Lysosome-related genes predict acute myeloid leukemia prognosis and response to immunotherapy.

Background: Acute myeloid leukemia (AML) is a highly aggressive and pathogenic hematologic malignancy with consistently high mortality. Lysosomes are organelles involved in cell growth and metabolism that fuse to form specialized Auer rods in AML, and their role in AML has not been elucidated. This study aimed to identify AML subtypes centered on lysosome-related genes and to construct a prognostic model to guide individualized treatment of AML. Methods: Gene expression data and clinical data from AML patients were downloaded from two high-throughput sequencing platforms. The 191 lysosomal signature genes were obtained from the database MsigDB. Lysosomal clusters were identified by unsupervised consensus clustering. The differences in molecular expression, biological processes, and the immune microenvironment among lysosomal clusters were subsequently analyzed. Based on the molecular expression differences between lysosomal clusters, lysosomal-related genes affecting AML prognosis were screened by univariate cox regression and multivariate cox regression analyses. Algorithms for LASSO regression analyses were employed to construct prognostic models. The risk factor distribution, KM survival curve, was applied to evaluate the survival distribution of the model. Time-dependent ROC curves, nomograms and calibration curves were used to evaluate the predictive performance of the prognostic models. TIDE scores and drug sensitivity analyses were used to explore the implication of the model for AML treatment. Results: Our study identified two lysosomal clusters, cluster1 has longer survival time and stronger immune infiltration compared to cluster2. The differences in biological processes between the two lysosomal clusters are mainly manifested in the lysosomes, vesicles, immune cell function, and apoptosis. The prognostic model consisting of six prognosis-related genes was constructed. The prognostic model showed good predictive performance in all three data sets. Patients in the low-risk group survived significantly longer than those in the high-risk group and had higher immune infiltration and stronger response to immunotherapy. Patients in the high-risk group showed greater sensitivity to cytarabine, imatinib, and bortezomib, but lower sensitivity to ATRA compared to low -risk patients. Conclusion: Our prognostic model based on lysosome-related genes can effectively predict the prognosis of AML patients and provide reference evidence for individualized immunotherapy and pharmacological chemotherapy for AML.

Engineering Gene-Specific DNAzymes for Accessible and Multiplexed Nucleic Acid Testing.

The accurate and timely detection of disease biomarkers at the point-of-care is essential to ensuring effective treatment and epidemiological surveillance. Here, we report the selection and engineering of RNA-cleaving DNAzymes that respond to specific genetic markers and amplify detection signals. Because the target-specific activation of gene-specific DNAzymes (gDz) is like the trans-cleavage activity of clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated (Cas) machinery, we further developed a CRISPR-like assay using RNA-cleaving DNAzyme coupled with isothermal sequence and signal amplification (CLARISSA) for nucleic acid detection in clinical samples. Building on the high sequence specificity and orthogonality of gDzs, CLARISSA is highly versatile and expandable for multiplex testing. Upon integration with an isothermal recombinase polymerase amplification, CLARISSA enabled the detection of human papillomavirus (HPV) 16 in 189 cervical samples collected from cervical cancer screening participants (n = 189) with 100% sensitivity and 97.4% specificity, respectively. A multiplexed CLARISSA further allowed the simultaneous analyses of HPV16 and HPV18 in 46 cervical samples, which returned clinical sensitivity of 96.3% for HPV16 and 83.3% for HPV18, respectively. No false positives were found throughout our tests. Besides the fluorescence readout using fluorogenic reporter probes, CLARISSA is also demonstrated to be fully compatible with a visual lateral flow readout. Because of the high sensitivity, accessibility, and multiplexity, we believe CLARISSA is an ideal CRISPR-Dx alternative for clinical diagnosis in field-based and point-of-care applications.

A Portable Nucleic Acid Testing Platform with Photosensitization, a Three-Dimensionally Printed Multipiece Chip, and Digital Color Sensing.

