Motile Phenotype Research Articles

A new in vitro study demonstrates that electronic cigarettes promote cellular carcinogenic characteristics.

Electronic cigarettes have been used as a new form of cigarettes, especially among young people, but the impact of these effects on mouth cancer is unknown. Therefore, there is a need for studies to evaluate their impact on health and oral mucosa. In this way, this study evaluates the risk of electronic cigarette liquid on in vitro cells of a panel: normal oral epithelium cell lines (NOE and HMK), oral squamous cell carcinoma human cell lines (CAL27 and HSC3), and a mouse oral cancer cell line (AT84). It was demonstrated that electronic cigarettes promote proliferation and anchorage-independent growth and induces morphological changes associated with enhanced motility and invasive phenotypes. Also, it was observed that the enhanced invasive migratory activity associated with the loss of epithelial markers, such as E-cadherin, and the acquisition of mesenchymal markers, strongly suggest that epithelial cells are undergoing to an aggressive phenotype within the framework of epithelial-mesenchymal transition. In conclusion, the liquid can promote carcinogenesis, in addition to promoting an aggressive phenotype in pre-existing lesions.

Polyhydroxyalkanoate production by the plant beneficial rhizobacterium Pseudomonas chlororaphis PCL1606 influences survival and rhizospheric performance

Pseudomonas chlororaphis PCL1606 (PcPCL1606) is a model rhizobacterium used to study beneficial bacterial interactions with the plant rhizosphere. Many of its beneficial phenotypes depend on the production of the antifungal compound 2-hexyl, 5-propyl resorcinol (HPR). Transcriptomic analysis of PcPCL1606 and the deletional mutant in HPR production ΔdarB strain, assigned an additional regulatory role to HPR, and allowed the detection of differentially expressed genes during the bacterial interaction with the avocado rhizosphere. Interestingly, the putative genes phaG (PCL1606_46820) and phaI (PCL1606_56560), with a predicted role in polyhydroxyalkanoate biosynthesis, were detected to be under HPR control. Both putative genes were expressed in the HPR-producing wild-type strain, but strongly repressed in the derivative mutant ΔdarB, impaired in HPR production. Thus, a derivative mutant impaired in the phaG gene was constructed, characterized and compared with the wild-type strain PcPCL1606 and with the derivative mutant ΔdarB. The phaG mutant had strongly reduced PHA production by PcPCL1606, and displayed altered phenotypes involved in bacterial survival on the plant roots, such as tolerance to high temperature and hydrogen peroxide, and decreased root survival, in a similar way that the ΔdarB mutant. On the other hand, the phaG mutant does not have altered resistance to desiccation, motility, biofilm formation or adhesion phenotypes, as displayed by the HPR-defective ΔdarB mutant have. Interestingly, the mutant defective in PHA production also lacked a biocontrol phenotype against the soilborne pathogenic fungus Rosellinia necatrix, even when the derivative mutant still produced the antifungal HPR compound, demonstrating that the final biocontrol phenotype of PcPCL1606 first requires bacterial survival and adaptation traits to the soil and rhizosphere environment.

Bacterial motility can govern the dynamics of antibiotic resistance evolution

Spatial heterogeneity in antibiotic concentrations is thought to accelerate the evolution of antibiotic resistance, but current theory and experiments have overlooked the effect of cell motility on bacterial adaptation. Here, we study bacterial evolution in antibiotic landscapes with a quantitative model where bacteria evolve under the stochastic processes of proliferation, death, mutation and migration. Numerical and analytical results show that cell motility can both accelerate and decelerate bacterial adaptation by affecting the degree of genotypic mixing and ecological competition. Moreover, we find that for sufficiently high rates, cell motility can limit bacterial survival, and we derive conditions for all these regimes. Similar patterns are observed in more complex scenarios, namely where bacteria can bias their motion in chemical gradients (chemotaxis) or switch between motility phenotypes either stochastically or in a density-dependent manner. Overall, our work reveals limits to bacterial adaptation in antibiotic landscapes that are set by cell motility.

Prognostic Nomogram for Lymph-Node Metastasis in Oral Squamous Cell Carcinoma (OSCC) Using Immunohistochemical Marker D2-40.

