Development of a mouse model expressing a bifunctional glutathione-synthesizing enzyme to study glutathione limitation in vivo.

Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are associated with inborn errors of metabolism, cancer, and neurodegenerative disorders, studying the limiting role of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus thermophilus (GshF), which possesses both glutamate-cysteine ligase and glutathione synthase activities. GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis induction, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes further revealed genes required for cell proliferation under cellular and mitochondrial GSH depletion. Among these, we identified the glutamate-cysteine ligase modifier subunit, GCLM, as a requirement for cellular sensitivity to buthionine sulfoximine, a glutathione synthesis inhibitor. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the limiting role of GSH in physiology and disease.

Longitudinal clonal dynamics of HIV-1 latent reservoirs measured by combination quadruplex polymerase chain reaction and sequencing.

HIV-1 infection produces a long-lived reservoir of latently infected CD4+ T cells that represents the major barrier to HIV-1 cure. The reservoir contains both intact and defective proviruses, but only the proviruses that are intact can reinitiate infection upon cessation of antiretroviral therapy (ART). Here we combine four-color quantitative PCR and next-generation sequencing (Q4PCR) to distinguish intact and defective proviruses and measure reservoir content longitudinally in 12 infected individuals. Q4PCR differs from other PCR-based methods in that the amplified proviruses are sequence verified as intact or defective. Samples were collected systematically over the course of up to 10 y beginning shortly after the initiation of ART. The size of the defective reservoir was relatively stable with minimal decay during the 10-y observation period. In contrast, the intact proviral reservoir decayed with an estimated half-life of 4.9 y. Nevertheless, both intact and defective proviral reservoirs are dynamic. As a result, the fraction of intact proviruses found in expanded clones of CD4+ T cells increases over time with a concomitant decrease in overall reservoir complexity. Thus, reservoir decay measurements by Q4PCR are quantitatively similar to viral outgrowth assay (VOA) and intact proviral DNA PCR assay (IPDA) with the addition of sequence information that distinguishes intact and defective proviruses and informs reservoir dynamics. The data are consistent with the notion that intact and defective proviruses are under distinct selective pressure, and that the intact proviral reservoir is progressively enriched in expanded clones of CD4+ T cells resulting in diminishing complexity over time.

Pyroptosis Tuning in Intestinal Cryptosporidiosis via the Natural Histone Deacetylase Inhibitor Romidepsin.

Cryptosporidium is an opportunistic protozoan, with many species of cross-human infectivity. It causes life-threatening diarrhoea in children and CD4-defective patients. Despite its limited efficacy, nitazoxanide remains the primary anti-cryptosporidial drug. Cryptosporidium infects the intestinal brush border (intracellular-extracytoplasmic) and down-regulates pyroptosis to prevent expulsion. Romidepsin is a natural histone deacetylase inhibitor that triggers pyroptosis. Romidepsin's effect on cryptosporidiosis was assessed in immunocompromised mice via gasdermin-D (GSDM-D) immunohistochemical expression, IFN-γ, IL-1ß and IL-18 blood levels by ELISA, and via parasite scanning by modified Ziehl-Neelsen staining and scanning electron microscopy (SEM). Oocyst deformity and local cytokines were also assessed in ex vivo ileal explants. Following intraperitoneal injection of romidepsin, oocyst shedding significantly reduced at the 9th, 12th and 15th d.p.i. compared with infected-control and drug-control (nitazoxanide-treated) mice. H&E staining of intestinal sections from romidepsin-treated mice showed significantly low intestinal scoring with marked reduction in epithelial hyperplasia, villous blunting and cellular infiltrate. SEM revealed marked oocyst blebbing and paucity (in vivo and ex vivo) after romidepsin compared with nitazoxanide. Regarding pyroptosis, romidepsin triggered significantly higher intestinal GSDM-D expression in vivo, and higher serum/culture IFN-γ, IL-1ß and IL-18 levels in romidepsin-treated mice than in the control groups. Collectively, in cryptosporidiosis, romidepsin succeeded in enhancing pyroptosis in the oocysts and infected epithelium, reducing infection and shifting the brush border towards normalisation.

