Tissue Suspensions Research Articles

Vertical Superior Vector Suspension of the Anterior Mobile Face with the V Plication Facelift: A Volumization and Lifting Technique.

In most of the published plication techniques in face lift surgery, the vectors of plication are not entirely superiorly and vertically directed. The same applies with the deep plane, SMAS elevation techniques in the majority of which the vectors of traction are not superiorly vertically directed. The aging symptoms are mostly prominent at the anterior mobile face due to the gravity effect, and this is the area where attention should be focused to correct these symptoms following a face lift surgery. We have evaluated the results of 58 patients who underwent the V plication face lift between January 2021 to January 2024 in terms of demographics, procedure, surgical technique, adjuvant treatments, final outcomes and complications. All 58 patients underwent primary face lift with the V plication for vertical superior traction and suspension of the facial tissues. The average age value was 64 years. Forty-six patients underwent concomitant upper and lower blepharoplasty, and 25 fat transfer of the upper periorbital region. There were one self-resolving neurapraxia of the right buccal branch of facial nerve and three minor self-absorbed localized blood collections. The extensive V plication face lift of the anterior face together with the progressively less extensive plication of the lateral face due to the shape of plication addressed satisfactorily the prominent symptoms of gravity effect on the face with the action of the vertical superior suspension of the facial tissues and corrected the malar deflation by repositioning the downwards sliding malar fat pad. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Benzo (A) pyrene exposure alters alveolar epithelial and macrophage cells diversity and induces antioxidant responses in lungs

This study was designed to investigate the toxic effects of benzo (a) pyrene (BaP) in the lungs. Mice were repeatedly treated orally with BaP (50 mg/kg body weight, twice a week for four weeks) to induce a tumour. After 4 months of BaP administration, tumours were visible beneath the skin. The histopathological section of the lungs shows congestion of pulmonary blood vessels, alveolar hyperplasia, and concurrent epithelial hyperplasia with infiltrates of inflammatory cells also seen. Thereafter, a single-cell suspension of lung tissues was stained with fluorescently conjugated antibodies for the demarcation of alveolar epithelial (anti-mouse CD74 and podoplanin) and macrophage (F4/80 and CD11b) cells and measured by flow cytometry. The expression of antioxidant genes was assessed by qRT–PCR. The number of alveolar epithelial cells 1 (AEC1) increased, but the number of alveolar epithelial cells 2 (AEC2) and transitional alveolar epithelial cells (TAEC) was significantly decreased in tumour-bearing mice. The proportion of CD11b+ alveolar macrophages (AM) and interstitial macrophages (IM) was increased, but the proportion of F4/80+ AM cells was reduced. The BaP administration significantly increased the ROS production in alveolar cells. The relative expression levels of antioxidant genes (SOD1, catalase, GPX1, and HIF-1α) were increased, but NRF2 expression was decreased in BaP-treated alveolar cells. The expression of anti-inflammatory (NF-κB) was also significantly increased. In conclusion, BaP exposure induced an inflammatory response, altered alveolar epithelial cell and macrophage diversity, and increased antioxidant responses in the lungs.

What happens after the free flap? Evaluation of aesthetic and functional elective revisions

BackgroundMicrovascular free flap reconstruction of a defect, as a single-stage procedure, does not always achieve the desired functional and esthetic result. Revisions may be necessary to achieve ideal contour, symmetry, and suspension of soft tissues, or to support dental rehabilitation. PurposeThe study purpose was to estimate the incidence and identify factors associated with elective free flap revisions.Study design, setting, sample: A retrospective cohort study of subjects who underwent free flap reconstruction was conducted to characterize the rate of elective free flap revision surgery. The study included subjects >18 years old, who underwent head and neck microvascular free flap surgery for any etiology, and both primary and secondary reconstructions, between 2014-2021. Subjects were excluded if they had incomplete records. Predictor variableThe predictor variable was composed of a set of variables, grouped into the following categories: subject demographic data, medical history, disease etiology, anatomical site, and postoperative complications. Main outcome variableThe outcome variable was time to elective revision surgery measured as months from the date of the initial operation to date of elective revision surgery. Elective revision surgery was defined as a procedure to enhance esthetics or function, which directly modifies the original surgical site, not including procedures to correct a complication, treat the original disease process, or emergencies. CovariatesNot applicable. AnalysisKaplan-Meier method was used to analyze the data, with p-value <0.05. Risk factors for elective revisions were analyzed with Cox Hazard ratio. ResultsThe sample was composed of 377 subjects that underwent free flap surgery, with a mean age of 55.9 +/- 15.9. Of these 67% were male, and 33% female, and sixty-two had an elective revision surgery (16.4%), with median follow-up period of 17 months (IQR 9-28). In the Cox regression model, only type of free flap was associated with an elective revision (p<0.05), where radial forearm had the lowest association with elective revision, and fasciocutaneous, latissimus dorsi, and scapula flaps had the highest. Conclusion and RelevanceAchieving basic wound coverage with free flaps can be a challenge, however, surgeons should prepare patients for the potential need for functional and esthetic revisions after free flap surgery to improve patient quality of life.