Nomograms are proven in "individualized risk prediction" in sarcomas and breast and prostate cancers. Incorporating immunohistochemical markers and histopathological parameters can enhance accuracy of these graphical representations of statistical predictive models concerning metastasis. D2-40, a monoclonal antibody to podoplanin (regulator of motility expressed in malignant epithelial cells), dually predicts metastatic potential of tumour by estimating the motile tumour phenotype and by detecting lymphatic vessels/density, both essential to metastasis in OSCC. Thus, we propose a model that incorporates D2-40 immunostaining of individual tumour cells (ITC) too with other variables (seen in H+E staining) as a predictive nomogram. Sixty cases of OSCC were selected with equal number of cases (n=30) of pN0 and pN+ status. Bryne's grading of invasive front of tumour (ITF) was done on H+E-stained slides followed by D2-40 immunostaining for ITCs at ITF and lymphatic vessels. Multivariate regression analysis was used to generate the nomogram of LNM where the predictive contribution of each covariate, namely depth of invasion, D2-40-stained ITCs, gender, histological grade, and worst pattern of invasion (WPOI), was plotted on a scale of 1-100 points. The nomogram showed that the strongest variable in OSCC was the WPOI in H+E-stained section followed by D2-40-positive ITCs and gender. Our predictive nomogram for LNM in OSCC surprisingly showed that a tumour with lower score of WPOI (islands vs ITC) showed numerous D2-40-positive ITCs, drastically increasing the probability of metastasis. The concept of "individualized risk prediction" can be used to predict lymph node metastasis using a variety of histopathological criteria that can be visualized in routine and immunohistochemical staining in OSCC with the aid of a nomogram.

Completed genomes for Rickettsia rickettsii isolated from ticks and quality controlled for motility phenotype.

Complete genomes of Rickettsia rickettsii were sequenced with Illumina and PacBio technologies from low-passage isolates from ticks. These isolates were quality controlled for intact roaM, a regulator of actin-based motility that is negatively selected for in culture. The Sheila Smith strain was re-sequenced using the same methodology.

Anti-virulence prospects of Metformin against Pseudomonas aeruginosa: A new dimension to a multifaceted drug

Metformin (MeT) is an FDA-approved drug with a myriad of health benefits. Besides being used as an anti-diabetic drug, MeT is also effective against various cancers, liver-, cardiovascular-, and renal diseases. This study was undertaken to examine its unique potential as an anti-virulence drug against an opportunistic bacterial pathogen, Pseudomonas aeruginosa. Due to the menace of multidrug resistance in pathogenic microorganisms, many novel or repurposed drugs with anti-virulence prospects are emerging as next-generation therapies with the aim to overshadow the application of existing antimicrobial regimens. The quorum sensing (QS) mechanisms of P. aeruginosa are an attractive drug target for attenuating bacterial virulence. In this context, the anti-QS potential of MeT was scrutinized using biosensor assays. MeT was comprehensively evaluated for its effects on different motility phenotypes, virulence factor production (phenotypic and genotypic expression) along with biofilm development in P. aeruginosa in vitro. At sub-lethal concentrations, MeT displayed prolific quorum quenching (QQ) ability and remarkably inhibited AHL biosynthesis in P. aeruginosa. Moreover, MeT (1/8 MIC) effectively downregulated the expression levels of various QS- and virulence genes in P. aeruginosa, which coincided with a notable reduction in the levels of alginate, hemolysin, pyocyanin, pyochelin, elastase, and protease production. In silico analysis through molecular docking also predicted strong associations between MeT and QS receptors of P. aeruginosa. MeT also compromised the motility phenotypes and successfully abrogated biofilm formation by inhibiting EPS production in P. aeruginosa. Hence, MeT may be repurposed as an anti-virulence drug against P. aeruginosa in clinical settings.