Potential i-Nos/Arg-1 Switch with NLRP3 and Parasitic Load Down Regulation in Experimental Schistosoma mansoni Infection via Chloroquine Repurposing.

In previous studies, the inhibitory effect of chloroquine on NLRP3 inflammasome and heme production was documented. This may be employed as a double-bladed sword in schistosomiasis (anti-inflammatory and parasiticidal). In this study, chloroquine's impact on schistosomiasis mansoni was investigated. The parasitic load (worm/egg counts and reproductive capacity index [RCI]), i-Nos/Arg-1 expression, splenomegaly, hepatic insult and NLRP3-immunohistochemical expression were assessed in infected mice after receiving early and late repeated doses of chloroquine alone or dually with praziquantel. By early treatment, the least RCI was reported in dually treated mice (41.48 ± 28.58) with a significant reduction in worm/egg counts (3.50 ± 1.29/2550 ± 479.58), compared with either drug alone. A marked reduction in the splenic index was achieved by prolonged chloroquine administration (alone: 43.15 ± 5.67, dually: 36.03 ± 5.27), with significantly less fibrosis (15 ± 3.37, 14.25 ± 2.22) than after praziquantel alone (20.5 ± 2.65). Regarding inflammation, despite the praziquantel-induced significant decrease in NLRP3 expression, the inhibitory effect was marked after dual and chloroquine administration (liver: 3.13 ± 1.21/3.45 ± 1.23, spleen: 5.7 ± 1.6/4.63 ± 2.41). i-Nos RNA peaked with early/late chloroquine administration (liver: 68.53 ± 1.8/57.78 ± 7.14, spleen: 63.22 ± 2.06/62.5 ± 3.05). High i-Nos echoed with a parasiticidal and hepatoprotective effect and may indicate macrophage-1 polarisation. On the flip side, the chloroquine-induced low Arg-1 seemed to abate immune tolerance and probably macrophage-2 polarisation. Collectively, chloroquine synergised the praziquantel-schistosomicidal effect and minimised tissue inflammation, splenomegaly and hepatic fibrosis.

Bioanalytical Validated Spectrofluorimetric Method for the Determination of Prucalopride succinate in Human Urine Samples and Its Greenness Evaluation.

An economical & eco-friendly spectrofluorometric method has been developed for the determination of prucalopride succinate (PRU) in human urine on the basis of the drug's native fluorescence. The type of solvent and the wavelengths of excitation and emission have been carefully selected for optimal experimental conditions. In deionized water, the fluorescence intensity was measured at λ emission 362 nm upon excitation at 310 nm. This bio-validated method was carried out using 30uL urine without any preliminary steps. The calibration curve for prucalopride succinate shows a linear relationship in a concentration range of 0.75-5.5 µg/mL. Accuracy and precision were obtained using 4 quality control samples which are: 0.75 µg/ mL (LLOQ), 2.25 µg/mL (QCL), 2.5 µg/mL (QCM) & 4.125 µg/mL (QCH). The validation of this proposed technique obeys European Medicines Agency (EMA) Guidelines for validating bioanalytical methods and the greenness assessment was evaluated according to the Analytical GAPI approach.

Controlling of Mycobacterium by Natural Degradant-Combination Models for Sequestering Mycolic Acids in Karish Cheese.

