Cell Detachment Research Articles

Covalent Immobilization of Collagen Type I to a Polydimethylsiloxane Surface for Preventing Cell Detachment by Retaining Collagen Molecules under Uniaxial Cyclic Mechanical Stretching Stress.

Surface modification of polydimethylsiloxane (PDMS) with an extracellular matrix (ECM) is useful for enhancing stable cell attachment. However, few studies have investigated the correlation between the stability of deposited ECM and cell behavior on the PDMS surfaces in external stretched cell culture systems. Herein, covalent collagen type I (Col)-immobilized PDMS surfaces were fabricated using 3-aminopropyl-trimethoxysilane, glutaraldehyde, and Col molecules. The immobilized collagen molecules on the PDMS surface were more stable and uniform than the physisorbed collagen. The cells stably adhered to the Col-immobilized surface and proliferated even under uniaxial cyclic mechanical stretching stress (UnCyMSt), whereas the cells gradually detached from the Col-physisorbed PDMS surface, accompanied by a decrease in the number of deposited collagen molecules. Moreover, the immobilization of collagen molecules enhanced cell alignment under the UnCyMSt. This study reveals that cell adhesion, proliferation, and alignment under the UnCyMSt can be attributed to the retention of collagen molecules on the PDMS surface.

SAT515 Searching For Markers To Identify Angioinvasion In Papillary Thyroid Cancer

Abstract Disclosure: A. Buczyńska: None. M. Kościuszko: None. I. Sidorkiewicz: None. A. Wiatr: None. K. Siewko: None. A. Adamska: None. A. Krętowski: None. A. Popławska-Kita: None. The incidence of Papillary Thyroid Cancer (PTC) has increased significantly over last decades. Despite the great progress in PTC clinical management, the association of angioinvasion with tumor aggressiveness in PTC remains as significant clinical problem, suggesting worse patients prognosis. Therefore cancer metastasis occurs through cell detachment and angioinvasion, epithelial–mesenchymal transition (EMT) and cell migration, changes in cell morphology are initiated through Rho-GTPase-dependent actin cytoskeleton polymerization. 8-hydroxydeoxyguanosine (8-OHdG) is generated accordingly to increased oxidative stress, however has been characterized by paradoxical anti-oxidative, anti-inflammatory and anti-mutagenic effects following Rho-GTPase inhibition, which were noted in various cancer models. Since oxidative stress has been recognized as one of the main risk factors for thyroid cancer development and progression, the total oxidative and antioxidant capacities may also be potentially involved in the process of angioinvasion. Regardless, the identification of angioinvasion in PTC remains controversial mainly due to loose criteria in judging vascular invasion. Thus, searching for laboratory biomarkers of angioinvasion is of paramount importance.In this study, the potential screening utility of oxidative stress-related markers, such as total oxidative capacity (TOC), total antioxidant capacity (TAC) and 8-OHdG was assessed to identify angioinvasion in PTC patients.For the purpose od this study, 50 patients diagnosed with angioinvasive PTC (study group) and 50 patients with very low risk PTC (control group) were enrolled. The TOC, TAC and 8-OHdG concentrations were evaluated using enzyme-linked immunosorbent assay (ELISA). Following the received results, the TAC and 8-OHdG concentrations were decresed among study group comparing to control group (p<0.001; p<0.02, respectively). However, there were no differences in TOC among studied groups (p>0.05). The AUC values for TAC=0.76 and AUC for 8-OHdG= 0.70 (all, p<0.05) suggest their screening utility. The results indicate that angioinvasion process is associated with decreased antioxidant defense in PTC patients which may insight new medical targets in the future. Presentation Date: Saturday, June 17, 2023

In situ hydrogel enhances non-efferocytic phagocytosis for post-surgical tumor treatment