Zingerone inhibits biofilm formation and enhances antibiotic efficacy against Salmonella biofilm

Zingerone inhibits biofilm formation and enhances antibiotic efficacy against Salmonella biofilm

Physiology of trans-translation deficiency in Bacillus subtilis - a comparative proteomics study.

trans-Translation is the most effective ribosome rescue system known in bacteria. While it is essential in some bacteria, Bacillus subtilis possesses two additional alternative ribosome rescue mechanisms that require the proteins BrfA or RqcH. To investigate the physiology of trans-translation deficiency in the model organism B.subtilis, we compared the proteomes of B. subtilis168 and a ΔssrA mutant in the mid-log phase using gel-free label-free quantitative proteomics. In chemically defined medium, the growth rate of the ssrA deletion mutant was 20% lower than that of B.subtilis 168. An 35 S-methionine incorporation assay demonstrated that protein synthesis rates were also lower in the ΔssrA strain. Alternative rescue factors were not detected. Among the 34 proteins overrepresented in the mutant strain were eight chemotaxis proteins. Indeed, both on LB agar and minimal medium the ΔssrA strain showed an altered motility and chemotaxis phenotype. Despite the lower growth rate, in the mutant proteome ribosomal proteins were more abundant while proteins related to amino acid biosynthesis were less abundant than in the parental strain. This overrepresentation of ribosomal proteins coupled with a lower protein synthesis rate and down-regulation of precursor supply reflects the slow ribosome recycling in the trans-translation-deficient mutant.

IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans.

Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice.

Modelling how curved active proteins and shear flow pattern cellular shape and motility.

Cell spreading and motility on an adhesive substrate are driven by the active physical forces generated by the actin cytoskeleton. We have recently shown that coupling curved membrane complexes to protrusive forces, exerted by the actin polymerization that they recruit, provides a mechanism that can give rise to spontaneous membrane shapes and patterns. In the presence of an adhesive substrate, this model was shown to give rise to an emergent motile phenotype, resembling a motile cell. Here, we utilize this "minimal-cell" model to explore the impact of external shear flow on the cell shape and migration on a uniform adhesive flat substrate. We find that in the presence of shear the motile cell reorients such that its leading edge, where the curved active proteins aggregate, faces the shear flow. The flow-facing configuration is found to minimize the adhesion energy by allowing the cell to spread more efficiently over the substrate. For the non-motile vesicle shapes, we find that they mostly slide and roll with the shear flow. We compare these theoretical results with experimental observations, and suggest that the tendency of many cell types to move against the flow may arise from the very general, and non-cell-type-specific mechanism predicted by our model.

Immunohistochemical expression of E-cadherin and N-cadherin in endometrioid endometrial carcinoma and its precursor lesions

ObjectivesEndometrial carcinoma (EC), majority being endometroid endometrial carcinoma (EEC), is the most common invasive gynecological cancer world-wide. Epithelial mesenchymal transition (EMT) is the process that cells undergo to switch from a polarized epithelial phenotype to a motile mesenchymal phenotype. Loss of E-cadherin and expression of N-cadherin are a critical step for development and progression of malignant tumors. In present study we evaluated the immunohistochemical (IHC) expression of E-cadherin and N-cadherin with clinicopathological parameters of EEC and its precursors lesions. MethodsIt was a prospective nested case-control study involving women aged 35–70 years, whose endometrial biopsy were obtained for histological diagnosis. Signed informed consent was obtained before enrolling the women in the study, and the approval to conduct the study was obtained from the institutional ethics committee. The IHC was done to measure the E-cadherin and N-cadherin. ResultsReduced expression of E-cadherin was observed in 69% of cancer and 65% of precancer, however only 32% of normal proliferative endometrium showed reduced E-cadherin expression. N-cadherin expression was observed in 82% of EEC and 81% of precancer cases, while 11% of normal proliferative endometrium showed N-cadherin expression. Statistically significant downregulation of E-cadherin and upregulation of N-cadherin were observed in EEC and precancerous lesions as compared to normal control. ConclusionSince downregulation of E-cadherin was also associated with upregulation of N-cadherin, the integration of immuno-expression of both E-cadherin and N-cadherin might offer a potential therapeutic target for EEC.

