bims-toxgon Biomed News
on Toxoplasma gondii metabolism
Issue of 2024–12–08
twenty papers selected by
Lakesh Kumar, BITS Pilani



  1. Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi. 2024 Sep 20. 36(5): 542-547
      Toxoplasma gondii is an obligatory intracellular parasite which infects a variety of warm-blooded animals and causes toxoplasmosis. Toxoplasmosis seriously endangers human health and animal husbandry production. As one of the effective gene editing tools, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been widely used for knockout of genes in T. gondii. This review summarizes the applications of the CRISPR/Cas9 technology in vaccines against single- and double-gene deletion strains of T. gondii, so as to provide insights into development of toxoplasmosis vaccines.
    Keywords:  CRISPR/Cas9 technology; Gene-deletion strain; Toxoplasma gondii; Vaccine
    DOI:  https://doi.org/10.16250/j.32.1374.2024026
  2. Parasitol Res. 2024 Dec 04. 123(12): 404
      The mitochondrial permeability transition pore (mPTP) significantly impacts mitochondrial responses to cell death signals through its structural opening. Cyclophilin D (CypD) serves as a key regulator of the mPTP and plays a pivotal role in governing mitochondrial responses to cell death. In this study, we have demonstrated that Toxoplasma expresses a homolog of cyclophilin D, named TgCypD, which is localized in the mitochondria. Depletion of TgCypD resulted in a modest inhibition of tachyzoite invasion and proliferation, with no notable effect on mitochondrial morphology. However, TgCypD deficiency led to the inhibition of cytochrome c release from mitochondria into the cytosol, thereby imparting resistance to oxidative stress-induced cell death. Our findings suggest that T. gondii contains the mPTP component protein TgCypD, which is intricately involved in regulating mitochondrial responses to cell death.
    Keywords:   Toxoplasma gondii ; Cyclophilin D; Mitochondrial permeability transition pore (mPTP); Oxidative stress damage response
    DOI:  https://doi.org/10.1007/s00436-024-08412-w
  3. iScience. 2024 Dec 20. 27(12): 111312
      Toxoplasma gondii (T. gondii) is an intracellular parasite, and its regulation of host cell apoptosis directly affects its parasitism. Studies link T. gondii-induced apoptosis to abnormal expression of mammalian STE20-like protein kinase 2 (MST2), but its precise role remains unclear. In this study, the regulatory roles in apoptosis and pathogenicity of T. gondii infection were identified in vitro and in vivo. Simultaneously, MST2 and Hippo signaling pathway activation induced by T. gondii were evaluated. MST2 overexpression and knockout were used to assess its regulatory role in apoptosis and Hippo signaling pathway. Results showed that T. gondii induced apoptosis and lung damage, with Hippo signaling pathway activation via MST2 phosphorylation. MST2 was demonstrated to regulate apoptosis and Hippo signaling pathway. Notably, MST2 knockout hindered the T. gondii-induced apoptosis and weakened Hippo signaling pathway activation. MST2 is an important target for T. gondii to control host cell fate and modulate immune responses.
    Keywords:  Microbiology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2024.111312
  4. Parasites Hosts Dis. 2024 Nov;62(4): 408-423
      Toxoplasma gondii primarily invades the central nervous system, causing latent infections. Cysts persist in the host for life and there is currently no effective treatment. T. gondii infects human hosts through contaminated meat, invading the intestinal tissue and leading to changes in the number and composition of the gut microbiota. Since probiotic ingestion modulates intestinal microbiota changes, we hypothesized that intestinal microbiota dysbiosis caused by T. gondii infection would be restored following probiotic supplementation. To this end, we orally infected C57BL/6 mice with 10 T. gondii cysts and administered supplemental probiotics daily. We analyzed the levels of T. gondii B1 gene DNA, indicative of T. gondii infection, in brain tissue. We investigated alterations in the gut microbiota composition and functional pathways between the probiotic and non-probiotic treatment groups via next-generation sequencing analysis of each fecal sample. The infection level in the probiotic-treated group was significantly reduced after 4 weeks (p<0.05). Probiotic supplementation notably changed the gut microbiota after 2 weeks of infection, increasing the relative abundance of Intestinimonas massiliensis and Lawsonibacter asaccharolyticus. Probiotic supplements appear to modulate the gut microbiota, activating functional pathways involved in intestinal short-chain fatty acid production and strengthening the intestinal barrier, thereby impeding T. gondii infection and subsequent proliferation. Our findings provide valuable insights into T. gondii infection control and future study directions.