Portable nucleic acid testing (NAT) holds great promise for point-of-care disease diagnosis and field-based applications but remains difficult to achieve. Herein, we describe a portable NAT that streamlines loop-mediated isothermal amplification with photosensitization-based color development in a fully sealed 3D-printed multipiece chip. Using a smartphone accessory and an APP, we also introduce a calibration-free quantification approach via digital color sensing and library matching. With these innovative approaches, our detection platform is highly accessible, allowing for rapid and sensitive NAT without requiring sophisticated instruments and well-trained personnel. The field applicability of our NAT platform was demonstrated by detecting tuberculosis infections in clinical sputum samples and food adulteration in commercial salmon meat products.

Integrating Paper-Based Microfluidics and Lateral Flow Strip into Nucleic Acid Amplification Device toward Rapid, Low-Cost, and Visual Diagnosis of Multiple Mycobacteria.

Efficient diagnosis of mycobacterial infections can effectively manage and prevent the transmission of infectious diseases. Unfortunately, existing diagnostic strategies are challenged by long assay times, high costs, and highly specialized expertise to distinguish between pulmonary tuberculosis (PTB) and nontuberculous mycobacterial pulmonary diseases (NTM-PDs). Herein, in this study, an optimized 3D paper-based analytical device (µPAD) is incorporated with a closed lateral flow (LF) strip into a loop-mediated isothermal amplification (LAMP) device (3D-µPAD-LF-LAMP) for rapid, low-cost, and visual detection of pathogenic mycobacteria. The platform's microfluidic feature enhanced the nucleic acid amplification, thereby reducing the costs and time as compared to boiling, easyMAG, and QIAGEN techniques. Moreover, the LF unit is specifically designed to minimize aerosol contamination for a user-friendly and visual readout. 3D-µPAD-LF-LAMP is optimized and assessed using standard strains, demonstrating a limit of detection (LOD) down to 10 fg reaction-1 . In a cohort of 815 patients, 3D-µPAD-LF-LAMP displays significantly better sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and diagnostic accuracy than conventional bacterial culture and Xpert techniques. Collectively, 3D-µPAD-LF-LAMP demonstrates enhanced accessibility, efficiency, and practicality for the diagnosis of multiple pathogenic mycobacteria, which can be applied across diverse clinical settings, thereby ultimately improving public health outcomes.

Utilization trend of prebiopsy multiparametric magnetic resonance imaging and its impact on prostate cancer detection: Real-world insights from a high-volume center in southwest China.

BACKGROUND: Data on the utilization and effects of prebiopsy prostate multiparametric magnetic resonance imaging (mpMRI) to support its routine use in real-world setting are still scarce. OBJECTIVE: To evaluate the change of clinical practice of prebiopsy mpMRI over time, and assess its diagnostic accuracy. DESIGN, SETTING, AND PARTICIPANTS: We retrospectively analyzed data from 6168 patients who underwent primary prostate biopsy (PBx) between January 2011 and December 2021 and had prostate-specific antigen (PSA) values ranging from 3 to 100 ng/mL. INTERVENTION: Prebiopsy MRI at the time of PBx. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We performed general linear regression and to elucidate trends in the annual use of prebiopsy mpMRI and conducted multivariable logistic regression to evaluate the potential benefits of incorporating prebiopsy mpMRI for prostate cancer (PCa) detection. RESULTS AND LIMITATIONS: The utilization of prebiopsy mpMRI significantly increased from 9.2% in 2011 to 75.0% in 2021 (p < 0.001). In addition, prebiopsy mpMRI significantly reduced negative PBx by 8.6% while improving the detection of clinically significant PCa (csPCa) by 7.0%. Regression analysis showed that the utilization of prebiopsy mpMRI was significantly associated with a 48% (95% confidence interval [CI]: 1.19-1.84) and 36% (95% CI: 1.12-1.66) increased PCa detection rate in the PSA 3-10 ng/mL and 10-20 ng/mL groups, respectively; and a 34% increased csPCa detection rate in the PSA 10-20 ng/mL group (95% CI: 1.09-1.64). The retrospective design and the single center cohort constituted the limitations of this study. CONCLUSIONS: Our study demonstrated a notable rise in the utilization of prebiopsy mpMRI in the past decade. The adoption of this imaging technique was significantly associated with an increased probability of detecting prostate cancer. PATIENT SUMMARY: From 2011 to 2021, we demonstrated a steady increase in the utilization of prebiopsy mpMRI among biopsy-naïve men. We also confirmed the positive impact of prebiopsy mpMRI utilization on the detection of prostate cancer.

