Force-Encoding DNA Nanomachines for Simultaneous and Direct Detection of Multiple Pathogenic Bacteria in Blood.

Pathogen detection is growing in importance in the early stages of bacterial infection and treatment due to the significant morbidity and mortality associated with bloodstream infections. Although various diagnostic approaches for pathogen detection have been proposed, most of them are time-consuming, with insufficient sensitivity and limited specificity and multiplexing capability for clinical use. Here, we report a force-encoding DNA nanomachine for simultaneous and high-throughput detection of multiple pathogens in blood through force-induced remnant magnetization spectroscopy (FIRMS). The force-encoding DNA nanomachines coupled with DNA walkers enable analytical sensitivity down to a single bacterium via a cascade signal amplification strategy. More importantly, it allows for rapid and specific profiling of various pathogens directly in blood samples, without being affected by factors such as light color and solution properties. We expect that this magnetic sensing platform holds great promise for various applications in biomedical research and clinical diagnostics.

Immunohistochemical assessment of HER2 low breast cancer: interobserver reproducibility and correlation with digital image analysis.

PURPOSE: The recent findings from the DESTINY-Breast04 trial highlighted the clinical importance of distinguishing between HER2 immunohistochemistry (IHC) scores 0 and 1 + in metastatic breast cancer (BC). However, pathologist interpretation of HER2 IHC scoring is subjective, and standardized methodology is needed. We evaluated the consistency of HER2 IHC scoring among pathologists and the accuracy of digital image analysis (DIA) in interpreting HER2 IHC staining in cases of HER2-low BC. METHODS: Fifty whole-slide biopsies of BC with HER2 IHC staining were evaluated, comprising 25 cases originally reported as IHC score 0 and 25 as 1 +. These slides were digitally scanned. Six pathologists with breast expertise independently reviewed and scored the scanned images, and DIA was applied. Agreement among pathologists and concordance between pathologist scores and DIA results were statistically analyzed using Kendall coefficient of concordance (W) tests. RESULTS: Substantial agreement among at least five of the six pathologists was found for 18 of the score 0 cases (72%) and 15 of the score 1 + cases (60%), indicating excellent interobserver agreement (W = 0.828). DIA scores were highly concordant with pathologist scores in 96% of cases (47/49), indicating excellent concordance (W = 0.959). CONCLUSION: Although breast subspecialty pathologists were relatively consistent in evaluating BC with HER2 IHC scores of 0 and 1 +, DIA may be a reliable supplementary tool to enhance the standardization and quantification of HER2 IHC assessment, especially in challenging cases where results may be ambiguous (i.e., scores 0-1 +). These findings hold promise for improving the accuracy and consistency of HER2 testing.

MYB expression by immunohistochemistry is highly specific and sensitive for detection of solid variant of adenoid cystic carcinoma of the breast among all triple-negative breast cancers.

BACKGROUND: Adenoid cystic carcinoma is a rare subtype of triple-negative breast carcinoma. These low-grade tumours, which are treated by simple mastectomy and have an excellent prognosis compared to other triple-negative breast carcinomas. Solid-variant adenoid cystic carcinomas have basaloid features and are difficult to distinguish morphologically from other triple-negative breast cancers. Breast adenoid cystic carcinoma exhibits MYB protein overexpression, which can be detected by immunohistochemistry (IHC). AIM: We compared the IHC expression of MYB in solid-variant adenoid cystic carcinoma with that in other triple-negative breast cancers. METHODS: We conducted IHC staining of 210 samples of triple-negative breast cancers, including solid-variant adenoid cystic carcinoma (n = 17), metaplastic breast carcinoma (n = 44), basaloid triple-negative breast cancer (n = 21), and other triple-negative invasive ductal carcinoma (n = 128). We classified nuclear staining of MYB as diffuse/strong (3+), focal moderate (2+), focal weak (1+), or none (0). RESULTS: All 17 solid/basaloid adenoid cystic carcinoma cases exhibited 3+ MYB expression. Of the 21 solid/basaloid triple-negative breast cancers, one (5%) had 2+ expression, seven (33%) 1+ expression, and 13 (62%) 0 expression. Of the 44 metaplastic carcinoma cases, 39 cases (89%) had no (0) staining, and the other five cases had focal weak (1+) or moderate (2+) staining. Among the 128 triple-negative invasive ductal carcinoma cases, 92 cases (72%) had no (0) staining, 36 cases (28%) exhibited focal weak (1+) or moderate (2+) staining. CONCLUSIONS: Our study revealed diffuse/strong MYB staining (3+) only in solid/basaloid adenoid cystic carcinomas. Thus, we recommend routine MYB IHC staining in triple-negative breast carcinoma with solid/basaloid morphology to improve diagnostic accuracy.

