Inactive Cells Research Articles

Antibiotics stimulates the development of persistent cells in biofilms of Candida albicans bloodstream isolates

Candida albicans invasive candidiasis is considered a global health problem. In such cases, biofilm formation on implanted devices represents a therapeutic challenge and the presence of metabolically inactive persistent cells (PCs) in these communities increases their tolerance to fungicidal drugs. This study investigated the influence of amoxicillin, AMX; cefepime, CEF; gentamicin, GEN; amikacin, AMK; vancomycin, VAN; and ciprofloxacin, CIP; on the production of PCs in biofilms of C. albicans bloodstream isolates. 48 h-mature biofilms (n = 6) grown in RPMI-1640 supplemented with antibiotics were treated with 100 μg ml−1 amphotericin B and then evaluated for PCs. Biofilms grown in the presence of antibiotics produced more PCs, up to 10×, when exposed to AMX and CIP; 5 × to CEF; and 6 × to GEN and VAN. The results indicate that antibiotics can modulate PC production in C. albicans biofilms. This scenario may have clinical repercussions in immunocompromised patients under broad-spectrum antibiotic therapy.

An insight of heart-like systems with percolation

We study the signal percolation through heart-like biological system. Starting from an initial distribution of waiting and inactive cells with probabilities p and (1−p) respectively, the signal propagation is observed in terms of active cells. As the signal enters the system from one end, the number of arrival of active sites at the other end is studied and analysis of the system behaviour is made by varying a few important parameters of the system like pswitch (switching probability from inactive to waiting) and pact (switching probability from waiting to active). In this connection, the non-regular heart rhythms are discussed. Fraction of paths percolating through the system shows a transition from 0 to 1 near p=pc. Some other important quantities like tortuosity and cluster distribution are discussed. Several critical exponents have been obtained and compared the exponents of standard percolation.

SUPRa is a dual-color reporter for unbiased quantification of the unfolded protein response with cellular resolution

The unfolded protein response (UPR) maintains proteostasis upon endoplasmic reticulum (ER) stress, and is initiated by a range of physiological and pathological processes. While there have been advances in developing fluorescent reporters for monitoring individual signaling pathways of the UPR, this approach may not capture a cell’s overall UPR activity. Here we describe a novel sensor of UPR activity, sUPRa, which is designed to report the global UPR. sUPRa displays excellent response characteristics, outperforms reporters of individual UPR pathways in terms of sensitivity and kinetics, and responds to a range of different ER stress stimuli. Furthermore, sUPRa’s dual promoter and fluorescent protein design ensures that both UPR-active and inactive cells are detected, and controls for reporter copy number. Using sUPRa, we reveal UPR activation in layer 2/3 pyramidal neurons of mouse cerebral cortex following a period of sleep deprivation. sUPRa affords new opportunities for quantifying physiological UPR activity with cellular resolution.

Growth, Antioxidant Capacity, and Liver Health in Largemouth Bass (Micropterus salmoides) Fed Multi-Strain Yeast-Based Paraprobiotic: A Lab-to-Pilot Scale Evaluation.

A multi-strain yeast-based paraprobiotic (MsYbP) comprising inactive cells and polysaccharides (β-glucan, mannan oligosaccharides, and oligosaccharides) derived from Saccharomyces cerevisiae and Cyberlindnera jadinii could ensure optimal growth and health in farmed fish. This study assessed the impact of an MsYbP on the growth, immune responses, antioxidant capacities, and liver health of largemouth bass (Micropterus salmoides) through lab-scale (65 days) and pilot-scale (15 weeks) experiments. Two groups of fish were monitored: one fed a control diet without the MsYbP and another fed 0.08% and 0.1% MsYbP in the lab-scale and pilot-scale studies, respectively (referred to as YANG). In the lab-scale study, four replicates were conducted, with 20 fish per replicate (average initial body weight = 31.0 ± 0.8 g), while the pilot-scale study involved three replicates with approximately 1500 fish per replicate (average initial body weight = 80.0 ± 2.2 g). The results indicate that the MsYbP-fed fish exhibited a significant increase in growth in both studies (p < 0.05). Additionally, the dietary MsYbP led to a noteworthy reduction in the liver function parameters (p < 0.05), such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (AKP), and hepatic nuclear density, indicating improved liver health. Furthermore, the dietary MsYbP elevated the antioxidative capacity of the fish by reducing their malondialdehyde levels and increasing their levels and gene expressions related to antioxidative markers, such as total antioxidant ca-pacity (T-AOC), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), catalase (CAT), nuclear factor erythroid 2-related factor 2 (nrf2) and kelch-1ike ech-associated protein (keap1) in both studies (p < 0.05). In terms of hepatic immune responses, the lab-scale study showed an increase in inflammation-related gene expressions, such as interleukin-1β (il-1β) and transforming growth factor β1 (tgf-β1), while the pilot-scale study significantly suppressed the expressions of genes related to inflammatory responses, such as tumor necrosis factor α (tnfα) and interleukin-10 (il-10) (p < 0.05). In summary, our findings underscore the role of dietary multi-strain yeast-based paraprobiotics in enhancing the growth and liver health of largemouth bass, potentially through increased antioxidative capacity and the modulation of immune responses, emphasizing the significance of employing yeast-based paraprobiotics in commercial conditions.