Anti-4-1BB×PDL1 Bispecific Antibody Reinvigorates Tumor-Specific Exhausted CD8+ T Cells and Enhances the Efficacy of Anti-PD1 Blockade.

To overcome the limited efficacy of immune checkpoint blockade, there is a need to find novel cancer immunotherapeutic strategies for the optimal treatment of cancer. The novel anti-4-1BB×PDL1 bispecific antibody-ABL503 (also known as TJ-L14B)-was designed to simultaneously target PDL1 and 4-1BB and demonstrated strong antitumor T-cell responses without considerable toxicity. In this study, we investigated the mechanisms by which the combination of ABL503 and anti-PD1 blockade affected the reinvigoration of exhausted tumor-infiltrating CD8+ T cells (CD8+ TIL) and antitumor efficacy. Single-cell suspensions of hepatocellular carcinoma and ovarian cancer tissues from treatment-naïve patients were used for immunophenotyping of CD8+ TILs and in vitro functional assays. Humanized hPD1/hPDL1/h4-1BB triple-knock-in mice were used to evaluate the effects of ABL503 and anti-PD1 blockade in vivo. We observed that ABL503 successfully restored the functions of 4-1BB+ exhausted CD8+ TILs, which were enriched for tumor-specific T cells but unresponsive to anti-PD1 blockade. Importantly, compared with anti-PD1 blockade alone, the combination of ABL503 and anti-PD1 blockade further enhanced the functional restoration of human CD8+ TILs in vitro. Consistently, the combination of ABL503 with anti-PD1 in vivo significantly alleviated tumor growth and induced enhanced infiltration and activation of CD8+ TILs. ABL503, a PDL1 and 4-1BB dual-targeting bispecific antibody, elicits pronounced additive tumor growth inhibition, with increased infiltration and functionality of exhausted CD8+ T cells, which in turn enhances the anticancer effects of anti-PD1 blockade. These promising findings suggest that ABL503 (TJ-L14B) in combination with PD1 inhibitors will likely further enhance therapeutic benefit in clinical trials. See related commentary by Molero-Glez et al., p. 3971.

Static reanimation of the paralyzed face using non-absorbable polypropylene mesh.

&lt;br&gt;&lt;b&gt;Introduction:&lt;/b&gt; There are 2 methods for correcting facial deformities resulting from facial nerve paralysis - dynamic and static. Although dynamic methods are the standard approach in selected patient groups, static facial suspension using synthetic material provides a minimally invasive alternative for those who do not qualify for them.&lt;/br&gt; &lt;br&gt;&lt;b&gt;Aim:&lt;b&gt; Presenting experiences with the use of non-absorbable polypropylene mesh in the Department of Plastic Surgery in Polanica-Zdrój and reviewing the literature related to static facial reanimation methods.&lt;/br&gt; &lt;br&gt;&lt;b&gt;Materials and methods:&lt;/b&gt; Fourteen patients underwent static facial tissue suspension using non-absorbable polypropylene mesh and 12 patients underwent surgery using classical autologous tissue techniques between 2019 and 2023. Indications for surgical treatment were analyzed, and preliminary results of treatment using both methods were compared.&lt;/br&gt; &lt;br&gt;&lt;b&gt;Results:&lt;/b&gt; Compared to autologous tissue methods, surgeries using synthetic mesh were associated with shorter hospitalization times. No long-term postoperative complications were observed in any patient. There were no differences in treatment outcomes between autologous tissue and synthetic mesh techniques, and all patients assessed the outcome as favorable.&lt;/br&gt; &lt;br&gt;&lt;b&gt;Discussion:&lt;/b&gt; Treatment of facial nerve paralysis sequelae using static techniques should be considered for patients not qualifying for surgery with dynamic methods and/or not consenting to such procedures. Using synthetic material as an alternative to autologous material reduces surgical time and avoids donor-site injuries and prolonged recovery.&lt;/br&gt; &lt;br&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Facial suspension with synthetic material is an effective, low-risk procedure for static correction of facial nerve paralysis sequelae.&lt;/br&gt; &lt;br&gt;&lt;b&gt;Study significance:&lt;/b&gt; Emphasizing the importance of static methods in facial reanimation.&lt;/br&gt.