Post-surgical efferocytosis of tumor associated macrophages (TAMs) originates an immunosuppressive tumor microenvironment and facilitates abscopal metastasis of residual tumor cells. Currently, few strategies could inhibit efferocytosis while recovering the tumor-eliminative phagocytosis of TAMs. Herein, we developed an in situ hydrogel that contains anti-CD47 antibody (aCD47) and apocynin (APO), an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase. This hydrogel amplifies the non-efferocytic phagocytosis of TAMs by (1) blocking the extracellular “Don’t eat me” signal of efferocytosis with aCD47, which enhances the receptor-mediated recognition and engulfment of tumor cells by TAMs in the post-surgical tumor bed, and (2) by utilizing APO to dispose of tumor debris in a non-efferocytic manner, which prevents acidification and maturation of efferosomes and allows for M1-polarization of TAMs, leading to improved antigen presentation ability. With the complementary intervention of extracellular and intracellular, this hydrogel reverses the immunosuppressive effects of efferocytosis, and induces a potent M1-associated Th1 immune response against tumor recurrence. In addition, the in situ detachment and distal colonization of metastatic tumor cells were efficiently restrained due to the intervention of efferocytosis. Collectively, the hydrogel potentiates surgery treatment of tumor by recovering the tumor-elimination ability of post-surgical TAMs.

Functional Profiling of Soft Tissue Sarcoma Using Mechanistic Models.

Soft tissue sarcoma is an umbrella term for a group of rare cancers that are difficult to treat. In addition to surgery, neoadjuvant chemotherapy has shown the potential to downstage tumors and prevent micrometastases. However, finding effective therapeutic targets remains a research challenge. Here, a previously developed computational approach called mechanistic models of signaling pathways has been employed to unravel the impact of observed changes at the gene expression level on the ultimate functional behavior of cells. In the context of such a mechanistic model, RNA-Seq counts sourced from the Recount3 resource, from The Cancer Genome Atlas (TCGA) Sarcoma project, and non-diseased sarcomagenic tissues from the Genotype-Tissue Expression (GTEx) project were utilized to investigate signal transduction activity through signaling pathways. This approach provides a precise view of the relationship between sarcoma patient survival and the signaling landscape in tumors and their environment. Despite the distinct regulatory alterations observed in each sarcoma subtype, this study identified 13 signaling circuits, or elementary sub-pathways triggering specific cell functions, present across all subtypes, belonging to eight signaling pathways, which served as predictors for patient survival. Additionally, nine signaling circuits from five signaling pathways that highlighted the modifications tumor samples underwent in comparison to normal tissues were found. These results describe the protective role of the immune system, suggesting an anti-tumorigenic effect in the tumor microenvironment, in the process of tumor cell detachment and migration, or the dysregulation of ion homeostasis. Also, the analysis of signaling circuit intermediary proteins suggests multiple strategies for therapy.

Indole-3-acetic acid impacts biofilm formation and virulence production of Pseudomonas aeruginosa

Bacterial pathogenesis involves complex mechanisms contributing to virulence and persistence of infections. Understanding the multifactorial nature of bacterial infections is crucial for developing effective interventions. The present study investigated the efficacy of indole-3-acetic acid (IAA) against Pseudomonas aeruginosa with various end points including antibacterial activity, minimum inhibitory concentration (MIC), virulence factor production, biofilm inhibition, bacterial cell detachment, and viability assays. Results showed significant biofilm inhibition, bacterial cell detachment, and modest effects on bacterial viability. Microscopic analysis confirmed the disintegrated biofilm matrix, supporting the inhibitory effect of IAA. Additionally, molecular docking studies revealed potential mechanisms of action through active bond interactions between IAA and virulence proteins. These findings highlight IAA as an effective antibiofilm agent against P. aeruginosa.

Evaluation of Imidacloprid Induced Changes in Thyroid Gland and its Amelioration with Withania somnifera in Wistar Rats

The study was conducted on a total of 48 female Wistar rats divided into four groups, each containing 12 rats. Group 1 was control, group 2 was imidacloprid control, group 3 was Withania somnifera control and group 4 was administered with both imidacloprid and Withania somnifera. The rats of group 3 and 4 were subjected to oral treatment of Withania somnifera (1 gm/kg feed) for a period of 30 days to measure the protective effect against thyroid toxicity induced by imidacloprid (30 mg/kg body weight/day for 30 days). Hormonal assays showed a significant (p<0.05) rise in thyroid stimulating hormone and fall in triidothyronine and thyroxine in imidacloprid administered rats when compared to control. No gross lesions were noticed in thyroid gland. Group 2 thyroid gland sections revealed congestion, degenerated thyroid follicles with loss of colloid substance, increase in inter-follicular space, atropic follicles, fibrosis, pyknotic nuclei, detachment of follicular epithelial cells, increase in number of micro-follicles and proliferation of follicular epithelium towards lumen. Electron microscopy of thyroid gland from group 2 rats revealed significant pathological changes. The degree of lesions was minimal in group 4 rats when compared to group 2 rats. These results indicate that Withania somnifera is beneficial in ameliorating the imidacloprid induced thyroid toxicity in rats.