Targeting lipid peroxidation-associated ferroptosis suppresses lung carcinoma progression by regulating cell cycle arrest

Lung carcinoma is a frequently encountered cancerous growth that affects the respiratory tract and has a high occurrence rate globally. In light of the ongoing worldwide health emergency, the significance of efficient therapeutic agents and strategies is of utmost importance. A meticulous control of the cell cycle is crucial for comprehending the pathophysiology and molecular causes of lung cancer, as well as for the formulation of efficacious therapeutic medicines. The mechanism by which cells synchronize cell cycle with cell survival and death is still not fully understood. In this study, we demonstrate that the halting of the cell cycle has a strong inhibitory impact on ferroptosis, a specific type of controlled cell death triggered by excessive lipid peroxidation at the membranes of cells. Ferroptosis is halted through the mechanism of cell cycle arrest, which involves the deposition of intracellular lipids mediated by diacylglycerol acyltransferase (DGAT). Excessive amounts of polyunsaturated fatty acids (PUFAs) are stored as triacylglycerols (TAGs) within inactive cells. As a result, inhibiting DGAT causes a rearrangement of PUFAs from TAGs to phospholipids and makes arrested cells more susceptible to ferroptosis. We demonstrate that certain lung cancer cells that are resistant to antimitotic drugs and have a slow-cycling behavior exhibit an increase in lipid droplets. Furthermore, we find that the growth of tumors resistant to 5-fluorouracil, lorlatinib, and docetaxel can be effectively suppressed by a combination treatment involving the use of ferroptosis inducers and DGAT inhibitors, which induces ferroptosis. Collectively, these findings demonstrate the involvement of cell cycle arrest in conferring resistance to ferroptosis and propose a potential therapeutic approach for addressing the challenge of slow-cycling malignancies that exhibit resistance to ferroptosis.

Use of a platform with lens-free shadow imaging technology to monitor natural killer cell activity

Natural killer (NK) cells are a crucial component of the innate immune system. This study introduces Cellytics NK, a novel platform for rapid and precise measurement of NK cell activity. This platform combines an NK-specific activation stimulator cocktail (ASC) and lens-free shadow imaging technology (LSIT), using optoelectronic components. LSIT captures digital hologram images of resting and ASC-activated NK cells, while an algorithm evaluates cell size and cytoplasmic complexity using shadow parameters. The combined shadow parameter derived from the peak-to-peak distance and width standard deviation rapidly distinguishes active NK cells from inactive NK cells at the single-cell level within 30 s. Here, the feasibility of the system was demonstrated by assessing NK cells from healthy donors and immunocompromised cancer patients, demonstrating a significant difference in the innate immunity index (I3). Cancer patients showed a lower I3 value (161%) than healthy donors (326%). I3 was strongly correlated with NK cell activity measured using various markers such as interferon-gamma, tumor necrosis factor-alpha, perforin, granzyme B, and CD107a. This technology holds promise for advancing immune functional assays, offering rapid and accurate on-site analysis of NK cells, a crucial innate immune cell, with its compact and cost-effective optoelectronic setup, especially in the post-COVID-19 era.

