Biotin-Pt(IV)-Ru(II)-Boron-Dipyrromethene Prodrug as "Platin Bullet" for Targeted Chemo- and Photodynamic Therapy.

Using the principle of "Magic Bullet", a cisplatin-derived platinum(IV) prodrug heterobimetallic Pt(IV)-Ru(II) complex, cis,cis,trans-[Pt(NH3)2Cl2{Ru(tpy-BODIPY)(tpy-COO)}(biotin)]Cl2 (Pt-Ru-B, 2), having two axial ligands, namely, biotin as water-soluble B-vitamin for enhanced cellular uptake and a BODIPY-ruthenium(II) (Ru-B, 1) photosensitizer having N,N,N-donor tpy (4'-phenyl-2,2':6',2″-terpyridine) bonded to boron-dipyrromethene (BODIPY), is developed as a "Platin Bullet" for targeted photodynamic therapy (PDT). Pt-Ru-B exhibited intense absorption near 500 nm and emission near 513 nm (λex = 488 nm) in a 10% dimethyl sulfoxide-Dulbecco's phosphate-buffered saline medium (pH 7.2). The BODIPY complex on light activation generates singlet oxygen as the reactive oxygen species (ROS) giving a quantum yield (ΦΔ) of ∼0.64 from 1,3-diphenylisobenzofuran experiments. Pt-Ru-B exhibited preferential cellular uptake in cancer cells over noncancerous cells. The dichlorodihydrofluorescein diacetate assay confirmed the generation of cellular ROS. Confocal images revealed its mitochondrial internalization. Pt-Ru-B showed submicromolar photocytotoxicity in visible light (400-700 nm) in A549 and multidrug-resistant MDA-MB-231 cancer cells. It remained nontoxic in the dark and less toxic in nontumorigenic cells. Cellular apoptosis and alteration of the mitochondrial membrane potential were evidenced from the respective Annexin V-FITC/propidium iodide assay and JC-1 dye assay. A wound healing assay using A549 cells and Pt-Ru-B revealed inhibition of cancer cell migration, highlighting its potential as an antimetastatic agent.

Measurement of Photorespiratory Cycle Enzyme Activities in Leaves Exposed to Abiotic Stress.

Photorespiration, an essential metabolic component, is a classic example of interactions between the intracellular compartments of a plant cell: the chloroplast, peroxisome, mitochondria, and cytoplasm. The photorespiratory pathway is often modulated by abiotic stress and is considered an adaptive response. Monitoring the patterns of key enzymes located in different subcellular components would be an ideal approach to assessing the modulation of the photorespiratory metabolism under abiotic stress. This chapter describes the procedures for assaying several individual enzyme activities of key photorespiratory enzymes and evaluating their response to oxidative/photooxidative stress. It is essential to ascertain the presence of stress in the experimental material. Therefore, procedures for typical abiotic stress induction in leaves by highlighting without or with menadione (an oxidant that targets mitochondria) are also included.

Protective Role of Nanoceria-Infused Nanofibrous Scaffold toward Bone Tissue Regeneration with Senescent Cells.

The presence of oxidative stress in bone defects leads to delayed regeneration, especially in the aged population and patients receiving cancer treatment. This delay is attributed to the increased levels of reactive oxygen species (ROS) in these populations due to the accumulation of senescent cells. Tissue-engineered scaffolds are emerging as an alternative method to treat bone defects. In this study, we engineered tissue scaffolds tailored to modulate the adverse effects of oxidative stress and promote bone regeneration. We used polycaprolactone to fabricate nanofibrous mats by using electrospinning. We exploited the ROS-scavenging properties of cerium oxide nanoparticles to alleviate the high oxidative stress microenvironment caused by the presence of senescent cells. We characterized the nanofibers for their physical and mechanical properties and utilized an ionization-radiation-based model to induce senescence in bone cells. We demonstrate that the presence of ceria can modulate ROS levels, thereby reducing the level of senescence and promoting osteogenesis. Overall, this study demonstrates that ceria-infused nanofibrous scaffolds can be used for augmenting the osteogenic activity of senescent progenitor cells, which has important implications for engineering bone tissue scaffolds for patients with low regeneration capabilities.

