Published online Jul PMID: Etienne Dague, Editor. Author information Article notes Copyright and License information Disclaimer. Received Mar 27; Accepted Jul 8. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
This article has been cited by other articles in PMC. Go to:. S1 Tables: Table A. S1 Table: RNAseq data. S1 Movie: Z-axis scan of an oil-in-water emulsions made with stationary L.
S2 Movie: Z-axis scan of an oil-in-water emulsions made with stationary L. Lactococcus lactis can stabilize oil-in-water emulsions In an earlier characterization of L. Open in a separate window. Fig 1. Emulsification of petroleum by L. Cell aggregation influences cell emulsification properties Pickering emulsification of petroleum was easily done with strain NCDO, however, with sunflower seed oil, which was free from natural emulsifiers, no or only little emulsification was observed Fig 2.
Fig 2. Emulsification of sunflower seed oil as a result of L. Transcriptome response of L. Fig 3. Oil-in-water emulsion stabilized by L. Fig 4. Clustering of RNAseq data based on normalized counts of all expressed genes in L.
Table 2 Differential gene expression in L. Lipid transport and metabolism Q. G Carbohydrate transport and metabolism. P Inorganic ion transport and metabolism. Bacterial strains, growth conditions and medium Bacterial strains and plasmids are presented in Table A in S1 Tables. Aggregation measurements Cell from overnight cultures were harvested by centrifuging at x g for 3 min in 50 ml tubes, washed twice with phosphate buffer 10 mM, pH 6.
Data analysis Each sample had on average 9. S1 Tables Table A. PDF Click here for additional data file. S1 Table RNAseq data. XLSX Click here for additional data file. TIF Click here for additional data file. S1 Movie Z-axis scan of an oil-in-water emulsions made with stationary L. MP4 Click here for additional data file. S2 Movie Z-axis scan of an oil-in-water emulsions made with stationary L.
Katsikogianni M, Missirlis YF. Concise review of mechanisms of bacterial adhesion to biomaterials and of techniques used in estimating bacteria-material interactions. Eur Cell Mater. Concise review of mechanisms of bacterial adhesion to biomaterial surfaces. J Biomed Mater Res. Navarre WW, Schneewind O. Surface proteins of Gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol Mol Biol Rev. Donlan RM.
Biofilms: microbial life on surfaces. Emerg Infect Dis. Decho A. Microbial biofilms in intertidal systems: an overview. Cont Shelf Res. Pili in Gram-positive bacteria: assembly, involvement in colonization and biofilm development.
Trends in Microbiology. Bacterial adhesion and biofilms on surfaces. Prog Nat Sci. Atlas RM. Microbial hydrocarbon degradation—bioremediation of oil spills. J Chem Technol Biotecnhnology. Das N, Chandran P. Microbial degradation of petroleum hydrocarbon contaminants: an overview. Biotechnol Res Int. Leroy F, De Vuyst L. Lactic acid bacteria as functional starter cultures for the food fermentation industry.
Trends Food Sci Technol. Tadros TF. Emulsion formation, stability, and rheology. Emuls Form Stab. Stabilization of oil-water emulsions by hydrophobic bacteria. Appl Environ Microbiol. Rosenberg M. Bacterial adherence to hydrocarbons: a useful technique for studying cell surface hydrophobicity. Dickinson E. Food emulsions and foams: stabilization by particles.
Curr Opin Colloid Interface Sci. Elsevier B. The role of bacterial cell wall hydrophobicity in adhesion. Firoozmand H, Rousseau D. Microbial cells as colloidal particles: Pickering oil-in-water emulsions stabilized by bacteria and yeast. Food Res Int. The most important sex hormones in men are androgens, which are involved in maintaining the normal functions of multiple tissues and organs in human physiology 3.
In older men, in addition to their effects on the reproductive system, low levels of androgens have been reported to increase the risk for many aging-related diseases in other systems, such as tumors, diabetes mellitus and cardiovascular diseases 4 , 5. Therefore, androgens are vital to older men.
