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Background: Patients with metastatic colorectal cancer (mCRC) carrying a deficit in the mismatch repair system/microsatellite instability (dMMR/MSI) show great responses to immune checkpoint inhibitors. However, 30% of patients with dMMR/MSI are primarily immunoresistant, and another 30% develop secondary resistance. Thus several combinations such as anti-programmed cell death protein 1 (anti-PD-1) and anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) are being pursued. The combination of radiotherapy and immunotherapy is another avenue of research that can increase the release of neoantigens resulting in the abscopal effect. This phenomenon has demonstrated promising potential activity in colon cancer preclinical studies; nevertheless, clinical results are limited to just a few case series. Patients and methods: We conducted a prospective interventional single-institution study to assess the feasibility, safety, and disease control rate of the combination of pembrolizumab and stereotactic ablative radiotherapy (SABR) in a cohort of 14 consecutive patients with dMMR/MSI mCRC. Results: Among the 14 patients enrolled, 11 received SABR in combination with pembrolizumab as the first to the fourth line. The disease control rate was 50% in the intention-to-treat population, with six patients still maintaining the response after >15 months. Any-grade treatment-related adverse events occurred in 50% of patients, with grade 3 (G3) events in three patients; no treatment-related death occurred. Conclusions: Our findings convey no signal of enhanced systemic efficacy compared with historical data on pembrolizumab alone even if the local control rate is high. To our knowledge, this represents the largest study conducted in this population; further studies could extend the knowledge on the toxicity profile of this combination.

Growing evidence suggests that many essential intracellular signaling events are compartmentalized within kinetically distinct microdomains in cells. Genetically encoded fluorescent biosensors are powerful tools to dissect compartmentalized signaling, but current approaches to probe these microdomains typically rely on biosensor fusion and overexpression of critical regulatory elements. Here, we present a novel class of biosensors named FluoSTEPs (fluorescent sensors targeted to endogenous proteins) that combine self-complementing split green fluorescent protein, CRISPR-mediated knock-in, and fluorescence resonance energy transfer biosensor technology to probe compartmentalized signaling dynamics in situ. We designed FluoSTEPs for simultaneously highlighting endogenous microdomains and reporting domain-specific, real-time signaling events including kinase activities, guanosine triphosphatase activation, and second messenger dynamics in live cells. A FluoSTEP for 3,5-cyclic adenosine monophosphate (cAMP) revealed distinct cAMP dynamics within clathrin microdomains in response to stimulation of G protein-coupled receptors, showcasing the utility of FluoSTEPs in probing spatiotemporal regulation within endogenous signaling architectures.

We designed an epi-illumination SPIM system that uses a single objective and has a sample interface identical to that of an inverted fluorescence microscope with no additional reflection elements. It achieves subcellular resolution and single-molecule sensitivity, and is compatible with common biological sample holders, including multi-well plates. We demonstrated multicolor fast volumetric imaging, single-molecule localization microscopy, parallel imaging of 16 cell lines and parallel recording of cellular responses to perturbations.

Spectral imaging is a powerful technique used to simultaneously study multiple fluorophore labels with overlapping emissions. Here, we present a computational spectral imaging method, which uses sample spatial fluorescence information as a reconstruction constraint. Our method addresses both the under-sampling issue of compressive spectral imaging and the low throughput issue of scanning spectral imaging. With simulated and experimental data, we have demonstrated the reconstruction precision of our method in two and three-color imaging. We have experimentally validated this method for differentiating cellular structures labeled with two red-colored fluorescent proteins, tdTomato and mCherry, which have highly overlapping emission spectra. Our method has the advantage of totally free wavelength choice and can also be combined with conventional filter-based sequential multi-color imaging to further improve multiplexing capability.

The major histocompatibility complex-class I chain related proteins A and B (MICA/B) is upregulated because of cellular stress and MICA/B shedding by cancer cells causes escape from NKG2D recognition favoring the emergence of cancers. Cholangiocarcinoma (CCA) is a relatively rare, though increasingly prevalent, primary liver cancer characterized by a late clinical presentation and a dismal prognosis. We explored the NKG2D-MICA/B axis in NK cells from 41 patients with intrahepatic cholangiocarcinoma (iCCA). The MICA/B-specific 7C6 mAb was used for ex vivo antibody-dependent cytotoxicity (ADCC) experiments using circulating, non tumor liver- and tumor-infiltrating NK cells against the HuCCT-1 cell line and patient-derived primary iCCA cells as targets. MICA/B were more expressed in iCCA than in non-tumoral tissue, MICA transcription being higher in moderately-differentiated compared with poorly-differentiated cancer. Serum MICA was elevated in iCCA patients in line with higher expression of ADAM10 and ADAM17 that are responsible for proteolytic release of MICA/B from tumor. Addition of 7C6 significantly boosted peripheral, liver- and tumor-infiltrating-NK cell degranulation and IFNγ production toward MICA/B-expressing established cell lines and autologous iCCA patient target cells. Our data show that anti-MICA/B drives NK cell anti-tumor activity, and provide preclinical evidence in support of 7C6 as a potential immunotherapeutic tool for iCCA.

