Pregnancy influences expression of interferon-stimulated genes, progesterone receptor and progesterone-induced blocking factor in ovine thyroid.

Objective: Embryonic interferon-tau (IFNT) and progesterone affect expression of interferon-stimulated genes (ISGs), progesterone receptor (PGR) and progesterone-induced blocking factor (PIBF) in the ovine thyroid. Methods: Thyroids of ewes were sampled at day 16 of nonpregnancy, days 13, 16 and 25 of pregnancy, and RT-qPCR assay, western blot and immunohistochemistry were used to detect expression of ISGs, PGR and PIBF. Results: Free ISG15 protein was undetected, but ISG15 conjugated proteins upregulated at day 16 of pregnancy, and expression levels of ISG15 conjugated proteins, PGR isoform (70 kDa), PIBF, interferon-gamma-inducible protein 10 and myxovirusresistance protein 1 peaked, but expression level of signal transducer and activator of transcription 1 was the lowest at day 16 of pregnancy. In addition, the expression levels of PGR isoform (70 kDa) and STAT1 decreased, but levels of PGR isoform (43 kDa), 2',5'-oligoadenylate synthetase, IP-10 and MX1 increased at day 25 of pregnancy comparing with day 16 of the estrous cycle. Conclusion: Early pregnancy affects expression of ISGs, PGR and PIBF in maternal thyroid through IFNT and progesterone, which may regulate thyroid autoimmunity and thyroid hormone secretion in ewes.

Effects of Regulating Hippo and Wnt on the Development and Fate Differentiation of Bovine Embryo.

The improvement of in vitro embryo development is a gateway to enhance the output of assisted reproductive technologies. The Wnt and Hippo signaling pathways are crucial for the early development of bovine embryos. This study investigated the development of bovine embryos under the influence of a Hippo signaling agonist (LPA) and a Wnt signaling inhibitor (DKK1). In this current study, embryos produced in vitro were cultured in media supplemented with LPA and DKK1. We comprehensively analyzed the impact of LPA and DKK1 on various developmental parameters of the bovine embryo, such as blastocyst formation, differential cell counts, YAP fluorescence intensity and apoptosis rate. Furthermore, single-cell RNA sequencing (scRNA-seq) was employed to elucidate the in vitro embryonic development. Our results revealed that LPA and DKK1 improved the blastocyst developmental potential, total cells, trophectoderm (TE) cells and YAP fluorescence intensity and decreased the apoptosis rate of bovine embryos. A total of 1203 genes exhibited differential expression between the control and LPA/DKK1-treated (LD) groups, with 577 genes upregulated and 626 genes downregulated. KEGG pathway analysis revealed significant enrichment of differentially expressed genes (DEGs) associated with TGF-beta signaling, Wnt signaling, apoptosis, Hippo signaling and other critical developmental pathways. Our study shows the role of LPA and DKK1 in embryonic differentiation and embryo establishment of pregnancy. These findings should be helpful for further unraveling the precise contributions of the Hippo and Wnt pathways in bovine trophoblast formation, thus advancing our comprehension of early bovine embryo development.

A rare malignant mesothelioma of the tunica vaginalis testis: A case report.

Malignant mesothelioma of the tunica vaginalis testis is a rare, highly invasive urogenital malignant tumor with no specific clinical manifestations. Reported cases of this disease are limited. Therefore, an early preoperative diagnosis is difficult. The current study presents a case of malignant mesothelioma of the tunica vaginalis testis and a literature review. A 52-year-old man was admitted to Xiaoshan Affiliated Hospital of Wenzhou Medical University (Hangzhou, China) in December 2022 and underwent radical resection of the right testicle and epididymis but did not undergo radiotherapy or chemotherapy. The patient was followed up for 5 months, and no recurrence or metastasis was found. The rarity of testicular mesothelioma poses a challenge to its etiology and diagnosis, which is rarely achieved preoperatively. Malignant mesothelioma of the testicular tunica vaginalis has a poor prognosis and is not sensitive to radiotherapy or chemotherapy, requiring close postoperative follow-up. This condition is rare in clinical practice; therefore, it needs to be reported to aid clinicians' decision-making regarding diagnosis and treatment.

Spatial Metabolomics Identifies LPC(18:0) and LPA(18:1) in Advanced Atheroma With Translation to Plasma for Cardiovascular Risk Estimation.