Sex-dependent intra-islet structural rearrangements affecting alpha-to-beta cell interactions lead to adaptive enhancements of Ca2+ dynamics in prediabetic beta cells

Aims/hypothesisPrediabetic pancreatic beta cells can adapt their function to maintain normoglycaemia for a limited period of time, after which diabetes mellitus will manifest upon beta cell exhaustion. Understanding sex-specific beta cell compensatory mechanisms and their failure in prediabetes (impaired glucose tolerance) is crucial for early disease diagnosis and individualised treatment. Our aims were as follows: (1) to determine the key time points of the progression from beta cells’ functional adaptations to their failure in vivo; and (2) to mechanistically explain in vivo sex-specific beta cell compensatory mechanisms and their failure in prediabetes.MethodsIslets from male and female transgenic Ins1CreERT2-GCaMP3 mice were transplanted into the anterior chamber of the eye of 10- to 12-week-old sex-matched C57BL/6J mice. Recipient mice were fed either a control diet (CD) or western diet (WD) for a maximum of 4 months. Metabolic variables were evaluated monthly. Beta cell cytoplasmic free calcium concentration ([Ca2+]i) dynamics were monitored in vivo longitudinally by image fluorescence of the GCaMP3 reporter islets. Global islet beta cell [Ca2+]i dynamics in line with single beta cell [Ca2+]i analysis were used for beta cell coordination studies. The glucagon receptor antagonist L-168,049 (4 mmol/l) was applied topically to the transplanted eyes to evaluate in vivo the effect of glucagon on beta cell [Ca2+]idynamics. Human islets from non-diabetic women and men were cultured for 24 h in either a control medium or high-fat/high-glucose medium in the presence or absence of the glucagon receptor antagonist L-168,049. [Ca2+]i dynamics of human islets were evaluated in vitro after 1 h exposure to Fura-10.ResultsMice fed a WD for 1 month displayed increased beta cell [Ca2+]i dynamics linked to enhanced insulin secretion as a functional compensatory mechanism in prediabetes. Recruitment of inactive beta cells in WD-fed mice explained the improved beta cell function adaptation observed in vivo; this occurred in a sex-specific manner. Mechanistically, this was attributable to an intra-islet structural rearrangement involving alpha cells. These sex-dependent cytoarchitecture reorganisations, observed in both mice and humans, induced enhanced paracrine input from adjacent alpha cells, adjusting the glucose setpoint and amplifying the insulin secretion pathway. When WD feeding was prolonged, female mice maintained the adaptive mechanism due to their intrinsically high proportion of alpha cells. In males, [Ca2+]i dynamics progressively declined subsequent to glucose stimulation while insulin secretion continue to increase, suggesting uncoordinated beta cell function as an early sign of diabetes.Conclusions/interpretationWe identified increased coordination of [Ca2+]i dynamics as a beta cell functional adaptation mechanisms in prediabetes. Importantly, we uncovered the mechanisms by which sex-dependent beta cell [Ca2+]i dynamics coordination is orchestrated by an intra-islet structure reorganisation increasing the paracrine input from alpha cells on beta cell function. Moreover, we identified reduced [Ca2+]i dynamics coordination in response to glucose as an early sign of diabetes preceding beta cell secretory dysfunction, with males being more vulnerable. Alterations in coordination capacity of [Ca2+]i dynamics may thus serve as an early marker for beta cell failure in prediabetes.Graphical

Cadherin Expression Is Regulated by Mechanical Phenotypes of Fibroblasts in the Perivascular Matrix.