    Keywords:  Toxoplasma gondii; microbiota; next-generation sequencing; probiotics
    DOI:  https://doi.org/10.3347/PHD.24068
  5. Int J Parasitol Parasites Wildl. 2024 Dec;25 101017
      Toxoplasma gondii is a ubiquitous parasite causing significant mortality in captive wildlife, especially marsupials. Historically, treatment has been unrewarding and no vaccine was available. An intranasal vaccine based on purified inactivated T. gondii was developed for toxoplasmosis prevention. A vaccination campaign started in early 2017 and was successful in preventing toxoplasma-related mortality in marsupials in many European and South American zoos. Amongst the vaccinated wallabies, about 30% were T. gondii seropositive before the vaccination, and no toxoplasma-related deaths were observed since the administration of the vaccine. The objective of this case study was to assess the potential effect of the vaccination on a seropositive wallaby. It is important to note that this vaccine doesn't induce any humoral response in sheep, and squirrel monkeys but induces a strong T-cell response. A T. gondii seropositive Yellow-footed rock wallaby (Petrogale xanthopus) from Copenhagen Zoo received two doses of the aforementioned intranasal vaccine. Blood samples were collected before each vaccination and used for peripheral blood mononuclear cell isolation. The impact of the vaccination on the lymphocyte phenotype was characterized by flow cytometry. Cell size, represented by forward scatter, and granularity, represented by side scatter parameters were analyzed. Two doses of the vaccine induced a respective 30.1 and 25.6% increase in cell size and granularity in lymphocytes stimulated with T. gondii antigens, as assessed by flow cytometry. These changes were likely correlated with T-cell activation, which indicates that two doses of the vaccine might have boosted the already-existing T-cell memory response against T. gondii in a seropositive animal. No morphological changes were observed in lymphocytes from an unvaccinated seronegative wallaby. This is the first documented case of boosting an already-existing cellular immune response against toxoplasmosis by the vaccine in a seropositive Yellow-footed rock wallaby.
    Keywords:  Inactivated vaccine; Marsupials; Nanoparticles; T. gondii; Toxoplasmosis; Wallabies
    DOI:  https://doi.org/10.1016/j.ijppaw.2024.101017
  6. Parasites Hosts Dis. 2024 Nov;62(4): 476-483
      Allyl isothiocyanate (AITC) is a natural product commonly used in food preservation and pharmaceutical applications. Toxoplasmosis, caused by the protozoan pathogen Toxoplasma gondii, is prevalent globally while the impact of AITC on toxoplasmosis is unclear. We explored the effect of AITC on acute toxoplasmosis. We infected C57BL/6 mice with T. gondii type I RH strain following AITC administration. On the 4th day after infection, which corresponds to the initial stage of infection, we collected serum for the determination of inflammatory cytokine levels. The mice serum of the AITC-administered group contained significantly lower levels of granulocyte colony-stimulating factor, interferon-gamma, interleukin (IL)-23 subunit p19, IL-4, IL-6, and monocyte chemoattractant protein-1. The lifespan of the mice in the AITC-administered group was significantly reduced. In vitro experiments showed that AITC promoted the proliferation of intracellular T. gondii accompanied by the inhibition of IL-4, IL-1β, and IL-6 production in RAW264.7 macrophages. Our results showed that AITC facilitated T. gondii infection in the early stage by inhibiting the production of several inflammatory cytokines.