BACKGROUND: The metabolic alterations occurring within the arterial architecture during atherosclerosis development remain poorly understood, let alone those particular to each arterial tunica. We aimed first to identify, in a spatially resolved manner, the specific metabolic changes in plaque, media, adventitia, and cardiac tissue between control and atherosclerotic murine aortas. Second, we assessed their translatability to human tissue and plasma for cardiovascular risk estimation. METHODS: In this observational study, mass spectrometry imaging (MSI) was applied to identify region-specific metabolic differences between atherosclerotic (n=11) and control (n=11) aortas from low-density lipoprotein receptor-deficient mice, via histology-guided virtual microdissection. Early and advanced plaques were compared within the same atherosclerotic animals. Progression metabolites were further analyzed by MSI in 9 human atherosclerotic carotids and by targeted mass spectrometry in human plasma from subjects with elective coronary artery bypass grafting (cardiovascular risk group, n=27) and a control group (n=27). RESULTS: MSI identified 362 local metabolic alterations in atherosclerotic mice (log2 fold-change ≥1.5; P≤0.05). The lipid composition of cardiac tissue is altered during atherosclerosis development and presents a generalized accumulation of glycerophospholipids, except for lysolipids. Lysolipids (among other glycerophospholipids) were found at elevated levels in all 3 arterial layers of atherosclerotic aortas. LPC(18:0) (lysophosphatidylcholine; P=0.024) and LPA(18:1) (lysophosphatidic acid; P=0.025) were found to be significantly elevated in advanced plaques as compared with mouse-matched early plaques. Higher levels of both lipid species were also observed in fibrosis-rich areas of advanced- versus early-stage human samples. They were found to be significantly reduced in human plasma from subjects with elective coronary artery bypass grafting (P<0.001 and P=0.031, respectively), with LPC(18:0) showing significant association with cardiovascular risk (odds ratio, 0.479 [95% CI, 0.225-0.883]; P=0.032) and diagnostic potential (area under the curve, 0.778 [95% CI, 0.638-0.917]). CONCLUSIONS: An altered phospholipid metabolism occurs in atherosclerosis, affecting both the aorta and the adjacent heart tissue. Plaque-progression lipids LPC(18:0) and LPA(18:1), as identified by MSI on tissue, reflect cardiovascular risk in human plasma.

Utility of enteral nutrition via percutaneous transhepatic cholangiography drainage catheterization in late-stage malignant obstructive jaundice.

Objective: The purpose of this study was to explore the clinical benefits of establishing an enteral nutrition (EN) pathway via percutaneous transhepatic cholangiography drainage (PTCD) catheterization in patients with late-stage malignant obstructive jaundice (MOJ).Methods: We selected 30 patients diagnosed as having late-stage MOJ with malnutrition. A dual-lumen biliary-enteral nutrition tube was placed via PTCD along with a biliary stent implantation. Postoperative EN was provided, and we observed the time taken for tube placement, its success rate, complications, and therapeutic efficacy.Results: Tube placement was successful in all 30 patients with an average procedural time of 5.7 ± 1.4 min with no tube placement complications. Compared to preoperative measures, there was a significant improvement in postoperative jaundice reduction and nutritional indicators one month after the procedure (p < 0.05). Post-placement complications included tube perileakage in 5 cases, entero-biliary reflux in 4 cases, tube blockage in 6 cases, tube displacement in 4 cases, accidental tube removal in 3 cases, and tube replacement due to degradation in 8 cases, with tube retention time ranging from 42 to 314 days, averaging 124.7 ± 37.5 days. All patients achieved the parameters for effective home-based enteral nutrition with a noticeable improvement in their quality of life.Conclusion: In this study, we found that the technique of establishing an EN pathway via PTCD catheterization was minimally invasive, safe, and effective; the tube was easy to maintain; and patient compliance was high. It is, thus, suitable for long-term tube retention in patients with late-stage MOJ.

Epigenomic analysis of the myometrium during late implantation revealed regulatory elements in genes related to the cellular zinc homeostasis pathway in pigs.