Degradation of the mycobacterial complex containing mycolic acids (MAs) by natural bioactive compounds is essential for producing safe and value-added foods with therapeutic activities. This study aimed to determine the degradation efficiency of natural organic acid extracts (i.e., citric, malic, tartaric, and lactic), quadri-mix extract from fruits and probiotics (i.e., lemon, apple, grape, and cell-free supernatant of Lactobacillus acidophilus), and synthetic pure organic acids (i.e., citric, malic, tartaric, and lactic), against MA in vitro in phosphate buffer solution (PBS) and Karish cheese models. The degradation effect was evaluated both individually and in combinations at different concentrations of degradants (1, 1.5, and 2%) and at various time intervals (0, 6, 12, 24, and 48 h). The results show that MA degradation percentage recorded its highest value at 2% of mixed fruit extract quadri-mix with L. acidophilus and reached 99.2% after 48 h both in PBS and Karish cheese, unlike other treatments (i.e., citric + malic + tartaric + lactic), individual acids, and sole extracts at all concentrations. Conversely, organic acid quadri-mix revealed the greatest MA degradation% of 95.9, 96.8, and 97.3% at 1, 1.5, and 2%, respectively, after 48 h. Citric acid was more effective in MA degradation than other acids. The fruit extract quadri-mix combined with L. acidophilus-fortified Karish cheese showed the highest sensorial characteristics; hence, it can be considered a novel food-grade degradant for MA and could be a promising biocontrol candidate against Mycobacterium tuberculosis (Mtb) in food matrices.

Use of bacteriophage to inactivate pathogenic bacteria from wastewater.

The aim of this study was to enhance the rhizobacterium potential in horizontal subsurface flow constructed wetland (CW) system planted by Phragmites australis using specific and lytic phages. The bioinoculation of specific bacteriophage for target bacteria; Salmonella typhi, and the monitoring of bacterial inactivation under different conditions showed the effectiveness of this methodology to enhance bacteria reduction and consequentially ameliorate purification performance of this studied biological treatment system. The injection of the phage at a concentration equal to 103 UFP/mL within the rhizosphere of the inoculated filter (F) was allowed 1 U-Log10 of improvement of bacterial inactivation compared to the control filter (T) nearly 1 logarithmic unit thus, a 90% improvement of bacteria reduction. When we increased the phage titer (105 UFP/mL), the bacterial reduction equal to 2.75 U-Log10 (N/N0) was registered that corresponds to a decrease of nearly 99.9%. According to the first-order model, the inactivation coefficient is equal to 2.29 min-1 (0.88 min-1 for the first experiment) and the bacterial reduction rate is 5 times higher than that determined for the control filter. This results show the positive impact of the phage in the bacterial inactivation and the improvement of water treatment of the biofilter C.

Niosomal versus nano-crystalline ivermectin against different stages of Trichinella spiralis infection in mice.

Ivermectin (IVM) is one of the competitive treatments used for trichinellosis. However, several studies linked its efficacy with early diagnosis and administration to tackle the intestinal phase with limited activity being recorded against encysted larvae. The aim of this study was to employ niosomes for enhancing effectiveness of oral IVM against different stages of Trichinella spiralis (T. spiralis) infection with reference to nano-crystalline IVM. Mice were randomized into four groups: group Ι, 15 uninfected controls; group ΙΙ, 30 infected untreated controls; group ΙΙΙ, 30 infected nano-crystalline IVM treated, and group ΙV, 30 infected niosomal IVM treated. All groups were equally subdivided into 3 subgroups; (a) treated on the 1st day post infection (dpi), (b) treated on the 10th dpi, and (c) treated on the 30th dpi. Assessment was done by counting adult worms and larvae plus histopathological examination of jejunum and diaphragm. Biochemical assessment of oxidant/antioxidant status, angiogenic, and inflammatory biomarkers in intestinal and muscle tissues was also performed. Both niosomes and nano-crystals resulted in significant reduction in adult and larval counts compared to the infected untreated control with superior activity of niosomal IVM. The superiority of niosomes was expressed further by reduction of inflammation in both jejunal and muscle homogenates. Biochemical parameters showed highly significant differences in all treated mice compared to infected untreated control at different stages with highly significant effect of niosomal IVM. In conclusion, niosomal IVM efficacy exceeded the nano-crystalline IVM in treatment of different phases of trichinellosis.

Bevacizumab as a potential anti-angiogenic therapy in schistosomiasis: A double-edged, but adjustable weapon.

