Impact of Region-of-Interest Size on Immune Profiling Using Multiplex Immunofluorescence Tyramide Signal Amplification for Paraffin-Embedded Tumor Tissues.

INTRODUCTION: Representative regions of interest (ROIs) analysis from the whole slide images (WSI) are currently being used to study immune markers by multiplex immunofluorescence (mIF) and single immunohistochemistry (IHC). However, the amount of area needed to be analyzed to be representative of the entire tumor in a WSI has not been defined. METHODS: We labeled tumor-associated immune cells by mIF and single IHC in separate cohorts of non-small cell lung cancer (NSCLC) samples and we analyzed them as whole tumor area as well as using different number of ROIs to know how much area will be need to represent the entire tumor area. RESULTS: For mIF using the InForm software and ROI of 0.33 mm2 each, we observed that the cell density data from five randomly selected ROIs is enough to achieve, in 90% of our samples, more than 0.9 of Spearman correlation coefficient and for single IHC using ScanScope tool box from Aperio and ROIs of 1 mm2 each, we found that the correlation value of more than 0.9 was achieved using 5 ROIs in a similar cohort. Additionally, we also observed that each cell phenotype in mIF influence differently the correlation between the areas analyzed by the ROIs and the WSI. Tumor tissue with high intratumor epithelial and immune cells phenotype, quality, and spatial distribution heterogeneity need more area analyzed to represent better the whole tumor area. CONCLUSION: We found that at minimum 1.65 mm2 area is enough to represent the entire tumor areas in most of our NSCLC samples using mIF.

Comprehensive multiplexed immune profiling of the ductal carcinoma in situ immune microenvironment regarding subsequent ipsilateral invasive breast cancer risk.

BACKGROUND: Ductal carcinoma in situ (DCIS) is treated to prevent subsequent ipsilateral invasive breast cancer (iIBC). However, many DCIS lesions will never become invasive. To prevent overtreatment, we need to distinguish harmless from potentially hazardous DCIS. We investigated whether the immune microenvironment (IME) in DCIS correlates with transition to iIBC. METHODS: Patients were derived from a Dutch population-based cohort of 10,090 women with pure DCIS with a median follow-up time of 12 years. Density, composition and proximity to the closest DCIS cell of CD20+ B-cells, CD3+CD8+ T-cells, CD3+CD8- T-cells, CD3+FOXP3+ regulatory T-cells, CD68+ cells, and CD8+Ki67+ T-cells was assessed with multiplex immunofluorescence (mIF) with digital whole-slide analysis and compared between primary DCIS lesions of 77 women with subsequent iIBC (cases) and 64 without (controls). RESULTS: Higher stromal density of analysed immune cell subsets was significantly associated with higher grade, ER negativity, HER-2 positivity, Ki67 ≥ 14%, periductal fibrosis and comedonecrosis (P < 0.05). Density, composition and proximity to the closest DCIS cell of all analysed immune cell subsets did not differ between cases and controls. CONCLUSION: IME features analysed by mIF in 141 patients from a well-annotated cohort of pure DCIS with long-term follow-up are no predictors of subsequent iIBC, but do correlate with other factors (grade, ER, HER2 status, Ki-67) known to be associated with invasive recurrences.