    Keywords:  Toxoplasma gondii; acute toxoplasmosis; allyl isothiocyanate; inflammatory cytokine; pathogen
    DOI:  https://doi.org/10.3347/PHD.24054
  7. Elife. 2024 Dec 05. pii: RP92499. [Epub ahead of print]12
      The environmental challenges the human malaria parasite, Plasmodium falciparum, faces during its progression into its various lifecycle stages warrant the use of effective and highly regulated access to chromatin for transcriptional regulation. Microrchidia (MORC) proteins have been implicated in DNA compaction and gene silencing across plant and animal kingdoms. Accumulating evidence has shed light on the role MORC protein plays as a transcriptional switch in apicomplexan parasites. In this study, using the CRISPR/Cas9 genome editing tool along with complementary molecular and genomics approaches, we demonstrate that PfMORC not only modulates chromatin structure and heterochromatin formation throughout the parasite erythrocytic cycle, but is also essential to the parasite survival. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) experiments suggests that PfMORC binds to not only sub-telomeric regions and genes involved in antigenic variation but may also play a role in modulating stage transition. Protein knockdown experiments followed by chromatin conformation capture (Hi-C) studies indicate that downregulation of PfMORC impairs key histone marks and induces the collapse of the parasite heterochromatin structure leading to its death. All together these findings confirm that PfMORC plays a crucial role in chromatin structure and gene regulation, validating this factor as a strong candidate for novel antimalarial strategies.
    Keywords:  P. falciparum; chromatin; chromosomes; epigenetic; gene expression; gene regulation; infectious disease; malaria; microbiology
    DOI:  https://doi.org/10.7554/eLife.92499
  8. Cancer Drug Resist. 2024 ;7 46
      The emergence of drug resistance leading to cancer recurrence is one of the challenges in the treatment of cancer patients. Several mechanisms can lead to drug resistance, including epigenetic changes. Histone deacetylases (HDACs) play a key role in chromatin regulation through epigenetic mechanisms and are also involved in drug resistance. The control of histone acetylation and the accessibility of regulatory DNA sequences such as promoters, enhancers, and super-enhancers are known mechanisms by which HDACs influence gene expression. Other targets of HDACs that are not histones can also contribute to resistance. This review describes the contribution of HDACs to the mechanisms that, in some cases, may determine resistance to chemotherapy or other cancer treatments.
    Keywords:  DNA damage; HDACs; PARP; acetylation; chemotherapy; hormone deprivation; palbociclib
    DOI:  https://doi.org/10.20517/cdr.2024.103
  9. J Biol Chem. 2024 Dec 03. pii: S0021-9258(24)02551-1. [Epub ahead of print] 108049
      The proteasome is considered an excellent drug target for many infectious diseases as well as cancer. Challenges with robust and safe supply of proteasomes from infectious agents, lack of structural information and complex pharmacology due to multiple active sites have hampered progress in the infectious disease space. We recombinantly expressed the proteasome of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease, and demonstrate pharmacological equivalence to the native T. cruzi proteasome. Active-site mutant recombinant proteasomes reveal substrate promiscuity for wild-type proteasomes, with important implications for assessing pharmacological responses of active-site selective inhibitors. Using these mutant proteasomes, we show that some selective parasite proteasome inhibitors only partially inhibit the chymotrypsin-like activity, including a newly developed 5-(phenoxymethyl)furan-2-carboxamide-based proteasome inhibitor. In spite of partial inhibition, these compounds remain potent inhibitors of intracellular T. cruzi growth. Drug-resistant mutants provide further insights in drug mode-of-inhibition. We also present the high-resolution CryoEM structures of both native and recombinantly-expressed T. cruzi proteasomes which reveal pharmacologically-relevant differences in the ligand-binding site compared to the related Leishmania proteasome. Furthermore, we show that the trypanosomatid β4/β5 selectivity pocket is not present in the proteasome structures of other protozoan parasites. This work highlights the need, and provides approaches, to precisely assess proteasome substrate selectivity and pharmacology. It enables structure-guided drug discovery for this promising Chagas disease drug target, provides a new chemical starting point for drug discovery, and paves the road for development of robust proteasome drug discovery programmes for other eukaryotic infectious diseases.