Androgens mainly include testosterone produced by Leydig cells, the testosterone metabolite dihydrotestosterone DHT , adrenal-derived dehydroepiandrosterone DHEA , androstenedione, androstenediol, and androstenone. The brain can synthesize neurosteroids using peripheral sex hormones, which can also be synthesized de novo by using cholesterol in the brain 6 , 7. Androgens can improve the biological activity of cells by increasing the production of ATP in the mitochondria and can also increase antioxidant activity to regulate redox homeostasis, thereby exerting a neuroprotective effect 9 , The cerebral vasculature is also the target of androgens, which increase vascular tone, enhance angiogenesis and cerebral vascular remodeling, reduce vascular damage by attenuating oxidative stress, and maintain the integrity of the blood—brain barrier, thereby exerting additional neuroprotective effects In the hippocampus of the brain, testosterone can improve synaptic plasticity and prevent neuronal cell death.
DHT induces circuit modifications by changing the number of excitatory spine synapses in a paracrine manner, which in turn affects the cognitive function of the brain Cognitive function is the result of high-level neural activity in the human brain. It mainly involves verbal, spatial and memory ability Therefore, the manifestations of cognitive impairment are diverse and often appear in multiple forms.
Among women, although androgen is not main sex hormone, it has been demonstrated that androgen deficiency is closely related to cognitive impairment For elderly men, in addition to brain aging causing cognitive dysfunction 15 , low androgen levels caused by aging can further aggravate cognitive decline in the brain Therefore, in older men, low levels of androgens and cognitive impairment also have a close relationship.
This article will objectively discuss the relationship between androgens and cognitive function in older men, potential mechanisms, and the roles of testosterone supplementation in improving cognition. The narrative review was conducted on extracted and summarized findings from relevant literature by an electronic search of the PubMed databases.
Based on the aim of this narrative review, the findings published after were recurrently reviewed by considering title and abstract and evaluating full texts, and relevant studies were selected. Additional studies were also selected from the reference lists in selected studies.
Sex hormones are regulated and produced by the gonads through the gonadal hypothalamus—pituitary—testis axis in males. Any factors that affect the gonadal axis, including aging and gonadal-related diseases, can cause an imbalance in the levels of sex hormones, such as testosterone, DHEA, luteinizing hormone LH and follicle-stimulating hormone FSH.
Hypogonadism is a clinical syndrome due to androgen deficiency caused by failure of testicular function, and its incidence was Clinically, hypogonadism due to aging is called late onset hypogonadism. Therefore, androgen deficiency caused by aging cannot be ignored due to the high incidence in older men. Although aging can directly lead to cognitive decline, androgen deficiency is considered to be another important cause of cognitive decline in older men These changes have been related to declines in cognitive ability However, interestingly, only serum testosterone, DHT and calculated FT are associated with decreased cognitive function after correcting for confounding factors This finding demonstrated that androgen levels are closely related to cognitive impairment in elderly men.
Various types of androgen have been found to be associated with cognition function. It has been shown that the decrease in TT and FT levels in plasma, which is independent of the relevant clinical and biochemical factors, is associated with an increased risk for dementia in older men 22 , and the risk ratios of TT and FT for dementia were 1. A correlation between serum DHEA-S levels and working memory has been found to be strong in males Thus, androgen plays an important role in cognitive function, and low androgen levels are an important cause of cognitive impairment in older men.
SHBG is the main transport for testosterone and regulation of total testosterone bioactivity In older men, with increasing age, the level of SHBG increases.
Interestingly, it has been found that in aging males, decreased bioavailable testosterone BT , a non-SHBG-bound form of testosterone, is closely correlated with cognitive function according to the Standardized Mini Mental Test for the diagnosis of dementia Low BT was associated with a greater risk for dementia HR 1.
Therefore, with aging, SHBG levels increase, BT may be a neglected important cause of cognitive impairment in older men. Accumulative studies have found that androgen levels decrease in the context of a variety of diseases and corresponding treatments, including schizophrenia, acquired immune deficiency syndrome, multiple sclerosis, hypogonadism, and acute lymphoblastic leukemia 23 , 30 — These clinical studies have also found that these populations with low androgen levels have a higher risk of cognitive impairment, such as spatial memory and attention deficit.
Prostatic hyperplasia is a common disease in older men and often causes lower urinary tract symptoms LUTS. They can reduce the level of DHT. Prostate cancer PCa is a common cancer in elderly men. ADT is an important treatment for inhibiting the growth of PCa that functions by lowering testosterone levels. Significant neurophysiological and psychological problems occur after ADT Accumulative studies have shown that compared with those of patients without ADT, the cognitive functions, including language ability, short-term memory, prospective memory, mental flexibility, inhibitory control and emotional psychology, of patients with ADT are significantly impaired 34 — After ADT treatment, cognitive impairment occurs within 6 and 12 months, and the risk for dementia is greatest when ADT exceeds 12 months 38 , Additionally, generally, the higher the dose of ADT is, the higher the risk of dementia AD is a serious type of dementia-related disease.