Design and Screening of Fluorescent Probes Based upon Hemicyanine Dyes to Sensitively Respond to HSO3- in Living Cells.

SO2, a gas signaling molecule, can be produced endogenously in mitochondria. Its hydrolysate, HSO3-, plays a key role in food preservation, cardiovascular relaxation, and other fields, suggesting that it is important to achieve its detection. Here, based on the Michael addition mechanism, four hemicyanine dye fluorescent probes (ETN, ETB, STB, and EIB) were designed and synthesized for responding to HSO3-. We evaluated the reaction ability of different probes with HSO3- and tried to explain the reasons for the significantly different response effects between probes and HSO3- according to the structure-activity relationship. The influence of different substituents of probes on the properties of mitochondria-targeting was also discussed. Finally, we screened out ETN as the optimal HSO3- probe due to its high sensitivity, rapid reactivity, and good mitochondria-targeting, and it could sensitively respond to HSO3- in living cells. The LODs of ETN for HSO3- were calculated by both absorption and fluorescence methods, respectively, which were 2.727 and 0.823 µM. Our work provided valuable references for designing strategies and potential tools for response to SO2 derivatives in biosystems.

"One stone, two birds": a mitochondria-targeted fluorescent probe for the detection of viscosity and HSO3- in living cells.

The material transport and physiological events of mitochondria need to be supported by a suitable microenvironment. For example, high viscosity will seriously hinder material exchange, and SO2, as the precursor of HSO3-, is an endogenous signal molecule that plays a key role in information transmission. It is very important to detect viscosity and HSO3- in mitochondria. Here, we developed a dual-responsive fluorescent probe (named Hcy-NT) to image the changes in mitochondrial viscosity and HSO3- in a "killing two birds with one stone" manner. Hcy-NT showed an OFF-ON fluorescence signal for the increase in cell viscosity induced by nystatin, while an ON-OFF fluorescence signal for intracellular and endogenous HSO3-. Its limits of detection for HSO3- were calculated by both absorption and fluorescence methods, which were 1.200 and 1.291 µM, respectively. This work provides a valuable tool for the study of viscosity and HSO3- related physiological processes and the diagnosis of potential diseases.

Gut Microbiota in Children with Hand Foot and Mouth Disease on 16S rRNA Gene Sequencing.

Hand foot and mouth disease (HFMD) is a contagious and seasonal viral disease in children. The gut microbiota of HFMD children is not clear now. The study aimed to explore the gut microbiota of HFMD children. The 16S rRNA gene of the gut microbiota of ten HFMD patients and ten healthy children were sequenced on the NovaSeq and PacBio platforms respectively. There were significant differences in gut microbiota between the patients and healthy children. The diversity and abundance of gut microbiota in HFMD patients were lower than that in healthy children. The species Roseburia inulinivorans and Romboutsia timonensis were more abundant in healthy children than those in HFMD patients, which suggests that the two species may be used as probiotics for adjusting the gut microbiota of HFMD patients. Meanwhile, the results of 16S rRNA gene sequences from the two platforms were different. The NovaSeq platform identified more microbiota and has the characteristics of high throughput, short time and low price. However, the NovaSeq platform has low resolution at the species level. The PacBio platform has high resolution based on its long reads length, which is more suitable for species-level analysis. But, the shortcomings of the high price and low throughput of PacBio still need to be overcome. With the development of sequencing technology, the reduction in sequencing price and the increase in throughput will promote the third-generation sequencing technology used in the study of gut microbes.

Comparison of noninvasive continuous arterial blood pressure measured by NICAP with arterial line in elderly patients.