    Keywords:  Chagas disease; drug discovery; enzyme inhibitor; enzyme structure; molecular pharmacology; proteasome; recombinant protein expression; substrate specificity
    DOI:  https://doi.org/10.1016/j.jbc.2024.108049
  10. Sci Adv. 2024 Dec 06. 10(49): eadp1840
      Conserved type B histone acetyltransferases are recognized for their role in acetylating newly synthesized histones in the cytoplasm of eukaryotes. However, their involvement in regulating chromatin within the nucleus remains unclear. Our study shows that the Arabidopsis thaliana type B histone acetyltransferase HAG2 interacts with the histone chaperones MSI2, MSI3, and NASP, as well as the histones H3 and H4, forming a complex in both the cytoplasm and the nucleus. Within this complex, HAG2 and MSI2/3 constitute a histone acetylation module essential for acetylating histone H4 in the cytoplasm. Furthermore, this module works together with NASP to regulate histone acetylation, chromatin accessibility, and gene transcription in the nucleus. This complex enhances chromatin accessibility near transcription start sites while reducing accessibility near transcription termination sites. Our findings reveal a distinct role for the Arabidopsis type B histone acetyltransferase in the nucleus, shedding light on the coordination between cytoplasmic histone acetylation and nuclear chromatin regulation in plants.
    DOI:  https://doi.org/10.1126/sciadv.adp1840
  11. Sci Rep. 2024 12 03. 14(1): 30043
      ATP citrate lyase (ACLY) and acyl-CoA short-chain synthetases 2 (ACSS2) are key enzymes in lipid metabolism. We explored the role of ACLY in gastric cancer (GC) and the effect of ACLY and ACSS2 compensation on GC growth. We used immunohistochemistry to verify the expression level of ACLY in GC, shRNA to stably knock down the expression level of ACLY in GC cells. The expression levels of lipid metabolizing enzymes were verified by qPCR and WB, and targeted lipidomics and quantification of lipid metabolism-related indicators helped us to understand the changes in lipid metabolism. Finally, subcutaneous graft tumors validate our findings from in vitro experiments. ACLY is upregulated in GC tissues, downregulation of ACLY reduced lipid accumulation and inhibited GC proliferation, migration, and invasion in vitro. ACSS2 maintains cell growth by compensatory elevation to maintain fatty acid synthesis activity in ACLY-depleted GC cells. Inhibition of ACSS2 enhanced the inhibitory effect of downregulation of ACLY on the growth of transplanted tumors in nude mice. Downregulation of ACLY inhibited GC cell growth in vitro and in vivo. ACSS2 was compensated to increase to maintain cell growth in ACLY-depleted GC cells.
    Keywords:  ACLY; ACSS2; Gastric cancer; Lipid metabolism; Treatment
    DOI:  https://doi.org/10.1038/s41598-024-81448-1
  12. Biochim Biophys Acta Bioenerg. 2024 Nov 28. pii: S0005-2728(24)00499-7. [Epub ahead of print]1866(2): 149529
      Alternative NADH dehydrogenase, also known as type II NADH dehydrogenase (NDH-2), catalyzes the same redox reaction as mitochondrial respiratory chain complex I. Specifically, it oxidizes reduced nicotinamide adenine dinucleotide (NADH) while simultaneously reducing ubiquinone to ubiquinol. However, unlike complex I, this enzyme is non-proton pumping, comprises of a single subunit, and is resistant to rotenone. Initially identified in bacteria, fungi and plants, NDH-2 was subsequently discovered in protists and certain animal taxa including sea squirts. The gene coding for NDH-2 is also present in the genomes of some annelids, tardigrades, and crustaceans. For over two decades, NDH-2 has been investigated as a potential substitute for defective complex I. In model organisms, NDH-2 has been shown to ameliorate a broad spectrum of conditions associated with complex I malfunction, including symptoms of Parkinson's disease. Recently, lifespan extension has been observed in animals expressing NDH-2 in a heterologous manner. A variety of mechanisms have been put forward by which NDH-2 may extend lifespan. Such mechanisms include the activation of pro-longevity pathways through modulation of the NAD+/NADH ratio, decreasing production of reactive oxygen species (ROS) in mitochondria, or then through moderate increases in ROS production followed by activation of defense pathways (mitohormesis). This review gives an overview of the latest research on NDH-2, including the structural peculiarities of NDH-2, its inhibitors, its role in the pathogenicity of mycobacteria and apicomplexan parasites, and its function in bacteria, fungi, and animals.