Targeted Strategies for Degradation of Key Transmembrane Proteins in Cancer.

Targeted protein degradation is an attractive technology for cancer treatment due to its ability to overcome the unpredictability of the small molecule inhibitors that cause resistance mutations. In recent years, various targeted protein degradation strategies have been developed based on the ubiquitin-proteasome system in the cytoplasm or the autophagy-lysosomal system during endocytosis. In this review, we describe and compare technologies for the targeted inhibition and targeted degradation of the epidermal growth factor receptor (EGFR), one of the major proteins responsible for the onset and progression of many types of cancer. In addition, we develop an alternative strategy, called alloAUTO, based on the binding of new heterocyclic compounds to an allosteric site located in close proximity to the EGFR catalytic site. These compounds cause the targeted degradation of the transmembrane receptor, simultaneously activating both systems of protein degradation in cells. Damage to the EGFR signaling pathways promotes the inactivation of Bim sensor protein phosphorylation, which leads to the disintegration of the cytoskeleton, followed by the detachment of cancer cells from the extracellular matrix, and, ultimately, to cancer cell death. This hallmark of targeted cancer cell death suggests an advantage over other targeted protein degradation strategies, namely, the fewer cancer cells that survive mean fewer chemotherapy-resistant mutants appear.

Therapeutic, histopathological, and non-specific immune status effect of Rhizophora mangle and Laguncularia racemose hydroalcoholic extracts against Vibrio harveyi and Vibrio campbellii in white shrimp (Penaeus vannamei)

The objective of the research was to evaluate in vitro and in vivo antibacterial efficacy of Rhizophora mangle and Laguncularia racemose when added to the feed of Penaeus vannamei in an experimental infection with Vibrio harveyi (CAIM 1792) and V. campbellii (CAIM 333). Results show that the minimum inhibitory concentration for both extracts was 1.65 g mL-1, with an inhibition zone of 25 ± 1 mm (V. campbellii); 18 ± 2 mm (V. harveyi) for L. racemose and of 21 ± 2 mm (V. campbellii); 20 ± 2 mm (V. harveyi) for R. mangle. R. mangle gave a higher total content of phenolic compounds (4.50 ± 0.26 mg GAE mL-1) and flavonoids (2.60 ± 0.15 mg QE mL-1). The challenge with V. harveyi resulted in 70% survival for organisms fed L. racemose extract and 53% for R. mangle extract. The challenge with V. campbellii resulted in 80% survival for organisms fed L. racemose and 90% R. mangle. Histopathological alterations were observed in the hepatopancreas with hemocytic infiltration within the intertubular connective tissue. Also, tubules with severe cell detachment and tubular atrophy were detected in the positive control organisms, and organisms treated with R. mangle and L. racemose only had vermiform structures in the tubular lumen, cell detachment, and infiltration hemolymph in intertubular connective tissue. According to the analysis of the studied variables, it can be concluded that the hydroalcoholic extracts of R. mangle and L. racemose reduced mortality, clinical signs, and organs and tissue alterations and improved P. vannamei immunity. It is possible to use these hydroalcoholic therapeutic extracts as treatments in shrimp-farm to enhance bacterial disease tolerance and prevent mortality.

Deciphering the Role of p60AmotL2 in Epithelial Extrusion and Cell Detachment.

Preserving an accurate cell count is crucial for maintaining homeostasis. Apical extrusion, a process in which redundant cells are eliminated by neighboring cells, plays a key role in this regard. Recent studies have revealed that apical extrusion can also be triggered in cells transformed by oncogenes, suggesting it may be a mechanism through which tumor cells escape their microenvironment. In previous work, we demonstrated that p60AmotL2 modulates the E-cadherin function by inhibiting its connection to radial actin filaments. This isoform of AmotL2 is expressed in invasive breast and colon tumors and promotes invasion in vitro and in vivo. Transcriptionally regulated by c-Fos, p60AmotL2 is induced by local stress signals such as severe hypoxia. In this study, we investigated the normal role of p60AmotL2 in epithelial tissues. We found that this isoform is predominantly expressed in the gut, where cells experience rapid turnover. Through time-lapse imaging, we present evidence that cells expressing p60AmotL2 are extruded by their normal neighboring cells. Based on these findings, we hypothesize that tumor cells exploit this pathway to detach from normal epithelia and invade surrounding tissues.

