A Critical Role of Culture Medium Selection in Maximizing the Purity and Expansion of Natural Killer Cells.

Natural killer (NK) cells hold promise in cancer treatment due to their ability to spontaneously lyse cancer cells. For clinical use, high quantities of pure, functional NK cells are necessary. Combining adherence-based isolation with specialized media showed the unreliability of the isolation method, but demonstrated the superiority of the NK MACS® medium, particularly in suboptimal conditions. Neither human pooled serum, fetal calf serum (FCS), human platelet lysate, nor chemically defined serum replacement could substitute human AB serum. Interleukin (IL-)2, IL-15, IL-21, and combined CD2/NKp46 stimulation were assessed. IL-21 and CD2/NKp46 stimulation increased cytotoxicity, but reduced NK cell proliferation. IL-15 stimulation alone achieved the highest proliferation, but the more affordable IL-2 performed similarly. The RosetteSep™ human NK cell enrichment kit was effective for isolation, but the presence of peripheral blood mononuclear cells (PBMCs) in the culture enhanced NK cell proliferation, despite similar expression levels of CD16, NKp46, NKG2D, and ICAM-1. In line with this, purified NK cells cultured in NK MACS® medium with human AB serum and IL-2 demonstrated high cytotoxicity against primary glioblastoma stem cells.

Molecular epidemiology, antibiotic resistance profile and frequency of integron 1 and 2 in adherent-invasive Escherichia coli isolates of colorectal cancer patients.

This study explores the prevalence of adherent-invasive Escherichia coli (AIEC) in colorectal cancer (CRC) patients and investigates the potential of effective intracellular antibiotics as a therapeutic strategy for CRC patients with AIEC infections. Considering the pivotal role of integrons in bacterial antibiotic resistance, the frequency of class 1 and 2 integrons in AIEC isolated from CRC patients, in one of the referenced 3 gastroenterology clinics in Isfahan, Iran was examined. AIEC strains were isolated from the colorectal biopsies and their antimicrobial sensitivity was assessed using the disc diffusion method. Polymerase chain reaction (PCR) was employed to detect intl1 and intl2. The multilocus sequence typing (MLST) method was utilized to type 10 selected isolates. Of the 150 samples, 24 were identified as AIEC, with the highest number isolated from CRC2 (33.4%) and CRC1 (29.16%), and the least from the FH group (8.3%) and control group (12.5%). int1 in 79.2% and int2 in 45.8% of AIEC strains were found and 41.6% of strains had both integrons. AIEC isolates with int1 exhibited the highest sensitivity to trimethoprim-sulfamethoxazole (57.9%), while those with int2 showed the highest sensitivity to ciprofloxacin (63.6%). A significant association between resistance to rifampin and integron 2 presence in AIEC isolates was observed. Furthermore, a significant correlation between integron 1 presence, invasion, survival, and replication within macrophages in AIEC strains was identified. MLST analysis revealed ST131 from CC131 with integron 1 as the most common sequence type (ST). The emergence of such strains in CRC populations poses a serious public health threat. The distribution pattern of STs varied among studied groups, with pandemic STs highlighting the importance of examining and treating patients infected with these isolates. Comprehensive prospective clinical investigations are warranted to assess the prognostic value of detecting this pathovar in CRC and to evaluate therapeutic techniques targeting drug-resistant AIECs, such as phage therapy, bacteriocins, and anti-adhesion compounds, for CRC prevention and treatment.

Two-component system RstAB promotes the pathogenicity of adherent-invasive Escherichia coli in response to acidic conditions within macrophages.