The influence of mechanical forces generated by stromal cells in the perivascular matrix is thought to be a key regulator in controlling blood vessel growth. Cadherins are mechanosensors that facilitate and maintain cell-cell interactions and blood vessel integrity, but little is known about how stromal cells regulate cadherin signaling in the vasculature. Our objective was to investigate the relationship between mechanical phenotypes of stromal cells with cadherin expression in 3D tissue engineering models of vascular growth. Stromal cell lines were subjected to a bead displacement assay to track matrix distortions and characterize mechanical phenotypes in 3D microtissue models. These cells included human ventricular cardiac (NHCF), dermal (NHDF), lung (NHLF), breast cancer-associated (CAF), and normal breast fibroblasts (NBF). Cells were embedded in a fibrin matrix (10 mg/mL) with fluorescent tracker beads; images were collected every 30 min. We also studied endothelial cells (ECs) in co-culture with mechanically active or inactive stromal cells and quantified N-Cad, OB-Cad, and VE-Cad expression using immunofluorescence. Bead displacement studies identified mechanically active stromal cells (CAFs, NHCFs, NHDFs) that generate matrix distortions and mechanically inactive cells (NHLFs, NBFs). CAFs, NHCFs, and NHDFs displaced the matrix with an average magnitude of 3.17 ± 0.11 μm, 3.13 ± 0.06 μm, and 2.76 ± 0.05 μm, respectively, while NHLFs and NBFs displaced the matrix with an average of 1.82 ± 0.05 μm and 2.66 ± 0.06 μm in fibrin gels. Compared to ECs only, CAFs + ECs as well as NBFs + ECs in 3D co-culture significantly decreased expression of VE-Cad; in addition, Pearson's Correlation Coefficient for N-Cad and VE-Cad showed a strong correlation (&gt;0.7), suggesting cadherin colocalization. Using a microtissue model, we demonstrated that mechanical phenotypes associated with increased matrix deformations correspond to enhanced angiogenic growth. The results could suggest a mechanism to control tight junction regulation in developing vascular beds for tissue engineering scaffolds or understanding vascular growth during developmental processes. Our studies provide novel data for how mechanical phenotype of stromal cells in combination with secreted factor profiles is related to cadherin regulation, localization, and vascularization potential in 3D microtissue models.

Anti-Biofilm and Anti-Quorum-Sensing Activity of Inula Extracts: A Strategy for Modulating Chromobacterium violaceum Virulence Factors.

The formation of microbial biofilm is a self-organizing process among bacterial cells, regulated by quorum-sensing (QS) mechanisms, contributing to development of infections. These processes, either separately or in combination, significantly contribute to bacterial resistance to antibiotics and disinfectants. A novel approach to addressing the challenge of treating infections due to antibacterial resistance involves the use of plant metabolites. In recent years, there has been increasing recognition of different phytochemicals as potential modulators. In our study, we evaluated the synergistic effect of chloroform and methanol extracts from Inula species against key virulence factors, including biofilm formation, violacein production, and swarming motility. Each of the 11 examined plant extracts demonstrated the ability to reduce biofilms and pigment synthesis in C. violaceum. Two of the extracts from I. britannica exhibited significant anti-biofilm and anti-quorum-sensing effects with over 80% inhibition. Their inhibitory effect on violacein synthesis indicates their potential as anti-QS agents, likely attributed to their high concentration of terpenoids (triterpenoids, sesquiterpene lactones, and diterpenoids). Scanning electron microscopy revealed a notable reduction in biofilm biomass, along with changes in biofilm architecture and cell morphology. Additionally, fluorescence microscopy revealed the presence of metabolically inactive cells, indicating the potent activity of the extracts during treatment. These new findings underscore the effectiveness of the plant extracts from the genus Inula as potential anti-virulent agents against C. violaceum. They also propose a promising strategy for preventing or treating its biofilm formation.

Intraocular mRNA delivery with endogenous MmPEG10-based virus-like particles

Virus-like particles (VLP) are a promising tool for intracellular gene delivery, yet their potential in ocular gene therapy remains underexplored. In this study, we bridged this knowledge gap by demonstrating the successful generation and application of vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped mouse PEG10 (MmPEG10)-VLP for intraocular mRNA delivery. Our findings revealed that PEG10-VLP can efficiently deliver GFP mRNA to adult retinal pigment epithelial cell line-19 (ARPE-19) cells, leading to transient expression. Moreover, we showed that MmPEG10-VLP can transfer SMAD7 to inhibit epithelial-mesenchymal transition (EMT) in RPE cells effectively. In vivo experiments further substantiated the potential of these vectors, as subretinal delivery into adult mice resulted in efficient transduction of retinal pigment epithelial (RPE) cells and GFP reporter gene expression without significant immune response. However, intravitreal injection did not yield efficient ocular expression. We also evaluated the transduction characteristics of MmPEG10-VLP following intracameral delivery, revealing transient GFP protein expression in corneal endothelial cells without significant immunotoxicities. In summary, our study established that VSVG pseudotyped MmPEG10-based VLP can transduce mitotically inactive RPE cells and corneal endothelial cells in vivo without triggering an inflammatory response, underscoring their potential utility in ocular gene therapy.