A portable all-in-one microfluidic device with real-time colorimetric LAMP for HPV16 and HPV18 DNA point-of-care testing.

Screening for high-risk human papillomavirus (HPV) infection is one of the most important preventative measures for cervical cancer. However, fast, convenient, and low-cost HPV detection remains challenging, especially in resource-limited settings. Here, we report a portable all-in-one device (PAD) for point-of-care testing (POCT) for HPV16 and HPV18 DNA in cervical swabs. The PAD was engineered to integrate modules for extraction-free sample lysis, loop-mediated isothermal amplification (LAMP) with lyophilized reagent beads, and real-time colorimetric signal sensing into a single miniaturized device, considerably shortening the sample-to-result time to 15 min. The precision liquid handling in the completely sealed microfluidic chip is achieved by a uniquely designed pressure-balanced automatic liquid flow mechanism, thereby eliminating the need for manual manipulation of liquids and thus the risk of biohazards. The PAD employs an improved real-time colorimetric LAMP (rcLAMP) assay with a limit of detection (LOD) of 1 copy/µL, enabled by enhanced assay chemistry to maximize the reaction kinetics. To validate this device for clinical application, we tested 206 clinical cervical swab samples and obtained a sensitivity of 92.1% and a specificity of 99.0%. This custom PAD enabled by microfluidic and electronic engineering techniques can be configured for the simultaneous detection of HPV16 and HPV18 or other pathogens in point-of-care applications.

Ultrasensitive detection of genetic variation based on dual signal amplification assisted by isothermal amplification and cobalt oxyhydroxide nanosheets/quantum dots.

PML/RARα fusion gene (P/R) is the characteristic signature genetic variation of acute promyelocytic leukemia (APL). Here, by integrating triple-stranded DNA hybridization-triggered strand displacement amplification (tri-HT SDA) and cobalt oxyhydroxide nanosheets/quantum dots (CoOOH/QD)-based amplification, we constructed a novel biosensor of easy-operating, time-saving and high sensitivity for detecting P/R to meet clinical needs. Owing to the specific recognition and efficient amplification of tri-HT SDA as well as impressive anti-interference and considerable amplification of CoOOH/QD, this biosensor demonstrated a wide dynamic range (10 fM to 10 nM) with a low limit of detection (5.50 fM) in P/R detection. Additionally, this biosensor could detect P/R spiked into human serum with good recoveries and relative standard deviation (RSD), thus potentially exhibiting ultrasensitive and specific nuclear acid sequence detection ability in clinical diagnosis owing to combing isothermal amplification and nanomaterials.

Coding Intrinsic Disorder into DNA Hybridization Probes Enables Discrimination of Single Nucleotide Variants over Wide and Tunable Temperature Ranges.

DNA hybridization probes are commonly used tools to discriminate clinically important single nucleotide variants (SNVs) but often work at elevated temperatures with very narrow temperature intervals (ΔT). Herein, we investigated the thermodynamic basis of the narrow ΔT both in silico and experimentally. Our study revealed that the high entropy penalty of classic hybridization probe designs was the key attributor for the narrow ΔT. Guided by this finding, we further introduced an entropy-compensate probe (Sprobe) design by coding intrinsic disorder into a stem-loop hybridization probe. Sprobe expanded ΔT from less than 10 °C to over 30 °C. Moreover, both ΔT and the optimal reaction temperature can be fine-tuned by simply altering the length of the loop domain. Sprobe was clinically validated by analyzing EGFR L858R mutation in 36 pairs of clinical tumor tissue samples collected from lung cancer patients, which revealed 100 % clinical sensitivity and specificity. We anticipate that our study will serve as a general guide for designing thermal robust hybridization probes for clinical diagnostics.