Development of a simplified human embryonic stem cell-based retinal pre-organoid model for toxicity evaluations of common pollutants

Objective To explore the retinal toxicity of pharmaceuticals and personal care products (PPCPs), flame retardants, bisphenols, phthalates, and polycyclic aromatic hydrocarbons (PAHs) on human retinal progenitor cells (RPCs) and retinal pigment epithelial (RPE) cells, which are the primary cell types at the early stages of retinal development, vital for subsequent functional cell type differentiation, and closely related to retinal diseases. Materials and methods After 23 days of differentiation, human embryonic stem cell (hESC)-based retinal pre-organoids, containing RPCs and RPE cells, were exposed to 10, 100, and 1000 nM pesticides (butachlor, terbutryn, imidacloprid, deltamethrin, pendimethalin, and carbaryl), flame retardants (PFOS, TBBPA, DBDPE, and TDCIPP), PPCPs (climbazole and BHT), and other typical pollutants (phenanthrene, DCHP, and BPA) for seven days. Then, mRNA expression changes were monitored and compared. Results (1) The selected pollutants did not show strong effects at environmental and human-relevant concentrations, although the effects of flame retardants were more potent than those of other categories of chemicals. Surprisingly, some pollutants with distinct structures showed similar adverse effects. (2) Exposure to pollutants induced different degrees of cell detachment, probably due to alterations in extracellular matrix and/or cell adhesion. Conclusions In this study, we established a retinal pre-organoid model suitable for evaluating multiple pollutants’ effects, and pointed out the potential retinal toxicity of flame retardants, among other pollutants. Nevertheless, the potential mechanisms of toxicity and the effects on cell detachment are still unclear and deserve further exploration. Additionally, this model holds promise for screening interventions aimed at mitigating the detrimental effects of these pollutants.

Influence of High-Frequency, Low-Voltage Alternating Electric Fields on Biofilm Development Processes of Escherichia coli and Pseudomonas aeruginosa

A non-chemical solution is needed to control biofilms in water and wastewater treatment systems. High-frequency alternating electric field application offers an alternative approach that does not involve undesired electrode surface reactions. However, the effect of high-frequency alternating electric fields on bacterial cells in the attached-growth mode remains unexplored. This study investigated the impact of such fields on two stages of the biofilm development process: the initial adhesion phase (stage 1) and the early development phase (stage 2). Experiments were conducted using Escherichia coli and Pseudomonas aeruginosa in a three-channel flow cell exposed to alternating electric fields (3.05 V/cm, 20 MHz). The primary outcome of this study demonstrated that alternating electric fields decreased adhered cell numbers at both stages due to their inhibitory effect on growth. The alternating electric fields also triggered cell detachment after the initial attachment stage but not in mature biofilms. Interestingly, despite a reduction in cell counts, the amount of total biofilm biomass remained unaffected, which was likely due to increased cell size via cell elongation compensating for the decrease in numbers. No synergistic effects with respect to hydrodynamic forces were observed. These findings highlight the potential applicability of alternating electric fields to biofilm control and provide implications for water and wastewater engineering applications.

Eupatilin Ameliorates Lipopolysaccharide-Induced Acute Kidney Injury by Inhibiting Inflammation, Oxidative Stress, and Apoptosis in Mice.

Acute kidney injury (AKI) is a common complication of sepsis. Eupatilin (EUP) is a natural flavone with multiple biological activities and has beneficial effects against various inflammatory disorders. However, whether EUP has a favorable effect on septic AKI remains unknown. Here, we examined the effect of EUP on lipopolysaccharide (LPS)-evoked AKI in mice. LPS-evoked renal dysfunction was attenuated by EUP, as reflected by reductions in serum creatinine and blood urea nitrogen levels. LPS injection also induced structural damage such as tubular cell detachment, tubular dilatation, brush border loss of proximal tubules, and upregulation of tubular injury markers. However, EUP significantly ameliorated this structural damage. EUP decreased serum and renal cytokine levels, prevented macrophage infiltration, and inhibited mitogen-activated protein kinase and NF-κB signaling cascades. Lipid peroxidation and DNA oxidation were increased after LPS treatment. However, EUP mitigated LPS-evoked oxidative stress through downregulation of NPDPH oxidase 4 and upregulation of antioxidant enzymes. EUP also inhibited p53-mediated apoptosis in LPS-treated mice. Therefore, these results suggest that EUP ameliorates LPS-evoked AKI through inhibiting inflammation, oxidative stress, and apoptosis.