Tumor cell enrichment by tissue suspension improves sensitivity to copy number variation in diffuse gastric cancer with low tumor content

The detection of copy number variations (CNVs) and somatic mutations in cancer is important for the selection of specific drugs for patients with cancer. In cancers with sporadic tumor cells, low tumor content prevents the accurate detection of somatic alterations using targeted sequencing. To efficiently identify CNVs, we performed tumor cell enrichment using tissue suspensions of formalin-fixed paraffin-embedded (FFPE) tissue sections with low tumor cell content. Tumor-enriched and residual fractions were separated from FFPE tissue suspensions of intestinal and diffuse-type gastric cancers containing sporadic tumor cells, and targeted sequencing was performed on 225 cancer-related genes. Sequencing of a targeted panel of cancer-related genes using tumor-enriched fractions increased the number of detectable CNVs and the copy number of amplified genes. Furthermore, CNV analysis using the normal cell-enriched residual fraction as a reference for CNV scoring allowed targeted sequencing to detect CNV characteristics of diffuse-type gastric cancer with low tumor content. Our approach improves the CNV detection rate in targeted sequencing with tumor enrichment and the accuracy of CNV detection in archival samples without paired blood.

Single-cell transcriptomic landscape and the microenvironment of normal adjacent tissues in hypopharyngeal carcinoma.

The cellular origin of hypopharyngeal diseases is crucial for further diagnosis and treatment, and the microenvironment in tissues may also be associated with specific cell types at the same time. Normal adjacent tissues (NATs) of hypopharyngeal carcinoma differ from non-tumor-bearing tissues, and can influenced by the tumor. However, the heterogeneity in kinds of disease samples remains little known, and the transcriptomic profile about biological information associated with disease occurrence and clinical outcome contained in it has yet to be fully evaluated. For these reasons, we should quickly investigate the taxonomic and transcriptomic information of NATs in human hypopharynx. Single-cell suspensions of normal adjacent tissues (NATs) of hypopharyngeal carcinoma were obtained and single-cell RNA sequencing (scRNA-seq) was performed. We present scRNA-seq data from 39,315 high-quality cells in the hypopharyngeal from five human donors, nine clusters of normal adjacent human hypopharyngeal cells were presented, including epithelial cells, endothelial cells (ECs), mononuclear phagocyte system cells (MPs), fibroblasts, T cells, plasma cells, B cells, mural cells and mast cells. Nonimmune components in the microenvironment, including epithelial cells, endothelial cells, fibroblasts and the subpopulations of them were performed. Our data provide a solid basis for the study of single-cell landscape in human normal adjacent hypopharyngeal tissues biology and related diseases.

Specialty-Specific Trends in Surgery for Apical Pelvic Organ Prolapse Post Vaginal Mesh.

Since transvaginal mesh (VM) kits for apical pelvic organ prolapse (APOP) were labeled "high-risk" in 2016 and subsequently banned in 2019 by the U.S. Food and Drug Administration, the most common remaining surgical options include abdominal mesh-augmented sacrocolpopexy (AS) and transvaginal native tissue suspension (VN). The objective of this study was to determine temporal trends in APOP procedures for urologists and gynecologists. Between 2011 and 2020, the American College of Surgeons National Surgical Quality Improvement Program database was queried for AS, VM, and VN. There were 26,477 cases of APOP repair (32% AS, 6% VM, 62% VN) included, 9% by urologists. Urologists operated on older (65 vs 61 years) patients with more medical comorbidities. Urologists performed significantly higher proportion of AS (65% vs 29%) and VM (8% vs 6%) relative to VN (27% vs 65%) than gynecologists ( P < 0.0001). Transvaginal mesh utilization has decreased over time for both specialties ( P < 0.05); nonsignificant trends toward increasing AS ( P = 0.1646) in urologists and VN ( P = 0.0913) in gynecologists concurrently occurred. Significant independent predictors of the operating surgeon being a urologist were surgery being performed in the latter half of the cohort (2016-2020; odds ratio [OR], 1.22), non-White patient race (OR < 1 for all), a concomitant sling being placed (OR = 0.89), the surgery being VM (OR = 2.95) or AS (OR = 4.36), the patient being older (OR > 1 for each age range), and having a higher frailty index score (OR = 1.16). Significant differences in APOP repair choices exist between specialties. Urologists operate on older, more medically complex patients while demonstrating a strong preference for mesh-augmented compared with transvaginal native tissue repairs.