Abstract 1158: Spatially resolved cell profiling unveils tumor metabolic states associated with immunotherapy response in NSCLC

Abstract Background. Only a subset of non-small cell lung cancer (NSCLC) patients benefit from immune checkpoint inhibitors (ICIs). Thus, there is a clinical need to develop predictive biomarkers for ICIs. Deeper insights from the tumor microenvironment (TME) can lead to identification of spatial features associated with clinical responses to immune checkpoint inhibitors (ICI). In this study, we investigated functional profiles within cell subtypes as a surrogate for ICI clinical endpoints. Methods. We profiled a retrospective cohort of 39 NSCLC tissue cores from 27 patients treated with ICI, using highly multiplexed immunofluorescence (mIF) stained by the Phenocycler Fusion platform (Akoya Biosciences) capturing 45 proteins. Utilizing a deep learning based multiplex imaging analysis pipeline, cells were classified to 15 cell types by known marker expression and were further subclassified by unsupervised clustering. Cells were assigned to the tumor area or TME and more than 1000 spatial features were calculated based on cell type, marker positivity, and area assignments, and were compared between responders and non-responders to ICI therapy using Fisher’s exact and logrank tests. Results. Unsupervised cell subtyping of the 15 cell types identified 43 cell subsets, which were mostly segregated by their metabolism and activation status. Interestingly, all lymphocytes showed a similar pattern of clustering resulting in two clusters of metabolically active and inactive cells. The most significantly differentially expressed proteins between these cell types were oxidative phosphorylation proteins such as CS, SDHA and ATPA5, and other metabolic enzymes such as HK1, GLUT1 and LDHA. We found a direct connection between metabolic state, effector functions and tissue localization as the metabolically active lymphocytes exhibited higher levels of PD-1, MHC class I and II and CD44 positivity, and were more abundant within tumor infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLS). Unsupervised clustering of tumor cells demonstrated segregation to three main metabolic states - OXPHOS+, OXPHOS- and a third cluster (PPP+) which was characterized by upregulation of ASCT2, a glutamine transporter, as well as pNRF2 and G6PD, regulators of the pentose phosphate pathway. These cells also exhibited higher proliferation rate and CD44, a tumor stemness marker, positivity. All tumors with high content of PPP+ tumor cells (&amp;gt;40%) were resistant to PD-1 blockade (0/8 vs. 10/18 response rate in other tumors, p=0.009) and showed reduced overall survival (OS) rates (median OS of 27.6 vs 90.3 months, p=0.02). Conclusions. Taken together, this study reveals connections between metabolic states, effector functions, and immunotherapy outcome and contributes to the evolving landscape of predictive biomarkers for ICI therapies. Citation Format: James Monkman, Rotem Czertok, Shai Bookstein, Becky Arbiv, Yuval Shachaf, Ron Elran, Kenneth Bloom, Oscar Puig, Ken O'Byrne, Ettai Markovits, Arutha Kulasinghe. Spatially resolved cell profiling unveils tumor metabolic states associated with immunotherapy response in NSCLC [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 1158.

QUANTITATIVE ANALYSIS OF PET IMAGING VIA THE FOUR-COMPARTMENT MODEL FOR LUNG CANCER ASSESSMENT

Lung cancer is a life-threatening disease that requires early detection for effective treatment. Positron emission tomography (PET) imaging has been instrumental in diagnosing and monitoring lung cancer, but it has some limitations. To overcome these limitations, the four-compartment model has been developed, which provides a more accurate representation of the cancer's metabolic activity. This model incorporates the vascular volume and tracer delivery rate into the traditional two-compartment model, resulting in improved diagnostic accuracy and treatment planning. Recent studies have shown that the four-compartment model outperforms conventional PET scans in predicting tumor response to treatment and differentiating between active and inactive cancer cells. The four-compartment model holds great promise in revolutionizing the diagnosis and monitoring of lung cancer, offering improved outcomes and renewed hope for patients worldwide. Keywords: Lung cancer, PET imaging, four-compartment model, metabolic activity, diagnosis, monitoring, limitations, vascular volume, tracer delivery rate, accuracy, treatment planning, tumor response, active cancer cells.