Abstract 1676: PRMT5 regulates the chemoresistnace of ovarian cancer cells

Abstract Ovarian cancer resistance to chemotherapy accounts for the most therapeutic challenge, leading to the high mortality rate of gynecological cancer worldwide. Meanwhile, cancer stem cells (CSCs) are identified as key features in regulating ovarian chemoresistant cancer. In this study, we first found that PRMT5 was upregulated in paclitaxel (PTX)-resistant ovarian cancer cells compared with non-resistant cancer cells. Then, we determined the oncogenic role of PRMT5 in chemoresistant ovarian cancer by using its potent and selective inhibitor, GSK3326595. Disruption of PRMT5 activity by GSK3326595 or depletion of PRMT5 in PTX-resistant ovarian cancer cells restored the sensitivity to chemotherapeutic drug via triggering cell apoptosis. Then, we also validated that inhibition of PRMT5 decreased cell migration ability through the transition of motile mesenchymal phenotypes into epithelial characteristics. Moreover, PRMT5 inhibitor significantly decreased the number of sphere formation and attenuated the stem cell-like properties in PTX-resistant ovarian cancer cells. Therefore, our study suggests that PRMT5 is a powerful target to overcome resistance to conventional chemotherapy for the treatment strategy in ovarian cancer. Citation Format: Yen Thi Do, Jin Young Kim, Seungmee Lee, Tam Thuy Lu Vo, Thi Tuyet Mai Pham, Eunyoung Ha, Chi-Heum Cho, So-Jin Shin, Ji Hae Seo. PRMT5 regulates the chemoresistnace of ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1676.

Abstract 428: The role of sphingosine kinase 2 in overcoming EGFR-tyrosine kinase inhibitor resistance, EMT and NSCLC tumor progression

Abstract Background: Tyrosine Kinase Inhibitors (TKIs) such as Erlotinib (ER) and Osimertinib (OR) are being used to treat lung cancer patients with EGFR mutations; however, patients develop resistance to these drugs within 10-18 months. EGFR-TKI resistance may be mediated by epithelial-mesenchymal transition (EMT), which causes a motile and invasive phenotype similar to mesenchymal cells. Sphingosine kinase 2 (SphK2), which is upregulated in Non-Small Cell Lung Cancer (NSCLC), phosphorylates sphingosine to sphingosine-1-phosphate (S1P) and increases cell proliferation, tumor survival and cell migration. The sphingolipid rheostat controls the cell fate by maintaining a balance between pro-survival and pro-apoptotic sphingolipids, which is regulated by SphK2. However, the role of SphK2 in TKI resistance and EMT is not well understood. Hypothesis: We hypothesize that the inhibition/downregulation of SphK2 will reverse EMT, reduce tumorigenicity and overcome ER and OR resistance. Study design and Results: We studied expression of SphK2 in both drug resistant (OR and ER) and drug sensitive (parental) H358, H2170, H3255 and PC9 NSCLC cell lines. In drug resistant cells, qPCR and immunoblotting studies showed that SphK2 was upregulated by 1.5-1.7 and 1.5-3.3 fold in ER and OR resistant cell lines respectively compared to parental cells. 48 hours after SphK2 knockdown with siRNA treatment, SphK2 was downregulated by 4.6-fold and EMT mediator E-cadherin expression was upregulated by 2.9-fold. EMT proteins Vimentin and N-cadherin were downregulated by 3.8 and 2.2 fold respectively (p≤0.01). Using an MTT assay, differences in percentage viability of cells with different treatments were calculated with respect to their viability in the presence of OR. We found that 81.43% of H358OR and 76.35% of the H3255OR cells were inhibited by a combination of Opaganib, a Sphk2 inhibitor, and OR, this is 27.74% and 12.28% higher compared to the additive effect of drug and inhibitor in H358OR and H3255 cells respectively. Furthermore, transwell migration assays, wound healing assays and spheroid assays were performed in OR resistant cells and the results indicated inhibition of the above assays by 94.97%, 82.36%, 88.67% respectively when treated with a combination of Opaganib and OR as compared to the diluent. This indicates the potential of SphK2 inhibition to overcome TKI resistance and EMT. To analyze the role of SphK2 expression in cancer progression, IHC was performed on 63 early and 48 late-stage lung cancer patient sections. We observed upregulation of SphK2 in late-stage lung cancer (p≤0.05), indicating its role in cancer progression. Conclusion: In this study, SphK2 was shown to decrease tumor survival, cancer cell proliferation, and expression of EMT markers. Thus, SphK2 may be a promising clinical target for overcoming EGFR-TKI resistance and NSCLC tumor progression. Citation Format: S M Nasifuzzaman, Namrata Dube, Katie Lam, Robert J. Hillwig, Neelu Puri. The role of sphingosine kinase 2 in overcoming EGFR-tyrosine kinase inhibitor resistance, EMT and NSCLC tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 428.