The myometrium, composed of the inner circular muscle (CM) and outer longitudinal muscle (LM), is crucial in establishing and maintaining early pregnancy. However, the molecular mechanisms involved are not well understood. In this study, we identified the transcriptomic features of the CM and LM collected from the mesometrial (M) and anti-mesometrial (AM) sides of the pig uterus on day 18 of pregnancy during the placentation initiation phase. Some genes in the cellular zinc ion level regulatory pathways (MT-1A, MT-1D, MT-2B, SLC30A2, and SLC39A2) were spatially and highly enriched in uterine CM at the mesometrial side. In addition, the histone modification profiles of H3K27ac and H3K4me3 in uterine CM and LM collected from the mesometrial side were characterized. Genomic regions associated with the expression of genes regulating the cellular zinc ion level were detected. Moreover, six highly linked variants in the H3K27ac-enriched region of the pig SLC30A2 gene were identified and found to be significantly associated with the total number born at the second parity (P < 0.05). In conclusion, the genes in the pathways of cellular zinc homeostasis and their regulatory elements identified have implications for pig reproduction trait improvement and warrant further investigations.

SMARCA4­deficient non­small cell lung cancer with an EGFR mutation: A case report.

SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4)-deficient non-small cell lung cancer (dNSCLC) is a rare malignant tumor that originates in the lungs. It occurs more frequently in male smokers, and the epidermal growth factor receptor (EGFR) gene is often mutation-free. In the present study, the case of a 60-year-old, non-smoking female patient diagnosed with SMARCA4-dNSCLC is reported. Biopsy of the tumor showed solid flaky, nest-like infiltrating growth. Immunohistochemistry revealed the following: SMARCA4/BRG1(-), SMARCB1/INI-1(+), cytokeratin7 (+), cytokeratin 5.2 (+), CK5/6(+) and calretinin(+). The Ki-67 positivity index was 75%, and the thyroid transcription factor-1, NapsinA, p40, nuclear protein in testis, CD34, Sal-like protein 4, SRY-box transcription factor 2 and synaptophysin were negative. Molecular analysis showed mutations in both EGFR and TP53. The pathological diagnosis was SMARCA4-dNSCLC with an EGFR gene mutation. The present case report could be used for broadening the pathological diagnosis of SMARCA4-dNSCLC and for selecting appropriate treatment approaches.

The association between albumin and C-reactive protein in older adults.

Albumin had been found to be a marker of inflammation. The purpose of our study was to investigate the relationship between albumin and C-reactive protein (CRP) in 3579 participants aged 60 to 80 years from the National Health and Nutrition Examination Survey (NHANES). In order to evaluate the association between albumin and CRP, We downloaded the analyzed data (2015-2018) from the NHANES in the United States, and the age of study population was limited to 60 to 80 years (n = 4051). After exclusion of subjects with missing albumin (n = 456) and CRP (n = 16) data, 3579 subjects aged 60 to 80 years were reserved for a cross-sectional study. All measures were calculated accounting for NHANES sample weights. We used the weighted χ2 test for categorical variables and the weighted linear regression model for continuous variables to calculate the difference among each group. The subgroup analysis was evaluated through stratified multivariable linear regression models. Fitting smooth curves and generalized additive models were also carried out. We found albumin negatively correlated with CRP after adjusting for other confounders in model 3 (ß = -0.37, 95% CI: -0.45, -0.28, P < .0001). After converting albumin from a continuous variable to a categorical variable (quartiles), albumin level was also negatively associated with serum CRP in all groups (P for trend < .001 for each). In the subgroup analysis stratified by gender, race/ethnicity, smoking, high blood pressure, the negative correlation of albumin with CRP was remained. We also found that the level of CRP further decreased in other race (OR: -0.72, 95% CI: -0.96, -0.47 P < .0001) and participants with smoking (OR: -0.61, 95% CI: -0.86, -0.36 P < .0001). Our findings revealed that albumin levels was negatively associated with CRP levels among in USA elderly. Besides, CRP level decreased faster with increasing albumin level in other race and participants with smoking. Considering this association, hypoalbuminemia could provide a potential predictive biomarker for inflammation. Therefore, studying the relationship between albumin and CRP can provide a screening tool for inflammation to guide therapeutic intervention and avoid excessive correction of patients with inflammation.

Bilateral femoral neck stress fractures in elderly individuals: A case report and literature review.