Establishment of a tilapia lake virus (TiLV)-susceptible cell line for anti-TiLV Chinese herbal compound screening

Tilapia lake virus (TiLV) has caused many deadly epidemics in wild and farmed tilapia, posing a great threat to the global tilapia-based industry. It is necessary and urgent to develop effective measures for TiLV control. Previously, we isolated a TiLV strain from naturally diseased tilapia, and its main target tissues are liver, spleen and brain. In the study, a TiLV-susceptive cell line is established based on the tissue tropism, and anti-TiLV activity of 11 Chinese herbal compounds (CHCs) is checked in vitro. A cell line from the brain of tilapia, designated as Nt-B, can be continuously subcultured for >100 passages and maintain a good proliferating state in L-15 media supplemented with 10% FBS at 28 °C. Following infection with TiLV, a series of cytopathic effects can be observed, including rounding, accumulation, vacuolation and detachment of cells. Enveloped viral particles can be observed in TiLV-infected cells, and TiLV-specific bands are identified by PCR analysis, confirming susceptibility of Nt-B cells to TiLV. For anti-TiLV activity evaluation, the selected CHCs show inhibition activity on TiLV-induced CPEs in different degrees except CHC-I. Two kinds of CHCs, CHC-II and CHC-X, have inhibition activity on TiLV-induced CPEs higher than 80%. Besides, the 90% inhibitory concentrations of CHC-II and CHC-X on TiLV are 51.12 ± 3.04 and 68.28 ± 6.37 mg/L, respectively. In summary, the newly established Nt-B cell line provides a useful tool for TiLV-related studies, moreover, CHC-II is a promising therapeutic agent against TiLV infection. Further studies will be required to reveal the active compounds and anti-TiLV mechanism of CHC-II.

Inherited chitinases enable sustained growth and rapid dispersal of bacteria from chitin particles.

Many biogeochemical functions involve bacteria utilizing solid substrates. However, little is known about the coordination of bacterial growth with the kinetics of attachment to and detachment from such substrates. In this quantitative study of Vibrio sp. 1A01 growing on chitin particles, we reveal the heterogeneous nature of the exponentially growing culture comprising two co-existing subpopulations: a minority replicating on chitin particles and a non-replicating majority which was planktonic. This partition resulted from a high rate of cell detachment from particles. Despite high detachment, sustained exponential growth of cells on particles was enabled by the enrichment of extracellular chitinases excreted and left behind by detached cells. The 'inheritance' of these chitinases sustains the colonizing subpopulation despite its reduced density. This simple mechanism helps to circumvent a trade-off between growth and dispersal, allowing particle-associated marine heterotrophs to explore new habitats without compromising their fitness on the habitat they have already colonized.

Reducing Cancer Cell Adhesion using Microtextured Surfaces.

For the past century, trypsin has been the primary method of cell dissociation, largely without any major changes to the process. Enzymatic cell detachment strategies for large-scale cell culturing processes are popular but can be labor-intensive, potentially lead to the accumulation of genetic mutations, and produce large quantities of liquid waste. Therefore, engineering surfaces to lower cell adhesion strength could enable the next generation of cell culture surfaces for delicate primary cells and automated, high-throughput workflows. In this study, a process for creating microtextured polystyrene (PS) surfaces to measure the impact of microposts on the adhesion strength of cells is developed. Cell viability and proliferation assays show comparable results in twocancer cell lines between micropost surfaces and standard cell culture vessels. However, cell image analysis on microposts reveals that cell area decreases by half, and leads to an average twofold increase in cell length per area. Using a microfluidic-based method up to a seven times greater percentage of cells are removed from micropost surfaces than the flat control surfaces. These results show that micropost surfaces enable decreased cell adhesion strength while maintaining similar cell viabilities and proliferation as compared to flat PS surfaces.