Abstract 5474: Biorepository yields potential for personalized medicine approach in pediatric brain cancer patients

Abstract Childhood brain cancer, with a 12% mortality rate, stands as the most lethal cancer among children. Out of the annual 4,000 diagnoses, 30% involve low-grade gliomas, with a troubling 10% progressing to lethal high-grade gliomas within 2 years. While surgery can often cure the remaining 90%, 50% necessitate adjuvant therapies like chemotherapy and molecular targeting, with radiation sparingly used. Alarmingly, 66% of survivors grapple with long-term health, mental, and cognitive issues due to treatment toxicity, emphasizing the urgent need for innovative biological therapies in pediatric glioma treatment.Our proposed strategy involves the acute analysis of freshly discarded tissue from brain cancer craniotomies, departing from the use of FFPE-preserved tissue, which may exhibit suboptimal quality. All patients provided informed consent, and by prospectively preserving viable tissue, we enable its utilization in ten or more subsequent analyses. These encompass cell-culture-based assays, viability assays, bulk, single cell, and single nucleus RNA sequencing, DNA sequencing, single-cell proteome analyses, flow cytometry, and immunofluorescence.Our findings highlight a direct correlation between surgical sample size and analysis feasibility. Bulk RNA sequencing proves valuable in acutely isolated surgical tissue and cryostored or flash-frozen tissue for identifying novel therapeutic targets in individual patients. Viable cells retrieved from cryopreserved tissue of the same patient allow ex vivo functional validation of therapeutic targets, eliminating the need for establishing patient-derived cell lines and xenografts, particularly challenging for low-grade gliomas. These findings establish the feasibility of a personalized bedside-to-bench approach, contingent on an adequate supply of surgical material.Expanding this approach to encompass all childhood brain cancer types, we plan to investigate using our comprehensive biorepository, housing viable tissue and single-cell suspensions from 122 childhood brain cancer patients. Among these, 51 cases yielded tissue for single-cell RNA-seq and viable cryoprotection. The most common tumor histology identified was low-grade glioma (32), primarily pilocytic astrocytoma (19), followed by embryonal (12) and ependymal tumors (4).In summary, we developed a standard operating procedure for analyzing fresh and cryopreserved surgical discards, identifying novel targets in childhood brain cancer tissue, and functionally validating them in patient-matched samples. This personalized medicine approach holds promise in circumventing the tedious development of patient-derived models. Citation Format: Anna Pronina, Emon Nasajpour, Caitlynn Tran, Troy Schouten, Eden Gibson, Jeffrey Nirschl, Laura Marie Prolo, Kelly Mahaney, Hannes Vogel, Claudia Katharina Petritsch. Biorepository yields potential for personalized medicine approach in pediatric brain cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5474.

Retrospective analysis of hand and foot function after application of cellular technologies in patients with post–traumatic ischemic and denervation–reinervation injuries of limb muscles

Objective. To determine the effect of cell therapy on the function of the hand and foot in patients with ischemic and denervation–reinervation injuries of the limb muscles.&#x0D; Materials and methods. Over 9 years (2014 – early 2023), 47 patients with posttraumatic ischaemia and/or peripheral nerve damage were treated at the Institute of Traumatology and Orthopedics. Cell therapy, namely the injection of bone marrow aspirate and adipose tissue suspension (cell substrates) into the target muscles, was used in 32 (68.1%) patients (main group), while 15 (31.9%) patients did not undergo cell therapy (control group). In 16 (50%) patients, cell therapy was applied to the upper limb, and in 16 (50%) – to the lower limb. Patients in the control and intervention groups were divided into three subgroups: with ischaemic contracture, peripheral nerve damage, and a combination of these pathologies.&#x0D; Results. The treatment with the use of cellular technology in patients with ischaemic and denervation–reinervation injuries of the muscles of the hand and foot had a statistically significant positive effect. In patients with ischaemic contracture of the hand 12 months after the introduction of cell substrates, the loss of its function decreased to less than 30%.&#x0D; Conclusions. Cell therapy helps to restore hand and foot function in patients with ischemic and/or denervation–reinervation injury of the limb muscles.