Effector memory cytotoxic CD3+/CD8+/CD45RO+ T cells are predictive of good survival and a lower risk of recurrence in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) with high tumour-infiltrating lymphocytes (TILs) has been associated with a promising prognosis. To better understand the prognostic value of immune cell subtypes in TNBC, we characterised TILs and the interaction between tumour cells and immune cell subtypes. A total of 145 breast cancer tissues were stained by multiplex immunofluorescence (mIF), including panel 1 (PD-L1, PD-1, CD3, CD8, CD68 and CK) and panel 2 (Foxp3, Granzyme B, CD45RO, CD3, CD8 and CK). Phenotypes were analysed and quantified by pathologists using InForm software. We found that in the ER-negative (ER <1% and HER2-negative) group and the ER/PR-low positive (ER 1-9% and HER2-negative) group, 11.2% and 7.1% of patients were PD-L1+ by the tumour cell score, 29.0% and 28.6% were PD-L1+ by the modified immune cell score and 30.8% and 32.1% were PD-L1+ by the combined positive score. We combined ER-negative and ER/PR-low positive cases for the survival analysis since a 10% cut-off is often used in clinical practice for therapeutic purposes. The densities of PD-L1+ tumour cells (HR: 0.366, 95% CI: 0.138-0.970; p = 0.043) within the tumour compartment and CD3+ immune cells in the total area (tumour and stromal compartments combined) (HR: 0.213, 95% CI: 0.070-0.642; p = 0.006) were favourable prognostic biomarkers for overall survival (OS) in TNBC. The density of effector/memory cytotoxic T cells (CD3+CD8+CD45RO+) in the tumour compartment was an independent prognostic biomarker for OS (HR: 0.232, 95% CI: 0.086-0.628; p = 0.004) and DFS (HR: 0.183, 95% CI: 0.1301-0.744; p = 0.009) in TNBC. Interestingly, spatial data suggested that patients with a higher density of PD-L1+ tumour cells had shorter cell-cell distances from tumour cells to cytotoxic T cells (p < 0.01). In conclusion, we found that phenotyping tumour immune cells by mIF is highly informative in understanding the immune microenvironment in TNBC. PD-L1+ tumour cells, total T cells and effector/memory cytotoxic T cells are promising prognostic biomarkers in TNBC.

Ablation and Inhibition of the Immunoproteasome Catalytic Subunit LMP7 Attenuate Experimental Abdominal Aortic Aneurysm Formation in Mice.

Low-molecular mass protein 7 (LMP7) is a proteolytic subunit of the immunoproteasome that is involved in regulating inflammatory responses. However, the role of LMP7 in the pathogenesis of abdominal aortic aneurysm (AAA) remains unknown. In this study, ApoE knockout (KO) or LMP7/ApoE double KO (dKO) mice were infused with angiotensin II (Ang II, 1000 ng/kg per minute) for up to 28 d. We found that LMP7 expression was significantly upregulated in AAA tissues from ApoE KO mice and human patients. Moreover, Ang II infusion markedly increased the incidence and severity of AAA in ApoE KO mice, which was considerably reduced in LMP7/ApoE dKO mice. Histological alterations, including aortic wall thickening, collagen deposition, elastin fragmentation, and vascular smooth muscle cell apoptosis in AAA tissue of ApoE KO mice, were also significantly attenuated in LMP7/ApoE dKO mice. Interestingly, LMP7/ApoE dKO mice showed a marked reduction of infiltration of CD3+ T cells, especially CD4+ T cells in AAA tissues compared with ApoE KO mice. Moreover, ablation of LMP7 substantially inhibited the differentiation of CD4+ T cells into Th1 and Th17 cells by reducing the activation of multiple transcriptional factors. We also investigated the effects of an LMP7-specific inhibitor PR-957 (also known as ONX 0914) on AAA formation in ApoE KO mice. PR-957 treatment could reduce the AAA incidence and severity. In conclusion, our results provide, to our knowledge, novel evidence that ablation or pharmacological inhibition of LMP7 attenuates Ang II-induced AAA formation, and LMP7 might be a novel therapeutic target for treating AAA in humans.

SOX4 promotes the growth and metastasis of breast cancer.