BACKGROUND: Non-Invasive Continuous Arterial Pressure system (NICAP) allows continuous monitoring, timely detection of hypotension, and avoiding risks from invasive procedures. A previous study showed good comparability of NICAP with arterial line in people with no evidence of cardiovascular disease. Therefore, the goal of this study was to investigate whether NICAP could be accurately applied to elderly patients. METHODS: In this single-centered observational study, forty-one patients above 65 undergoing elective surgeries requiring artery catheterizations were enrolled from July 17, 2020, to June 25, 2021. Radial artery cannulation and NICAP monitoring were started before anesthesia. Blood pressure during the anesthesia induction and the whole surgery, trend of blood pressure changes, time needed for establishing continuous monitoring, and complications were recorded. RESULTS: A total of 6751 valid pairs of blood pressure measurements were analyzed. In the Bland-Altman analysis, the arithmetic means for systolic, diastolic, and mean arterial pressure were 2.2, 3.3, and 2.8 mmHg, respectively. NICAP and arterial line correlation coefficients for systolic, diastolic, and mean arterial pressure were 0.49, 0.33, and 0.45, respectively. In the trending analysis, the polar concordance rates at 30 degrees were 70.9% for systolic, 67.7% for diastolic, and 69.3% for mean arterial blood pressure. During the anesthesia induction, the arithmetic means for systolic, diastolic, and mean arterial pressure in the Bland-Altman analysis were 1.7, -0.2, and 0.5 mmHg, respectively. NICAP and arterial line correlation coefficients for systolic, diastolic, and mean arterial pressure were 0.78, 0.61 and 0.75, respectively. No severe complications occurred. CONCLUSIONS: NICAP has a poor correlation with the arterial line in elderly patients for the whole surgery or during anesthesia induction. Moreover, it showed poor comparability in the detection of blood pressure change trends with arterial lines. Our findings suggest that NICAP might not be sufficiently accurate to be applied clinically in elderly patients with comorbidities. More accurate calibration and iteration are needed.

Assessing genomic diversity and signatures of selection in Jiaxian Red cattle using whole-genome sequencing data.

BACKGROUND: Native cattle breeds are an important source of genetic variation because they might carry alleles that enable them to adapt to local environment and tough feeding conditions. Jiaxian Red, a Chinese native cattle breed, is reported to have originated from crossbreeding between taurine and indicine cattle; their history as a draft and meat animal dates back at least 30 years. Using whole-genome sequencing (WGS) data of 30 animals from the core breeding farm, we investigated the genetic diversity, population structure and genomic regions under selection of Jiaxian Red cattle. Furthermore, we used 131 published genomes of world-wide cattle to characterize the genomic variation of Jiaxian Red cattle. RESULTS: The population structure analysis revealed that Jiaxian Red cattle harboured the ancestry with East Asian taurine (0.493), Chinese indicine (0.379), European taurine (0.095) and Indian indicine (0.033). Three methods (nucleotide diversity, linkage disequilibrium decay and runs of homozygosity) implied the relatively high genomic diversity in Jiaxian Red cattle. We used θπ, CLR, FST and XP-EHH methods to look for the candidate signatures of positive selection in Jiaxian Red cattle. A total number of 171 (θπ and CLR) and 17 (FST and XP-EHH) shared genes were identified using different detection strategies. Functional annotation analysis revealed that these genes are potentially responsible for growth and feed efficiency (CCSER1), meat quality traits (ROCK2, PPP1R12A, CYB5R4, EYA3, PHACTR1), fertility (RFX4, SRD5A2) and immune system response (SLAMF1, CD84 and SLAMF6). CONCLUSION: We provide a comprehensive overview of sequence variations in Jiaxian Red cattle genomes. Selection signatures were detected in genomic regions that are possibly related to economically important traits in Jiaxian Red cattle. We observed a high level of genomic diversity and low inbreeding in Jiaxian Red cattle. These results provide a basis for further resource protection and breeding improvement of this breed.

Design, Synthesis, and Structure-Activity Relationship Study of Potent MAPK11 Inhibitors.