Adherent-invasive Escherichia coli (AIEC) strain LF82, isolated from patients with Crohn's disease, invades gut epithelial cells, and replicates in macrophages contributing to chronic inflammation. In this study, we found that RstAB contributing to the colonization of LF82 in a mouse model of chronic colitis by promoting bacterial replication in macrophages. By comparing the transcriptomes of rstAB mutant- and wild-type when infected macrophages, 83 significant differentially expressed genes in LF82 were identified. And we identified two possible RstA target genes (csgD and asr) among the differentially expressed genes. The electrophoretic mobility shift assay and quantitative real-time PCR confirmed that RstA binds to the promoters of csgD and asr and activates their expression. csgD deletion attenuated LF82 intracellular biofilm formation, and asr deletion reduced acid tolerance compared with the wild-type. Acidic pH was shown by quantitative real-time PCR to be the signal sensed by RstAB to activate the expression of csgD and asr. We uncovered a signal transduction pathway whereby LF82, in response to the acidic environment within macrophages, activates transcription of the csgD to promote biofilm formation, and activates transcription of the asr to promote acid tolerance, promoting its replication within macrophages and colonization of the intestine. This finding deepens our understanding of the LF82 replication regulation mechanism in macrophages and offers new perspectives for further studies on AIEC virulence mechanisms.

Reprogramming anchorage dependency to develop cell lines for recombinant protein expression.

As the biopharmaceutical industry continues to mature in its cost-effectiveness and productivity, many companies have begun employing larger-scale biomanufacturing and bioprocessing protocols. While many of these protocols require cells with anchorage-independent growth, it remains challenging to induce the necessary suspension adaptations in many different cell types. In addition, although transfection efficiency is an important consideration for all cells, especially for therapeutic protein production, cells in suspension are generally more difficult to transfect than adherent cells. Thus, much of the biomanufacturing industry is focused on the development of new human cell lines with properties that can support more efficient biopharmaceutical production. With this in mind, we identified a set of "Adherent-to-Suspension Transition" (AST) factors, IKZF1, BTG2 and KLF1, the expression of which induces adherent cells to acquire anchorage-independent growth. Working from the HEK293A cell line, we established 293-AST cells and 293-AST-TetR cells for inducible and reversible reprogramming of anchorage dependency. Surprisingly, we found that the AST-TetR system induces the necessary suspension adaptations with an accompanying increase in transfection efficiency and protein expression rate. Our AST-TetR system therefore represents a novel technological platform for the development of cell lines used for generating therapeutic proteins.

Anchorage Dependence and Cancer Metastasis.

The process of cancer metastasis is dependent on the cancer cells' capacity to detach from the primary tumor, endure in a suspended state, and establish colonies in other locations. Anchorage dependence, which refers to the cells' reliance on attachment to the extracellular matrix (ECM), is a critical determinant of cellular shape, dynamics, behavior, and, ultimately, cell fate in nonmalignant and cancer cells. Anchorage-independent growth is a characteristic feature of cells resistant to anoikis, a programmed cell death process triggered by detachment from the ECM. This ability to grow and survive without attachment to a substrate is a crucial stage in the progression of metastasis. The recently discovered phenomenon named "adherent-to-suspension transition (AST)" alters the requirement for anchoring and enhances survival in a suspended state. AST is controlled by four transcription factors (IKAROS family zinc finger 1, nuclear factor erythroid 2, BTG anti-proliferation factor 2, and interferon regulatory factor 8) and can detach cells without undergoing the typical epithelial-mesenchymal transition. Notably, AST factors are highly expressed in circulating tumor cells compared to their attached counterparts, indicating their crucial role in the spread of cancer. Crucially, the suppression of AST substantially reduces metastasis while sparing primary tumors. These findings open up possibilities for developing targeted therapies that inhibit metastasis and emphasize the importance of AST, leading to a fundamental change in our comprehension of how cancer spreads.

Differentiating placenta accreta spectrum from scar dehiscence with underlying, non-adherent placenta: A systematic review of scoring systems and primary data analysis.