    Keywords:  Hypoxia/reoxygenation; Mitochondrial diseases; Quinolones; Rossmann fold; Type II NADH dehydrogenase; iron‑sulfur clusters
    DOI:  https://doi.org/10.1016/j.bbabio.2024.149529
  13. Front Immunol. 2024 ;15 1495221
      Histones play crucial roles in both promoting and repressing gene expression, primarily regulated through post-translational modifications (PTMs) at specific amino acid residues. Histone PTMs, including methylation, acetylation, ubiquitination, phosphorylation, lactylation, butyrylation, and propionylation, act as important epigenetic markers. These modifications influence not only chromatin compaction but also gene expression. Their importance extends to the treatment and prevention of various human diseases, particularly cancer, due to their involvement in key cellular processes. Abnormal histone modifications and the enzymes responsible for these alterations often serve as critical drivers in tumor cell proliferation, invasion, apoptosis, and stemness. This review introduces key histone PTMs and the enzymes responsible for these modifications, examining their impact on tumorigenesis and cancer progression. Furthermore, it explores therapeutic strategies targeting histone PTMs and offers recommendations for identifying new potential therapeutic targets.
    Keywords:  HDAC; HMTs; KDMs; acetylation; cancer; histone PTMs; methylation
    DOI:  https://doi.org/10.3389/fimmu.2024.1495221
  14. Exp Mol Med. 2024 Dec 04.
      TP53-induced glycolysis and apoptosis regulator (TIGAR) regulates redox homeostasis and provides the intermediates necessary for cell growth by reducing the glycolytic rate. During cellular senescence, cells undergo metabolic rewiring towards the glycolytic pathway, along with the development of the senescence-associated secretory phenotype (SASP), also known as the secretome. We observed that TIGAR expression increased during replicative senescence following the in vitro expansion of human mesenchymal stromal cells (MSCs) and that TIGAR knockout (KO) decreased SASP factors and triggered premature senescence with decelerated progression. Additionally, TIGAR KO impaired flexible lysosomal movement to the perinuclear region and decreased the autophagic flux of MSCs. Research on the mechanism of lysosomal movement revealed that, while native senescent MSCs presented low levels of Ac-α-tubulin (lysine 40) and increased sirtuin 2 (SIRT2) activity compared with those in growing cells, TIGAR KO-MSCs maintained Ac-α-tubulin levels and exhibited decreased SIRT2 activity despite being in a senescent state. The overexpression of SIRT2 reduced Ac-α-tubulin as a protein target of SIRT2 and induced the positioning of lysosomes at the perinuclear region, restoring the cytokine secretion of TIGAR KO-MSCs. Furthermore, TIGAR expression was positively correlated with SIRT2 activity, indicating that TIGAR affects SIRT2 activity partly by modulating the NAD+ level. Thus, our study demonstrated that TIGAR provides a foundation that translates the regulation of energy metabolism into lysosome positioning, affecting the secretome for senescence development. Considering the functional value of the cell-secretome in aging-related diseases, these findings suggest the feasibility of TIGAR for the regulation of secretory phenotypes.
    DOI:  https://doi.org/10.1038/s12276-024-01362-4
  15. Parasit Vectors. 2024 Dec 02. 17(1): 497
       BACKGROUND: People can acquire Toxoplasma gondii infection by ingestion of sporulated oocysts passed in cat feces; whether this route is common in cats is unknown. The primary objectives of this study were to (a) adapt a commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of T. gondii tachyzoite IgG antibodies in feline sera to detect T. gondii sporozoite IgG antibodies, (b) utilize the ELISA to confirm that exposed cats can mount an antibody response to sporozoites, (c) estimate the prevalence of sporozoite antibodies in naturally exposed cats, and (d) evaluate associations between the serologic status of naturally exposed cats and clinical signs that could be caused by toxoplasmosis.