Gene therapy designed to drive expression of ILBP, a secreted binding protein that inhibits IL signaling, reduces tumor burden in mice Again, caution is warranted as some studies indicate disruption of Th17 and Th22 cell function can promote tumor development and progression The cause of these disparate outcomes is not fully understood, but may relate to the specific mechanism of CRC pathogenesis and the role of T cells in promoting appropriate versus chronic, dysregulated inflammatory responses.
Further elucidation of the role of these cells in CRC is required. Interventions designed to modulate these factors could influence functional heterogeneity within multiple lineages simultaneously. They are potentially powerful therapeutic targets. Expression of T-bet in both effector and regulatory lineages correlates with enhanced tumor response and improved outcome.
Co-expression with T-bet, however, confers potent anti-tumor activity. A balance may have to be found. The mechanisms by which to exert this pressure must also be determined. Empiric evidence indicates potential utility. Concurrent inhibition of IL-6 and PD-1 leads to elevated Th1 levels and enhances response to checkpoint blockade in multiple mouse models , Blockade of IL may therefore blunt pathogenic Th17 differentiation and, as with STAT3 inhibition, redirect developing cells to a Th1-like phenotype.
Interference with this pathway also has the potential for unintended consequences. When administered to mice harboring a toxigenic strain of B. Co-administration of IL also reduces tumor Th17 numbers, and dramatically improves tumor burden This cooperative effect is promising, and suggests additional interactions could be similarly exploited.
But its mechanism is incompletely understood, and it is difficult to anticipate which additional combinations will prove beneficial. Selective amplification of Tfh may represent an alternative potential therapeutic avenue. ICOS ligation may additionally modulate the effector response. Exploitation of Treg biology represents one of the most promising mechanisms for combatting CRC. Unfortunately, very little is known about the signaling and transcriptional events that guide this transition.
As with other proposed interventions, targeted approaches localizing effects to specific cell populations might be required. Bi-specific antibodies, for example, could be used to block signaling events in specific subsets of T cells, including Tregs.
Even so, these interventions are highly speculative. Our understanding of eTreg cell states is limited. The molecular determinants guiding their development must be elucidated before viable interventions can be developed. Tumors preferentially develop in the distal colon and rectum, which harbors the highest concentration of microbial species Early studies using germ free animals confirmed a role for microbial organisms in the development of CRC This dysbiosis is transferable, as fecal transplantation from tumor-bearing mice to conventionalized germ-free mice results in increased colon inflammation and tumorigenesis Fecal transplants from CRC patients into germ-free mice also results in increased tumor burden Interestingly, microbial patterns and signatures vary substantially between colon cancer tissue and adjacent non-malignant colon tissues Thus, localized dysbiosis of intestinal microbiota can trigger inflammation leading to an increased permeability of the epithelial barrier and enhanced bacterial translocation, which in turn, promotes chronic inflammation by provoking a persistent immune response.
This generates reactive oxygen and nitrogen species that lead to oxidative stress, DNA damage, and abnormal cellular proliferation, eventually culminating in the development of CRC. While disparities between studies preclude the identification of a CRC-specific microbiome, substantial evidence supports causal roles for some species, including Fusobacterium nucleatum and Bacteroides fragilis.
Fusobacterium is enriched in human CRC mucosa, predicts poor response to chemotherapy and prognosis and promotes tumor development in mice Colonization persists even in distal metastases Toxigenic Bacteroides fragilis is also enriched in CRC lesions, and promotes tumor development in mice. Interestingly, while toxigenic strains of B. Interventions should be designed to alter microbial populations to promote a beneficial immune response.
Due to the localized nature of dysbiosis, direct sampling of colonic mucosa may be required to identify relevant organisms. Species level identification may not be sufficient given the strain dependent effects of B. In addition, commensal organisms form a complicated, inter-dependent network. Manipulations affecting single species could prove insufficient to alter function.
More sophisticated approaches should be considered. The potential therapeutic utility is apparent but, as before, our ability to exploit this potential is hampered by an abridged appreciation of biology.
The gastrointestinal GI tract is a large surface lined by a single layer of epithelium exposed to trillions of microbes and innocuous substances from the diet. It harbors the largest collection of immune cells in the body. The gut immune system maintains a state of dynamic equilibrium, monitoring luminal contents to sustain tolerance to dietary and commensal antigens while retaining the ability to rapidly respond to invading pathogens.