Novel pyrazole‐biphenyl‐carboxamides for SARS‐CoV2 entry‐level restriction and microbial infections

AbstractMicrobial diseases including viral infection are big issues globally. Effective medicinal discovery for them is the need for the day. In this study, we report pyrazole‐biphenyl‐carboxamides (4a‐l) validated for their SARS‐CoV2 entry‐level restriction effect over studying the protein–protein interaction of SARS‐CoV2 with human ACE protein. Their extended antimicrobial properties were also evaluated. Online and offline software tools predicted MD simulation and ADMET druggability in silico. The antimicrobial efficacy of all compounds was also evaluated against Gram+ve Streptococcus pneumoniae (MTCC 1936), Staphylococcus aureus (MTCC 737) and Gram‐ve Escherichia coli (MTCC 443), Pseudomonas aeruginosa (MTCC 424) (bacteria). In the results, compounds 4g and 4i were evenly active against both bacteria at a low concentration range (MIC: 1.00 to 9.5 μg/mL) and displayed lesser toxicity to tested mammalian cells (EC100 = 75 μg/mL). Furthermore, it was able to kill metabolically inactive bacterial cells and eradicate established biofilms of Methicillin‐resistant Staphylococcus aureus (MRSA). Both the compounds inhibited DNA gyrase well with an IC50 0.25 μM (96% relative activity) and 0.52 μM (97% relative activity) respectively. Compounds (4a‐l) showed restrictive efficiency of SARS‐CoV2 spike protein (SC2SP) and human angiotensin‐converting enzyme 2 (hACE2) entry‐level association in COVID‐19 in silico. To assess this ability, firstly, we identified the crucial amino acid residues involved in the interface of SARS‐CoV‐2 and hACE2 virtually. We recognized the ability of 4a‐l binding to the binding interface to SARS‐CoV2; thus, the interaction of SC2SP‐hACE2 was effectively inhibited.

Prediction of the risk of recurrence of the leprosy process

The aim of the study was to study the degree of neutrophil granulocyte myeloperoxidase activity in peripheral blood of patients with leprosy in order to predict the possible recurrence of the infectious process.Materials and methods. Electron grams of biopsies of the affected skin and blood smears of 8 (5 women and 3 men) patients with borderline (BB), 9 (6 women and 3 men) patients with subpolar lepromatous (LLs) and 7 (4 women and 3 men) patients with polar lepromatous (LLr) were used as the object of the study forms of leprosy. At the ultramicroscopic level, the degree of MP activity detected by the electron-cytochemical method in the Mf of biopsies of the affected skin, both first registered before the start of therapy and with the activation of the process during treatment, was taken into account. The percentage of mitochondria with MP Mf activity relative to the MP of inactive cells was calculated. In peripheral blood smears, the activity of neutrophilic granulocytes (NG) MP was evaluated by the mean cytochemical coefficient (SCC).Results. It was found that in patients with the highest percentage of peroxidase-active mitochondria (79-92%), the presence of MP activity on the membranes of phagosomes and in the electron-transparent zone (around M. leprae) correlated with rapid and persistent regression of the leprosy process and long periods of relapse-free period, which was determined by comparing the data of medical histories with the results obtained in during our research. It was found that in patients without relapse with forms of BB and LLs leprosy, the activity of MP in neutrophils was 1.3 times higher (p&lt;0.05), and the percentage of peroxidase-active mitochondria was 3 times higher (p&lt;0.01) compared with these indicators of patients with relapse of the disease. When comparing the studied markers of groups of patients without relapse with the form of leprosy LLp, the degree of MP NG activity increased by 1.4 times (p&lt;0.05), the percentage of mitochondria with MP Mf activity increased by 3 times (p&lt;0.01) relative to patients with relapses.Conclusion. Thus, the determination of myeloperoxidase activity of neutrophil granulocytes of peripheral blood can act as a prognostic marker for determining the effectiveness of anti-leprosy therapy and the risk of relapses of the disease during and after treatment.