Comparison of the transcriptome, lipidome, and c-di-GMP production between BCGΔBCG1419c and BCG, with Mincle- and Myd88-dependent induction of proinflammatory cytokines in murine macrophages.

We have previously reported the transcriptomic and lipidomic profile of the first-generation, hygromycin-resistant (HygR) version of the BCGΔBCG1419c vaccine candidate, under biofilm conditions. We recently constructed and characterized the efficacy, safety, whole genome sequence, and proteomic profile of a second-generation version of BCGΔBCG1419c, a strain lacking the BCG1419c gene and devoid of antibiotic markers. Here, we compared the antibiotic-less BCGΔBCG1419c with BCG. We assessed their colonial and ultrastructural morphology, biofilm, c-di-GMP production in vitro, as well as their transcriptomic and lipidomic profiles, including their capacity to activate macrophages via Mincle and Myd88. Our results show that BCGΔBCG1419c colonial and ultrastructural morphology, c-di-GMP, and biofilm production differed from parental BCG, whereas we found no significant changes in its lipidomic profile either in biofilm or planktonic growth conditions. Transcriptomic profiling suggests changes in BCGΔBCG1419c cell wall and showed reduced transcription of some members of the DosR, MtrA, and ArgR regulons. Finally, induction of TNF-α, IL-6 or G-CSF by bone-marrow derived macrophages infected with either BCGΔBCG1419c or BCG required Mincle and Myd88. Our results confirm that some differences already found to occur in HygR BCGΔBCG1419c compared with BCG are maintained in the antibiotic-less version of this vaccine candidate except changes in production of PDIM. Comparison with previous characterizations conducted by OMICs show that some differences observed in BCGΔBCG1419c compared with BCG are maintained whereas others are dependent on the growth condition employed to culture them.

Role of mitochondria and chloroplasts during stomatal closure: Subcellular location of superoxide and H2O2 production in guard cells of Arabidopsis thaliana.

Stomatal guard cells are unique in that they have more mitochondria than chloroplasts. Several reports emphasized the importance of mitochondria as the major energy source during stomatal opening. We re-examined their role during stomatal closure. The marked sensitivity of stomata to both menadione (MD) and methyl viologen (MV) demonstrated that both mitochondria and chloroplasts helped to promote stomatal closure in Arabidopsis. As in the case of abscisic acid (ABA), a plant stress hormone, MD and MV induced stomatal closure at micromolar concentration. All three compounds generated superoxide and H2O2, as indicated by fluorescence probes, BES-So-AM and CM-H2DCFDA, respectively. Results from tiron (a superoxide scavenger) and catalase (an H2O2 scavenger) confirmed that both the superoxide and H2O2 were requisites for stomatal closure. Co-localization of the superoxide and H2O2 in mitochondria and chloroplasts using fluorescent probes revealed that exposure to MV initially triggered higher superoxide and H2O2 generation in mitochondria. In contrast, MD elevated superoxide/H2O2 levels in chloroplasts. However, with prolonged exposure, MD and MV induced ROS production in other organelles. We conclude that ROS production in mitochondria and chloroplasts leads to stomatal closure. We propose that stomatal guard cells can be good models for examining inter-organellar interactions.

Advanced 3D In Vitro Lung Fibrosis Models: Contemporary Status, Clinical Uptake, and Prospective Outlooks.

Fibrosis has been characterized as a global health problem and ranks as one of the primary causes of organ dysfunction. Currently, there is no cure for pulmonary fibrosis, and limited therapeutic options are available due to an inadequate understanding of the disease pathogenesis. The absence of advanced in vitro models replicating dynamic temporal changes observed in the tissue with the progression of the disease is a significant impediment in the development of novel antifibrotic treatments, which has motivated research on tissue-mimetic three-dimensional (3D) models. In this review, we summarize emerging trends in preparing advanced lung models to recapitulate biochemical and biomechanical processes associated with lung fibrogenesis. We begin by describing the importance of in vivo studies and highlighting the often poor correlation between preclinical research and clinical outcomes and the limitations of conventional cell culture in accurately simulating the 3D tissue microenvironment. Rapid advancement in biomaterials, biofabrication, biomicrofluidics, and related bioengineering techniques are enabling the preparation of in vitro models to reproduce the epithelium structure and operate as reliable drug screening strategies for precise prediction. Improving and understanding these model systems is necessary to find the cross-talks between growing cells and the stage at which myofibroblasts differentiate. These advanced models allow us to utilize the knowledge and identify, characterize, and hand pick medicines beneficial to the human community. The challenges of the current approaches, along with the opportunities for further research with potential for translation in this field, are presented toward developing novel treatments for pulmonary fibrosis.