PURPOSE: Increasing evidence has shown that the transcription factor SOX4 is closely associated with the development and progression of many malignant tumors. However, the effect of SOX4 on breast cancer is unclear. In this study, we purposed to investigate the role of SOX4 in the growth and metastasis in breast cancer and the underlying mechanism. Moreover, the effect of SOX4 on cancer cell resistance to chemotherapeutic agents was also evaluated in vitro and in vivo. METHODS: We used lentivirus technique to ectopically express SOX4 in MDA-MB-231 and SUM149 cells or knockdown SOX4 in BT474 cells, and examined the effect of these changes on various cellular functions. MTT assay was used to determine the cell viability as well as resistance to chemotherapeutic agents. The regulation of SOX4 on epithelial-mesenchymal transition (EMT)-related genes was analyzed using qRT-PCR. The binding of SOX4 to the CXCR7 gene was demonstrated using chromatin immunoprecipitation assay and dual-luciferase reporter activity assay. The effect of SOX4/CXCR7 axis on metastasis was examined using Transwell migration and Matrigel invasion assays. The expression of SOX4/CXCR7 in primary tumors and metastatic foci in lymph nodes was assessed using immunohistochemistry. Cellular morphology was investigated under phase contrast microscope and transmission electron microscopy. Moreover, the effect of SOX4 on tumor growth, metastasis, and resistance to chemotherapy was also studied in vivo by using bioluminescent imaging. RESULTS: SOX4 increased breast cancer cell viability, migration, and invasion in vitro and enhanced tumor growth and metastasis in vivo. It regulated EMT-related genes and bound to CXCR7 promoter to upregulate CXCR7 transcription. Both SOX4 and CXCR7 were highly expressed in human primary tumors and metastatic foci in lymph nodes. Treatment of breast cancer cells with the CXCR7 inhibitor CCX771 reversed the SOX4 effect on cell migration and invasion. Ectopic expression of SOX4 increased the susceptibility of cells to paclitaxel. CONCLUSIONS: SOX4 plays an important role in the growth and metastasis of breast cancer. SOX4/CXCR7 may serve as potential therapeutic targets for the treatment. Paclitaxel may be a good therapeutic option if the expression level of SOX4 is high.

Screening Analysis of Sirtuins Family Expression on Anti-Inflammation of Resveratrol in Endothelial Cells.

BACKGROUND Resveratrol has been shown to possess beneficial activities including antioxidant, anti-inflammatory, and cardioprotective effects through activating a nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. The current study was undertaken to investigate the role of sirtuin family members (SIRT1-SIRT7) on the anti-inflammation activities of resveratrol in endothelial cells. MATERIAL AND METHODS Primary human umbilical vein endothelial cells (HUVECs) were pretreated with resveratrol before tumor necrosis factor (TNF)-alpha (10-20 µg/L) stimulation. Cell viability was measured using the Cell Counting Kit-8 method. Total RNA was extracted after different treatments and the NimbleGen Human 12×135K Gene Expression Array was applied to screen and analyze SIRTs expression. Quantitative real-time polymerase chain reaction and western blot were applied to verify the results of the gene expression microarrays. Reactive oxygen species (ROS) production was examined using flow cytometry analysis. RESULTS Microarray analysis showed that the expressions of SIRT1, SIRT2, SIRT3, SIRT5, SIRT6, and SIRT7 showed the tendency to increase while SIRT4 showed the tendency to decrease. SIRT1, SIRT2, SIRT5, and SIRT7 gene expression could be upregulated by pretreatment with resveratrol compared with TNF-alpha alone while there were no obvious differences of SIRT3, SIRT4, and SIRT6 expressions observed in TNF-alpha alone treated cells and resveratrol-TNF-alpha co-treated cells. Interestingly, SIRT1, SIRT2, SIRT3, SIRT4, and SIRT5 siRNA could reverse the effect of resveratrol on ROS production; SIRT1 and SIRT5 siRNA could significantly increase CD40 expression inhibited by resveratrol in TNF-a treated cells. CONCLUSIONS Our results suggest that resveratrol inhibiting oxidative stress production is associated with SIRT1, SIRT2, SIRT3, SIRT4, and SIRT5 pathways; attenuating CD40 expression was only associated with SIRT1 and SIRT5 pathways in TNF-alpha-induced endothelial cells injury.