AIM: Investigating the anti-angiogenic effect of bevacizumab on chronic schistosomiasis mansoni in a trial to hinder the Schistosome-induced angiogenesis and porto-systemic shunting complications. METHODS: The immunohistochemical expression of CD34, VEGF-R1, PCNA and α-SMA (angiogenesis markers) was analysed in the lung, liver and gastrointestinal junctions of chronic S mansoni infected mice after intraperitoneal injection of bevacizumab. The effect of prolonged administration of bevacizumab with praziquantel was also assessed through parasitic load, protective index, granuloma and fibrous tissue evaluation. RESULTS: A regression in the vascular activity and microvascular density was observed in the infected mice after receiving bevacizumab. They had a significantly less VEGF-R1, PCNA, CD-34 and α-SMA expression in comparison to the infected untreated mice. The least tissue egg count was reported in mice received bevacizumab for 6 weeks (Mean = 27 120). However, they had persistent liver granulomas, and massively amalgamated fibrosis. Interestingly, the least faecal egg and tissue worms counts (Mean = 112, 13.4), and the highest protection index (39.26) were reported in mice received bevacizumab for 3 weeks, with marked granuloma, and fibrous tissue resolution. CONCLUSIONS: Bevacizumab has a promising protective effect against the Schistosoma-induced angiogenesis. As an adjuvant to praziquantel, it is important to adjust the appropriate duration of administration that achieves the best schistosomicidal effect without impeding granuloma and fibrous tissue resolution.

Detection of active pathogenic bacteria under stress conditions using lytic and specific phage.

In this study, we have monitored the potential activity of a foodborne and waterborne pathogenic bacterium, Salmonella typhi, under starvation conditions. The interaction between lytic phage and starved-VBNC pathogenic bacteria was studied to establish reliable methods for the detection of active cells before resuscitation. The analysis of phage kinetic parameters has demonstrated the flexibility of lytic with the quantity and mainly the quality of host cells. After 2 h of phage-starved-VBNC bacteria interaction, the reduction of phage amplification rate can reveal the ability of specific-lytic phage to recognize and to attach to their host cells with a probability of burst and release of infectious phages by active bacteria. After an extension of the latent period, the boost of the phage amplification rate was directly related to the positive interaction between potential intracellular 'engaged' phages and potential active bacteria. Furthermore, the modeling of the Salmonella-specific phage growth cycle in relationship with starved host cells can highlight the impact of the viability and the activity state of the host cells on the phage's growth cycle.

Enhancement of rhizocompetence in pathogenic bacteria removal of a constructed wetland system.

The main goal of the present study was to enhance the rhizobacterium potential in a horizontal subsurface flow constructed wetland system planted with Phragmites australis, through environmentally friendly biological approaches. The bioinoculation of antagonist bacteria has been used to promote higher rhizosphere competence and improve pathogenic bacteria removal from wastewater. The experiment was performed both with single and sequential bioinoculation. The results showed that strain PFH1 played an active role in pathogenic bacteria removal, remarkably improving inactivation kinetics of the pathogenic tested bacterium Salmonella typhi in the plant rhizosphere. The single bioinoculation of selected bacteria into the rhizosphere of P. australis improved the kinetics of S. typhi inactivation by approximately 1 U-Log10 (N/N0) (N is the number of viable cultured bacteria at time t, N0 is the number of viable and cultivable bacteria at time t0) compared to the control. By a series of multi-bioinoculations, the enhancement of pathogenic bacteria reduction compared to the inhibition rate in the pilot-scale control was of 2 U-Log10(N/N0). These findings suggested that this strain represents a promising candidate to enhance water purification in constructed wetlands.

Acidic mammalian chitinase tuning after enteric helminths eradication in inflammatory respiratory disease patients.