Huntington's disease (HD) is a rare single-gene neurodegenerative disease, which can only be treated symptomatically. Currently, there are no approved drugs for HD on the market. Studies have found that MAPK11 can serve as a potential therapeutic target for HD. Regrettably, no MAPK11 small molecule inhibitors have been approved at present. This paper presents three series of compounds that were designed and synthesized based on the structure of skepinone-L, a known MAPK14 inhibitor. Among the synthesized compounds, 13a and 13b, with IC50 values of 6.40 nM and 4.20 nM, respectively, displayed the best inhibitory activities against MAPK11. Furthermore, the structure-activity relationship (SAR) is discussed in detail, which is constructive in optimizing the MAPK11 inhibitors for better activity and effect against HD.

Real-time monitoring of DNA G-quadruplexes in living cells with a small-molecule fluorescent probe.

G-quadruplex DNA has been viewed as a prospective anti-cancer target owing to its potential biological relevance. Real-time monitoring of DNA G-quadruplex structures in living cells can provide valuable insights into the relationship between G-quadruplex formation and its cellular consequences. However, the probes capable of detecting DNA G-quadruplexes in living cells are still very limited. Herein, we reported a new fluorescent probe, IMT, for real-time visualization of DNA G-quadruplex structures in living cells. Using IMT as a fluorescent indicator, the quantity changes of DNA G-quadruplex at different points in time during continuous cellular progression responding to Aphidicolin and Hydroxyurea treatment have been directly visualized. Our data demonstrate that IMT will be a valuable tool for exploring DNA G-quadruplexes in live cells. Further application of IMT in fluorescence imaging may reveal more information on the roles of DNA G-quadruplexes in biological systems.

Association of regenerating gene 1A single-nucleotide polymorphisms and nasopharyngeal carcinoma susceptibility in southern Chinese population.

OBJECTIVE: Nasopharyngeal carcinoma (NPC) is a common malignancy in Southern China and Southeast Asia. Genetic susceptibility is a major contributing factor in determining the individual risk of NPC in these areas. To test the association between NPC and variants in regenerating gene 1A (REG1A), we conducted a hospital-based case-control study in a Cantonese-speaking population from Guangdong province. METHODS: We endeavored to determine whether genetic variants of the REG1A gene were associated with the risk of NPC amidst the Cantonese population in a hospital-based case-control study using polymerase chain reaction-restriction and direct sequencing analysis in 211 NPC patients and 150 healthy controls. The association between NPC risk and the 14C/T, 20C/T, 369G/T, 1201A/G, and 2922C/T polymorphisms was examined after adjustment for age and sex. RESULTS: We found an increased risk of developing NPC in individuals with REG1A 2922C/T variant genotype (p = 0.003, OR 0.419, 95% CI 0.235-0.746), and after adjustment for sex and age (p = 0.003, OR 0.406, 95% CI 0.226-0.732). No association between other polymorphisms (14C/T, 20C/T, 369G/T, and 1201A/G) and the risk of NPC was observed, before or after adjustment for age and sex. CONCLUSION: Our findings suggest that the REG1A 2922C/T polymorphism is associated with an increased risk of developing NPC in a Cantonese population from Guangdong province. Larger studies are required to confirm our findings and unravel the underlying mechanisms.

Direct visualization of nucleolar G-quadruplexes in live cells by using a fluorescent light-up probe.

BACKGROUND: Direct detection of G-quadruplexes in human cells has become an important issue due to the vital role of G-quadruplex related to biological functions. Despite several probes have been developed for detection of the G-quadruplexes in cytoplasm or whole cells, the probe being used to monitor the nucleolar G-quadruplexes is still lacking. METHODS: Formation of the nucleolar G-quadruplex structures was confirmed by using circular dichroism (CD) spectroscopy. The binding affinity and selectivity of Thioflavin T (ThT) towards various DNA/RNA motifs in solution and gel system were measured by using fluorescence spectroscopy and polyacrylamide gel electrophoresis (PAGE), respectively. G-quadruplex imaging in live cells was directly captured by using confocal laser scanning microscopy (CLSM). RESULTS: Formation of the rDNA and rRNA G-quadruplex structures is demonstrated in vitro. ThT is found to show much higher affinity and selectivity towards these G-quadruplex structures versus other nucleic acid motifs either in solution or in gel system. The nucleolar G-quadruplexes in living cells are visualized by using ThT as a fluorescent probe. G-quadruplex-ligand treatments in live cells lead to sharp decrease of ThT signal. CONCLUSIONS: The natural existence of the G-quadruplexes structure in the nucleoli of living cells is directly visualized by using ThT as an indicator. GENERAL SIGNIFICANCE: The research provides substantive evidence for formation of the rRNA G-quadruplex structures, and also offers an effective probe for direct visualization of the nucleolar G-quadruplexes in living cells.