Mismatch-Guided Deoxyribonucleic Acid Assembly Enables Ultrasensitive and Multiplex Detection of Low-Allele-Fraction Variants in Clinical Samples.

Somatic mutations are important signatures in clinical cancer treatment. However, accurate detection of rare somatic mutations with low variant-allele frequencies (VAFs) in clinical samples is challenging because of the interference caused by high concentrations of wild-type (WT) sequences. Here, we report a post amplification SNV-specific DNA assembly (PANDA) technology that eliminates the high concentration pressure caused by WT through a mismatch-guided DNA assembly and enables the ultrasensitive detection of cancer mutations with VAFs as low as 0.1%. Because it generates an assembly product that only exposes a single-stranded domain with the minimal length for signal readout and thus eliminates possible interferences from secondary structures and cross-interactions among sequences, PANDA is highly versatile and expandable for multiplex testing. With ultrahigh sensitivity, PANDA enabled the quantitative analysis of EGFR mutations in cell-free DNA of 68 clinical plasma samples and four pleuroperitoneal fluid samples, with test results highly consistent with NGS deep sequencing. Compared to digital PCR, PANDA returned fewer false negatives and ambiguous cases of clinical tests. Meanwhile, it also offers much lower upfront instrumental and operational costs. The multiplexity was demonstrated by developing a 3-plex PANDA for the simultaneous analysis of three EGFR mutations in 54 pairs of tumor and the adjacent noncancerous tissue samples collected from lung cancer patients. Because of the ultrahigh sensitivity, multiplexity, and simplicity, we anticipate that PANDA will find wide applications for analyzing clinically important rare mutations in diverse devastating diseases.

Enzyme-free dual amplification biosensor based on functional nucleic acid and CDs/CoOOH for detection of leukemia fusion gene.

Acute promyelocytic leukemia (APL) is an acute myeloid leukemia (AML) with a specific fusion gene target, PML/RARα fusion gene (PML/RARα), which is formed by the translocation of chromosomes 15 and 17. Detection of PML/RARα is the most reliable parameter for the diagnosis, treatment adjustment, efficacy evaluation, prognosis analysis and relapse prediction of APL. In this study, a novel biosensor was constructed for rapid enzyme-free detection of PML/RARα using DNAzyme and carbon dots/cobalt oxhydroxide nanosheet complexs (CDs/CoOOH). In the detection system, the separated DNAzymes could specifically recognize and bind together by the PML/RARα to form a complete DNAzyme for shearing hairpin probe (HP), then generated trigger, which was the first signal amplification. Then, trigger could hybridize with the capture probe (CP) anchored to streptavidin (SA) modified microplate as well as fluorescence quenching signal probe (SP@CDs/CoOOH). Finally, ascorbic acid (AA) was added to decompose CoOOH and the fluorescence of CDs was released, which was the second signal amplification. Through the dual signal amplification of DNAzyme and CDs/CoOOH, PML/RARα could be detected quickly and sensitively, which overcame the limitation of protein enzyme in traditional fluorescence methods, showing potential clinical application value in the diagnosis and treatment of leukemia.

Enabling a universal lateral flow readout for DNA strand displacement via disassembling chemical labels.

Herein, we describe a novel strategy that enables lateral flow readout for DNA strand displacement via disassembling chemical labels (DCL). Comparing it to a classic fluorogenic assay, we demonstrate that our DCL-based lateral flow assay is highly sensitive and specific, capable of discriminating single nucleotide variants in buccal swab samples.

Progressive brain abnormalities in schizophrenia across different illness periods: a structural and functional MRI study.