Transient mutation bias increases the predictability of evolution on an empirical genotype-phenotype landscape.

Predicting how a population will likely navigate a genotype-phenotype landscape requires consideration of selection in combination with mutation bias, which can skew the likelihood of following a particular trajectory. Strong and persistent directional selection can drive populations to ascend toward a peak. However, with a greater number of peaks and more routes to reach them, adaptation inevitably becomes less predictable. Transient mutation bias, which operates only on one mutational step, can influence landscape navigability by biasing the mutational trajectory early in the adaptive walk. This sets an evolving population upon a particular path, constraining the number of accessible routes and making certain peaks and routes more likely to be realized than others. In this work, we employ a model system to investigate whether such transient mutation bias can reliably and predictably place populations on a mutational trajectory to the strongest selective phenotype or usher populations to realize inferior phenotypic outcomes. For this we use motile mutants evolved from ancestrally non-motile variants of the microbe Pseudomonas fluorescens SBW25, of which one trajectory exhibits significant mutation bias. Using this system, we elucidate an empirical genotype-phenotype landscape, where the hill-climbing process represents increasing strength of the motility phenotype, to reveal that transient mutation bias can facilitate rapid and predictable ascension to the strongest observed phenotype in place of equivalent and inferior trajectories. This article is part of the theme issue 'Interdisciplinary approaches to predicting evolutionary biology'.

Knockdown Experiment Reveals an Essential GTPase CgtA's Involvement in Growth, Viability, Motility, Morphology, and Persister Phenotypes in Vibrio cholerae.

CgtA is an essential bacterial GTPase consisting of a highly conserved N-terminal Spo0B-associated GTP-binding protein (Obg) domain, a central GTPase domain, and a variable C-terminal domain (CTD). This study reports global changes in the proteome and transcriptome of wild-type (Wt) versus full-length CgtA-depleted Vibrio cholerae in minimal media. Comparative transcriptome sequencing (RNA-Seq), followed by comparative proteomic analyses, revealed that the knockdown of cgtA significantly altered expressions of 311 proteins involved in diverse cellular activities, many of which are associated with the survival of V. cholerae. Various intracellular functional roles of CgtA in growth, viability, motility, morphology, and persister phenotypes in V. cholerae are revealed based on subsequent confirmatory experiments. Furthermore, a more sustained mRNA expression pattern of cgtA in a minimal medium than in a rich medium was also observed for Wt V. cholerae, where the highest level of mRNA expression of cgtA was observed during the logarithmic growth phase. Thereby, we propose that minimal medium-associated reduced growth rate coupled with cgtA depletion aggravates the intracellular stress in V. cholerae, interrupting vital cellular processes. The functional role of the CTD in V. cholerae is not fully understood. Hence, to specifically investigate the intracellular role of the 57-amino-acid-long CTD of CgtAVC, the CTD-only portion of CgtA was deleted. Subsequent proteomics studies revealed an altered expression of 240 proteins in the CgtA(ΔCTD) mutant, having major overlap with the full-length cgtA-deleted condition. Overall, our study reveals an alternative facet of the survival mechanism of V. cholerae during nutritional downshift as per the concomitant consequences of cgtA depletion. IMPORTANCE It is very important that we must find new drug target proteins from multidrug-resistant human-pathogenic organisms like V. cholerae. CgtA is among such potential candidates, and here, we are reporting about some newly identified cellular roles of it that are important for the survival of V. cholerae. Briefly, we knocked down the full-length cgtA gene, as well as did a partial deletion of this gene from the V. cholerae genome followed by RNA-Seq and proteomics studies. Results from our study revealed up- and downregulation of several known and unknown genes and proteins as the effect of the cgtA knockdown experiment. Also, we have presented some interesting observations that are linked with cgtA for growth, viability, motility, morphology, and persister phenotypes in V. cholerae. Our study enhances the importance of CgtA and paves the way for further exploration based on our provided data.