Synergistic activity of BET protein antagonist-based combinations in mantle cell lymphoma cells sensitive or resistant to ibrutinib.

Mantle cell lymphoma (MCL) cells exhibit increased B-cell receptor and nuclear factor (NF)-κB activities. The bromodomain and extra-terminal (BET) protein bromodomain 4 is essential for the transcriptional activity of NF-κB. Here, we demonstrate that treatment with the BET protein bromodomain antagonist (BA) JQ1 attenuates MYC and cyclin-dependent kinase (CDK)4/6, inhibits the nuclear RelA levels and the expression of NF-κB target genes, including Bruton tyrosine kinase (BTK) in MCL cells. Although lowering the levels of the antiapoptotic B-cell lymphoma (BCL)2 family proteins, BA treatment induces the proapoptotic protein BIM and exerts dose-dependent lethality against cultured and primary MCL cells. Cotreatment with BA and the BTK inhibitor ibrutinib synergistically induces apoptosis of MCL cells. Compared with each agent alone, cotreatment with BA and ibrutinib markedly improved the median survival of mice engrafted with the MCL cells. BA treatment also induced apoptosis of the in vitro isolated, ibrutinib-resistant MCL cells, which overexpress CDK6, BCL2, Bcl-xL, XIAP, and AKT, but lack ibrutinib resistance-conferring BTK mutation. Cotreatment with BA and panobinostat (pan-histone deacetylase inhibitor) or palbociclib (CDK4/6 inhibitor) or ABT-199 (BCL2 antagonist) synergistically induced apoptosis of the ibrutinib-resistant MCL cells. These findings highlight and support further in vivo evaluation of the efficacy of the BA-based combinations with these agents against MCL, including ibrutinib-resistant MCL.

Tyrosine kinase inhibitors induce mesenchymal stem cell-mediated resistance in BCR-ABL+ acute lymphoblastic leukemia.

Tyrosine kinase inhibitors (TKIs) are used as a frontline therapy for BCR-ABL(+) acute lymphoblastic leukemia (ALL). However, resistance to TKI therapy arises rapidly, and its underlying molecular mechanisms are poorly understood. In this study, we identified a novel cascade of events initiated by TKIs and traversing through mesenchymal stem cells (MSCs) to leukemic cells, leading to resistance. MSCs exposed to TKIs acquired a new functional status with the expression of genes encoding for chemo-attractants, adhesion molecules, and prosurvival growth factors, and this priming enabled leukemic cells to form clusters underneath the MSCs. This cluster formation was associated with the protection of ALL cells from therapy as leukemic cells switched from BCR-ABL signaling to IL-7R/Janus kinase signaling to survive in the MSC milieu. Our findings illustrate a novel perspective in the evolution of TKI resistance and provide insights for advancing the treatment of BCR-ABL(+) ALL.

STAT3 potentiates the ability of airway smooth muscle cells to promote angiogenesis by regulating VEGF signalling.