Many fundamental questions will have to be addressed before a new model can be developed. The increasing complexity of lineage diversity and functional heterogeneity have made these questions harder to answer. But they must be answered. It will be very difficult to wield this tool for clinical benefit without understanding how it works. What underlies the seemingly contradictory roles played by some cells? Both nTregs and pTregs are beneficial in controlling the inflammation that serves as the nidus for CRC, but are harmful after inflammation leads to cancer.
And yet some Tregs shed their suppressive role, become eTregs, and participate in anti-cancer immune responses, much as effector cells do. Similarly, Th17 and Th22 cells promote pathogen clearance and epithelial barrier function, respectively.
Effective clearance and barrier integrity minimize exposure of epithelial cells to noxious inflammatory stimuli. But the sustained activity of these cells promotes tumor development. Similarly, the concerted influence of follicular T cells and the colonic microbiota can both promote and oppose CRC. The development of these populations, and their influence on inflammatory responses to CRC, must be resolved in greater detail so that they can be exploited to improve disease outcomes.
Regardless of the target, interventions must be designed with pleiotropic, combinatorial effects in mind. Independent effects on both effector and regulatory cell populations must be examined carefully. Potential effects on follicular T cells should also be considered, as should interactions with innate, epithelial and tumor cells.
Given potentially counterproductive effects on disparate cell types, targeted interventions may afford enhanced efficacy. Current attempts are limited by an incomplete understanding of the underlying biology.
A more nuanced understanding of lineage diversity and plasticity in inflammatory responses during CRC is needed. The contributions of specific cell populations must be better delineated to understand the best way to implement therapeutic approaches. The relationships between these cells, and the molecular determinants guiding their development, must be understood. Much remains to be done. But we are close enough to see the reward far outweighs the cost.
DD and RB contributed equally. All authors contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.
Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Inflammation and Colorectal Cancer. Curr Colorectal Cancer Rep — Annu Rev Immunol — J Immunol Baltimore Md: — Google Scholar. Cell — These innovative strategies increased drug accumulation into TME EPR effect compared with the poor solubility, penetration capability and bio-availability of standard chemotherapeutic drugs.
Moreover, the decoration of nanocarrier shells with targeted ligands active targeting facilitates the interaction with tumor cells Figure 4. Several nanosystems for the delivery of therapeutics at melanoma site have been studied. They include lipid and polymer-based nanoparticles designed to improve delivery and release of drugs to melanoma cells, thus exhibiting robust effects against melanoma cells proliferation and angiogenesis both in vitro and in vivo , To overcome PTX poor water solubility, new drug delivery strategies have been investigated Notably, liposomal systems able to directly deliver PTX to melanoma cells have been developed and studied both in vitro and in vivo.
Furthermore, liposomes surface can be functionalized by adding peptides able to both recognize integrins expressed by melanoma cells and promote low pH-mediated internalization of liposomes, with increased anti-tumoral effects and survival compared to free PTX Moreover, liposomes can be functionalized with Fibroblast Growth Factor FGF -derived peptides to improve melanoma cells targeting and favor their accumulation within TME One of the most important barriers which compromises the efficacy of nanocarriers is associated with the ability of immune cells to recognize and engulf nanoparticles.
To overcome this problem, the possibility to deliver immunoregulating agents for specific immune cell populations has been investigated This combination is useful to activate killer cells facilitating the elimination of metastatic cells during the invasion and metastasization process and could potentiate the efficacy of immunotherapy by increasing delivery and retention and by reducing immunomodulation toxicity , Nanocarriers for the delivery of IL are generated to improve tumor infiltration of killer cells, while other peptides may specifically modulate T cells proliferation, infiltration, and activation In particular, the injection of microspheres in B16 melanoma-bearing mice provoked the induction of a tumor-specific memory T cell response and consequently the tumor rejection Recently, the role of NK cell-derived exosomes has gained attention since they are equipped with the same cytotoxic molecules found in NK cells, such as perforin and FasL.
Therefore, they can eliminate tumor cells by the same mechanisms as NK cells. In melanoma, NK cell-derived exosomes have been shown to induce apoptosis in vitro as well as to limit tumor growth in vivo Taken together, these data underlie the role of nano-based drug delivery systems combined with chemoimmunotherapy as a future strategy to treat metastatic melanoma by boosting immune response. However, the failure of a fraction of patients to respond to therapy spurred the investigation of novel approaches in order to improve the success rate.