Fibronectin fragments generated by pancreatic trypsin act as endogenous inhibitors of pancreatic tumor growth

BackgroundThe pancreatic microenvironment has a defensive role against cancer but it can acquire tumor-promoting properties triggered by multiple mechanisms including alterations in the equilibrium between proteases and their inhibitors. The identification of proteolytic events, targets and pathways would set the basis for the design of new therapeutic approaches.Methods and resultsHere we demonstrate that spheroids isolated from human and murine healthy pancreas and co-transplanted orthotopically with pancreatic ductal adenocarcinoma (PDAC) in mouse pancreas inhibited tumor growth. The effect was mediated by trypsin-generated fibronectin (FN) fragments released by pancreatic spheroids. Tumor inhibition was observed also in a model of acute pancreatitis associated with trypsin activation. Mass spectrometry proteomic analysis of fragments and mAb against different FN epitopes identified the FN type III domain as responsible for the activity. By inhibiting integrin α5β1, FAK and FGFR1 signaling, the fragments induced tumor cell detachment and reduced cell proliferation. Consistent with the mutual relationship between the two pathways, FGF2 restored both FGFR1 and FAK signaling and promoted PDAC cell adhesion and proliferation. FAK and FGFR inhibitors additively inhibited PDAC growth in vitro and in orthotopic in vivo models.ConclusionsThis study identifies a novel role for pancreatic trypsin and fibronectin cleavage as a mechanism of protection against cancer by the pancreatic microenvironment. The finding of a FAK-FGFR cross-talk in PDAC support the combination of FAK and FGFR inhibitors for PDAC treatment to emulate the protective effect of the normal pancreas against cancer.

An adhesion signaling axis involving Dystroglycan, β1-Integrin, and Cas adaptor proteins regulates the establishment of the cortical glial scaffold.

The mature mammalian cortex is composed of 6 architecturally and functionally distinct layers. Two key steps in the assembly of this layered structure are the initial establishment of the glial scaffold and the subsequent migration of postmitotic neurons to their final position. These processes involve the precise and timely regulation of adhesion and detachment of neural cells from their substrates. Although much is known about the roles of adhesive substrates during neuronal migration and the formation of the glial scaffold, less is understood about how these signals are interpreted and integrated within these neural cells. Here, we provide in vivo evidence that Cas proteins, a family of cytoplasmic adaptors, serve a functional and redundant role during cortical lamination. Cas triple conditional knock-out (Cas TcKO) mice display severe cortical phenotypes that feature cobblestone malformations. Molecular epistasis and genetic experiments suggest that Cas proteins act downstream of transmembrane Dystroglycan and β1-Integrin in a radial glial cell-autonomous manner. Overall, these data establish a new and essential role for Cas adaptor proteins during the formation of cortical circuits and reveal a signaling axis controlling cortical scaffold formation.

Assessment of the water quality and toxicity effects on zebrafish (Danio rerio) of a stream near a phosphorus chemical plant in Guizhou Province, southwestern China.

To reveal the influence of the phosphorus chemical industry (PCI) on regional water environmental quality and safety, the water quality and ecotoxicological effects of a stream near a phosphorus chemical plant (PCP) in Guizhou Province, southwestern China, were investigated based on water samples collected from the stream. The results showed that the average concentrations of NH3-N, TN, P, F-, Hg, Mn, and Ni were 3.14mg/L, 30.09mg/L, 3.34mg/L, 1.18mg/L, 1.06μg/L, 45.82μg/L, and 11.30μg/L, respectively. The overall water quality of the stream was in the heavily polluted category, and NH3-N, TN, P, F-, and Hg were the main pollution factors. The degree of pollution was in the order of rainy period > transitional period > dry period, and the most polluted sample site was 1100m from the PCP. After 28 days of exposure to stream water, there was no significant change in the growth parameters of zebrafish. The gills of zebrafish showed a small amount of epithelial cell detachment and a small amount of inflammatory cell infiltration, and the liver tissue displayed a large amount of hepatocyte degeneration with loose and lightly stained cytoplasm. Compared with the control group, the%DNA intail, tail length, tail moment, and olive tail moment were significantly increased (p < 0.05), indicating that the water sample caused DNA damage in the peripheral blood erythrocytes of zebrafish. The stream water in the PCI area was found to be polluted and exhibited significant toxicity to zebrafish, which could pose a threat to regional ecological security.