AIM: This study aimed at investigating the presence of intestinal parasitic infections in inflammatory respiratory diseases patients during the disease attack, and measuring the acidic mammalian chitinase (AMCase) gene expression in blood before and after infection eradication. METHODOLOGY: This case-control study included 123 inflammatory respiratory diseases patients and 120 apparently healthy individuals. Repeated stool examination was done, while total and specific IgE were measured. AMCase gene expression was analysed by real time-polymerase chain reaction (RT-PCR). RESULTS: Infection was detected in 32.5% of the diseased and 23.25% of the healthy individuals. Higher rate of the helminthic infection was detected (23.57) in comparison to the protozoal (12.19%) in the patients. A significantly higher rate of infection with the chitin-rich helminths "Enterobius vermicularis & Hymenolepis nana" and level of anti-Dermatophagoide-IgE were reported in the patients (14.63%, 6.5% and 23.57%, respectively). AMCase expression was significantly higher in helminths-infected patients than the noninfected, or protozoa infected. After infection eradication, AMCase expression significantly declined in the previously helminth-infected patients (mean ± SD = 13.9 ± 3.918 before and 4.515 ± 1.93 after), but insignificantly affected in the protozoa infected (mean ± SD = 2.095 ± .285 before and 2.675 ± 1.181 after). CONCLUSION: Chitin-rich intestinal helminths are suspected to precipitate Th2-immune response in remote tissues by enhancing systemic AMCase expression through intestinal mucosa and macrophages irritation.

Trichinella Spiralis Impact on Mesenchymal Stem Cells: Immunohistochemical Study by Image Analyzer in Murine Model.

This study aims to elucidate whether Trichinella spiralis infection or its crude antigen administration can stimulate recruitment of CD105+ve/CD45-ve cells that could represent MSCs in intestine and skeletal muscle of experimental BALB/c albino mice compared to healthy control mice. Studied mice were divided into: 20 healthy control, 20 with orally induced T. spiralis infection, 20 received adult worm crude antigen orally and 20 received larval crude antigen intramuscular. According to specific timing schedule, mice were sacrificed and tissue sections were examined for CD105 and CD45 immunohistochemical expression using image J image analyzing software, to compare different study groups. T. spiralis infection induced a significant increase in density of CD105+ve/CD45-ve cells that could represent MSCs in both intestinal and muscle sections, similarly the intramuscular injected larval crude antigen caused more infiltration of such cells in muscles compared to muscle sections from healthy control mice. However, no significant difference was noticed in intestinal sections after oral adult crude antigen administration compared to healthy control mice. So, injected T. spiralis crude antigen might be a successful stimulant to MSCs attraction and recruitment in tissues nearby injection site. This could be beneficial for cell regeneration and tissue repair in case of presence of a disease induced damage.

Fluorescent Nanocomposite of Embedded Ceria Nanoparticles in Electrospun Chitosan Nanofibers.

This paper introduces a detailed optical characterization for a novel fluorescent biodegradable nanocomposite of electro-spun chitosan nanofibers with in-situ embedded cerium oxide (ceria) nanoparticles as the nanocomposite optical fluorescent material. Under near ultra-violet excitation, this synthesized nanocomposite emits a visible green wavelength at nearly 520nmwith different intensities according to the concentration of the embedded fluorescent material; i.e. ceria nanoparticles. This emission is due to the synthesized ceria nanoparticles optical tri-valiant cerium ions ce3+, associated with formed oxygen vacancies with a direct allowed bandgap around 3.5 eV. Optical characteristics such as fluorescence emission intensity, absorbance dispersion, and direct bandgap are presented besides structural characteristics such as FTIR spectroscopy, and SEM analysis. The synthesized optical nanocomposite could be helpful in many further applications such as bio-imaging, biomedical engineering, and environmental optical sensors.

FoxP3+ T regulatory cells and immunomodulation after Schistosoma mansoni egg antigen immunization in experimental model of inflammatory bowel disease.