NEW FINDINGS: What is the central question of this study? Airway angiogenesis occurs in asthma, and airway smooth muscle (ASM) cells have been reported to be capable of promoting airway angiogenesis. What is the potential mechanism by which ASM cells harvested from patients with asthma are capable of promoting airway angiogenesis? What is the main finding and its importance? Endogenous STAT3 mediated the pro-angiogenic ability of ASM cells by directly activating VEGF signalling. These findings contribute to the understanding of airway angiogenesis in pathology and could represent a possible therapeutic target for asthma. Airway angiogenesis indicates the specific vascular structure remodelling that occurs in asthma. Airway smooth muscle (ASM) cells have been reported to be capable of promoting airway angiogenesis; however, the potential mechanism is not yet fully defined. Herein, we investigated the role of signal transducer and activator of transcription 3 (STAT3) in the progress of airway angiogenesis. Western blot analysis showed that STAT3 activation was aberrantly upregulated in ASM tissues of patients with asthma and ASM cells that were exposed to cytokines to imitate the airway conditions in patients with asthma. Compared with the control group, both the inhibition of STAT3 activation and the silencing of endogenous STAT3 in ASM cells significantly reduced the proliferation, migration and tube-forming ability of human lung microvascular endothelial cells induced by the conditioned medium (CM) of ASM cells. The increased proliferation and migration of human aortic vascular smooth muscle cells were also repressed by inhibition of STAT3 in ASM cells. Besides, the increased activity of VEGF signalling was observed in ASM cells and the CM by RT-PCR and Western blotting assay, whereas this increased activity was reduced by STAT3 silencing. Further studies indicated that STAT3 regulated VEGF activation by directly interacting with the binding site on the 5' region of the VEGF gene. The increase in STAT3-induced pro-angiogenic activity of ASM cells was significantly decreased by administration of VEGF neutralizing antibody. In conclusion, we provided evidence that endogenous STAT3 mediates the pro-angiogenic ability of ASM cells by directly activating VEGF signalling, which could represent a possible therapeutic target for asthma.

CLDN-1 promoted the epithelial to migration and mesenchymal transition (EMT) in human bronchial epithelial cells via Notch pathway.

Claudin-1 (CLDN-1) is one of main tight junction components that play an important role in epithelial-mesenchymal transition (EMT). However, the effects of CLDN-1 on the migration and EMT induced by TGF-ß1 in primary normal human bronchial epithelial (NHBE) and BEAS-2B cells have not been clear. The expression of CLDN-1 was quantified by Western blotting in NHBE and BEAS-2B cells. Cell migration and invasion were detected using transwell assays. The expression level of E-cadherin, N-cadherin, α-SMA, and Vimentin was evaluated by quantitative real-time PCR and Western blotting. Here we showed that the protein expression of CLDN-1 was increased exposed to TGF-ß1 in a dose- and time-dependent manner. Knockdown of CLDN-1 using small interfering CLDN-1 RNA (siCLDN-1) prevented the migration and invasion in NHBE and BEAS-2B cells. Moreover, depletion of CLDN-1 promoted the E-cadherin expression and decreased the mRNA and protein levels of N-cadherin, α-SMA, and Vimentin induced by TGF-ß1. Furthermore, CLDN-1 silencing resulted in the reduction of the Notch intracellular domain (NICD) and hairy enhancer of split-1 (Hes-1) in mRNA and protein level. Jagged-1, an activator of Notch signaling pathway, abrogated the protective function of siCLDN-1 in migration and EMT. In conclusion, CLDN-1 promoted the migration and EMT through the Notch signaling pathway.

Integrated genetic approaches identify the molecular mechanisms of Sox4 in early B-cell development: intricate roles for RAG1/2 and CK1ε.

Commitment of hematopoietic stem cells to B lineage precursors and subsequent development of B lineage precursors into mature B cells is stringently controlled by stage-specific transcription factors. In this study, we used integrated genetic approaches and systematically determined the role of Sry-related high mobility group box (Sox) 4 and the underlying molecular mechanisms in early B-cell development. We found that Sox4 coordinates multilevel controls in the differentiation of early stage B cells. At the molecular level, Sox4 orchestrates a unique gene regulatory program, and its function was predominantly mediated through a conventional Sox4-binding motif as well as an unconventional GA-binding protein α chain binding motif. Our integrated gene network and functional analysis indicated that Sox4 functions as a bimodular transcription factor and ensures B lineage precursor differentiation through 2 distinct mechanisms. It positively induces gene rearrangements at immunoglobulin heavy chain gene loci by transcriptionally activating the Rag1 and Rag2 genes and negatively regulates Wnt signaling, which is critical for self-renewal, by inducing the expression of casein kinase 1 ε. Our findings illustrate that Sox4 mediates critical fine-tuning of the 2 opposing forces in early B-cell development and also set forth a model for characterization of critical genes whose deficiency, like Sox4 deficiency, is detrimental to this process.