RATIONALE: Bilateral femoral neck stress fractures are relatively rare injuries that occur frequently in military recruits, athletes and patients with osteoporosis, renal bone disease, metabolic bone disease, and chronic steroid use. Herein, a case of an elderly patient with bilateral femoral neck stress fractures is reported. PATIENT CONCERNS: A 65-year-old man presented to the author's hospital with right hip pain for over a month. The patient was a farmer, had a long history of field labor before the onset of pain, denied any history of trauma. DIAGNOSIS: The patient was diagnosed with a right subcapital fracture of the femoral neck after examination. The patient complained of only right hip symptoms, and hip computed tomography showed no abnormalities in the left hip. A tension fracture of the left femoral neck was missed due to unawareness of the abnormal signal of the left femoral neck seen on right hip magnetic resonance imaging. INTERVENTIONS: During the first hospitalization, the patient underwent total hip arthroplasty (THA) on the right hip. Two months after the operation, the patient started to have pain in the left hip and underwent left THA again for a displaced left femoral neck fracture. OUTCOMES: The patient eventually underwent bilateral THA surgery and had a satisfactory functional recovery. But the oversight in the diagnostic process led to the patient undergoing left THA that could have been avoided. LESSONS: For patients who complain of hip pain but deny a history of trauma, we should be concerned about the presence of a hip fracture even if the patient's radiograph does not report a positive result. The most sensitive method is bilateral magnetic resonance imaging examination of the hip. Femoral neck stress fractures require early diagnosis and treatment to prevent complications.

Small-diameter PCL/PU vascular graft modified with heparin-aspirin compound for preventing the occurrence of acute thrombosis.

The occurrence of acute thrombosis, directly related to platelet aggregation and coagulant system, is a considerable reason for the failure of small-diameter vascular grafts. Heparin is commonly used as a functional molecule for graft modification due to the strong anticoagulant effect. Unfortunately, heparin cannot directly resist the adhesion and aggregation of platelets. Therefore, we have prepared a heparin-aspirin compound by coupling heparin with aspirin, an antiplatelet drug, and covalently grafted it onto the surface of polycaprolactone/polyurethane composite tube. In this way, the graft not only showed a dual function of both anticoagulation and antiplatelet, but also effectively avoided the rapid drug release and excessive toxicity to other organs caused by simple blending the medicine with material matrix. The compound retained the original function of heparin, showing good hydrophilicity and biocompatibility, which could promote the adhesion and proliferation of endothelial cells (ECs) and facilitate the process of tissue regeneration. What's more, the compound showed more effective than heparin in reducing platelet activation and preventing thrombosis. The graft modified by this compound maintained completely unobstructed for one month of implantation, while severe obstruction or stenosis occurred in PCL/PU and PCL/PU-Hep lumen at the first week, verifying the effect of the compound on preventing acute thrombosis. In general, this study proposed a designing method for small-diameter vascular graft which could prevent acute thrombosis and promote intimal construction.

Expression of IkappaB Family in the Ovine Liver during Early Pregnancy.

During normal pregnancy, there is a dynamic regulation of the maternal immune system, including the liver, to accommodate the presence of the allogeneic foetus in the uterus. However, it was unclear that the expression of the IkappaB (IκB) family was regulated in the ovine maternal liver during early pregnancy. In this study, sheep livers were collected at day 16 of the oestrous cycle (NP16), and days 13, 16 and 25 of gestation (DP13, DP16 and DP25), and RT-qPCR, Western blot and immunohistochemistry analysis were used to analyse the expression of the IκB family, including B cell leukemia-3 (BCL-3), IκBα, IκBß, IκBε, IKKγ, IκBNS and IκBζ. The results revealed that expression of BCL-3, IκBß, IκBε and IKKγ peaked at DP16, and the expression of IκBα was increased during early pregnancy. In addition, the expression of IκBζ peaked at DP13 and DP16, and IκBNS peaked at DP13. IκBß and IKKγ proteins were located in the endothelial cells of the proper hepatic arteries and portal veins, and hepatocytes. In conclusion, early pregnancy changed the expression of the IκB family, suggesting that the modulation of the IκB family may be related to the regulation of maternal hepatic functions, which may be favourable for pregnancy establishment in sheep.

Early Pregnancy Modulates Expression of the Nod-like Receptor Family in Lymph Nodes of Ewes.