To assess the effect of Schistosoma mansoni egg antigen immunization on the immunomodulation in dextran sodium sulfate (DSS) induced colitis as an experimental model of IBD in comparison to non immunization and healthy control. The study was performed on 180 mice; 25 healthy control, 15 to identify the inflammatory peak of DSS, 25 received DSS for 7 days; 90 infected with S. mansoni cercariae to collect eggs for antigen preparation, and 25 immunized with the prepared antigen then received DSS course. Disease activity index, macroscopic & microscopic inflammatory scores, FoxP3+ T regulatory cell count, myeloperoxidase activity, and Th1/Th2 cytokine profile were compared in studied groups. Immunization induced both FoxP3+ T(regs) and Th2 cytokines to establish a state of immune homeostasis and create a quiescent steadier immune response to DSS. S. mansoni egg antigen succeeded in acting like a prophylactic helminthic therapy as it has a profitable modulatory effect on DSS-induced colitis model.

Non-destructive detection and identification of plasticizers in PVC objects by means of machine learning-assisted Raman spectroscopy.

Vibrational spectroscopic techniques, such as Raman spectroscopy, as a non-destructive method combined with machine learning (ML), were successfully tested as a quick method of plasticizer identification in poly(vinyl chloride) - PVC objects in heritage collection. ML algorithms such as Convolutional Neural Network (CNN), Random Forest (RF), Support Vector Machines (SVM), and Linear Discriminant Analysis (LDA) were applied to the classification and identification of the most common plasticizers used in the case of PVC. The CNN model was able to successfully classify the five plasticizers under study from their Raman spectra with a high accuracy of (98%), whereas the highest accuracy (100%) was observed with the RF algorithm. The finding opens doors for the development of robust and economical tools for conservators and museum professionals for fast identification of materials in heritage collections.

A novel treatment approach using vitamin B12-conjugated sericin for mitigating nanodiamond-induced toxicity in darkling beetles.

The escalating use of nanodiamonds (NDs) has raised concerns about their ecotoxicological impact, prompting exploration of therapeutic interventions. This paper pioneers the examination of Vitamin B12-conjugated sericin (VB12-SER) as a potential therapeutic approach against ND-induced toxicity in darkling beetles (Blaps polychresta). The study analyzes mortality rates and organ-specific effects, covering the testis, ovary, and midgut, before and after treatments. Following exposure to 10 mg NDs/g body weight, within a subgroup of individuals termed ND2 with a mortality rate below 50%, two therapeutic treatments were administered, including pure sericin (SER) at 10 mg/mL and VB12-SER at 10.12 mg/mL. Consequently, five experimental groups (control, SER, ND2, ND2+SER, ND2+SER+VB12) were considered. Kaplan-Meier survival analysis was performed to assess the lifespan distribution of the insects in these groups over a 30-d period. Analyses revealed increased mortality and significant abnormalities induced by NDs within the examined organs, including cell death, DNA damage, enzyme dysregulation, antioxidant imbalances, protein depletion, lipid peroxidation, and morphological deformities. In contrast, the proposed treatments, especially (ND2+SER+VB12), demonstrated remarkable recovery, highlighting VB12-conjugated SER's potential in mitigating ND-triggered adverse effects. Molecular docking simulations affirmed binding stability and favorable interactions of the VB12-SER complex with target proteins. This research enhances understanding of NDs' effects on B. polychresta, proposing it as an effective bioindicator, and introduces VB12-conjugated SER as a promising therapeutic strategy in nanotoxicological studies.

Comprehensive toxicity assessment of nanodiamond on Blaps polychresta: implications and novel findings.