Seawater biodesalination treatment using Phormidium keutzingianum in attached growth-packed bed continuous flow stirred tank reactor

Water scarcity is increasing worldwide due to rising population which is creating opportunities to unlock alternative green desalination techniques for seawater, such as biodesalination. Therefore, this study presents the utilization of the Phormidium keutzingianum strain in an attached growth-packed bed reactor to treat seawater in real-time in a continuous-flow stirred tank reactor for biodesalination. Two reactors were designed and developed, in which zeolites were used as the support media for the attached growth. The experiment was conducted in an open outdoor environment with a continuous air flow rate of 3 mL/min and two hydraulic retention times (HRT) of 7 and 15 d. Parameters such as the pH, chloride ion concentration, total organic carbon (TOC), and optical density were monitored regularly. The pH change was not significant in either reactor and remained within the range of 7.25–8.0. Chloride ion removal was the most crucial component of biodesalination efficiency, with d 7 removal efficiencies of approximately 40% and 32% for reactors 1 and 2, respectively. Reactor 1 exhibited a TOC reduction of 36% within the first 10 d at a HRT of 7, and when the HRT was set to 15 d, a TOC removal efficiency of 89% was achieved on d 53. For reactor 2, a TOC removal efficiency of approximately 81% was achieved on d 11 at HRT 7, and it reduced to less than 50% at an HRT of 15. The chloride ion and TOC removal phenomena were similar in both reactors. The optical density (OD) showed low measurement recordings, ranging from 0.005 to 0.01, indicating low cell detachment in the seawater effluent. Therefore, using the attached growth method for the biodesalination of seawater is feasible. Furthermore, biomass harvesting in attached growth systems is easier than that in suspension growth systems.

An innovative design for trophectoderm biopsy without laser pulses: a step-by-step demonstration

To present a novel trophectoderm biopsy method, independent of laser pulses, using innovatively designed micropipettes on blastocysts at different stages and show variable characteristics. A step-by-step demonstration of this method with narrated video. Invitro laboratory fertilization. Individuals whose embryos underwent preimplantation genetic testing. Trophectoderm biopsy is accomplished using micropipettes that contain a set of innovative designs. Both biopsy and holding pipettes are characterized by sharp, flat opening ends. The holding pipette is designed with an inclined plane on the outer wall surface of its opening end. It is used to help the biopsy pipette make contact with the holding pipette with increased stability, preventing slipping during the detachment of the trophectoderm cells. There is a narrow structure inside the biopsy pipette, which is designed to trap released fragments and prevent sample loss. A trophectoderm biopsy for fully expanded blastocysts starts from artificial shrinkage, followed by zona pellucida drilling. Then, 5-10 trophectoderm cells are aspirated into a biopsy pipette. The blastocyst is released from the holding pipette, the edge of the opening end of the biopsy pipette is tightly pressed onto the inclined plane of the holding pipette, and the biopsy pipette is flicked directly without laser pulses or pulling off the trophectoderm cells. The aspirated trophectoderm cells are subsequently detached by mechanical friction between the edges of the biopsy and holding pipettes. Apart from drilling the zona pellucida for fully expanded blastocysts, the remaining steps do not require lasers. For hatching (including peanut-shaped and 8-shaped) and hatched blastocysts, a trophectoderm biopsy is accomplished by aspirating the cells without securing the blastocyst with a holding pipette, followed by detachment using the direct flicking method. The biopsy time, sample loss rate, successful DNA amplification rate, and survival rate. The innovatively designed micropipettes facilitate the successful detachment of trophectoderm cells through a single direct flicking procedure. This eliminates thermal damage caused by laser pulses, notably simplifying operational steps and shortening the biopsy time. Significant differences were noted between the direct flicking and conventional methods, wherein laser pulses and pulling of trophectoderm cells are prerequisites for cell detachment. When comparing the average biopsy time of fully expanded blastocyst (61 ± 8s vs. 104 ± 9s, P<.05), peanut-shaped hatching blastocyst (35 ± 6s vs. 113 ± 13s, P<.05), 8-shaped hatching blastocyst (32 ± 4s vs. 59 ± 6s, P<.05), and hatched blastocyst (34 ± 4s vs. 67 ± 8s, P<.05), the direct flicking method shows a significantly decreased biopsy time. The narrow structure inside the biopsy pipette effectively prevents sample loss, showing a significantly reduced sample loss rate (0%) compared with the conventional biopsy method (18%) for trainees. Moreover, a satisfactory survival rate (100%) and successful DNA amplification rate (99.5%) were achieved using the direct flicking method. This innovative trophectoderm biopsy method, independent of laser pulses, has wide applicability and a satisfactory, stable performance. Moreover, the simplicity of the method makes it easy to master.