Comparison of preparation methods of rat kidney single-cell suspensions

Preparation of kidney tissue single-cell suspensions is the basis of single-cell sequencing, flow cytometry and primary cell culture, but it is difficult to prepare high quality whole kidney single-cell suspensions because of the complex structure of the kidney. We explored a technique called stepwise enzymatic digestion (StE) method for preparing a single-cell suspension of rat whole kidney tissue which contained three main steps. The first step is to cut the kidney into a homogenate. The second step is the digestion of renal tubules using Multi Tissue Dissociation Kit 2 and the last step is the digestion of glomeruli using type IV collagenase. We also compared it with two previous techniques, mechanical grinding method and simple enzymatic digestion method. The StE method had the advantages of high intrinsic glomerular cells and immune cells harvest rate, high singlets rate and high cell viability compared with the other two techniques. In conclusion, the StE method is feasible, highly efficient, and worthy of further research and development.

First Report of Pseudomonas oryzihabitans Causing Walnut Leaf Spot Disease in China.

China ranks first in the production and harvest area of walnut (Juglans regia L.) worldwide. Currently, the poor health and low yield of walnut caused by pathogen infection is of concern. In 2022, severe walnut leaf spot disease was observed on the seedlings of four walnut nurseries (0.08 to 0.23 ha) in Liaocheng, Shandong, China, with an average incidence of 48.6% (from 34.6% to 65.3% on the cultivar Xiangling). From August to October, leaf spots mainly appeared on the edges of the leaflets, and occasionally between veins. The lesions were initially soft and rotten, and then light brown, round to semi-circular. Subsequently, the adjacent lesions fused, and the edges of the leaflets and entire leaflets showed symptoms of browning and wilting. For pathogen isolation, five leaflets with representative symptoms from one of the nurseries were collected and wiped three times with sterile absorbent cotton dipped in 75% alcohol and washed with distilled water. Leaflet pieces at the junction of the lesion and healthy tissues were removed, crushed in a sterile mortar, and soaked in a small amount of distilled water for 10 min. The diseased tissue suspension was streaked on a nutrient agar medium (NA) with a sterile inoculation ring and incubated at 28°C for 24 to 72 h. The bacterial colonies obtained were further cultured on NA. The purified colonies were uniform in shape, round, and yellow, with a raised, shiny surface and smooth margin. The isolates were Gram-negative, and the electron microscope analysis showed that the pathogens were short rods (0.35 to 0.52 × 0.90 to 1.24 μm, average = 0.44 ± 0.05 × 1.08 ± 0.11 μm, n = 25). For bacterial species identification, a single-colony culture was subjected to genomic DNA extraction and gene amplification and sequencing of 16S rRNA, rpoD, and gyrB. The universal primers 27F/1492R (Lane 1991) were used to amplify the 16S rRNA gene and the specific primers 70F/70R and UP-1E/APrU (Yamamoto et al. 2000) were used to amplify the rpoD and gyrB genes, respectively. In the BLAST analysis, the 16S rRNA sequence (GenBank OR195734) of the isolate shared 99% similarity (1409/1410 bp) with Pseudomonas oryzihabitans strain IAM 1568T (AM262973.1), and the rpoD (OR709708) and gyrB (OR709707) sequences showed >98% identity to rpoD (707/717 bp; FN554494.1) and gyrB (787/801 bp; FN554210.1) of P. oryzihabitans strain LMG 7040T. Based on the above results, the isolated bacterium was identified as P. oryzihabitans. For the pathogenicity test, healthy leaflets from 10 two-year-old potted walnut seedlings (cv. Xiangling) were used as inoculation materials. The leaflets were punctured with a sterile inoculation needle of 0.4 mm, and three small holes on each leaflet at an interval of about 5 mm were covered with a piece of sterile cotton. A bacterial suspension (1 ml) at 107 CFU/ml was spread onto the cotton, and wrapped with plastic film for 24 h. Water was used as a negative control. The inoculations were performed five times. Plants were grown outdoors at a daily average temperature of 22°C with relative humidity over 45%. Two days after inoculation, the disease began to develop in the leaflets with similar symptoms to those observed in the field. In contrast, control plants remained healthy and symptomless. Bacteria were reisolated from the inoculated walnut plants, and the morphology and 16S rRNA gene sequences of the isolates were the same as those of the original strains. Since it was discovered as an opportunistic human pathogenic bacterium in the 1970s (Keikha et al. 2019), P. oryzihabitans has also been shown to cause certain plant diseases, such as panicle blight and grain discoloration on rice (Hou et al. 2020), fruit black rot on prickly ash (Liu et al. 2021), and stem and leaf rot on muskmelon (Li et al. 2021). As far as we know, this is the first report of P. oryzihabitans causing walnut leaf spot disease in China. Leaf spot caused by P. oryzihabitans may be a threat to walnut cultivation, and this report of its occurrence is the first step in determining potential spread and effective control measures.