INTRODUCTION: Accurate discrimination between placenta accreta spectrum (PAS) and scar dehiscence with underlying non-adherent placenta is challenging both on prenatal ultrasound and intraoperatively. This can lead to overdiagnosis of PAS and unnecessarily aggressive management of scar dehiscence which increases the risk of morbidity. Several scoring systems have been published which combine clinical and ultrasound information to help diagnose PAS in women at high risk. This research aims to provide insights into the reliability and utility of existing accreta scoring systems in differentiating these two closely related but different conditions to contribute to improved clinical decision making and patient outcomes. MATERIAL AND METHODS: A literature search was performed in four electronic databases. The references of relevant articles were also assessed. The articles were then evaluated according to the predefined inclusion criteria. Primary data for testing each scoring system were obtained retrospectively from two hospitals with specialized PAS services. Each scoring system was used to evaluate the predicted outcome of each case. RESULTS: The literature review yielded 15 articles. Of these, eight did not have a clearly described diagnostic criteria for accreta, hence were excluded. Of the remaining seven studies, one was excluded due to unorthodox diagnostic criteria and two were excluded as they differed from the other systems hindering comparison. Four scoring systems were therefore tested with the primary data. All the scoring systems demonstrated higher scores for high-grade PAS compared to scar dehiscence (p < 0.001) with an excellent Area Under the receiver operator characteristic Curve ranging from 0.82 (95% CI 0.71-0.92) to 0.87 (95% CI 0.79-0.96) in differentiating between these two conditions. However, no statistically significant differences were noted between the low-grade PAS and scar dehiscence on all scoring systems. CONCLUSIONS: Most published scoring systems have no clearly defined diagnostic criteria. Scoring systems can differentiate between scar dehiscence with underlying non-adherent placenta from high-grade PAS with excellent diagnostic accuracy, but not for low-grade PAS. Hence, relying solely on these scoring systems may lead to errors in estimating the risk or extent of the condition which hinders preoperative planning.

Doxycycline adherence for the management of Chlamydia trachomatis infections.

BACKGROUND: The updated 2021 CDC treatment guidelines recommend a single dose of 500 mg intramuscular ceftriaxone for Neisseria gonorrhea and doxycycline 100 mg by mouth twice daily for 7 days for Chlamydia trachomatis coinfection. However, there is a significant public health concern regarding patient non-adherence to the 7-day course of doxycycline. To date, there are no studies assessing this concern. Therefore, the objective of this study was to evaluate a patient's adherence to doxycycline for chlamydial infections after discharge from the Emergency Department (ED). METHODS: This was an IRB-approved, single-center, retrospective cohort study evaluating the adherence to doxycycline for Chlamydia trachomatis infections. Patients who received treatment and were discharged from the ED with a doxycycline e-prescription between May 2021 and September 2022 were included. Patients were excluded if <18 years of age, pregnant, a sexual assault victim, or admitted inpatient. The primary endpoint was the incidence of doxycycline prescription pick-up after discharge from the ED. The secondary endpoint was the incidence of repeat ED visits for the same chief complaint within 28 days. Descriptive statistics were computed for all study variables and Fisher's Exact tests were used to assess the outcomes. RESULTS: A review of 144 patients who tested positive for chlamydia and were discharged from the ED with an e-prescription for doxycycline revealed that 18% of patients did not pick up their prescription (N = 26). Non-adherent patients were more likely to return to the ED with the same chief complaint within 28 days (23.1% vs 7.6%, OR 3.6 [1.2-11.3], p = 0.026). No differences were detected in baseline demographics, housing status, insurance type, sexual orientation, or Sexually Transmitted Infection history. CONCLUSION: For patients with a positive chlamydia infection who were discharged from the ED on doxycycline, an 18% non-adherence rate was found and a 3.6-fold higher likelihood of returning to the ED with the same chief complaint if the prescription was not picked up.

Predicting the presence of adherent perinephric fat using MRI radiomics combined with machine learning.

OBJECTIVES: Adherent perinephric fat (APF) poses significant challenges to surgical procedures. This study aimed to evaluate the usefulness of machine learning algorithms combined with MRI-based radiomics features for predicting the presence of APF. MATERIALS AND METHODS: Patients with renal cell carcinoma who underwent surgery between April 2019 and February 2022 at Chonnam National University Hwasun Hospital were retrospectively screened, and 119 patients included. Twenty-one and seventeen patients were set aside for the internal and external test sets, respectively. Pre-operative T1-weighted MRI acquired at 60 s following a contrast injection (T1w-60) were collected. For each T1w-60 data, two regions of interest (ROIs) were manually drawn: the perinephric fat tissue and an aorta segment on the same level as the targeted kidney. Preprocessing steps included resizing voxels, N4 Bias Correction filtering, and aorta-based normalization. For each patient, 851 radiomics features were extracted from the ROI of perinephric fat tissue. Gender and BMI were added as clinical factors. Least Absolute Shrinkage and Selection Operator was adopted for feature selection. We trained and evaluated five models using a 4-fold cross validation. The final model was chosen based on the highest mean AUC across four folds. The performance of the final model was evaluated on the internal and external test sets. RESULTS: A total of 15 features were selected in the final set. The final model achieved the accuracy, sensitivity, specificity, and AUC of 81% (95% confidence interval, 61.9-95.2%), 72.7% (42.9-100%), 90% (66.7-100%), and 0.855 (0.615-1.0), respectively on the internal test set, and 88.2% (70.6-100%), 100% (100-100%), 80% (50%-100%), 0.971 (0.871-1.0), respectively on the external test set. CONCLUSIONS: Our study demonstrated the feasibility of machine learning algorithms trained with MRI-based radiomics features for APF prediction. Further studies with a multi-center approach are necessary to validate our findings.