In this context, NK cells gained increasing attention because of their alternative and complementary capability to kill tumor cells compared to T cells. Most of the ICIs that are currently used or under development target inhibitory receptors also expressed by NK cells. Indeed, it has been shown that their blockade is able to reactivate NK cell-mediated cytotoxicity and that NK cells are essential for ICIs anti-tumoral activity An alternative strategy to improve NK cell-mediated killing is the usage of bi- and trispecific Killer Cell Engagers, which engage activating receptors on NK cells and specific antigens on tumor cells, thus favoring immune synapse formation and NK cell degranulation In addition, NK cells have been evaluated for adoptive cell transfer both in autologous and allogenic settings.
Compared to CAR-T cells, CAR-NK cells are not antigen-restricted and maintain their capability to recognize the target through multiple pathways, ensuring effective activation. Moreover, CAR-NK cells are short-living cells that do not generate memory, thus displaying a very low risk of adverse reactions and a high safety profile The usage of chemotherapeutics to treat cancer relied on their capability to counteract uncontrolled tumor cells proliferation.
However, their action is not cell-specific, which means that all the cell types with high rates of replication, including immune cells, are negatively affected by chemotherapy. Indeed, immune suppression is one of the most important collateral effect of standard-dosage chemotherapy and thus was regarded as the major mechanism making chemotherapy incompatible with immunotherapy.
However, more recent evidence challenged this assumption, showing that sub-toxic concentrations of chemotherapeutics exert immunomodulatory activities able to reactivate immune responses. Additionally, it has been shown that suppressive immune subsets display a higher sensitivity to chemotherapy-mediated cytotoxicity, making low doses of chemotherapeutics a potential strategy to deplete specific immune cell populations Thus, the usage of chemotherapy is nowadays gaining attention as therapeutic strategy to improve effectiveness rates of current immunotherapy.
The evidence discussed here indicate that metastatic melanoma chemotherapy affects not only tumor cells but also the TME, thus having the potential to unleash the anti-tumor activities of the immune system. However, the combination between chemo- and immunotherapy has recently begun to be investigated and there is still not a consensus about the schedule to be used.
Indeed, while some reports suggested that chemotherapy should be administrated before immunotherapy 11 — 13 , other studies demonstrated that the survival benefit could be achieved also by the concomitant administration of the two kinds of drugs 14 , Moreover, some case reports indicated that chemotherapy could be effectively used after immunotherapy in non-responding patients 16 , Although in theory initial chemotherapy would represent the best strategy to reactivate the immune system and sensitize patients to immunotherapy, the lack of biomarkers predicting immunotherapy response makes the subsequent usage of chemotherapy a more feasible approach.
Overall, additional studies are needed in order to define the best therapeutic strategy to combine chemo- and immunotherapy as well as to identify those patients actually needing the combinatorial therapy. However, cell-intrinsic pathways in melanoma cells as well as their capability to recruit and activate suppressive subsets can profoundly impair NK cells cytolytic and anti-proliferative functions. The evidences reported here clearly suggest that chemotherapeutic drugs may support NK cell-mediated elimination of melanoma cells by four different, complementary mechanisms: 1 increase of melanoma cells immunogenicity, mainly occurring through the up-regulation of NKG2D ligands but also involving other activating molecules specifically induced by the different drugs, thus boosting the natural cytotoxic activity of NK cells; 2 induction of NK cells infiltration within TME, which would contribute to overcome an important limitation in the usage of autologous, allogenic as well as CAR-NK cells; 3 promotion of myeloid cells activation and differentiation towards anti-cancer phenotypes that largely contribute to develop and support NK cells functions; 4 specific impairment and depletion of immune suppressive cells, which play the major role in generating and maintaining the suppressive conditions blunting NK cells cytotoxicity within TME.
Moreover, these effects could be further enhanced by nano-based drug delivery systems designed to improve pharmacokinetics behaviors and to increase drugs stability in vivo , thus allowing the effective usage of sub-toxic amounts of drugs that keep the immunomodulatory properties avoiding at the same time the immune suppression commonly associated to high-dosage of chemotherapeutics Overall, the association of immunotherapies with low dosages of chemotherapeutics possibly delivered by nanosystems could restore NK cells anti-tumor properties thus representing an effective alternative therapeutic strategy for those melanoma patients that fail to respond to the current immunotherapeutic treatments.