Sox4 is required for the survival of pro-B cells.

The development of mature B cells from hematopoietic stem cells is a strictly orchestrated process involving multiple regulatory genes. The transcription factor Sox4 is required for this process, but its role has not been systematically studied, and the underlying mechanisms remain unknown. To determine when and how Sox4 functions in the stepwise process of B cell development, we used mice harboring conditional null alleles for Sox4 and a Cre transgene. Sox4 deletion in hematopoietic stem cells almost entirely eliminated pro-B cells in both fetal livers and adult bone marrow, resulting in a severe deficiency in later stage B cells, including circulating mature B cells. Sox4-deficient pro-B cells, particularly those expressing the stem cell factor receptor c-Kit, readily underwent apoptosis, and even more so when c-Kit activity was inhibited by imatinib. C-Kit-expressing pro-B cells showed decreased activation of the c-Kit downstream protein Src upon Sox4 deletion. Likewise, the level of the anti-apoptotic Bcl2 protein was decreased in residual pro-B cells, and its restoration using a Bcl2 transgene allowed not only partial rescue of pro-B cell survival but also B cell maturation in the absence of Sox4. Our findings indicate that Sox4 is required for the survival of pro-B cells and may functionally interact with c-Kit and Bcl2.

[Characteristics of subgroups classified by pulmonary function test in chronic obstructive pulmonary disease patients].

OBJECTIVE: To compare the clinical characteristics and the therapeutic responses in chronic obstructive pulmonary disease (COPD) subgroups, classified by diffusing capacity of the lung for carbon monoxide (D(L)CO) and inspiratary capacity (IC). METHODS: A total of 105 mild-severe patients (70 males, 35 females, 44 to 85 years of age), with stable COPD were recruited in Cangzhou Central Hospital from June 2012 to June 2014. According to baseline D(L)CO and IC, the patients were divided into normal D(L)CO/IC group (group A, n=10), low D(L)CO/normal IC group (group B, n=18), normal D(L)CO/low IC group (group C, n=22) and low D(L)CO/IC group (group D, n=55). We compared the clinical characteristics, induced sputum cells and the therapeutic responses to 3-month treatment of budesonide/formoterol(320 µg/9 µg, inhale, bid)among the 4 groups. RESULTS: Group D showed the highest CAT scores(27.6 ± 6.4). Group C showed a higher prevalence of patients with wheezing(81.8)% and the highest percentage of sputum eosinophils (7.6 ± 3.2)%. Group C showed the greatest FEV(1) increase(0.214 ± 0.053)L. Group D showed a greater FEV(1) increase than Group B [(0.137 ± 0.063) vs (0.092 ± 0.048)L]. Group C showed a greater FVC[(0.342 ± 0.073), (0.190 ± 0.081), (0.223 ± 0.094)L] increase and CAT[(4.4 ± 2.0), (2.3 ± 1.3), (3.9 ± 1.9)] decrease than Group B and Group D(P<0.05). CONCLUSION: These findings suggest that COPD subgroups classified by D(L)CO and IC show several clinical characteristics and may be helpful to predict responses to treatment.

The Sox4/Tcf7l1 axis promotes progression of BCR-ABL-positive acute lymphoblastic leukemia.

