Based Recommendations (CBRs) were formulated. The recommendations focus on the clinical and practical aspects of care for older people with frailty, and promote person-centred care. Recommendations for Screening and Assessment: The task force recommends that health practitioners case identify/screen all older adults for frailty using a validated instrument suitable for the specific setting or context (strong recommendation). Ideally, the screening instrument should exclude disability as part of the screening process. For individuals screened as positive for frailty, a more comprehensive clinical assessment should be performed to identify signs and underlying mechanisms of frailty (strong recommendation). Recommendations for Management: A comprehensive care plan for frailty should address polypharmacy (whether rational or nonrational), the management of sarcopenia, the treatable causes of weight loss, and the causes of exhaustion (depression, anaemia, hypotension, hypothyroidism, and B12 deficiency) (strong recommendation). All persons with frailty should receive social support as needed to address unmet needs and encourage adherence to a comprehensive care plan (strong recommendation). First-line therapy for the management of frailty should include a multi-component physical activity programme with a resistance-based training component (strong recommendation). Protein/caloric supplementation is recommended when weight loss or undernutrition are present (conditional recommendation). No recommendation was given for systematic additional therapies such as cognitive therapy, problem-solving therapy, vitamin D supplementation, and hormone-based treatment. Pharmacological treatment as presently available is not recommended therapy for the treatment of frailty.

Sputum long non-coding RNA biomarkers for diagnosis of lung cancer.

BACKGROUND: Analysis of molecular changes in sputum may help diagnose lung cancer. Long non-coding RNAs (lncRNAs) play vital roles in various biological processes, and their dysregulations contribute to the development and progression of lung tumorigenesis. Herein, we determine whether aberrant lncRNAs could be used as potential sputum biomarkers for lung cancer. METHODS: Using reverse transcription PCR, we measure expressions of lung cancer-associated lncRNAs in sputum of a discovery cohort of 67 lung cancer patients and 65 cancer-free smokers with benign diseases and a validation cohort of 59 lung cancer patients and 60 cancer-free smokers with benign diseases. RESULTS: In the discovery cohort, four of the lncRNAs displayed a significantly different level in sputum of lung cancer patients vs.cancer-free smokers with benign diseases (all P< 0.001). From the four lncRNAs, three lncRNAs (SNHG1, H19, and HOTAIR) are identified as a biomarker panel, producing 82.09% sensitivity and 89.23% specificity for diagnosis of lung cancer. Furthermore, the biomarker panel has a higher sensitivity (82.09% vs. 52.24%, P= 0.02) and a similar specificity compared with sputum cytology (89.23% vs. 90.77%, P= 0.45). In addition, the lncRNA biomarker panel had a higher sensitivity (87.50% vs. 70.07%, p= 0.03) for diagnosis of squamous cell carcinoma compared with adenocarcinoma of the lung, while maintaining the same specificity (89.23%). The potential of the sputum lncRNA biomarkers for lung cancer detection is confirmed in the validation cohort. CONCLUSION: We have for the first time shown that the analysis of lncRNAs in sputum might be a noninvasive approach for diagnosis of lung cancer.