Schizophrenia is a chronic brain disorder, and neuroimaging abnormalities have been reported in different stages of the illness for decades. However, when and how these brain abnormalities occur and evolve remains undetermined. We hypothesized structural and functional brain abnormalities progress throughout the illness course at different rates in schizophrenia. A total of 115 patients with schizophrenia were recruited and stratified into three groups of different illness periods: 5-year group (illness duration: ≤5 years), 15-year group (illness duration: 12-18 years), and 25-year group (illness duration: ≥25 years); 230 healthy controls were matched by age and sex to the three groups, respectively. All participants underwent resting-state MRI scanning. Each group of patients with schizophrenia was compared with the corresponding controls in terms of voxel-based morphometry (VBM), fractional anisotropy (FA), global functional connectivity density (gFCD), and sample entropy (SampEn) abnormalities. In the 5-year group we observed only SampEn abnormalities in the putamen. In the 15-year group, we observed VBM abnormalities in the insula and cingulate gyrus and gFCD abnormalities in the temporal cortex. In the 25-year group, we observed FA abnormalities in nearly all white matter tracts, and additional VBM and gFCD abnormalities in the frontal cortex and cerebellum. By using two structural and two functional MRI analysis methods, we demonstrated that individual functional abnormalities occur in limited brain areas initially, functional connectivity and gray matter density abnormalities ensue later in wider brain areas, and structural connectivity abnormalities involving almost all white matter tracts emerge in the third decade of the course in schizophrenia.

Toehold-Exchange-Based Activation of Aptamer Switches Enables High Thermal Robustness and Programmability.

Aptamer switches are attractive nature-inspired tools for developing smart materials and nanodevices. However, the thermal robustness and programmability of current aptamer switches are often limited by their activation processes that are coupled with high reaction enthalpy. Here, we present an enthalpy-independent activation approach that harnesses toehold-exchange as a general framework to design aptamer switches. We demonstrate mathematically and experimentally that this approach is highly effective in improving thermal robustness and thus leads to better analytical performances of aptamer switches. Enhanced programmability is also demonstrated through fine-grained and dynamic tuning of effective affinities and dynamic ranges, as well as the construction of a synthetic DNA network that resembled biological signaling cascades. Our study not only enriches the current toolbox for engineering and controlling synthetic molecular switches but also offers new insights into their thermodynamic basis, which is critical for diverse synthetic biological designs and applications.

[lncRNA CRNDE promotes proliferation and inhibits apoptosis of U937 cells by downregulating miR-136-5p and upregulating MCM5].

Objective To investigate the effect of lncRNA CRNDE on proliferation, apoptosis, and cell cycle of U937 cells and its mechanism. Methods The expression level of CRNDE in bone marrow cells of AML patients was analyzed by GEPIA database; the mRNA expression levels of miR-136-5p, CRNDE, and minichromosome maintenance 5(MCM5) in AML cell lines were detected by quantitative real-time PCR (qRT-PCR). The lentiviral vector with CRNDE knocked down was constructed and transfected into U937 cells which were randomized into CRNDE knockdown group (sh-CRNDE group) and negative control group (sh-NC group); miR-136-5p mimic and miR-136-5p inhibitor were transfected respectively to overexpress and knock down miR-136-5p in U937 cells which were randomized into miR-136-5p-mimic group, NC-mimic group, miR-136-5p-inhibitor group, and NC-inhibitor group. The effect of CRNDE and miR-136-5p on proliferation was detected by CCK-8 assay and cell counting assay, and the effect of them on cell cycle and apoptosis was detected by flow cytometry. The mRNA expressions of miR-136-5p, CRNDE, and MCM5 were detected by qRT-PCR, and the protein expressions of MCM5, Bcl2, cyclin D1, and cyclin A2 were detected by Western blotting. Results CRNDE was highly expressed in the bone marrow and cell lines of AML patients. Knockdown of CRNDE upregulated miR-136-5p, inhibited the MCM5 mRNA and protein expressions and the cell proliferation, promoted the cell apoptosis, and blocked the cell cycle in G1 phase. Overexpression of miR-136-5p also inhibited the expression of MCM5 at both mRNA and protein levels, while knockdown of miR-136-5p reversed those effects. Conclusion CRNDE promotes the proliferation and inhibits the apoptosis of U937 cells by downregulating miR-136-5p and upregulating MCM5.