Ethanol Extract from Brucea Javanica Seed Inhibits Angiogenesis Mediated by Platelet Derived Growth Factor Receptor-beta.

The present study aimed to investigate the effects of ethanol extract from Brucea javanicaseed (EEBJS) on the angiogenesis of human umbilical vein endothelial cells (HUVECs) and the possible molecular signal involved. Firstly, a Matrigel-based in vitro angiogenesis assay demonstrated that EEBJS inhibited the angiogenesis of HUVECs in a dose-dependent manner. Then by using porcine aortic endothelial cells which stably express human PDGFR-beta, we found that the inhibition of angiogenesis was mediated by PDGFR-beta. Taken together, we conclude that EEBJS inhibited the angiogenesis function of the vascular endothelial cells mediated by PDGFR-beta, and postulate that it might contribute to the therapeutic effects of EEBJS on malignant tumors.

Reproducible evaluation of tumor-infiltrating lymphocytes (TILs) using the recommendations of International TILs Working Group 2014.

Tumor infiltrating lymphocytes (TILs) in breast cancer play an important role in predicting the outcome of breast cancer. The goal of our current study is to investigate the consistency and reproducibility of the recommendations published by the International TILs Working Group 2014 among pathology trainees and pathologists. Hematoxylin & Eosin (H&E) slides from 129 breast cancer cases (one slide each) from 2009 to 2014 were evaluated. Each case was blindly and independently reviewed by two observers following the International TILs Working Group 2014 recommendations. Three pathology trainees (PGY2, PGY3 and PGY4) and three pathologists (2 general pathologists and 1 breast pathologist) were involved in this study. Of the 129 cases, 10 (10/129, 7.8%) cases had TILs >50%, 90 (90/129, 69.8%) cases had <10% of TILs, and 29 (29/129, 22.4%) cases had TILs ranging from 10 to 50%. Our results showed that in 104 cases (104/129, 80.6%) the TILs percentage was identical between the 2 observers. In 18 cases (18/129, 14%), the difference between the two observers was by 10% and in 7 cases (7/129, 5.4%) there was a difference of 20% or more. The inter-observer kappa value was 0.776 between two observers, and the kappa score improved to 0.86 if using the 3 categoric groups (<10%, 10-50%, and >50%). Our study showed that the recommendations and instructions for TILs evaluation by the International TILs Working Group 2014 were sufficiently detailed to be applied for TILs evaluation in breast cancer.

Synthesis, Design, and Structure⁻Activity Relationship of the Pyrimidone Derivatives as Novel Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase.

The inhibition of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) potentially represents a new treatment option for malaria, as P. falciparum relies entirely on a de novo pyrimidine biosynthetic pathway for survival. Herein, we report a series of pyrimidone derivatives as novel inhibitors of PfDHODH. The most potent compound, 26, showed high inhibition activity against PfDHODH (IC50 = 23 nM), with >400-fold species selectivity over human dihydroorotate dehydrogenase (hDHODH). The brand-new inhibitor scaffold targeting PfDHODH reported in this work may lead to the discovery of new antimalarial agents.

Counterclockwise maxillomandibular advancement: a choice for Chinese patients with severe obstructive sleep apnea.