Predictive Value of the Mayo Adhesive Probability (MAP) Score in Laparoscopic Partial Nephrectomies: A Systematic Review from the EAU Section of Uro-Technology (ESUT).

The Mayo Adhesive Probability (MAP) score is a radiographic scoring system that predicts the presence of adherent perinephric fat (APF) during partial nephrectomies (PNs). The purpose of this systematic review is to summarize the current literature on the application of the MAP score for predicting intraoperative difficulties related to APF and complications in laparoscopic PNs. Three databases, PubMed, Scopus and Cochrane, were screened, from inception to 29 October 2023, taking into consideration the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Guidelines. All the inclusion criteria were met by eight studies. The total operative time was around two hours in most studies, while the warm ischemia time was <30 min in all studies and <20 min in four studies. Positive surgical margins, conversion and transfusion rates ranged from 0% to 6.3%, from 0% to 5.0% and from 0.7% to 7.5%, respectively. Finally, the majority of the complications were classified as Grade I-II, according to the Clavien-Dindo Classification System. The MAP score is a useful tool for predicting not only the presence of APF during laparoscopic PNs but also various intraoperative and postoperative characteristics. It was found to be significantly associated with an increased operative time, estimated blood loss and intraoperative and postoperative complication rates.

Identification of autophagy receptors for the Crohn's disease-associated adherent-invasive Escherichia coli.

Introduction: Crohn's disease (CD) is a chronic inflammatory bowel disease, of which the etiology involves genetic, environmental and microbial factors. Adherent-invasive Escherichia coli (AIEC) and polymorphisms in autophagy-related genes have been implicated in CD etiology. Autophagy is a key process for the maintenance of cellular homeostasis, which allows the degradation of damaged cytoplasmic components and pathogens via lysosome. We have shown that a functional autophagy is necessary for AIEC clearance. Here, we aimed at identifying the autophagy receptor(s) responsible to target AIEC to autophagy for degradation. Methods: The levels of autophagy receptors p62, NDP52, NBR1, TAX1BP1 and Optineurin were knocked down in human intestinal epithelial cells T84 using siRNAs. The NDP52 knock-out (KO) and p62 KO HeLa cells, as well as NDP52 KO HeLa cells expressing the wild-type NDP52 or the mutated NDP52Val248Ala protein were used. Results and discussion: We showed that, among the tested autophagy receptors (p62, NDP52, NBR1, TAX1BP1 and Optineurin), diminished expression of p62 or NDP52 increased the number of the clinical AIEC LF82 strain inside epithelial cells. This was associated with increased pro-inflammatory cytokine production. Moreover, p62 or NDP52 directly colocalized with AIEC LF82 and LC3, an autophagy marker. As the NDP52Val248Ala polymorphism has been associated with increased CD susceptibility, we investigated its impact on AIEC control. However, in HeLa cell and under our experimental condition, no effect of this polymorphism neither on AIEC LF82 intracellular number nor on pro-inflammatory cytokine production was observed. Together, our results suggest that p62 and NDP52 act as autophagy receptors for AIEC recognition, controlling AIEC intracellular replication and inflammation.

Unveiling Therapeutic Avenues for Crohn's Disease Management: Exploring Inhibitors for Adherent-Invasive Escherichia coli Propanediol Dehydratase.