With the increasing development of nanomaterials, the use of nanodiamonds (NDs) has been broadly manifested in many applications. However, their high penetration into the ecosystem indubitably poses remarkable toxicological risks. This paper investigates the toxic effects of NDs on the darkling beetle, Blaps polychresta Forskal, 1775 (Coleoptera: Tenebrionidae). Survival analysis was carried out by monitoring the beetles for 30 d after the injection of four different doses of NDs. A dose of 10.0 mg NDs/g body weight, causing less than 50% mortality effect, was assigned in the analysis of the different organs of studied beetles, including testis, ovary, and midgut. Structural and ultrastructural analyses were followed using light, TEM, and SEM microscopes. In addition, a variety of stress markers and enzyme activities were assessed using spectrophotometric methods. Furthermore, cell viability and DNA damage were evaluated using cytometry and comet assay, respectively. Compared to the control group, the NDs-treated group was exposed to various abnormalities within all the studied organs as follows. Significant disturbances in enzyme activities were accompanied by an apparent dysregulation in the antioxidant system. The flow cytometry results indicated a substantial decrease of viable cells along with a rise of apoptotic and necrotic cells. The comet assay demonstrated a highly increased level of DNA damage. Likewise, histological analyses accentuated the same findings showing remarkable deformities in the studied organs. Prominently, the research findings substantially contribute for the first time to evaluating the critical effects of NDs on B. polychresta, adopted as the bioindicator in this paper.

Dual roles of the conditional extracellular vesicles derived from Pseudomonas aeruginosa biofilms: Promoting and inhibiting bacterial biofilm growth.

Antibiotic-resistant biofilm infections have emerged as public health concerns because of their enhanced tolerance to high-dose antibiotic treatments. The biofilm life cycle involves multiple developmental stages, which are tightly regulated by active cell-cell communication via specific extracellular signal messengers such as extracellular vesicles. This study was aimed at exploring the roles of extracellular vesicles secreted by Pseudomonas aeruginosa at different developmental stages in controlling biofilm growth. Our results show that extracellular vesicles secreted by P. aeruginosa biofilms during their exponential growth phase (G-EVs) enhance biofilm growth. In contrast, extracellular vesicles secreted by P. aeruginosa biofilms during their death/survival phase (D-EVs) can effectively inhibit/eliminate P. aeruginosa PAO1 biofilms up to 4.8-log10 CFU/cm2. The inhibition effectiveness of D-EVs against P. aeruginosa biofilms grown for 96 h improved further in the presence of 10-50 µM Fe3+ ions. Proteomic analysis suggests the inhibition involves an iron-dependent ferroptosis mechanism. This study is the first to report the functional role of bacterial extracellular vesicles in bacterial growth, which depends on the developmental stage of the parent bacteria. The finding of D-EV-activated ferroptosis-based bacterial death may have significant implications for preventing antibiotic resistance in biofilms.

Comprehensive and Validated Chromatographic Techniques for the Estimation of Lercanidipine Hydrochloride and Atenolol in Bulk and Combined Dosage Form in the Presence of Lercanidipine Degradation Products with LC/MS Characterization.

Two rapid, smart and validated stability indicating HPLC and TLC techniques were developed to determine atenolol (ATE) and lercanidipine HCl (LER) simultaneously in their pharmaceutical formulation. HPLC chromatographic separation was implemented by using Microsorb C18 (250 × 4.6 mm, 5 µm) column, with mobile phase of acetonitrile and 20 mM potassium dihydrogen phosphate buffer pH 3.5 adjusted by orthophosphoric acid in the ratio of (65:35, v/v) at a flow rate of 1.2 mL/min at 240 nm also the injection volume adjusted to be 30 µL. These selected conditions effectively separated ATE and LER at a retention time of 2 and 6.7 min, respectively, by isocratic elution mode without any interference from the obtained degradation products of LER. The densitometric determination was performed by using precoated silica gel 60F254 aluminum plates and chloroform, methanol and triethylamine (11.3:1.3: 0.3, by volume) as a developing system. The detection wavelength for simultaneous estimation of both drugs was 240 nm in the presence of the oxidative product of LER. The RF values for ATE and LER were 0.22 and 0.78, respectively. The calibration curves of both techniques were constructed with linearity ranges of (5-55) µg.mL-1 and (1-55) µg.mL-1 for both ATE and LER, respectively, for HPLC determination. While for TLC, the linearity ranges were (1-4) µg/band and (0.2-1.4) µg/band for ATE and LER, respectively. LER degradation products were characterized using UPLC/MS and the suggested mechanisms and degradation pathways were introduced.