A simple and rapid preparation of smooth muscle myosin 2 for the electron microscopic analysis.

There has been an increase in the demand for purified protein as a result of recent developments in the structural biology of myosin 2. Although promising, current practices in myosin purification are usually time-consuming and cumbersome. The reported increased actin to myosin ratio in smooth muscles adds to the complexity of the purification process. Present study outlines a streamlined approach to isolate smooth muscle myosin 2 molecules from actomyosin suspension of chicken gizzard tissues. The procedure entails treating actomyosin for a brief period with actin-binding peptide phalloidin, followed by co-sedimentation and short column size exclusion chromatography. Typical myosin molecule with heavy and light chains and approximately 95% purity was examined using gel electrophoresis. Negative staining electron microscopy and image processing showed intact 10S myosin 2 molecules, proving that phalloidin is effective at eliminating majority of actin in the form of F-actin without dramatic alteration in the structure of myosin. The entire purification discussed here can be completed in a few hours, and further analysis can be done the same day. Thus, by offering quick and fresh supplies of native myosin molecules suited for structural research, specially cryo-electron microscopy, this innovative approach can be adapted to get around the drawbacks of time-intensive myosin purifying processes.

Prostate Cancer Organoids for Tumor Modeling and Drug Screening.

Prostate cancer (PCa) is the second most common malignancy and the fifth leading cause of cancer death in men worldwide. Despite its prevalence, the highly heterogenic PCa has shown difficulty to establish representative cell lines that reflect the diverse phenotypes and different stages of the disease in vitro and hence hard to model in preclinical research. The patient-derived organoid (PDO) technique has emerged as a groundbreaking three-dimensional (3D) tumor modeling platform in cancer research. This versatile assay relies on the unique ability of cancer stem cells (CSCs) to self-organize and differentiate into organ-like mini structures. The PDO culture system allows for the long-term maintenance of cancer cells derived from patient tumor tissues. Moreover, it recapitulates the parental tumor features and serves as a superior preclinical model for in vitro tumor representation and personalized drug screening. Henceforth, PDOs hold great promise in precision medicine for cancer. Herein, we describe the detailed protocol to establish and propagate organoids derived from isolated cell suspensions of PCa patient tissues or cell lines using the 3D semisolid Matrigel™-based hanging-drop method. In addition, we highlight the relevance of PDOs as a tool for evaluating drug efficacy and predicting tumor response in PCa patients.

In Vitro Tumorigenic Assay: A Tumor Sphere Assay for Cancer Stem Cells.

Cancer stem cells (CSCs) represent a subpopulation of tumor cells that are thought to be responsible for therapy resistance, recurrence, and metastasis through their capacity to self-renew and differentiate into heterogeneous downstream lineages of cancer cells. Understanding the features of CSCs is crucial for managing cancer disease and establishing potential targeted therapeutics. Tumor sphere formation assay is a widely used in vitro method that selects and enriches the CSC subpopulation from the total population of cancer cells, based on their inherent ability to grow and clonally expand in serum-free and nonadherent culture conditions. Here we provide a detailed methodology to generate and propagate spheres from isolated cell suspensions of tumor tissues and cell lines using a semisolid MatrigelTM-based three-dimensional (3D) culture system.

Analysis of cancer-associated fibroblasts in cervical cancer by single-cell RNA sequencing.