    METHODS: To generate positive control sera, three male cats were orally inoculated with approximately 100,000 sporulated oocysts of the ME49 strain of T. gondii. A human antisporozoite antibody ELISA was then adapted for use with cat sera. Detectable levels of antisporozoite IgG were found in two of the three experimentally inoculated cats. The sera of 100 healthy cats and 295 clinically ill cats were assessed in the prototype sporozoite ELISA and a commercially available tachyzoite ELISA.
    RESULTS: The ELISA estimated that prevalence of antisporozoite IgG was 2% in healthy cats and 3.1% in clinically ill cats; in contrast, the overall estimated prevalence of antitachyzoite IgG was 15%. Only two of 395 cats (0.5%) had both antisporozoite and antitachyzoite IgG.
    CONCLUSIONS: While experimentally infected and naturally exposed cats developed antisporozoite antibodies, the low prevalence did not allow for the evaluation of associations among clinical signs.
    Keywords:   Toxoplasma gondii ; Cat; Oocyst; Sporozoite
    DOI:  https://doi.org/10.1186/s13071-024-06553-6
  16. PLoS One. 2024 ;19(12): e0311691
      Chromosome movement speeds during anaphase are regulated by depolymerization of microtubules. Several models describe chromosome movement during cell division but none of them consider post-translational modifications of tubulin, even though such modifications help specify microtubules for unique cellular activities. Among these modifications, acetylation of Lysine 40 is one of the common post-translational modifications. Acetylation of microtubules greatly improves their stability, especially when subjected to cooling or drug treatment. Since kinetochore microtubules are acetylated in a variety of eukaryote cells, we wondered whether deacetylation of kinetochore microtubules was necessary in order for microtubules to be able to depolymerize during anaphase. HDAC6 (Histone Deacetylase 6) deacetylates acetylated tubulin. To study whether tubulin must be deacetylated during anaphase, we added to living cells two different HDAC6 inhibitors (Tubacin and Trichostatin A), separately, as chromosomes moved poleward in anaphase. Both HDAC6 inhibitors altered chromosome movement: chromosomes either completely stopped moving, or moved more slowly, or sometimes continued movement without speed changes. The effects of the inhibitors on chromosome movement are reversible: half-bivalents either restarted anaphase movement by themselves before washing out the inhibitor or resumed their poleward movement after the inhibitor was washed out. We suggest that kinetochore microtubules need to be deacetylated in order for normal anaphase movements to occur.
    DOI:  https://doi.org/10.1371/journal.pone.0311691
  17. Biol Rev Camb Philos Soc. 2024 Dec 01.
      Parasites have a rich and long natural history among biological entities, and it has been suggested that parasites are one of the most significant factors in the evolution of their hosts. However, it has been emphasized less frequently how co-evolution has undoubtedly also shaped the paths of parasites. It may seem safe to assume that specific differences among the array of potential hosts for particular parasites have restricted and diversified their evolutionary pathways and strategies for survival. Nevertheless, if one looks closely enough at host and parasite, one finds commonalities, both in terms of host defences and parasite strategies to out-manoeuvre them. While such analyses have been the source of numerous reviews, they are generally limited to interactions between, at most, one kingdom of parasite with two kingdoms of host (e.g. similarities in animal and plant host responses against fungi). With the aim of extending this view, we herein critically evaluate the similarities and differences across all four eukaryotic host kingdoms (plants, animals, fungi, and protists) and their parasites. In doing so, we show that hosts tend to share common strategies for defence, including both physical and behavioural barriers, and highly evolved immune responses, in particular innate immunity. Parasites have, similarly, evolved convergent strategies to counter these defences, including mechanisms of active penetration, and evading the host's innate and/or adaptive immune responses. Moreover, just as hosts have evolved behaviours to avoid parasites, many parasites have adaptations to manipulate host phenotype, physiologically, reproductively, and in terms of behaviour. Many of these strategies overlap in the host and parasite, even across wide phylogenetic expanses. That said, specific differences in host physiology and immune responses often necessitate different adaptations for parasites exploiting fundamentally different hosts. Taken together, this review facilitates hypothesis-driven investigations of parasite-host interactions that transcend the traditional kingdom-based research fields.