INTRODUCTION: Inflammatory Bowel Disease (IBD) encompasses a group of chronic disorders distinguished by inflammation of the gastrointestinal tract. Among these, Crohn's Disease (CD) stands out as a complex and impactful condition due to challenges for both diagnosis and management, making it a cynosure of research. METHOD: In CD, there is the predominance of proinflammatory bacteria, including the Adherentinvasive Escherichia coli (AIEC) with virulence-associated metabolic enzyme Propanediol Dehydratase (pduC), which has been identified as a therapeutic target for the management of CD. Herein, molecular modeling techniques, including molecular docking, Molecular Mechanics with Generalized Born and Surface Area (MMGBSA), drug-likeness, and pharmacokinetics profiling, were utilized to probe the potentials of eighty antibacterial compounds to serve as inhibitors of pduC. RESULT: The results of this study led to the identification of five compounds with promising potentials; the results of the molecular docking simulation revealed the compounds as possessing better binding affinities for the target compared to the standard drug (sulfasalazine), while Lipinski's rule of five-based assessment of their drug-likeness properties revealed them as potential oral drugs. MMGBSA free energy calculation and Molecular Dynamics (MD) simulation of the complexes formed a sequel to molecular docking, revealing the compounds as stable binders in the active site of the protein. CONCLUSION: Ultimately, the results of this study have revealed five compounds to possess the potential to serve as inhibitors of pduC of AIEC. However, experimental studies are still needed to validate the findings of this study.

Microplate-Based Cryopreservation of Adherent-Cultured Human Cell Lines Using Amino Acids and Proteins.

With the progression of regenerative medicine and cell therapy, the importance of cryopreservation techniques for cultured cells continues to rise. Traditional cryoprotectants, such as dimethyl sulfoxide and glycerol, are effective in cryopreserving suspended cells, but they do not demonstrate sufficient efficacy for two-dimensional (2D)-cultured cells. In the past decade, small molecules and polymers have been studied as cryoprotectants. Some L-amino acids have been reported to be natural and biocompatible cryoprotectants. However, the cryoprotective effects of D-amino acids have not been investigated for such organized cells. In the present study, the cryoprotective effects of D- and L-amino acids and previously reported cryoprotectants were assessed using HepG2 cells cultured on a microplate without suspending the cells. d-Proline had the highest cryoprotective effect on 2D-cultured cells. The composition of the cell-freezing solution and freezing conditions were then optimized. The d-proline-containing cell-freezing solution also effectively worked for other cell lines. To minimize the amount of animal-derived components, fetal bovine serum in the cell freezing solution was substituted with bovine serum albumin and StemFit (a commercial supplement for stem cell induction). Further investigations on the mechanism of cryopreservation suggested that d-proline protected enzymes essential for cell survival from freeze-induced damage. In conclusion, an effective and xeno-free cell-freezing solution was produced using d-proline combined with dimethyl sulfoxide and StemFit for 2D-cultured cells.

The Role of Ultrasonography and Magnetic Resonance Imaging in the Diagnosis of the Adherent Placenta: An Observational Study.

Introduction Placenta accreta is an important factor responsible for maternal morbidity and mortality and is commonly associated with emergent postpartum hysterectomy. The precise prenatal diagnosis of affected pregnancies allows optimal obstetric management. Ultrasonography (USG) and magnetic resonance imaging (MRI) are the only diagnostic modalities available for the prenatal diagnosis of placenta accreta. Objective This study aims to evaluate the accuracy of USG and MRI in diagnosing adherent placenta. Methods Thirty females with placenta previa or a history of previous cesarean sections were evaluated with USG at 28-30 weeks, followed by MRI. The findings of USG and MRI were compared with the intra-operative findings (gold standard) as determined at surgery and by pathological examination. Results Abnormal bridging vessel (n = 24; 80%) was the most common finding seen on USG, whereas abnormal bulge (n = 22; 73.3%) and heterogenous placenta (n = 21; 70%) were the most common findings seen on MRI. The sensitivity of USG and MRI was in the range of 86.7%-92.9% and 92.9%-100%, respectively, in diagnosing three types of adherent placenta. The positive predictive values (PPV) of USG and MRI were in the range of 86.7%-86.7% and 93.8%-100%, respectively, in diagnosing three types of adherent placenta. The accuracy of USG and MRI was in the range of 86.7%-96.7% and 96.7%-100%, respectively, in diagnosing three types of adherent placenta. Conclusion MRI helps to accurately classify placental invasion according to depth, as can be seen from the results of the present study, where the MRI technique was more accurate in diagnosing three types of adherent placenta.