CMC conceived the work. CG and CMC reviewed the relevant literature. All the authors critically read and revised the manuscript, contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.
Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. This manuscript is dedicated to Prof. Ennio Carbone, who was our tutor and suddenly passed away on March , too early for his age.
He always focused his interest on NK cells and their role in viral diseases as well as in tumor immunology, providing valuable achievements to Immunology research. As our mentor, he widely contributed to the development of our scientific expertise. We hope this work may contribute to honor his memory. Atallah E, Flaherty L. Treatment of Metastatic Malignant Melanoma. Curr Treat Options Oncol 6 3 — Metastatic Melanoma: Chemotherapy. Semin Oncol 29 5 — Eur J Cancer 47 10 — N Engl J Med 8 — N Engl J Med 4 — Pembrolizumab Versus Ipilimumab in Advanced Melanoma.
N Engl J Med 26 — N Engl J Med 16 — N Engl J Med 21 — Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell 5 — Immunity 39 1 — Int J Cancer 1 —9. Cancer Res 70 18 — J Dermatol 47 8 — Invest N Drugs 29 3 — Cancer Immunol Immunother 66 10 — Melanoma Res 29 6 —7.
Oncotarget 11 52 — Hazenberg MD, Spits H. Human Innate Lymphoid Cells. Blood 5 —9. Blood Rev 20 3 — Scand J Immunol 91 4 :e Pigment Cell Melanoma Res 22 2 — Hum Pathol 42 12 —4. Cancer Res 72 5 — Cancer Immunol Res 7 5 — Int Immunol 21 7 — Nat Immunol 9 5 — Annu Rev Immunol — Cancer Immunol Immunother 58 9 — Nature —9.
NK Cells to Cure Cancer. Semin Immunol Science —9. Immunity 14 2 — J Exp Med 4 — J Immunol 6 — See also Figure 8—figure supplement 1 , Videos 9 , 10 , 11 and The movies were captured at a speed of 0. Movies are depicted at 60fps.
Time is shown as mm:ss. Consequently, it is conceivable that the TgFRM2-mediated actin polymerisation centre anterior to the nucleus contributes to the parasite peripheral actin pool, making Formin-2 a key player in regulating actin distribution within intracellular parasites. Intriguingly, but perhaps not surprisingly, F-actin was still measurable and visible at the apical and the basal poles, as well as in the residual body of TgFRM2-cKO parasites.
Tosetti and colleagues proposed peripheral actin flux towards the basal end of extracellular parasites Tosetti et al. In a complementary approach, we applied kymograph analysis Mangeol et al.
Kymographs of wild-type parasites showed trajectories representing CB-EME particle movement towards the apical and basal poles red signal represents forward displacement, while green signal represents backward displacement on kymographs, Figure 8D , Figure 8—figure supplements 1 and 2 ; Videos 9 , 10 , 11 and 12 , indicating bi-directional actin flow all along the lateral parasite axis. Time-averaged intensity profiling was also performed on T.
In TgAct cKO parasites, no distinguishable tracks could be identified by kymograph analysis, when compared to background noise Figure 8—figure supplements 1 and 2B , Video TgADF cKO kymographs represent the previously described phenotype of strong static actin accumulation at the basal end of the parasite with no obvious F-actin dynamics at the periphery Figure 8—figure supplement 2D , blue signal on kymographs, Video This is most likely due to the abundance of actin in the periphery and perhaps due to the contribution of TgFRM1.
We conclude that loss of TgFRM2 causes a significant decrease of peripheral actin flow in intracellular parasites. Due to the unconventional behaviour of apicomplexan actin, visualisation of actin filaments in P. Therefore, previous attempts to use established indicators from other eukaryotic systems, such as Life-Act, failed Tardieux, In a recent study, it was shown that actin-binding nanobodies fused to epitope tags could be expressed in Toxoplasma gondii, allowing for the first time to analyse F-actin localisation and dynamics in living parasites Periz et al.
Another recent study showed that these nanobodies also bind to P. Here we successfully adapted this technology to live P. This allowed us to visualise F-actin throughout the asexual life-cycle of P. A recent study also used actin-binding chromobodies to analyse actin polymerisation centres in T. Importantly, validation of this reagent using either F-actin modulating drugs or a conditional mutant for PfACT1 led to expected results and phenotypes Figures 1 and 2 , demonstrating that F-actin dynamics are finely balanced in the parasite.
