Immunosuppressive effects of morphine on macrophage polarization and function.

Macrophages play a pivotal role in safeguarding against a broad spectrum of infections, from viral, bacterial, fungal to parasitic threats and contributing to the immune defense against cancer. While morphine's immunosuppressive effects on immune cells are extensively documented, a significant knowledge gap exists regarding its influence on macrophage polarization and differentiation. Hence, we conducted a study that unveils that prior exposure to morphine significantly impedes the differentiation of bone marrow cells into macrophages. Furthermore, the polarization of macrophages toward the M1 phenotype under M1-inducing conditions experiences substantial impairment, as evidenced by the diminished expression of CD80, CD86, CD40, iNOS, and MHCII. This correlates with reduced expression of M1 phenotypical markers such as iNOS, IL-1ß, and IL-6, accompanied by noticeable morphological, size, and phagocytic alterations. Further, we also observed that morphine affected M2 macrophages. These findings emphasize the necessity for a more comprehensive understanding of the impact of morphine on compromising macrophage function and its potential ramifications for therapeutic approaches.

Hypoxia drives shared and distinct transcriptomic changes in two invasive glioma stem cell lines.

Glioblastoma (GBM) is the most common primary malignant cancer of the central nervous system. Insufficient oxygenation (hypoxia) has been linked to GBM invasion and aggression, leading to poor patient outcomes. Hypoxia induces gene expression for cellular adaptations. However, GBM is characterized by high intertumoral (molecular subtypes) and intratumoral heterogeneity (cell states), and it is not well understood to what extent hypoxia triggers patient-specific gene responses and cellular diversity in GBM. Here, we surveyed eight patient-derived GBM stem cell lines for invasion phenotypes in 3D culture, which identified two GBM lines showing increased invasiveness in response to hypoxia. RNA-seq analysis of the two patient GBM lines revealed a set of shared hypoxia response genes concerning glucose metabolism, angiogenesis, and autophagy, but also a large set of patient-specific hypoxia-induced genes featuring cell migration and anti-inflammation, highlighting intertumoral diversity of hypoxia responses in GBM. We further applied the Shared GBM Hypoxia gene signature to single cell RNA-seq datasets of glioma patients, which showed that hypoxic cells displayed a shift towards mesenchymal-like (MES) and astrocyte-like (AC) states. Interestingly, in response to hypoxia, tumor cells in IDH-mutant gliomas displayed a strong shift to the AC state, whereas tumor cells in IDH-wildtype gliomas mainly shifted to the MES state. This distinct hypoxia response of IDH-mutant gliomas may contribute to its more favorable prognosis. Our transcriptomic studies provide a basis for future approaches to better understand the diversity of hypoxic niches in gliomas.

The impact of aging-induced gut microbiome dysbiosis on dendritic cells and lung diseases.

Aging is an inevitable natural process that impacts every individual, and understanding its effect on the gut microbiome and dendritic cell (DC) functionality in elderly subjects is crucial. DCs are vital antigen-presenting cells (APCs) that orchestrate the immune response, maintaining immune tolerance to self-antigens and bridging innate and adaptive immunity. With aging, there is a shift toward nonspecific innate immunity, resulting in a decline in adaptive immune responses. This alteration raises significant concerns about managing the health of an elderly population. However, the precise impact of aging and microbiome changes on DC function and their implications in lung-associated diseases remain relatively understudied. To illuminate this subject, we will discuss recent advancements in understanding the connections between aging, gut dysbiosis, DCs, and lung diseases. Emphasizing the key concepts linking age-related gut microbiome changes and DC functions, we will focus on their relevance to overall health and immune response in elderly individuals. This article aims to improve our understanding of the intricate relationship between aging, gut microbiome, and DCs, potentially benefiting the management of age-associated diseases and promoting healthy aging.

Influence of chronic administration of morphine and its withdrawal on the behaviour of zebrafish.