E-liquid alters oral epithelial cell function to promote epithelial to mesenchymal transition and invasiveness in preclinical oral squamous cell carcinoma

The gaining popularity of tobacco and nicotine delivery products, such as electronic cigarettes (e-cigarettes) being perceived as relatively safe is of a medical concern. The long-term safety of these new products remains uncertain for oral health. In this study, in vitro effects of e-liquid were assessed in a panel of normal oral epithelium cell lines (NOE and HMK), oral squamous cell carcinoma (OSCC) human cell lines (CAL27 and HSC3), and a mouse oral cancer cell line (AT84) using cell proliferation, survival/cell death, and cell invasion assays. In addition, signaling pathways underlying the pro-invasive activity of e-cigarettes were evaluated by gene and protein expression analysis. We demonstrated that e-liquid promotes proliferation and anchorage-independent growth of OSCC and induces morphological changes associated with enhanced motility and invasive phenotypes. Furthermore, e-liquid-exposed cells express significantly reduced cell viability, regardless of e-cigarette flavour content. At the gene expression level, e-liquid induces changes in gene expression consistent with epithelial to mesenchymal transition (EMT) revealed by reduced expression of cell epithelial markers such as E-cadherin and enhanced expression of mesenchymal proteins like vimentin and B-catenin seen both in OSCC cell lines and normal oral epithelium cells. In summary, the ability of e-liquid to induce proliferative and invasive properties along the activation of the EMT process can contribute to the development of tumorigenesis in normal epithelial cells and promote aggressive phenotype in pre-existing oral malignant cells.

Hedgehog-GLI and Notch Pathways Sustain Chemoresistance and Invasiveness in Colorectal Cancer and Their Inhibition Restores Chemotherapy Efficacy

Colorectal cancer (CRC) is a leading cause of cancer-related mortality and chemoresistance is a major medical issue. The epithelial-to-mesenchymal transition (EMT) is the primary step in the emergence of the invasive phenotype and the Hedgehog-GLI (HH-GLI) and NOTCH signaling pathways are associated with poor prognosis and EMT in CRC. CRC cell lines harboring KRAS or BRAF mutations, grown as monolayers and organoids, were treated with the chemotherapeutic agent 5-Fluorouracil (5-FU) alone or combined with HH-GLI and NOTCH pathway inhibitors GANT61 and DAPT, or arsenic trioxide (ATO) to inhibit both pathways. Treatment with 5-FU led to the activation of HH-GLI and NOTCH pathways in both models. In KRAS mutant CRC, HH-GLI and NOTCH signaling activation co-operate to enhance chemoresistance and cell motility, while in BRAF mutant CRC, the HH-GLI pathway drives the chemoresistant and motile phenotype. We then showed that 5-FU promotes the mesenchymal and thus invasive phenotype in KRAS and BRAF mutant organoids and that chemosensitivity could be restored by targeting the HH-GLI pathway in BRAF mutant CRC or both HH-GLI and NOTCH pathways in KRAS mutant CRC. We suggest that in KRAS-driven CRC, the FDA-approved ATO acts as a chemotherapeutic sensitizer, whereas GANT61 is a promising chemotherapeutic sensitizer in BRAF-driven CRC.

Challenges in using transcriptome data to study the c-di-GMP signaling network in Pseudomonas aeruginosa clinical isolates.

In the Pseudomonas aeruginosa type strain PA14, 40 genes are known to encode for diguanylate cyclases (DGCs) and/or phosphodiesterases (PDEs), which modulate the intracellular pool of the nucleotide second messenger c-di-GMP. While in general, high levels of c-di-GMP drive the switch from highly motile phenotypes towards a sessile lifestyle, the different c-di-GMP modulating enzymes are responsible for smaller and in parts nonoverlapping phenotypes. In this study, we sought to utilize previously recorded P. aeruginosa gene expression datasets on 414 clinical isolates to uncover transcriptional changes as a result of a high expression of genes encoding DGCs. This approach might provide a unique opportunity to bypass the problem that for many c-di-GMP modulating enzymes it is not known under which conditions their expression is activated. However, while we demonstrate that the selection of subgroups of clinical isolates with high versus low expression of sigma factor encoding genes served the identification of their downstream regulons, we were unable to confirm the predicted DGC regulons, because the high c-di-GMP associated phenotypes were rapidly lost in the clinical isolates,. Further studies are needed to determine the specific mechanisms underlying the loss of cyclase activity upon prolonged cultivation of clinical P. aeruginosa isolates.