Super-resolution imaging revealed a complex F-actin network in P. We show that the primary nucleator of F-actin in intracellular P. Importantly, our previous characterisation of a conditional mutant for PfACT1 highlighted three primary functions of actin during the asexual life cycle of the parasite Das et al.
PfACT1 is essential for P. Our data suggest that early signalling events just after egress are a trigger for actin-polymerisation at the apical end. This is likely to be mediated by an apically resident nucleator of F-actin, a likely candidate being Formin-1 Baum et al.
Therefore, the expression of chromobodies in P. F-actin can be clearly visualised during growth, in invading merozoites and in gametocytes — opening up many avenues for further research. It is conceivable that this novel tool could also be used to investigate other motile and developmental stages of the Plasmodium parasite. Using actin-binding chromobodies combined with powerful reverse genetics made possible by the DiCre system Andenmatten et al. Using bioinformatic searches within alveolates, we found the presence of a PTEN-C2-like domain only in apicomplexan Formin-2 sequences Figure 3.
This domain has been demonstrated in rice to be responsible for Formin-2 targeting to chloroplast membranes Zhang et al. It is therefore likely that the apicomplexan PTEN-C2-like domain is used for apicoplast recruitment of apicomplexan Formin We found that the function of Formin-2 is partially conserved in T.
This network appears to be critical for material exchange, synchronised replication of parasites and host cell egress Periz et al. Furthermore, using kymograph analysis, we gained new detailed insights into actin distribution and dynamics in Toxoplasma tachyzoites. In addition to the previously reported actin polymerisation centres anterior to the nucleus and the residual body Periz et al.
Interestingly, our data suggest that these sites of actin accumulation interact with each other. We therefore propose that a steady flow of actin connects different sites of actin polymerisation, allowing for particle transport and exchange Figure 9.
Correspondingly, in a recent report it was elegantly demonstrated that forces set up by waves of actin polymerisation, along with actin comet formation, aided in segregation of yolk granules towards the vegetal pole of zebrafish oocytes Shamipour et al.
B Daughter cell formation in P. C Apicoplast inheritance in both P. In Toxoplasma, Formin-3 can still make extracellular F-actin connections between daughter cells. Establishment of bidirectional peripheral F-actin flow is disrupted. Since Tg FRM2 cKO parasites still show actin polymerisation at the apical pole and in the residual body, and PfFRM2 cKOs can polymerise actin at the apical end, our findings support previous studies which reported non-overlapping functions for Formin-1,—2 Baum et al.
Our study supports the view that Formin-1 is active during gliding and invasion, while Formin-2 drives actin translocation and flow in intracellular apicomplexan parasites, achieving apicoplast inheritance in both Toxoplasma and Plasmodium , and efficient daughter formation, observed only in P. In conclusion, we show here that chromobodies can be used to determine F-actin dynamics in apicomplexan parasites and will form the basis for functional in-vivo studies of other actin regulatory proteins found in apicomplexans.
Parasites were selected by use of appropriate drug medium. The culture medium was subsequently exchanged every day for the next 4 days to remove cell debris which accumulates during electroporation and then twice a week until parasites were detected by Giemsa smear.
Drug-resistant parasites were generally detectable in thin blood films 2—3 weeks post-transfection. Lines were then cloned by limiting dilution using a simple plaque assay Thomas et al.
Lines were selected with 2. The slides were then permeabilised with 0. Deconvolution was performed using SoftWoRx Suite 2. Video microscopy of P. Images were routinely collected at 5 s intervals, beginning 6 min 30 s after washing off C1, over a total of 30 min. These were concatenated into a single proteome sequence dataset. The proteome sequence dataset was searched for FH2 domains FH2. These sequences were retrieved from the dataset, and subjected to alignment against the FH2.
The profile conformant subsequences were extracted from the alignment and this sequence set subjected to alignment using: 1 hmmalign to FH2. A rooted neighbour-joining tree of Formin Homology type two domains FH2 was constructed from this alignment using the SplitsTree program [version 1. The program converged after three iterations. To obtain conditional truncation of the pffrm2 gene we used silent loxP sites within a heterologous P.
This was transfected into the DiCre expressing strain B11 Perrin et al. Various floxed parasite strains were synchronised by Percoll and sorbitol as previously described Collins et al.