The transcription factor Sox4 plays an indispensable role in the development of early progenitor B cells from hematopoietic stem cells. However, its role in B-cell acute lymphoblastic leukemia, a malignant counterpart of normal progenitor B cells, is not fully understood. Here we show that SOX4 is highly expressed in human acute lymphoblastic leukemia cells. To systematically study the function of Sox4 in acute lymphoblastic leukemia, we established a genetically defined mouse leukemia model by transforming progenitor B cells carrying a floxed Sox4 allele and inducing deletion of the allele by the self-excising Cre recombinase. This model allowed us to work with two groups of leukemic cells that had either one copy or both copies of Sox4 deleted. We found that depletion of Sox4 in transformed cells in vitro reduced cell growth in vitro and the progression of leukemia in vivo. Moreover, depletion of Sox4 in leukemic cells in vivo prolonged the survival of the mice, suggesting that it could be a potential target in acute lymphoblastic leukemia therapy. Our microarray and bioChIP studies revealed that Tcf7l1 was the key gene directly regulated by Sox4. Knockdown of Tcf7l1 reduced cell proliferation, just as did knockout of Sox4, and ectopic expression of Tcf7l1 could reverse the effect of Sox4 knockout on cell proliferation. These data suggest that Sox4 and Tcf7l1 form a functional axis that promotes the progression of BCR-ABL-positive acute lymphoblastic leukemia.

An indirect estimation of x-ray spectrum via convolutional neural network and transmission measurement.

Objective.In this work, we aim to propose an accurate and robust spectrum estimation method by synergistically combining x-ray imaging physics with a convolutional neural network (CNN).Approach.The approach relies on transmission measurements, and the estimated spectrum is formulated as a convolutional summation of a few model spectra generated using Monte Carlo simulation. The difference between the actual and estimated projections is utilized as the loss function to train the network. We contrasted this approach with the weighted sums of model spectra approach previously proposed. Comprehensive studies were performed to demonstrate the robustness and accuracy of the proposed approach in various scenarios.Main results.The results show the desirable accuracy of the CNN-based method for spectrum estimation. The ME and NRMSE were -0.021 keV and 3.04% for 80 kVp, and 0.006 keV and 4.44% for 100 kVp, superior to the previous approach. The robustness test and experimental study also demonstrated superior performances. The CNN-based approach yielded remarkably consistent results in phantoms with various material combinations, and the CNN-based approach was robust concerning spectrum generators and calibration phantoms.Significance. We proposed a method for estimating the real spectrum by integrating a deep learning model with real imaging physics. The results demonstrated that this method was accurate and robust in estimating the spectrum, and it is potentially helpful for broad x-ray imaging tasks.

A new approach for deducing rms proton radii from charge-changing reactions of neutron-rich nuclei and the reaction-target dependence.

We report the charge-changing cross sections (σcc) of 24 p-shell nuclides on both hydrogen and carbon at about 900A MeV, of which 8,9Li, 10-12Be, 10,14,15B, 14,15,17-22N and 16O on hydrogen and 8,9Li on carbon are for the first time. Benefiting from the data set, we found a new and robust relationship between the scaling factor of the Glauber model calculations and the separation energies of the nuclei of interest on both targets. This allows us to deduce proton radii (Rp) for the first time from the cross sections on hydrogen. Nearly identical Rp values are deduced from both target data for the neutron-rich carbon isotopes; however, the Rp from the hydrogen target is systematically smaller in the neutron-rich nitrogen isotopes. This calls for further experimental and theoretical investigations.

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples.

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Microbiome alteration via fecal microbiota transplantation is effective for refractory immune checkpoint inhibitor-induced colitis.

Immune checkpoint inhibitors (ICIs) target advanced malignancies with high efficacy but also predispose patients to immune-related adverse events like immune-mediated colitis (IMC). Given the association between gut bacteria with response to ICI therapy and subsequent IMC, fecal microbiota transplantation (FMT) represents a feasible way to manipulate microbial composition in patients, with a potential benefit for IMC. Here, we present a large case series of 12 patients with refractory IMC who underwent FMT from healthy donors as salvage therapy. All 12 patients had grade 3 or 4 ICI-related diarrhea or colitis that failed to respond to standard first-line (corticosteroids) and second-line immunosuppression (infliximab or vedolizumab). Ten patients (83%) achieved symptom improvement after FMT, and three patients (25%) required repeat FMT, two of whom had no subsequent response. At the end of the study, 92% achieved IMC clinical remission. 16S rRNA sequencing of patient stool samples revealed that compositional differences between FMT donors and patients with IMC before FMT were associated with a complete response after FMT. Comparison of pre- and post-FMT stool samples in patients with complete responses showed significant increases in alpha diversity and increases in the abundances of Collinsella and Bifidobacterium, which were depleted in FMT responders before FMT. Histologically evaluable complete response patients also had decreases in select immune cells , including CD8+ T cells, in the colon after FMT when compared with non-complete response patients (n = 4). This study validates FMT as an effective treatment strategy for IMC and gives insights into the microbial signatures that may play a critical role in FMT response.