Since scRNA-seq is an effective tool to study tumor heterogeneity, this paper intends to reveal the differences of cervical cancer in patients at the individual cell level by scRNA-seq, and focus on the biological functions of cancer-associated fibroblasts (CAFs) in cervical cancer, facilitating the provision of a new interpretation of the heterogeneity of the microenvironment of cervical cancer, and an in-depth exploration of the pathogenesis of cervical cancer as well as pursuit of effective means of treatment intake. 3 cervical cancer specimens were collected by clinical surgery for single-cell RNA sequencing. Cell suspensions of fresh cervical cancer tissues were prepared, and cDNA libraries were created and sequenced on the machine. Furthermore, the sequencing data were analyzed using bioinformatics, including descending clustering of cells, identification of cell populations, mimetic time series analysis, inferCNV, cell communication analysis, and identification of transcription factors. A total of 9 cell types were identified, encompassing T cells, epithelial cells, smooth muscle cells, CAFs, endothelial cells, macrophages, B cells, lymphocytes, and plasma cells. CAFs were further divided into three cell subtypes, named type1 cells, type2 cells, and type3 cells. With key transcription factors for the three cells, TCF21, ZC3H11A, and MYEF2 obtained, this research revealed the communication relationship between CAFs and several other cells, and found an important role of CAFs in the MK signaling pathway. scRNA-seq technology contributed to exploring the tumor heterogeneity of cervical cancer more deeply, and also further gaining insight into the biological functions of CAFs in cervical cancer.

Aggregation-Induced Emission-Based Macrophage-Like Nanoparticles for Targeted Photothermal Therapy and Virus Transmission Blockage in Monkeypox.

The recent prevalence of monkeypox has led to the declaration of a Public Health Emergency of International Concern. Monkeypox lesions are typically ulcers or pustules (containing high titers of replication-competent virus) in the skin and mucous membranes, which allow monkeypox virus to transmit predominantly through intimate contact. Currently, effective clinical treatments for monkeypox are lacking, and strategies for blocking virus transmission are fraught with drawbacks. Herein, this work constructs a biomimetic nanotemplate (termed TBD@M NPs) with macrophage membranes as the coat and polymeric nanoparticles loading a versatile aggregation-induced emission featured photothermal molecule TPE-BT-DPTQ as the core. In a surrogate mouse model of monkeypox (vaccinia-virus-infected tail scarification model), intravenously injected TBD@M NPs show precise tracking and near-infrared region II fluorescence imaging of the lesions. Upon 808nm laser irradiation, the virus is eliminated by the photothermal effect and the infected wound heals rapidly. More importantly, the inoculation of treated lesion tissue suspensions does not trigger tail infection or inflammatory activation in healthy mice, indicating successful blockage of virus transmission. This study demonstrates for the first time monkeypox theranostics using nanomedicine, and may bring a new insight into the development of a viable strategy for monkeypox management in clinical trials.

Single-Cell RNA Sequencing Reveals Roles of Fibroblasts During Intestinal Injury and Repair in Rats with Severe Acute Pancreatitis.

To explore the molecular mechanisms of intestinal injury and treatment by analyzing changes in cellular heterogeneity and composition in rat ileal tissue during injury and treatment processes. We constructed a rat model of SAP and evaluated treatment with an injected of monoacylglycerol lipase (MAGL) inhibitor (JZL184) solution using three experimental groups: healthy male Sprague-Dawley (SD) rats injected with vehicle (CON), male SD SAP model rats injected with vehicle (SAP), and male SAP rats injected with JZL184. We obtained and prepared a single-cell suspension of ileal tissue of each rat for single-cell transcriptome sequencing. This project classified changes in cellular heterogeneity and composition in rat ileal tissue during SAP-induced intestinal injury and MAGL treatment. We found that the number of fibroblast clusters was decreased in the SAP group relative to the CON group, and increased after JZL184 treatment. Further analysis of differences in gene expression between cell clusters in each group reveals that fibroblasts had the greatest number of differentially expressed genes. Most notably, expression of genes involved in communication between cells was found to vary during SAP-induced intestinal injury and JZL184 treatment. Among these changes, the degree of difference in expression of genes involved in communication between fibroblasts and other cells was the highest, indicating that fibroblasts in rat ileal tissue affect intestinal injury and repair through cell-to-cell communication. In addition, our results reveal that differentially expressed RNA-binding proteins in fibroblasts may affect their functions in intestinal injury and treatment by affecting the expression of genes regulating communication between cells. These findings emphasize the importance of understanding the interactions between fibroblasts and other cells in the context of intestinal injury, providing valuable insights for further exploring molecular mechanisms and insight for discovering new treatment targets and strategies.