NOD receptors (NLRs) mediate adaptive immune responses and immune tolerance. Nevertheless, it is not clear if gestation modulates the NLR signaling pathway in lymph nodes of ewes. In this study, lymph nodes of ewes were collected at day 16 of the estrous cycle, and at days 13, 16 and 25 of gestation (n = 6 for each group). RT-qPCR, Western blot and immunohistochemistry analysis were used to analyze the expression of the NLR family, including NOD1, NOD2, CIITA, NAIP, NLRP1, NLRP3 and NLRP7. The data showed that early gestation enhanced expression of NOD1, CIITA, NLRP1, NLRP3 and NLRP7 mRNA, as well as proteins at day 16 of gestation, and the expression levels of NOD2, CIITA, NLRP1 and NLRP7 were higher at days 13 and 25 of gestation than day 16 of the estrous cycle. However, NOD1 expression was lower on days 13 and 25 of gestation compared to day 16 of the estrous cycle, and early gestation suppressed NAIP expression. In summary, early pregnancy modulated expression of the NLR family in ovine lymph nodes, which participates in immune regulation, and this modulation may be necessary for pregnancy establishment in ewes.

Characterization and attribution of vegetation dynamics in the ecologically fragile South China Karst: Evidence from three decadal Landsat observations.

Plant growth and its changes over space and time are effective indicators for signifying ecosystem health. However, large uncertainties remain in characterizing and attributing vegetation changes in the ecologically fragile South China Karst region, since most existing studies were conducted at a coarse spatial resolution or covered limited time spans. Considering the highly fragmented landscapes in the region, this hinders their capability in detecting fine information of vegetation dynamics taking place at local scales and comprehending the influence of climate change usually over relatively long temporal ranges. Here, we explored the spatiotemporal variations in vegetation greenness for the entire South China Karst region (1.9 million km2) at a resolution of 30m for the notably increased time span (1987-2018) using three decadal Landsat images and the cloud-based Google Earth Engine. Moreover, we spatially attributed the vegetation changes and quantified the relative contribution of driving factors. Our results revealed a widespread vegetation recovery in the South China Karst (74.80%) during the past three decades. Notably, the area of vegetation recovery tripled following the implementation of ecological engineering compared with the reference period (1987-1999). Meanwhile, the vegetation restoration trend was strongly sustainable beyond 2018 as demonstrated by the Hurst exponent. Furthermore, climate change contributed only one-fifth to vegetation restoration, whereas major vegetation recovery was highly attributable to afforestation projects, implying that anthropogenic influences accelerated vegetation greenness gains in karst areas since the start of the new millennium during which ecological engineering was continually established. Our study provides additional insights into ecological restoration and conservation in the highly heterogeneous karst landscapes and other similar ecologically fragile areas worldwide.

Large Fragment InDels Reshape Genome Structure of Porcine Alveolar Macrophage 3D4/21 Cells.

The porcine monomyeloid cell line, or 3D4/21 cells, is an effective tool to study the immune characteristics and virus infection mechanism of pigs. Due to the introduction of the neomycin resistance gene and the SV40 large T antigen gene, its genome has undergone essential changes, which are still unknown. Studying the variation in genome structure, especially the large fragments of insertions and deletions (InDels), is one of the proper ways to reveal these issues. In this study, an All-seq method was established by combining Mate-pair and Shotgun sequencing methods, and the detection and verification of large fragments of InDels were performed on 3D4/21 cells. The results showed that there were 844 InDels with a length of more than 1 kb, of which 12 regions were deletions of more than 100 kb in the 3D4/21 cell genome. In addition, compared with porcine primary alveolar macrophages, 82 genes including the CD163 had lost transcription in 3D4/21 cells, and 72 genes gained transcription as well. Further referring to the Hi-C structure, it was found that the fusion of the topologically associated domains (TADs) caused by the deletion may lead to abnormal gene function. The results of this study provide a basis for elaborating the genome structure and functional variation in 3D4/21 cells, provide a method for rapid and convenient detection of large-scale InDels, and provide useful clues for the study of the porcine immune function genome and the molecular mechanism of virus infection.

Long-range interaction within the chromatin domain determines regulatory patterns in porcine skeletal muscle.