Cellular Senescence Program is Sensitive to Physical Differences in Polymeric Tissue Scaffolds.

A typical cellular senescence program involves exposing cells to DNA-damaging agents such as ionization radiation or chemotherapeutic drugs, which cause multipronged changes, including increased cell size and volume, the onset of enhanced oxidative stress, and inflammation. In the present study, we examined if the senescence onset decision is sensitive to the design, porosity, and architecture of the substrate. To address this, we generated a library of polymeric scaffolds widely used in tissue engineering of varied stiffness, architecture, and porosity. Using irradiated A549 lung cancer cells, we examined the differences between cellular responses in these 3D scaffold systems and observed that senescence onset is equally diminished. When compared to the two-dimensional (2D) culture formats, there were profound changes in cell size and senescence induction in three-dimensional (3D) scaffolds. We further establish that these observed differences in the senescence state can be attributed to the altered cell spreading and cellular interactions on these substrates. This study elucidates the role of scaffold architecture in the cellular senescence program.

Moderate modulation by S-nitrosoglutathione of photorespiratory enzymes in pea (Pisum sativum) leaves, compared to the strong effects of high light.

When plants are exposed to water stress, photosynthesis is downregulated due to enhanced reactive oxygen species (ROS) and nitric oxide (NO). In contrast, photorespiratory metabolism protected photosynthesis and sustained yield. Modulation of photorespiration by ROS was established, but the effect of NO on photorespiratory metabolism was unclear. We, therefore, examined the impact of externally added NO by using S-nitrosoglutathione (GSNO), a natural NO donor, in leaf discs of pea (Pisum sativum) under dark or light: moderate or high light (HL). Maximum NO accumulation with GSNO was under high light. The presence of 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO scavenger, prevented the increase in NO, confirming the release of NO in leaves. The increase in S-nitrosothiols and tyrosine-nitrated proteins on exposure to GSNO confirmed the nitrosative stress in leaves. However, the changes by GSNO in the activities and transcripts of five photorespiratory enzymes: glycolate oxidase, hydroxypyruvate reductase, catalase, glycerate kinase, and phosphoglycolate phosphatase activities were marginal. The changes in photorespiratory enzymes caused by GSNO were much less than those with HL. Since GSNO caused only mild oxidative stress, we felt that the key modulator of photorespiration might be ROS, but not NO.

The senescent mesothelial matrix accentuates colonization by ovarian cancer cells.

Ovarian cancer is amongst the most morbid of gynecological malignancies due to its diagnosis at an advanced stage, a transcoelomic mode of metastasis, and rapid transition to chemotherapeutic resistance. Like all other malignancies, the progression of ovarian cancer may be interpreted as an emergent outcome of the conflict between metastasizing cancer cells and the natural defense mounted by microenvironmental barriers to such migration. Here, we asked whether senescence in coelom-lining mesothelia, brought about by drug exposure, affects their interaction with disseminated ovarian cancer cells. We observed that cancer cells adhered faster on senescent human and murine mesothelial monolayers than on non-senescent controls. Time-lapse epifluorescence microscopy showed that mesothelial cells were cleared by a host of cancer cells that surrounded the former, even under sub-confluent conditions. A multiscale computational model predicted that such colocalized mesothelial clearance under sub-confluence requires greater adhesion between cancer cells and senescent mesothelia. Consistent with the prediction, we observed that senescent mesothelia expressed an extracellular matrix with higher levels of fibronectin, laminins and hyaluronan than non-senescent controls. On senescent matrix, cancer cells adhered more efficiently, spread better, and moved faster and persistently, aiding the spread of cancer. Inhibition assays using RGD cyclopeptides suggested the adhesion was predominantly contributed by fibronectin and laminin. These findings led us to propose that the senescence-associated matrisomal phenotype of peritoneal barriers enhances the colonization of invading ovarian cancer cells contributing to the metastatic burden associated with the disease.