Diversity and Potential Multifunctionality of Archaeal CetZ Tubulin-like Cytoskeletal Proteins.

Tubulin superfamily (TSF) proteins are widespread, and are known for their multifaceted roles as cytoskeletal proteins underpinning many basic cellular functions, including morphogenesis, division, and motility. In eukaryotes, tubulin assembles into microtubules, a major component of the dynamic cytoskeletal network of fibres, whereas the bacterial homolog FtsZ assembles the division ring at midcell. The functions of the lesser-known archaeal TSF proteins are beginning to be identified and show surprising diversity, including homologs of tubulin and FtsZ as well as a third archaea-specific family, CetZ, implicated in the regulation of cell shape and possibly other unknown functions. In this study, we define sequence and structural characteristics of the CetZ family and CetZ1 and CetZ2 subfamilies, identify CetZ groups and diversity amongst archaea, and identify potential functional relationships through analysis of the genomic neighbourhoods of cetZ genes. We identified at least three subfamilies of orthologous CetZ proteins in the archaeal class Halobacteria, including CetZ1 and CetZ2 as well as a novel uncharacterized subfamily. CetZ1 and CetZ2 were correlated to one another as well as to cell shape and motility phenotypes across diverse Halobacteria. Among other known CetZ clusters in orders Archaeoglobales, Methanomicrobiales, Methanosarcinales, and Thermococcales, an additional uncharacterized group from Archaeoglobales and Methanomicrobiales is affiliated strongly with Halobacteria CetZs, suggesting that they originated via horizontal transfer. Subgroups of Halobacteria CetZ2 and Thermococcales CetZ genes were found adjacent to different type IV pili regulons, suggesting potential utilization of CetZs by type IV systems. More broadly conserved cetZ gene neighbourhoods include nucleotide and cofactor biosynthesis (e.g., F420) and predicted cell surface sugar epimerase genes. These findings imply that CetZ subfamilies are involved in multiple functions linked to the cell surface, biosynthesis, and motility.

Identification of bi-allelic KIF9 loss-of-function variants contributing to asthenospermia and male infertility in two Chinese families.

Asthenozoospermia (AZS) is a leading cause of male infertility, affecting an estimated 18% of infertile patients. Kinesin proteins function as molecular motors capable of moving along microtubules. The highly conserved kinesin family member 9 (KIF9) localizes to the central microtubule pair in the flagella of Chlamydomonas cells. The loss of KIF9 expression in mice has been linked to AZS phenotypes. Variant screening was performed by whole exome sequencing from 92 Chinese infertile patients with AZS. Western blot was used to was used for analyzing of candidate proteins expression. Patients' sperm samples were stained with immunofluorescent to visualise proteins localization and were visualised by transmission electron microscopy (TEM) to determine axoneme structures. Co-immunoprecipitation assay was used to verify the binding proteins of KIF9. In vitro fertilization (IVF) was used to evaluate the efficiency of clinical treatment. Bi-allelic KIF9 loss-of-function variants were identified in two unrelated Chinese males exhibiting atypical sperm motility phenotypes. Both of these men exhibited typical AZS and suffered from infertility together with the complete absence of KIF9 expression. In contrast to these KIF9-deficient patients, positive KIF9 staining was evident throughout the flagella of sperm from normal control individuals. KIF9 was able to interact with the microtubule central pair (CP) component hydrocephalus-inducing protein homolog (HYDIN) in human samples. And KIF9 was undetectable in spermatozoa harboring CP deletions. The morphologicy of KIF9-deficient spermatozoa appeared normal under gross examination and TEM. Like in mice, in vitro fertilization was sufficient to overcome the fertility issues for these two patients. These findings indicate that KIF9 associates with the central microtubules in human sperm and that it functions to specifically regulate flagellar swinging. Overall, these results offer greater insight into the biological functions of KIF9 in the assembly of the human flagella and its role in male fertility.