Spatial chromatin structure is crucial for understanding the early growth and development of porcine skeletal muscle. However, its characteristic of 3D architecture and elaborate regulation of gene transcription remains unclear. In this study, ChIA-PET method is used to study the changes of early chromatin three-dimensional structure in skeletal muscle of lean type Yorkshire pig and fat type Meishan pig. Integrating the in situ Hi-C data revealed the 3D architecture and long-range interaction of the porcine muscle. The results showed the CTCF/RNAPII mediated long-range interaction shapes the different chromatin architecture and dominates the unique regulation of enhancers. In addition, the results revealed that key myogenic genes like ssc-mir-1 had a unique enhancer regulation function in myogenesis. Interestingly, the FGF6 gene is of breed-specific regulation, implying the difference between two breeds in skeletal muscle development. Our research thus may provide a clue for the porcine genetic improvement of skeletal muscle.

[Design and application of a new type of biliary-intestinal nutrient tube].

The establishment of a nutritional pathway is the premise and basis of nutritional therapy for patients with malignant tumor. The nasogastric tube, nasoenteric tube, and percutaneous endoscopic gastric/jejunostomy are commonly used clinical pathways for enteral nutrition (EN) therapy. However, these EN pathways are often difficult to establish in patients with malignant obstructive jaundice (MOJ) with pyloric or duodenum primary obstruction. For this reason, a new type biliary-intestinal nutrient tube placed through percutaneous transhepatic cholangiography drainage (PTCD) pathway was designed by the medical staff of hepatobiliary surgery department of Yinchuan First People's Hospital, and National Utility Model Patent of China were obtained (ZL 2020 2 0283951.5, ZL 2020 2 0288938.9). The new biliary-intestinal nutrient tube has two types: double-lumen tube and single-lumen tube, which consists of tube head, tube body, tail ring and developing ring. The double lumen tube realizes bile internal drainage and EN simultaneously through the double lumen structure of the tube body. The single-lumen tube is used for nutrient infusion after bile duct metal stent implantation, which is not limited by the type of nutrient solution. The tail ring of the two types of nutrient tube is placed in the upper jejunum to reduce retrograde infection and unexpected extubation. Compared with the prior art, the utility model has the advantages of simple structure, reasonable design, safe and effective placement through PTCD pathway, and opens up a new EN path for MOJ patients.

C/EBP-ß contributes to pig endometrial LE receptivity by targeting cell remodeling genes during implantation.

In brief: Transforming the endometrial luminal epithelium (LE) into a receptive state is a requisite event for successful embryo implantation. This study suggests the role of a transcription factor in regulating endometrial LE receptivity. Abstract: The endometrial luminal epithelium (LE) undergoes extensive remodeling during implantation to establish receptivity of the uterus in response to the conceptus signals, such as interleukin 1ß (IL1B). But the mechanisms remain to be fully understood. This study investigated the role of CCAAT/enhancer-binding protein ß (C/EBP-ß) in regulating pig endometrial LE receptivity. Our results showed that C/EBP-ß was expressed and activated only in the endometrial LE in an implantation-dependent manner. In addition, C/EBP-ß was highly activated at the pre-attachment stage compared to the attachment stage, and its activation was correlated with the expression of IL1B-dependent extracellular signal-regulated kinases1/2-p90 ribosomal S6 kinase signaling axis. Subsequent chromatin immunoprecipitation (ChIP)-sequencing analysis revealed that the binding of C/EBP-ß within the promoter was positively associated with the transcription of genes related to cell remodeling. One such gene is matrix metalloproteinase 8 (MMP8), which is responsible for extracellular matrix degradation. The expression of MMP8 was abundant at the pre-attachment stage but dramatically declined at the attachment stage in the endometrial LE. Consistent with C/EBP-ß, the expression and activation of MMP8 were limited to the endometrial LE in an implantation-dependent manner. Using ChIP-qPCR and electrophoresis mobility shift assay approaches, we demonstrated that C/EBP-ß regulated the expression of the MMP8 gene during implantation. Furthermore, we detected that MMP8 and one of its substrates, type II collagen, showed a mutually exclusive expression pattern in pig endometrial LE during implantation. Our findings indicate that C/EBP-ß plays a role in pig endometrial LE receptivity by regulating cell remodeling-related genes, such as MMP8, in response to conceptus signals during implantation.

Characterization and perturbation of CTCF-mediated chromatin interactions for enhancing myogenic transdifferentiation.