CAR-T-Cell Therapy in Multiple Myeloma: B-Cell Maturation Antigen (BCMA) and Beyond.

Significant progress has been achieved in the realm of therapeutic interventions for multiple myeloma (MM), leading to transformative shifts in its clinical management. While conventional modalities such as surgery, radiotherapy, and chemotherapy have improved the clinical outcomes, the overarching challenge of effecting a comprehensive cure for patients afflicted with relapsed and refractory MM (RRMM) endures. Notably, adoptive cellular therapy, especially chimeric antigen receptor T-cell (CAR-T) therapy, has exhibited efficacy in patients with refractory or resistant B-cell malignancies and is now also being tested in patients with MM. Within this context, the B-cell maturation antigen (BCMA) has emerged as a promising candidate for CAR-T-cell antigen targeting in MM. Alternative targets include SLAMF7, CD38, CD19, the signaling lymphocyte activation molecule CS1, NKG2D, and CD138. Numerous clinical studies have demonstrated the clinical efficacy of these CAR-T-cell therapies, although longitudinal follow-up reveals some degree of antigenic escape. The widespread implementation of CAR-T-cell therapy is encumbered by several barriers, including antigenic evasion, uneven intratumoral infiltration in solid cancers, cytokine release syndrome, neurotoxicity, logistical implementation, and financial burden. This article provides an overview of CAR-T-cell therapy in MM and the utilization of BCMA as the target antigen, as well as an overview of other potential target moieties.

Paradoxical behaviour of rainfall and temperature over ecologically sensitive areas along the Western Ghats.

Initial reports signify some specific isolated locations in different latitudes, revealing a paradoxical increase in both heavy and very heavy rainfall events and also an increment in total, i.e., in both rainfall and temperature, over ecologically sensitive areas along the Western Ghats (WG). This paper presents a coherent study of the full-scale of daily rainfall and temperature over 27 well-spaced stations in the study area to determine its extent and investigate whether or not this contradictory behaviour is real. Also, an attempt has been made to assess the differential behaviour of rainfall, temperature, and heavy rainfall events in association with land use and land cover change (LULC). The analysis revealed that rainfall and temperature over the study area are increasing, whereas heavy rainfall events have increased during 1981-2020 with strong peaks after 2000 around 18-19°N (Mumbai metropolitan region), 14-16°N (mining and quarrying regions in Goa), and 9-12°N (a narrow strip of land spanning across the coastal towns of Karnataka and Kerala) latitudes. The majority of the rainfall excess years coincided with El Nino years, indicating that El Nino does not affect rainfall negatively. However, rainfall over the WG is influenced by local relief and cascading topography. The spatial pattern of average annual rainfall shows a decreasing trend from south to north because the elevation and span of rainfall occurrence are higher in the southern part of WG. The findings of the current research will help in building a strategy to address trends and patterns of climatic variables in association with LULC.

ß-Galactosidase-activated nitroxyl (HNO) donors provide insights into redox cross-talk in senescent cells.

The cross-talk among reductive and oxidative species (redox cross-talk), especially those derived from sulfur, nitrogen and oxygen, influence several physiological processes including aging. One major hallmark of aging is cellular senescence, which is associated with chronic systemic inflammation. Here, we report a chemical tool that generates nitoxyl (HNO) upon activation by ß-galactosidase, an enzyme that is over-expressed in senescent cells. In a radiation-induced senescence model, the HNO donor suppressed reactive oxygen species (ROS) in a hydrogen sulfide (H2S)-dependent manner. Hence, the newly developed tool provides insights into redox cross-talk and establishes the foundation for new interventions that modulate levels of these species to mitigate oxidative stress and inflammation.