Morphine is a potent analgesic opiate used to treat chronic pain, mostly in cancer patients. In addition, it is widely used as a drug of abuse. Due to the continuous rise of morphine-associated addiction, there is an urgent need to develop pre-clinical animal models to understand the behavioural pattern of drug dependence and its withdrawal. Recently, the experimental use of zebrafish has attained significance in behavioural neuroscience studies. The literature on zebrafish is conflicting with regard to morphine withdrawal symptoms. Unfortunately, no single model provides comprehensive details to evaluate zebrafish behaviour on opiate exposure. Further, the current models have various limitations, such as short duration, complexity of phenotypes, intricate quantification, and difficulty in studying withdrawal symptoms. Consequently, a firm standardization of the protocol to understand the influence of opiates on physiological and psychological behaviours is required. In this study, we have tried to overcome the shortcomings associated with the existing models and to optimize the protocols involving an array of parameters. We observed that the administration of morphine caused a significant increase in zebrafish behavioural patterns of spiral movements, circular movements, erratic movements, upper transitions, water surface transitions, wall licking, wall licking with upper transitions, wall licking with lower transitions, absolute angle changes, and time spent in the upper compartment. A decline in the freezing bouts and time spent in the lower compartment were noticed. In essence, this study offers a zebrafish model to comprehensively examine changes in behaviour of animals on opiate dependence and its withdrawal. The present study also reported that in zebrafish, the influence of chronic exposure of morphine modulates key gene targets involved in behaviour, neuroinflammation, and autophagy, which directly or indirectly are associated with morphine addiction in a chronic morphine model.

Sulfonyl Diazaborine 'Click' Chemistry Enables Rapid and Efficient Bioorthogonal Labeling.

Finding an ideal bioorthogonal reaction that responds to a wide range of biological queries and applications is of great interest in biomedical applications. Rapid diazaborine (DAB) formation in water by the reactions of ortho-carbonyl phenylboronic acid with α-nucleophiles is an attractive conjugation module. Nevertheless, these conjugation reactions demand to satisfy stringent criteria for bioorthogonal applications. Here we show that widely used sulfonyl hydrazide (SHz) offers a stable DAB conjugate by combining with ortho-carbonyl phenylboronic acid at physiological pH, competent for an optimal biorthogonal reaction. Remarkably, the reaction conversion is quantitative and rapid (k2 >103  M-1 s-1 ) at low micromolar concentrations, and it preserves comparable efficacy in a complex biological milieu. DFT calculations support that SHz facilitates DAB formation via the most stable hydrazone intermediate and the lowest energy transition state compared to other biocompatible α-nucleophiles. This conjugation is extremely efficient on living cell surfaces, enabling compelling pretargeted imaging and peptide delivery. We anticipate this work will permit addressing a wide range of cell biology queries and drug discovery platforms exploiting commercially available sulfonyl hydrazide fluorophores and derivatives.

Targeting dendritic cells with TLR-2 ligand-coated nanoparticles loaded with Mycobacterium tuberculosis epitope induce antituberculosis immunity.

Novel vaccination strategies are crucial to efficiently control tuberculosis, as proposed by the World Health Organization under its flagship program "End TB Strategy." However, the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), particularly in those coinfected with HIV-AIDS, constitutes a major impediment to achieving this goal. We report here a novel vaccination strategy that involves synthesizing a formulation of an immunodominant peptide derived from the Acr1 protein of Mtb. This nanoformulation in addition displayed on the surface a toll-like receptor-2 ligand to offer to target dendritic cells (DCs). Our results showed an efficient uptake of such a concoction by DCs in a predominantly toll-like receptor-2-dependent pathway. These DCs produced elevated levels of nitric oxide, proinflammatory cytokines interleukin-6, interleukin-12, and tumor necrosis factor-α, and upregulated the surface expression of major histocompatibility complex class II molecules as well as costimulatory molecules such as CD80 and CD86. Animals injected with such a vaccine mounted a significantly higher response of effector and memory Th1 cells and Th17 cells. Furthermore, we noticed a reduction in the bacterial load in the lungs of animals challenged with aerosolized live Mtb. Therefore, our findings indicated that the described vaccine triggered protective anti-Mtb immunity to control the tuberculosis infection.

Fiction and Facts about BCG Imparting Trained Immunity against COVID-19.