The rings were then washed and returned to culture. Phenotypic analysis was performed primarily 44 hr post RAP-treatment unless stated otherwise. Cultured cells and parasites were regularly screened against mycoplasma contamination using the LookOut Mycoplasma detection kit Sigma and cured with Mycoplasma Removal Agent Bio-Rad if necessary.
Further image processing was performed using ImageJ software Schindelin et al. Afterwards coverslips were permeabilised in 0. Using a different set of primers, the complete upstream LoxP site was amplified via PCR and verified by sequencing. Disruption of the target genes was confirmed by sequencing Eurofins Genomics.
Subsequently, parasites were mechanically lysed and inoculated on glass bottom dishes MaTek for another 24 hr. Images were taken using a x oil objective lens. Videos were processed using ImageJ Schneider et al. In short, we used this application to define a track on a maximum intensity image that was calculated from an image sequence. A kymograph was then generated depicting particle movement alongside the chosen track. Fourier filtering done by the plugin enables the distinction between forward-moving red , backward-moving green and static blue particles in the kymograph.
Intensity profiles depict Cb-Emerald intensity along the measured axis over the entire duration of the movie. Background corrections were performed for all imported kymographs. Prior to skeletonization, thresholding was performed on the movie stacks to create binary images defining signal and no signal.
These binary images were then processed by the skeletonization plugin, converting the signal into pixels that can be followed through the time-lapse.
Skeletonized images in this study represent collapsed t-stacks. All data generated or analysed during this study are included in the manuscript and supporting files. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. The first decision letter after peer review is shown below.
Thank you for submitting your work entitled "Formin-2 drives polymerisation of actin filaments enabling segregation of apicoplasts in P. Your article has been reviewed a Senior Editor, a Reviewing Editor, and two reviewers. The following individual involved in the review of your submission has agreed to reveal their identity: Isabelle Tardieux Reviewer 1. Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.
This work builds on a previous study aimed at visualizing the actin cytoskeleton in Apicomplexa using chromobodies. As you will see from the reviewer reports appended below, both reviewers recognized the importance of the topic. The reviewers discussed your work in detail, and the consensus was that although the studied problem is challenging and the described work has some potential, in the current form the conclusions are not sufficiently supported by data, such as complementary approaches to provide sound evidence for the rather strong statements on the identity of the studied structures.
Another strong point of criticism, on which both reviewers agreed, was the lack of quantitation throughout the paper. Since these concerns cannot be easily addressed within a short time frame, we advise you to seek publication elsewhere. The manuscript entitled "Formin-2 drives polymerisation of actin filaments enabling segregation of apicoplasts in P.
While the authors have already obtained T. Using this tool to characterize putative F actin structures, they then investigated how intracellular dividing parasites P. They confirmed for P. The manuscript is then centered on the key role of actin dynamics to promote apicoplast segregation.
Given this observation the authors established an association between pools of actin chromobody-bound actin and apicoplasts using confocal and super resolution imaging. To start addressing how actin dynamics could control apicoplast, they raised the hypothesis that the actin nucleator Formin here Formin-2 would be a relevant candidate.
They showed using both epitope tagging at the endogenous locus and conditional silencing, that i main pools of Formin-2 are indeed juxtaposed to apicoplasts and ii loss of formin induces the breakdown of the F-actin structure and eventually apicoplast segregation defect. It is noteworthy that this study relies on a combination of recently introduced but still challenging molecular genetics in the Apicomplexa field.
The authors have engineered several lines of interest in both T. In addition, the view that Formin-2 could play a key role in controlling actin at specific locations during cell cycle is also quite interesting and rather convincingly documented. However, in my opinion the work should be strengthened by few additional datasets and by careful reevaluation of several statements prior to publication.
Real time and static SR imaging should allow capturing the kinetics of the segregation process as well as spatial details Z stacks thus 3D reconstitution will reveal them.
If they want to characterize filopodia occurrence, they need to provide a marker for membrane and to analyze whether the parasite actin protrusions coincide with membrane projections. This work continues some previous studies that showed in Toxoplasma that one could get some visualisation of the actin biology by expressing fluorescent probes called chromobodies.
Here the authors use this same technology to reanalyse the distribution of actin in both Plasmodium and Toxoplasma. The authors show some potentially interesting results — mainly built around interpretation of images relating to the apicoplast and implicating Formin-2 in the polymerisation of actin filaments that are known from previous work to be involved in apicoplast division. At the heart of the work are some statements that define the fluorescent dots or areas of brightness as actin filamentous structures or apicoplasts etc.
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