Polyurethane foam dressing with non-adherent membrane improves negative pressure wound therapy in pigs.

OBJECTIVE: Negative pressure wound therapy (NPWT) is considered to be an effective technique to promote the healing of various wounds. The aim of this study was to evaluate different wound dressings combined with NPWT in treating wounds in Wuzhishan pigs. METHOD: Excisions were made in the backs of the pigs and were covered with polyvinyl alcohol (PVA) dressing, polyurethane (PU) dressing or PU dressing with non-adherent membrane (PU-non-ad). NPWT was applied to the wound site. In the control group, basic occlusive dressing (gauze) without NPWT was applied. On days 0, 3, 7, 14, 21 and 28 post-surgery, the wound size was measured during dressing change, and wound healing rate (WHR) was calculated. In addition, blood perfusion within 2cm of the surrounding wound was measured by laser doppler flowmetry. Dressing specimen was collected and microbiology was analysed. Granulation tissues from the central part of the wounds were analysed for histology, vascular endothelial growth factor (VEGF) and cluster of differentiation 31 (CD31) mRNA expression. RESULTS: The PU-non-ad-NPWT significantly (p<0.01) accelerated wound healing in the pigs. Further pathological analysis revealed that the non-adherent membrane effectively protected granulation tissue formation in PU-NPWT treated wounds. The blood perfusion analysis suggested that the non-adherent membrane improved the blood supply to the wound area. Microbiological analysis showed that non-adherent membrane decreased the bacterial load in the PU-NPWT dressing. VEGF and CD31 mRNA expression was upregulated in the wound tissue from the PU-non-ad-NPWT treated groups. CONCLUSION: In this study, the PU dressing with non-adherent membrane was an ideal dressing in NPWT-assisted wound healing.

Trimester-specific diagnostic accuracy of ultrasound for detection of placenta accreta spectrum: systematic review and meta-analysis.

OBJECTIVE: To assess the diagnostic accuracy of ultrasound for detecting placenta accreta spectrum (PAS) during the first trimester of pregnancy and compare it with the accuracy of second- and third-trimester ultrasound examination in pregnancies at risk for PAS. METHODS: PubMed, EMBASE and Web of Science databases were searched to identify relevant studies published from inception until 10 March 2023. Inclusion criteria were cohort, case-control or cross-sectional studies that evaluated the accuracy of ultrasound examination performed at < 14 weeks of gestation (first trimester) or ≥ 14 weeks of gestation (second/third trimester) for the diagnosis of PAS in pregnancies with clinical risk factors. The primary outcome was the diagnostic accuracy of sonography in detecting PAS in the first trimester, compared with the accuracy of ultrasound examination in the second and third trimesters. The secondary outcome was the diagnostic accuracy of each sonographic marker individually across the trimesters of pregnancy. The reference standard was PAS confirmed at pathological or surgical examination. The potential of ultrasound and different ultrasound signs to detect PAS was assessed by computing summary estimates of sensitivity, specificity, diagnostic odds ratio and positive and negative likelihood ratios. RESULTS: A total of 37 studies, including 5764 pregnancies at risk of PAS, with 1348 cases of confirmed PAS, were included in our analysis. The meta-analysis demonstrated that ultrasound had a sensitivity of 86% (95% CI, 78-92%) and specificity of 63% (95% CI, 55-70%) during the first trimester, and a sensitivity of 88% (95% CI, 84-91%) and specificity of 92% (95% CI, 85-96%) during the second/third trimester. Regarding sonographic markers examined in the first trimester, lower uterine hypervascularity exhibited the highest sensitivity (97% (95% CI, 19-100%)), and uterovesical interface irregularity demonstrated the highest specificity (99% (95% CI, 96-100%)). In the second/third trimester, loss of clear zone had the highest sensitivity (80% (95% CI, 72-86%)), and uterovesical interface irregularity exhibited the highest specificity (99% (95% CI, 97-100%)). CONCLUSIONS: First-trimester ultrasound examination has similar accuracy to second- and third-trimester ultrasound examinations for the diagnosis of PAS. Routine first-trimester ultrasound screening for patients at high risk of PAS may improve detection rates and allow earlier referral to tertiary care centers for pregnancy management. © 2024 International Society of Ultrasound in Obstetrics and Gynecology.