Biotin and boron-dipyrromethene-tagged platinum(IV) prodrug for cellular imaging and mito-targeted photocytotoxicity in red light.

A platinum(IV) prodrug, cis,cis,trans-[Pt(NH3)2Cl2(biotin)(L)] (1), derived from cisplatin, where HL is the PEGylated red-light active boron-dipyrromethene (BODIPY) ligand, was synthesized, characterized and its photocytotoxicity evaluated. The complex showed a near-IR absorption band at 653 nm (ε ∼9.19 × 104 M-1 cm-1) in dimethyl sulfoxide and Dulbecco's phosphate-buffered saline (1 : 1 v/v) at pH 7.2. When excited at 630 nm, it showed an emission band at 677 nm in DMSO with a fluorescence quantum yield of 0.13. The 1,3-diphenylisobenzofuran titration experiment gave a singlet oxygen quantum yield (ΦΔ) of ∼0.32. A mechanistic DNA photocleavage study revealed singlet oxygen as the reactive oxygen species (ROS). The complex with biotin and PEGylated-distyryl-BODIPY showed significantly higher cellular uptake in A549 cancer cells as compared to non-cancerous Beas-2B cells from flow cytometry, indicating selectivity towards cancer cells. A dichlorodihydrofluorescein diacetate assay showed cellular ROS generation. Confocal images revealed predominant internalization in the mitochondria. The prodrug showed remarkable photodynamic therapy (PDT) activity in cancerous A549 and multidrug-resistant MDA-MB-231 cells with a high photocytotoxicity index value (half-maximal inhibitory concentration (IC50): 0.61-1.54 µM in red light), while being non-toxic in the dark. The chemo-PDT activity was significantly less in non-tumorigenic lung epithelial cells (Beas-2B). The prodrug effectively triggered cellular apoptosis, which was confirmed by the Annexin V-FITC/propidium iodide assay, and the alteration of the mitochondrial membrane potential was substantiated by the JC-1 dye assay. The ß-tubulin immunofluorescence assay confirmed that incubating the cells with a light-treated complex resulted in the rapture of the cytoskeletal structure and the formation of apoptotic bodies. The results demonstrate that the prodrug triggered apoptosis via DNA damage, a reduction in mitochondrial function and disruption of the cytoskeletal framework.

A Platinum(II) Boron-dipyrromethene Complex for Cellular Imaging and Mitochondria-targeted Photodynamic Therapy in Red Light.

Cisplatin-derived platinum(II) complexes [Pt(NH3 )2 (pacac)](NO3 ) (1, DPP-Pt) and [Pt(NH3 )2 (Acac-RB)](NO3 ) (2, Acacplatin-RB), where Hpacac is 1,3-diphenyl-1,3-propanedione and HAcac-RB is a red-light active distyryl-BODIPY-appended acetylacetone ligand, are prepared, characterized and their photodynamic therapy (PDT) activity studied (RB abbreviated for red-light BODIPY). Complex 2 displayed an intense absorption band at λ=652 nm (ϵ=7.3×104  M-1  cm-1 ) and 601 nm (ϵ=3.1×104  M-1  cm-1 ) in 1 : 1 DMSO-DPBS (Dulbecco's Phosphate Buffered Saline). Its emission profile includes a broad maximum at ~673 nm (λex =630 nm). The fluorescence quantum yield (ΦF ) of HAcac-RB and 2 are 0.19 and 0.07, respectively. Dichlorodihydrofluorescein diacetate and 1,3-diphenylisobenzofuran assay of complex 2 indicated photogeneration of singlet oxygen (ΦΔ : 0.36) as reactive oxygen species (ROS). Light irradiation caused only minor extent of ligand release forming chemo-active cisplatin analogue. The complex showed ~70-100 fold enhancement in cytotoxicity on light exposure in A549 lung cancer cells and MDA-MB-231 multidrug resistant breast cancer cells, giving half maximal inhibitory concentration (IC50 ) of 0.9-1.8 µM. Confocal imaging showed its mitochondrial localization and complex 2 exhibited anti-metastasis properties. Immunostaining of ß-tubulin and Annexin V-FITC/propidium iodide staining displayed complex 2 induced photo-selective microtubule rupture and cellular apoptosis, respectively.