The Application Value of Metagenomic and Whole-Genome Capture Next-Generation Sequencing in the Diagnosis and Epidemiological Analysis of Psittacosis.

Background: To evaluate the value of metagenomic next-generation sequencing (mNGS) for the early diagnosis of psittacosis, and to investigate its epidemiology by whole-genome capture. Methods: Twenty-one bronchoalveolar lavage fluid (BALF) and blood samples of 16 psittacosis patients from multiple centers during August 2019 to September 2021 were analyzed retrospectively. mNGS with normal datasets (10 M 75-bp single-end reads after sequencing) and larger datasets (30 M 150-bp paired-end reads after sequencing) as well as quantitative real-time polymerase chain reaction (qPCR) were used to detect the pathogen. Also, whole-genome capture of Chlamydophila psittaci was applied to draw the phylogenetic tree. Results: mNGS successfully detected the pathogen in all 16 cases (100%), while qPCR was positive only in 5 out of 10 cases (50%), indicating a significantly higher sensitivity of mNGS than qPCR (p < 0.01). BALF-mNGS performed better than blood-mNGS (16/16 versus 3/5, p < 0.05). In addition, larger datasets (the read counts have tripled, and the base number was 12-fold larger compared to clinical mNGS with a normal dataset) of mNGS showed significantly increased contents of human DNA (p < 0.05) and decreased reads per million of the pathogen, suggesting no improvement. Whole-genome capture results of five samples (>60% coverage and >1 depth) were used to construct the phylogenetic tree. Conclusion: Significant advantages of mNGS with normal datasets were demonstrated in early diagnosing psittacosis. It is the first study to use whole-genome capture to analyze C. psittaci epidemiological information.

MicroRNA-140-3p represses the proliferation, migration, invasion and angiogenesis of lung adenocarcinoma cells via targeting TYMS (thymidylate synthetase).

MicroRNA (miR)-140-3p has been proved to repress lung adenocarcinoma (LUAD), and our study aims to further evaluate the mechanism. Bioinformatic analyses were performed. The viability, proliferation, migration, invasion and angiogenesis of transfected LUAD cells were all determined via Cell Counting Kit-8, colony formation, Scratch, Transwell, and tube formation assays. The targeting relationship between miR-140-3p and thymidylate synthetase (TYMS) was confirmed by dual-luciferase reporter assay. Relative expressions of miR-140-3p, TYMS, epithelial-to-mesenchymal transition- (E-cadherin, N-cadherin, vimentin), angiogenesis- (vascular endothelial growth factor (VEGF)), and apoptosis-related factors (cleaved caspase-3, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax)) were quantified by quantitative real-time polymerase chain reaction or Western blot. TYMS was high-expressed yet miR-140-3p was low-expressed in LUAD cells. Upregulation of miR-140-3p inhibited TYMS expression, viability, colony formation, migration, invasion, and tube length within LUAD cells, while downregulation of miR-140-3p did oppositely. Silenced TYMS, the downstream target gene of miR-140-3p, reversed the effects of miR-140-3p downregulation on TYMS expression, cell viability, colony formation, migration, invasion, and tube length as well as the metastasis-, apoptosis- and angiogenesis-related proteins in LUAD cells. Upregulation of miR-140-3p inhibited the proliferation, migration, invasion and angiogenesis of LUAD cells via targeting TYMS.