CRNDE enhances the expression of MCM5 and proliferation in acute myeloid leukemia KG-1a cells by sponging miR-136-5p.

The long-noncoding RNA colorectal neoplasia differentially expressed (CRNDE) gene has been considered to be crucial in tumor malignancy. Although CRNDE is highly expressed in acute myeloid leukemia (AML), its mechanism of action remains unknown. In this study, GEPIA and qRT-PCR were performed to confirm the expression of CRNDE in AML samples and cell lines, respectively. CRNDE shRNA vectors were transfected to explore the biological functions of CRNDE. The cell proliferation was assessed by the CCK8 assay, while apoptosis and cell cycle distribution were measured by flow cytometry and Western blotting. The results showed that CRNDE was overexpressed in both AML samples and cell lines. CRNDE silencing inhibited proliferation and increased apoptotic rate and cell cycle arrest of KG-1a cells. The luciferase reporter assay coupled with RIP assay revealed that CRNDE act as a ceRNA. Rescue assays demonstrated that the effects of CRNDE silencing could be reversed by miR-136-5p inhibitors. In conclusion, our results expound that the CRNDE/miR-136-5p/MCM5 axis modulates cell progression and provide a new regulatory network of CRNDE in KG-1a cells.

P50, N100, and P200 Auditory Sensory Gating Deficits in Schizophrenia Patients.

BACKGROUND: Sensory gating describes neurological processes of filtering out redundant or unnecessary stimuli during information processing, and sensory gating deficits may contribute to the symptoms of schizophrenia. Among the three components of auditory event-related potentials reflecting sensory gating, P50 implies pre-attentional filtering of sensory information and N100/P200 reflects attention triggering and allocation processes. Although diminished P50 gating has been extensively documented in patients with schizophrenia, previous studies on N100 were inconclusive, and P200 has been rarely examined. This study aimed to investigate whether patients with schizophrenia have P50, N100, and P200 gating deficits compared with control subjects. METHODS: Control subjects and clinically stable schizophrenia patients were recruited. The mid-latency auditory evoked responses, comprising P50, N100, and P200, were measured using the auditory-paired click paradigm without manipulation of attention. Sensory gating parameters included S1 amplitude, S2 amplitude, amplitude difference (S1-S2), and gating ratio (S2/S1). We also evaluated schizophrenia patients with PANSS to be correlated with sensory gating indices. RESULTS: One hundred four patients and 102 control subjects were examined. Compared to the control group, schizophrenia patients had significant sensory gating deficits in P50, N100, and P200, reflected by larger gating ratios and smaller amplitude differences. Further analysis revealed that the S2 amplitude of P50 was larger, while the S1 amplitude of N100/P200 was smaller, in schizophrenia patients than in the controls. We found no correlations between sensory gating indices and schizophrenia positive or negative symptom clusters. However, we found a negative correlation between the P200 S2 amplitude and Bell's emotional discomfort factor/Wallwork's depressed factor. CONCLUSION: Till date, this study has the largest sample size to analyze P50, N100, and P200 collectively by adopting the passive auditory paired-click paradigm without distractors. With covariates controlled for possible confounds, such as age, education, smoking amount and retained pairs, we found that schizophrenia patients had significant sensory gating deficits in P50-N100-P200. The schizophrenia patients had demonstrated a unique pattern of sensory gating deficits, including repetition suppression deficits in P50 and stimulus registration deficits in N100/200. These results suggest that sensory gating is a pervasive cognitive abnormality in schizophrenia patients that is not limited to the pre-attentive phase of information processing. Since P200 exhibited a large effect size and did not require additional time during recruitment, future studies of P50-N100-P200 collectively are highly recommended.