A COMPARATIVE STUDY OF THE WATER QUALITY BEFORE AND AFTER TREATMENT OF CONTAMINATED WATER USING THE PEELS OF MUSA PARASIDIACA IN RELATION TO SEASONAL VARIATION

Chromium contamination can alter genetic materials and cause hazardous health effect in life. Bio absorption method relies on the ability of biological materials to remove the heavy metals by binding metal in various ways. The bio-adsorbents consist of dead and metabolically inactive cells. The residues of banana peels can be processed and converted to be adsorbents because they have large surface areas, high swelling capacities, excellent mechanical strengths, and are convenient to use and have great potential to adsorb harmful contaminants such as heavy metals. In the present study, contaminated water was treated using the powdered peels of Musa parasidiaca, then the physico-chemical factors and the amount of chromium before and after treatment were compared in relation to winter and summer. The amount of heavy metal chromium concentration was reduced drastically. Based on the results obtained the peels of Musa parasidiaca is proved to be a low cost and effective bio-adsorbent in bioremediation of chromium.

TMET-16. ELUCIDATING MECHANISMS OF ENHANCED OXIDATIVE PHOSPHORYLATION IN MYC-AMPLIFIED MEDULLOBLASTOMA

Abstract Medulloblastoma (MBL) is the most commonly diagnosed pediatric brain tumour and the leading cause of childhood cancer deaths. MYC overexpression often accompanies poor prognosis in MBL patients, as MYC-driven transformation is known to promote mitochondrial oxidative phosphorylation (OXPHOS). However, it also induces a high metabolic demand that impairs cell survival under acute nutrient deprivation. We previously reported that eukaryotic Elongation Factor Kinase 2 (eEF2K), the master regulator of mRNA translation elongation, is overexpressed in MYC-driven MBL. Preliminary data suggests a relationship between eEF2K-mediated selective translation of mRNAs needed for efficient OXPHOS, and for the progression of MYC-driven MBL. Multiplexed enhanced Protein Dynamic Mass Spectrometry was carried out in eEF2K knockdown MYC-overexpressing D425 MB cells to identify mRNAs selectively translated upon eEF2K activation. 9 of 10 detected components of the OXPHOS pathway were selectively translated in eEF2K-active MBL cells. Genetic eEF2K knockout led to the disassembly of electron transport chain (ETC) complexes I-V and reduced complex activity, as observed via Blue Native PAGE and colorimetric assays. Furthermore, siRNA knockdown of Cytochrome c oxidase assembly factor 7 (COA7), a mitochondrial OXPHOS assembly factor differentially expressed in eEF2K active or inactive D425 cells, produces the same pattern of supercomplex (SC) loss as the inactivation of eEF2K in wild-type D425 cells. The assembly of ETC SC across D425 eEF2K active and inactive cells was further studied using blue native gel-based mass spectrometry to further characterize patterns in SC loss. In summary, control of translation elongation by eEF2K is critical for mitochondrial ETC complex assembly and efficient OXPHOS in MYC-overexpressing MBL, likely representing an adaptive response against acute metabolic stress. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetics.

Distinct Effects of Moxifloxacin and Bedaquiline on Growing and 'Non-Culturable' Mycobacterium abscessus.

Mycobacterium abscessus has recently emerged as the cause of an increasing number of human infections worldwide. Unfortunately, it is highly resistant to existing drugs, and new specific agents to combat M. abscessus have not yet been found. The discovery of antibiotics that are effective not only against replicating but also against dormant and often recalcitrant cells is a daunting challenge. In this study, we developed a model of non-replicating M. abscessus, which represents a valuable screening tool for antibacterial agents. Thus, we demonstrated that, under a deficiency of potassium ions in the growth media and prolonged incubation, M. abscessus entered a 'non-culturable' state with a significant loss of colony-forming ability, but it retained viability, as confirmed using the most-probable-number (MPN) assay. The 'non-culturable' mycobacteria possessed decelerated cellular metabolism and noticeable differences in cell morphology from actively growing mycobacteria. 'Non-culturable' cells were used in a comprehensive screening of the efficacy of antibiotics, along with actively growing cells. Both CFU and MPN tests confirmed the prominent bactericidal effect of moxifloxacin on actively growing and 'non-culturable' M. abscessus, as proven by less than 0.01% of cells surviving after antibiotic treatment and prolonged storage. Bedaquiline exhibited a comparable bactericidal effect only on metabolically inactive non-culturable cells aged for 44 days. There were reductions ranging from 1000 to 10,000-fold in CFU and MPN, but it was not so efficient with respect to active cells, resulting in a bacteriostatic effect. The demonstrated specificity of bedaquiline in relation to inert non-replicating M. abscessus offers a new and unexpected result. Based on the findings of this research, moxifloxacin and bedaquiline can be regarded as potential treatments for infections caused by M. abscessus. In addition, a key outcome is the proposal to include the combination of viability assays for comprehensive testing of drug candidates. Relying on CFU-based assays alone resulted in overestimates of antibacterial efficacy, as demonstrated in our experiments.