Analysis of the Adherent Cell Response to the Substrate Stiffness Using Tensegrity.

Cell's shape is dependent on the cytoskeleton mechanical properties. Hybrid models were developed that combine the discrete structure for the cytoskeleton and continuum parts for other cell organelles. Tensegrity-based structures that consist of tensile and compression elements are useful models to understand the cytoskeleton mechanical behavior. In this study, we are looking to examine the reaction of the cell to a variety of substrate stiffnesses and explain the relationship between cell behavior and substrate mechanical properties. However, which tensegrity structure is appropriate for modeling a living cell? Is the structure's complexity play a major role? We used two spherical tensegrities with different complexities to assess the impact of the structure on the cell's mechanical response versus substrate's stiffness. Six- and twelve-strut tensegrities together with membrane, cytoplasm, nucleoskeleton, and nucleus envelope were assembled in Abaqus package to create a hybrid cell model. A compressive load was applied to the cell model and the reaction forces versus deflection curves were analyzed for number of substrate stiffness values. By analyzing the difference due to two different tensegrities it became clear that the lower density structure is a better choice for modeling stiffer cells. It was also found that the six-strut tensegrity is sensitive to higher range of substrate stiffness.

Medical therapy has similar hemostatic efficacy with endoscopic treatment for PUB patients with adherent clot (FIIb ulcers).

BACKGROUND: Currently, there is no clear consensus on whether medical treatment or endoscopic treatment should be used for peptic ulcer bleeding patients with adherent clot. The aim of this study is to investigate the hemostatic effects of medical treatment, single endoscopic treatment, and combination endoscopic treatment for peptic ulcer bleeding (PUB) patients with adherent clot. METHODS: We retrospectively analyzed PUB patients with adherent clot who underwent endoscopic examination or treatment in our center from March 2014 to January 2023 and received intravenous administration of proton pump inhibitors. Patients were divided into medical treatment (MT) group, single endoscopic treatment (ST) group, and combined endoscopic treatment (CT) group. Subsequently, inverse probability of treatment weighting (IPTW) was performed to calculate the rebleeding rate. RESULTS: A total of 605 eligible patients were included in this study. After IPTW, the rebleeding rate in the MT group on days 3, 7, 14, and 30 were 13.3 (7.3), 14.2 (7.8), 14.5 (7.9), and 14.5 (7.9), respectively; the rebleeding rates in the ST group were 17.4 (5.1), 20.8 (6.1), 20.8 (6.1), and 20.8 (6.1), respectively; the rebleeding rates in the CT group were 0.4 (0.9), 1.7 (3.3), 2.3 (4.5), and 2.3 (4.5), respectively. Although the rebleeding rate in the medical treatment group was higher, there was no significant difference among the three groups on days 3, 7, 14, and 30 (P = 0.132, 0.442, 0.552, and 0.552). CONCLUSIONS: Medical therapy has similar hemostatic efficacy with endoscopic treatment for PUB patients with adherent clot (FIIb ulcers). However, for patients with more risk factors and access to well-equipped endoscopy centers, endoscopic treatment may be considered. The choice of treatment approach should be based on the individual conditions of the patient, as well as other factors such as medical resources available.

Cyclic-di-AMP confers an invasive phenotype on Escherichia coli through elongation of flagellin filaments.