Anti-microbial resistance and aging-A design for evolution.

The emergence of resistance to anti-infective agents poses a significant threat to successfully treating infections caused by bacteria. Bacteria acquire random mutations due to exposure to environmental stresses, which may increase their fitness to other selection pressures. Interestingly, for bacteria, the frequency of anti-microbial resistance (AMR) seems to be increasing in tandem with the human lifespan. Based on evidence from previous literature, we speculate that increased levels of free radicals (Reactive Oxygen Species-ROS and Reactive Nitrosative Species-RNS), elevated inflammation, and the altered tissue microenvironment in aged individuals may drive pathogen mutagenesis. If these mutations result in the hyperactivation of efflux pumps or alteration in drug target binding sites, it could confer AMR, thus rendering antibiotic therapy ineffective while leading to the selection of novel drug-resistant variants. This article is categorized under: Immune System Diseases > Genetics/Genomics/Epigenetics Infectious Diseases > Environmental Factors Metabolic Diseases > Environmental Factors.

Sequestration of histidine kinases by non-cognate response regulators establishes a threshold level of stimulation for bacterial two-component signaling.

Bacterial two-component systems (TCSs) consist of a sensor histidine kinase (HK) that perceives a specific signal, and a cognate response regulator (RR) that modulates the expression of target genes. Positive autoregulation improves TCS sensitivity to stimuli, but may trigger disproportionately large responses to weak signals, compromising bacterial fitness. Here, we combine experiments and mathematical modelling to reveal a general design that prevents such disproportionate responses: phosphorylated HKs (HK~Ps) can be sequestered by non-cognate RRs. We study five TCSs of Mycobacterium tuberculosis and find, for all of them, non-cognate RRs that show higher affinity than cognate RRs for HK~Ps. Indeed, in vitro assays show that HK~Ps preferentially bind higher affinity non-cognate RRs and get sequestered. Mathematical modelling indicates that this sequestration would introduce a 'threshold' stimulus strength for eliciting responses, thereby preventing responses to weak signals. Finally, we construct tunable expression systems in Mycobacterium bovis BCG to show that higher affinity non-cognate RRs suppress responses in vivo.

Asymmetric total synthesis of diosniponols A and B.

A concise asymmetric total synthesis of diosniponols A and B has been achieved based on an enantioselective Jacobsen kinetic resolution of racemic epoxide and the important 2,3-dihydro-4H-pyran-4-one moiety being installed by the metal-free δ-hydroxyalkynone rearrangement catalyzed by p-TsOH. A diastereoselective catalytic hydrogenation set the required all-syn stereochemistry leading to diosniponol A, which then, under the Mitsunobu inversion conditions, provided diosniponol B. The structure and absolute stereochemistry of the natural products were further confirmed.

ZMYM2 is essential for methylation of germline genes and active transposons in embryonic development.

ZMYM2 is a transcriptional repressor whose role in development is largely unexplored. We found that Zmym2-/- mice show embryonic lethality by E10.5. Molecular characterization of Zmym2-/- embryos revealed two distinct defects. First, they fail to undergo DNA methylation and silencing of germline gene promoters, resulting in widespread upregulation of germline genes. Second, they fail to methylate and silence the evolutionarily youngest and most active LINE element subclasses in mice. Zmym2-/- embryos show ubiquitous overexpression of LINE-1 protein as well as aberrant expression of transposon-gene fusion transcripts. ZMYM2 homes to sites of PRC1.6 and TRIM28 complex binding, mediating repression of germline genes and transposons respectively. In the absence of ZMYM2, hypermethylation of histone 3 lysine 4 occurs at target sites, creating a chromatin landscape unfavourable for establishment of DNA methylation. ZMYM2-/- human embryonic stem cells also show aberrant upregulation and demethylation of young LINE elements, indicating a conserved role in repression of active transposons. ZMYM2 is thus an important new factor in DNA methylation patterning in early embryonic development.