Identification and Quantification of Lipopeptide Homologues Induced and Produced by Bacillus amyloliquefaciens

Cyclic lipopeptides (LPs) are potentially promising in the agricultural, industrial and pharmaceutical sectors. LPs have a variable hydrophilic cyclic peptide part attached to a variable fatty acid chain. One limitation of these compounds is their low availability due to their limited production by bacteria. The objective of this study was to identify and quantify homologues of LPs biosynthesized by Bacillus amyloliquefaciens using ultra-performance liquid chromatography (UPLC–MS/MS) after inducing the synthesis of these secondary metabolites using different inducers, including chemical compounds and inactive cells of Colletotrichum sp. Four homologues were identified in the iturin family (bacillomycin D), and the iturin homologue with the highest synthesis was the molecular ion m/z 1031.54, with 173.1 µg mg−1 crude extract. In addition, seven homologues were detected in the fengycin family (four of fengycin A and three of fengycin B), and the homologue with the highest content was the molecular ion m/z 1463.69 (fengycin A), with 3288 ± 528.5 ng mg−1 crude extract. Finally, five homologues were identified in the surfactin family, where the highest concentration was observed for the molecular ion m/z 1036.68, with 61.5 ± 3.01 µg mg−1 crude extract. The highest concentration of LP homologues (iturin, fengycin and surfactin) synthesized by B. amyloliquefaciens was detected in the presence of inactive cells of Coletotrichum sp., suggesting that the inducing substance is associated with the inducer’s cell envelope and could be a single protein or a structure that includes protein components.

Electron transport bifurcation in bioanode with the metabolic shift to nitrate reduction

Electron transport bifurcation in bioanode determines the performance of microbial electrochemical technologies with the presence of an alternative electron acceptor. Here, the bioanode responses including electron transfer efficiency, microbial community, and microbial structure are investigated with the metabolic shift from current production to denitrification. Electrochemical measurements including cyclic voltammetry and electrochemical impedance spectra are performed to identify the change of electron transfer pathways in bioanode. Electron transfer efficiency for electrode reduction decreases ∼17 % with nitrate reduction. Biofilm resistance and charge transfer resistance increase from 23.3 Ω and 22.5 Ω to 36.6 Ω and 61.4 Ω with the metabolic shift, respectively. These results are mainly due to the loss of exoelectrogens inhabited in bioanode. Confocal imaging results indicate the elevated proportion of inactive cells in bioanode as the denitrification. Our results propose a possible mechanism for electron transfer bifurcation in bioanode with the metabolic shift from electrode reduction to soluble electron acceptor reduction.

Naringin Attenuates CCl4-Induced Hepatocyte Damage by Inhibiting Endoplasmic Reticulum Stress

Objective: Exposure of the liver to diverse chemicals induce hepatic damage. Carbon tetrachloride (CCl4) is widely used toxin to investigate hepatic injury. In our study, the effects of naringin (NRG), a flavanone abundant in citrus fruits, on endoplasmic reticulum (ER) stress and stress-mediated apoptosis in CCl4-induced liver injury were investigated. Materials and methods: THLE-3 cells were exposed to varying concentrations of CCl4 for 24 hours and then treated with different doses of NRG for 4 hours. The effects of varying concentrations on cell viability were determined. Then, protein expressions of ER stress markers were detected in hepatocytes. Finally, Bcl2 active / inactive cell ratios were determined by flow cytometry. Results: NRG treatment (5 and 10 μM) significantly increased cell viability, which decreased with CCl4 administration. Similarly, the increased levels of ER stress markers as a result of CCl4 application were significantly reduced with NRG treatment. Finally, NRG prevented apoptosis by significantly reducing ratio of Bcl2 inactive cells. Conclusion: NRG treatment is effective in suppressing ER stress in CCl4-induced hepatocyte damage and preventing ER stress-induced apoptosis.