PURPOSE: Maxillomandibular advancement (MMA) is an effective alternative for treating severe obstructive sleep apnea (OSA). However, the promotion of MMA in China is limited by many Chinese patients having a convex facial profile. To achieve maximal upper airway enlargement without an esthetic disaster, we added counterclockwise rotation of the maxillomandibular complex (MMC) in the routine MMA. In this paper, we have evaluated the objective and subjective outcomes of this technology. METHODS: In total, 33 severe OSA patients who accepted counterclockwise maxillomandibular advancement (CMMA) were investigated in this study. Genioplasty, uvulopalatopharyngoplasty (UPPP), and turbinate reduction were also performed on selected patients. Polysomnography (PSG) and Epworth sleepiness scale (ESS) were chosen to evaluate the effectiveness of this technology in treating OSA. Patients' facial appearances were evaluated by cephalometric analysis and 5-point Likert scales. RESULTS: After CMMA, the apnea-hypopnea index (AHI) decreased from 59.3 ± 14.6 to 10.2 ± 6.7 (P < 0.001), minimum SpO2 (pulse oxygen saturation, %) increased from 74.0 ± 11.7 to 88.8 ± 4.4 (P < 0.001), and ESS decreased from 12.5 ± 2.3 to 7.3 ± 2.1. It is encouraging that soft-tissue cephalometric measurements such as facial convexity angle, nasolabial angle, and labiomental fold were not worsening after surgery. The Likert scales revealed that 28 patients (85%) were satisfied or very satisfied with their facial changes. In addition, no patient complained about dental function after surgery. CONCLUSIONS: These findings indicate that CMMA is an effective way to achieve a balance between airway enlargement and facial appearance for Chinese patients with severe OSA.

Directly lighting up RNA G-quadruplexes from test tubes to living human cells.

RNA G-quadruplexes (G4s) are one of the key components of the transcriptome that act as efficient post-transcriptional regulatory elements in living cells. To conduct further studies of the unique biological functions of RNA G4s, techniques need to be developed that can efficiently recognize RNA G4 structures under various conditions, in fixed cells and living cells, as well as in vitro. This paper presents the development of such a method, a new technique using a cyanine dye called CyT, which can detect both canonical and non-canonical RNA G4 structures from test tubes to living human cells. The ability of CyT to distinguish between G4 and nonG4 RNA offers a promising tool for future RNA G4-based biomarker discovery and potential diagnostic applications.

Combined Counterclockwise Maxillomandibular Advancement and Uvulopalatopharyngoplasty Surgeries for Severe Obstructive Sleep Apnea.

As the most effective surgical technique maxillomandibular advancement (MMA) has been used to treat severe obstructive sleep apnea (OSA) in adults, particularly for those who are intolerant of continuous positive airway pressure. Yet for large-scale advancement, it is faced with esthetic problems with marked skeletal protrusion especially for people with convex facial profile. In this study, the authors performed counterclockwise MMA combined with quantified uvulopalatopharyngoplasty (UPPP) surgeries on Chinese adult patients with severe OSA, in order to initially explore the efficacy of these procedures on Chinese populations and provide evidence for esthetic advantages. As the primary procedure counterclockwise MMA was applied on 10 patients, achieving a forward distance of the mandible and the maxilla for 10.6 and 6.7 mm, respectively, and the occlusion plane rotated counterclockwise of 6.2°. After a follow-up of beyond 12 months, polysomnography results showed the apnea-hypopnea index (AHI) significantly decreased from 64.3 to 11.0 per hour, achieving surgical success of 90%. Upper airway measurements demonstrated that the retropalatal and retrolingual spaces got enlarged greatly, resulting in significant AHI reduction and oxygen saturation elevation. More importantly, cephalometric analysis revealed that SNA and SNB were enlarged but in well control without visual abnormalities. Follow-up results showed large-scale advancement of the maxilla and mandible were stable in treating severe OSA. Quantified UPPP surgeries guaranteed no functional insufficiency in pronouncing and swallowing and played auxiliary role in enlarging the upper airway. Thus, procedures of counterclockwise MMA combined with quantified UPPP surgeries might find more application especially in patients with severe OSA with convex facial profile in future.

Stabilization of G-quadruplex DNA and inhibition of Bcl-2 expression by a pyridostatin analog.

The G-quadruplexes located in the P1 promoter of B-cell lymphoma-2 (Bcl-2) gene are implicated to regulate Bcl-2 expression. Here, we designed a new pyridostatin analog named PDF, which exhibited high specificity and stabilizing effect toward G-quadruplexes. The luciferase assay demonstrated that PDF could significantly suppress Bcl-2 transcriptional activation in human laryngeal squamous carcinoma cells (Hep-2) cells. Besides, PDF also induced cell apoptosis in vitro assays. These results provide an excellent G-quadruplex specific ligand as an efficient Bcl-2 inhibitor. These results also implicate that PDF may be a potential anticancer drug to head neck cancer.