The Bacille Calmette-Guérin or BCG vaccine, the only vaccine available against Mycobacterium tuberculosis can induce a marked Th1 polarization of T-cells, characterized by the antigen-specific secretion of IFN-γ and enhanced antiviral response. A number of studies have supported the concept of protection by non-specific boosting of immunity by BCG and other microbes. BCG is a well-known example of a trained immunity inducer since it imparts 'non-specific heterologous' immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the recent pandemic. SARS-CoV-2 continues to inflict an unabated surge in morbidity and mortality around the world. There is an urgent need to devise and develop alternate strategies to bolster host immunity against the coronavirus disease of 2019 (COVID-19) and its continuously emerging variants. Several vaccines have been developed recently against COVID-19, but the data on their protective efficacy remains doubtful. Therefore, urgent strategies are required to enhance system immunity to adequately defend against newly emerging infections. The concept of trained immunity may play a cardinal role in protection against COVID-19. The ability of trained immunity-based vaccines is to promote heterologous immune responses beyond their specific antigens, which may notably help in defending against an emergency situation such as COVID-19 when the protective ability of vaccines is suspicious. A growing body of evidence points towards the beneficial non-specific boosting of immune responses by BCG or other microbes, which may protect against COVID-19. Clinical trials are underway to consider the efficacy of BCG vaccination against SARS-CoV-2 on healthcare workers and the elderly population. In this review, we will discuss the role of BCG in eliciting trained immunity and the possible limitations and challenges in controlling COVID-19 and future pandemics.

Hypoxia-induced miR-210-3p expression in lung adenocarcinoma potentiates tumor development by regulating CCL2-mediated monocyte infiltration.

In most cancers, tumor hypoxia downregulates the expression of C-C motif chemokine 2 (CCL2), and this downregulation has been implicated in monocyte infiltration and tumor progression; however, the molecular mechanism is yet not clear. We compared non-cancerous and lung-adenocarcinoma human samples for hypoxia-inducible factor 1-alpha (HIF-1A), microRNA-210-3p (mir-210-3p) and CCL2 levels. Mechanistic studies were performed on lung adenocarcinoma cell lines and 3D tumor spheroids to understand the role of hypoxia-induced miR-210-3p in the regulation of CCL2 expression and macrophage polarization. HIF-1 A stabilization increases miR-210-3p levels in lung adenocarcinoma and impairs monocyte infiltration by inhibiting CCL2 expression. Mechanistically, miR-210-3p directly binds to the 3'untranslated region (UTR) of CCL2 mRNA and silences it. Suppressing miR-210-3p substantially downregulates the effect of hypoxia on CCL2 expression. Monocyte migration is significantly hampered in miR-210-3p mimic-transfected HIF-1A silenced cancer cells. In contrast, inhibition of miR-210-3p in HIF-1A-overexpressed cells markedly restored monocyte migration, highlighting a direct link between miR-210-3p level and tumor monocyte burden. Moreover, miR-210-3p inhibition in 3D tumor spheroids promotes monocyte recruitment and skewing towards an anti-tumor M1 phenotype. Anti-hsa-miR-210-3p-locked nucleic acid (LNA) delivery in a lung tumor xenograft zebrafish model caused tumor regression, suggesting that miR-210-3p could be a promising target for immunomodulatory therapeutic strategies against lung adenocarcinoma.

Studies on efficient production of a novel l-asparaginase by a newly isolated Pseudomonas resinovorans IGS-131 and its heterologous expression in Escherichia coli.

In the current study, the production of novel glutaminase free l-asparaginase from a new microbial source (Pseudomonas resinovorans IGS-131) is reported. Optimization of l-asparaginase production using conventional and statistical optimization techniques resulted in an enzyme yield of 37.63 IU/mL, which was 3.45-fold higher than the initial enzyme activity (i.e., 10.91 IU/mL). l-Asparaginase production from P. resinovorans IGS-131 was successfully carried out at the bioreactor level and investigations on the effect of agitation rates showed a maximum asparaginase yield of 38.88 IU/mL after 24 h fermentation at 400 rpm. The l-asparaginase gene from this source, showing 78% identity with a reported sequence in GenBank, was expressed in Escherichia coli rosetta DE3. The molecular weight of the recombinant protein was determined as 35.6 kDa. Downstream processing of recombinant l-asparaginase resulted in a purified protein concentration of 62.53 mg/L, which showed good free radical scavenging activity of 62%. The current findings provide promising results for a process of l-asparaginase production from P. resinovorans IGS-131. Furthermore, the recombinant production of this enzyme could help in avoiding the complexity of down streaming processes associated with the purification of this enzyme from wild-type organisms.