Expression of key transcription factors can induce transdifferentiation in somatic cells; however, this conversion is usually incomplete due to undefined intrinsic barriers. Here, we employ MyoD-induced transdifferentiation of fibroblasts as a model to illustrate the chromatin structures that impede the cell-fate transition. Focusing on the three-dimensional (3D) chromatin interactions, we show that MyoD directly establishes chromatin loops to activate myogenic transcriptional program. Similarly, dynamic changes of CTCF-mediated chromatin interactions are favorable for fibroblast-to-myoblast conversion. However, a substantial portion of CTCF-mediated chromatin interactions remain stable, and the associated genes are steady in expression and enriched for fibroblast function that may restrict cell-identity transformation. Temporal CTCF depletion can interrupt the resistant chromatin loops to enhance myogenic transdifferentiation in mice, pig, and chicken fibroblasts. Therefore, during induced transdifferentiation, the transcription factor can directly reorganize the 3D chromatin interactions, and perturbation of CTCF-mediated genome topology may resolve the limitations of cell fate transitions.

Mass spectrometry imaging identifies altered hepatic lipid signatures during experimental Leishmania donovani infection.

Introduction: Spatial analysis of lipids in inflammatory microenvironments is key to understand the pathogenesis of infectious disease. Granulomatous inflammation is a hallmark of leishmaniasis and changes in host and parasite lipid metabolism have been observed at the bulk tissue level in various infection models. Here, mass spectrometry imaging (MSI) is applied to spatially map hepatic lipid composition following infection with Leishmania donovani, an experimental mouse model of visceral leishmaniasis. Methods: Livers from naïve and L. donovani-infected C57BL/6 mice were harvested at 14- and 20-days post-infection (n=5 per time point). 12 µm transverse sections were cut and covered with norhamane, prior to lipid analysis using MALDI-MSI. MALDI-MSI was performed in negative mode on a Rapiflex (Bruker Daltonics) at 5 and 50 µm spatial resolution and data-dependent analysis (DDA) on an Orbitrap-Elite (Thermo-Scientific) at 50 µm spatial resolution for structural identification analysis of lipids. Results: Aberrant lipid abundances were observed in a heterogeneous distribution across infected mouse livers compared to naïve mouse liver. Distinctive localized correlated lipid masses were found in granulomas and surrounding parenchymal tissue. Structural identification revealed 40 different lipids common to naïve and d14/d20 infected mouse livers, whereas 15 identified lipids were only detected in infected mouse livers. For pathology-guided MSI imaging, we deduced lipids from manually annotated granulomatous and parenchyma regions of interests (ROIs), identifying 34 lipids that showed significantly different intensities between parenchyma and granulomas across all infected livers. Discussion: Our results identify specific lipids that spatially correlate to the major histopathological feature of Leishmania donovani infection in the liver, viz. hepatic granulomas. In addition, we identified a three-fold increase in the number of unique phosphatidylglycerols (PGs) in infected liver tissue and provide direct evidence that arachidonic acid-containing phospholipids are localized with hepatic granulomas. These phospholipids may serve as important precursors for downstream oxylipin generation with consequences for the regulation of the inflammatory cascade. This study provides the first description of the use of MSI to define spatial-temporal lipid changes at local sites of infection induced by Leishmania donovani in mice.

Integrating multiplex immunofluorescent and mass spectrometry imaging to map myeloid heterogeneity in its metabolic and cellular context.

Cells often adopt different phenotypes, dictated by tissue-specific or local signals such as cell-cell and cell-matrix contacts or molecular micro-environment. This holds in extremis for macrophages with their high phenotypic plasticity. Their broad range of functions, some even opposing, reflects their heterogeneity, and a multitude of subsets has been described in different tissues and diseases. Such micro-environmental imprint cannot be adequately studied by single-cell applications, as cells are detached from their context, while histology-based assessment lacks the phenotypic depth due to limitations in marker combination. Here, we present a novel, integrative approach in which 15-color multispectral imaging allows comprehensive cell classification based on multi-marker expression patterns, followed by downstream analysis pipelines to link their phenotypes to contextual, micro-environmental cues, such as their cellular ("community") and metabolic ("local lipidome") niches in complex tissue. The power of this approach is illustrated for myeloid subsets and associated lipid signatures in murine atherosclerotic plaque.