BACKGROUND: Adherent-invasive Escherichia coli (AIEC) is isolated from patients with Crohn's disease (CD). AIEC can invade the intestinal epithelium, suggesting that it is involved in the development and pathogenesis of CD. However, the mechanism by which AIEC acquired the invasive phenotype remains unknown. RESULTS: This study was designed to examine the mechanisms of AIEC invasiveness. We found that the flagellin (fliC) expression in AIEC was two-fold higher than that in non-AIEC strains, and this overexpression induced the formation of long-filament flagellin. Deletion of fliC in the AIEC LF82 strain resulted in the disappearance of flagellar filaments and attenuated the motility and invasive ability of the bacterium, suggesting that the formation of long filament flagellin induced by increased fliC expression is required by AIEC to invade the intestinal epithelium. In AIEC and non-AIEC K12 strains cultured in the presence of cyclic-di-AMP (c-di-AMP), the expression of fliC was enhanced, and flagellar filaments were elongated. Stimulation with c-di-AMP enhanced the bacterial motility and ability to invade epithelial cells, even in the non-AIEC K12 strain. CONCLUSIONS: Our findings show that c-di-AMP confers an AIEC-like phenotype on non-AIEC strains by enhancing the expression of fliC. The results should be useful for understanding the pathogenesis of CD.

Human intestinal organoid-derived PDGFRα + mesenchymal stroma enables proliferation and maintenance of LGR4 + epithelial stem cells.

BACKGROUND: Intestinal epithelial cells derived from human pluripotent stem cells (hPSCs) are generally maintained and cultured as organoids in vitro because they do not exhibit adhesion when cultured. However, the three-dimensional structure of organoids makes their use in regenerative medicine and drug discovery difficult. Mesenchymal stromal cells are found near intestinal stem cells in vivo and provide trophic factors to regulate stem cell maintenance and proliferation, such as BMP inhibitors, WNT, and R-spondin. In this study, we aimed to use mesenchymal stromal cells isolated from hPSC-derived intestinal organoids to establish an in vitro culture system that enables stable proliferation and maintenance of hPSC-derived intestinal epithelial cells in adhesion culture. METHODS: We established an isolation protocol for intestinal epithelial cells and mesenchymal stromal cells from hPSCs-derived intestinal organoids and a co-culture system for these cells. We then evaluated the intestinal epithelial cells and mesenchymal stromal cells' morphology, proliferative capacity, chromosomal stability, tumorigenicity, and gene expression profiles. We also evaluated the usefulness of the cells for pharmacokinetic and toxicity studies. RESULTS: The proliferating intestinal epithelial cells exhibited a columnar form, microvilli and glycocalyx formation, cell polarity, and expression of drug-metabolizing enzymes and transporters. The intestinal epithelial cells also showed barrier function, transporter activity, and drug-metabolizing capacity. Notably, small intestinal epithelial stem cells cannot be cultured in adherent culture without mesenchymal stromal cells and cannot replaced by other feeder cells. Organoid-derived mesenchymal stromal cells resemble the trophocytes essential for maintaining small intestinal epithelial stem cells and play a crucial role in adherent culture. CONCLUSIONS: The high proliferative expansion, productivity, and functionality of hPSC-derived intestinal epithelial cells may have potential applications in pharmacokinetic and toxicity studies and regenerative medicine.

Bladder-Adherent Primary Appendiceal Carcinoma Masquerading as a Carpeting Rectal Lesion Detected by a Fecal Immunochemical Test: A Case Report.

Introduction: Primary appendiceal carcinoma is rare and comprises up to 1% of all colorectal malignancies. Its invasion into adjacent organs, such as the bladder and rectum, especially as a presenting characteristic, is even less common. Case Presentation: A 75-year-old asymptomatic male tested positive on a screening fecal immunochemical test (FIT). Colonoscopy showed a rectosigmoid tumor and normal appendiceal orifice. Staging MRI surprisingly showed that the cancer was, in fact, of appendiceal origin, coursed posteriorly to invade the rectosigmoid and form adhesions with the urinary bladder. Staging CT did not show metastatic disease. Low anterior resection, en bloc appendectomy, and right hemicolectomy were performed along with cystectomy and ileal conduit. Hematoxylin and eosin stains showed appendiceal adenocarcinoma invading through the appendiceal wall into the rectal muscularis and submucosa. Features of neuroendocrine carcinoma were not identified on immunohistochemistry. This was a colonic type of adenocarcinoma of the appendix. Conclusion: This is a rare case of appendiceal carcinoma invading the rectum and presenting as a positive screening fecal immunochemical test in an asymptomatic individual. We effectively demonstrate the use of preoperative MRI to identify the appendiceal origin of the tumor, as well as to demonstrate the extent of tumor spread, which assisted with operative management and treatment planning.