A comparative study of establishing rabbit vertebral tumor model by two ways of CT-guided percutaneous puncture

ObjectiveTo investigate the difference of tumor formation rate of rabbit vertebral tumor model established by percutaneous injection of V×2 tumor tissue suspension and tumor mass under computed tomography (CT) guidance, and the imaging findings of CT, magnetic resonance images (MRI) and positron emission tomography with computed tomography (PET/CT) at 7 days, 14 days and 21 days after implantation, and preliminarily verify the safety and feasibility of microwave ablation (MWA), percutaneous vertebroplasty (PVP) and microwave ablation combined with percutaneous vertebroplasty (MWA + PVP) in rabbit VX2 vertebral tumor model. MethodsThirty healthy New Zealand rabbits were randomly allocated to tissue suspension group and tumor block group, with 15 rabbits for each group. The VX2 tumor block and mixed suspension were inoculated into the L5 vertebral body under CT-guided percutaneous puncture. The PET/CT, MRI and CT examinations were performed at 7, 14 and 21 days after implantation. Fisher exact probability test was used to compare the success rate of the two implantation methods and the tumor display rate at each time point of the three examination methods. Observe the paralysis of tumor-forming rabbits, and immediately perform MWA/PVP/MWA + PVP treatment according to groups after paralysis to verify the safety and feasibility of treatment. ResultsA total of 18 experimental rabbits were successfully modeled in two groups, of which the success rate was 26.6% (4/15) in tissue suspension group and 93.3% (14/15) in tumor block group, with statistically significant differences between two groups (P < 0.01). The tumor display rates by PET/CT, MRI and CT at each time point after implantation were: 83.3% (15/18), 16.6% (3/18), and 0% (0/18) at 7 days after implantation; 100% (18/18), 88.8% (16/18), and 11.1% (2/18) at 14 days after implantation; and 100% (18/18), 100% (18/18), 77.7% (14/18) at 21 days after implantation. The average paralysis time of 18 experimental rabbits successfully modeled was 24.44 ± 2.38 days, and MWA/PVP/MWA + PVP treatment was performed in groups immediately after paralysis. Except for 2 rabbits who died due to anesthesia overdose during anesthesia before treatment, the remaining 16 rabbits were successfully treated with MWA/PVP/MWA + PVP, and the technical success rate was 100% (16/16). In MWA group, one experimental rabbit was randomly selected and killed after ablation, and histopathological examination (H and E staining) was performed together with 2 experimental rabbits who died of anesthesia. The pathological changes before and after ablation were compared. The survival time of the remaining 15 experimental rabbits varied from 3 to 8 days after treatment. ConclusionThe success rate of establishing rabbit vertebral tumor model by injecting tumor masses under the CT-guided percutaneous puncture is high, and the following MWA and PVP treatment can be successfully conducted. PET/CT is the most sensitive method for early detection of tumor compared with MRI and CT. Spectral Presaturation with Inversion Recovery (SPIR) sequence can significantly improve the detection rate of smaller tumors by MRI and shorten the detection time.

Rapid detection of tetracycline residues at clinically relevant levels via a novel, inexpensive and analytically-sensitive magneto-immunoassay – A feasibility study

Expensive, time-consuming and labour-intensive solvent-extraction and liquid-chromatography methods are the industry's current gold standard for antibiotic residue quantification. A novel immunoassay methodology and system for the rapid detection of clinically relevant levels of tetracycline residues found in food-producing animal tissues is described. Anti-tetracycline antibody-coated paramagnetic particles were used for the specific capture of tetracycline in spiked buffer (with and without a 1% pork muscle tissue suspension) and quantified via an analytically-sensitive in-house magnetometer instrument. Detection of tetracycline between 0.1 μg/mL - 1 μg/mL was achieved, with a readout time (including sample treatment) presented in 20 min. The magneto-immunoassay described provides a rapid, low-cost, de-skilled and analytically-sensitive solution for tetracycline screening at the point-of-sampling, with potential applications for other prevalent antibiotic families used in the international farming and food industry.