    Keywords:  adaptive immunity; behavioural modification of hosts; innate immunity; parasite effectors; parasites
    DOI:  https://doi.org/10.1111/brv.13169
  18. Eng Microbiol. 2023 Dec;3(4): 100094
      The methylotrophic yeast Pichia pastoris (also known as Komagataella phaffii) is widely used as a yeast cell factory for producing heterologous proteins. Recently, it has gained attention for its potential in producing chemicals from inexpensive feedstocks, which requires efficient genetic engineering platforms. This review provides an overview of the current advances in developing genetic tools for metabolic engineering of P. pastoris. The topics cover promoters, terminators, plasmids, genome integration sites, and genetic editing systems, with a special focus on the development of CRISPR/Cas systems and their comparison to other genome editing tools. Additionally, this review highlights the prospects of multiplex genome integration, fine-tuning gene expression, and single-base editing systems. Overall, the aim of this review is to provide valuable insights into current genetic engineering and discuss potential directions for future efforts in developing efficient genetic tools in P. pastoris.
    Keywords:  CRISPR/Cas9; Genetic tools; Metabolic engineering; Pichia pastoris
    DOI:  https://doi.org/10.1016/j.engmic.2023.100094
  19. Proc Natl Acad Sci U S A. 2024 Dec 10. 121(50): e2412157121
      Proliferating tumor cells take up glutamine for anabolic processes, engendering glutamine deficiency in the tumor microenvironment. How this might impact immune cells is not well understood. Using multiple mouse models of soft tissue sarcomas, glutamine antagonists, as well as genetic and pharmacological inhibition of glutamine utilization, we found that the number and frequency of conventional dendritic cells (cDCs) is dependent on microenvironmental glutamine levels. cDCs comprise two distinct subsets-cDC1s and cDC2s, with the former subset playing a critical role in antigen cross-presentation and tumor immunity. While both subsets show dependence on glutamine, cDC1s are particularly sensitive. Notably, glutamine antagonism did not reduce the frequency of DC precursors but decreased the proliferation and survival of cDC1s. Further studies suggest a role of the nutrient sensing mechanistic target of rapamycin (mTOR) signaling pathway in this process. Taken together, these findings uncover glutamine dependence of cDC1s that is coopted by tumors to escape immune responses.
    Keywords:  dendritic cells; glutamine; tumor microenvironment
    DOI:  https://doi.org/10.1073/pnas.2412157121
  20. Chin J Dent Res. 2024 Dec 06. 27(4): 303-310
       OBJECTIVE: To assess the role of microtubule acetylation in the transportation of amorphous calcium phosphate (ACP)-containing vesicles that mediate the osteogenic differentiation process of rat bone mesenchymal stem cells (BMSCs).
    METHODS: Rat BMSCs were cultured and transfected with sirtuin 2 (SIRT2) overexpression plasmids for an in vitro model. The microtubule acetylation-related protein levels were detected by western blots. The microtubule acetylation and the secretion rate of extracellular ACPcontaining vesicles were observed with immunofluorescence and live cell fluorescence imaging. The secretion of ACP was observed by transmission electron microscopy. The mineralised nodule formation was stained with Alizarin Red S staining and observed by microscopy.
    RESULTS: Microtubule acetylation was increased during osteogenic differentiation of BMSCs, and microtubule transport efficiency was enhanced. Mechanically, microtubule acetylation is the key reason for the increased transportation rate of ACP-containing vesicles and enhanced osteogenic differentiation, as both were blocked after SIRT2-mediated microtubule acetylation inhibition.
    CONCLUSION: Microtubule acetylation mainly promotes the transportation and secretion of ACP vesicles, and ultimately promotes the osteogenic differentiation process.
    Keywords:  biomineralization; intracellular transport; microtubule acetylation; osteogenic differentiation
    DOI:  https://doi.org/10.3290/j.cjdr.b5860280