Natural killer (NK) cells are emerging as unique players in the immune response against cancer; however, only limited data are available on tumor infiltrating NK cells in head and neck squamous cell carcinoma (HNSCC), one of the most common cancer. Occurrence of HNSCC is closely related to the immune microenvironment, and immunotherapy is increasingly being applied to this setting. However, the limited success of this type of treatment in this tumor calls for further investigation in the field.Surgical HNSSC specimens of 32 consecutive patients were mechanically and enzymatically dissociated. Tumor cells were separated from infiltrating cells by short centrifugation and infiltrating NK cells were phenotypically and functionally characterized by multiple antibody staining and flow cytometry. Tumor infiltrating NK cells in HNSCC showed a peculiar phenotype predominantly characterized by increased NKG2A and reduced Siglec-7, NKG2D, NKp30 and CD16 expression. This phenotype was associated with a decreased ability to perform antibody-dependent cellular cytotoxicity (ADCC). However, NK, CD4 and CD8 shared an increment of glucocorticoid-induced tumor necrosis factor-related (GITR) costimulatory receptor which could be exploited for immunotherapy with agonistic anti-GITR antibodies combined with checkpoint inhibitors.

Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact. To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen21-10/11 that improves the ratio of complemented signal to the background of FP1-10-expressing cells compared to the commonly used split GFP1-10/11; as well as a 10-fold brighter red-colored split-sfCherry21-10/11. Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry211 and GFP11, revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes.Split fluorescent proteins (FPs) have been widely used to visualise proteins in cells. Here the authors develop a screen for engineering new split FPs, and report a yellow-green split-mNeonGreen2 with reduced background, a red split-sfCherry2 for multicolour labeling, and its photoactivatable variant for super-resolution use.

Labeling proteins with high specificity and efficiency is a fundamental prerequisite for microscopic visualization of subcellular protein structures and interactions. Although the comparatively small size of epitope tags makes them less perturbative to fusion proteins, they require the use of large antibodies that often limit probe accessibility and effective resolution. Here we use the covalent SpyTag-SpyCatcher system as an epitope-like tag for fluorescent labeling of intracellular proteins in fixed cells for both conventional and super-resolution microscopy. We also applied this method to endogenous proteins by gene editing, demonstrating its high labeling efficiency and capability for isoform-specific labeling.

G-protein-coupled receptors (GPCRs) are increasingly recognized to operate from intracellular membranes as well as the plasma membrane. The β2-adrenergic GPCR can activate Gs-linked cyclic AMP (Gs-cAMP) signaling from endosomes. We show here that the homologous human β1-adrenergic receptor initiates an internal Gs-cAMP signal from the Golgi apparatus. By developing a chemical method to acutely squelch G-protein coupling at defined membrane locations, we demonstrate that Golgi activation contributes significantly to the overall cellular cAMP response. Golgi signaling utilizes a preexisting receptor pool rather than receptors delivered from the cell surface, requiring separate access of extracellular ligands. Epinephrine, a hydrophilic endogenous ligand, accesses the Golgi-localized receptor pool by facilitated transport requiring the organic cation transporter 3 (OCT3), whereas drugs can access the Golgi pool by passive diffusion according to hydrophobicity. We demonstrate marked differences, among both agonist and antagonist drugs, in Golgi-localized receptor access and show that β-blocker drugs currently used in the clinic differ markedly in ability to antagonize the Golgi signal. We propose 'location bias' as a new principle for achieving functional selectivity of GPCR-directed drug action.

Abstract Background Apomixis, an asexual mode of plant reproduction, is a genetically heritable trait evolutionarily related to sexuality, which enables the fixation of heterozygous genetic combinations through the development of maternal seeds. Recently, reference floral transcriptomes were generated from sexual and apomictic biotypes of Paspalum notatum, one of the most well-known plant models for the study of apomixis. However, the transcriptome dynamics, the occurrence of apomixis vs. sexual expression heterochronicity across consecutive developmental steps and the orientation of transcription (sense/antisense) remain unexplored. Results We produced 24 Illumina TruSeq®/ Hiseq 1500 sense/antisense floral transcriptome libraries covering four developmental stages (premeiosis, meiosis, postmeiosis, and anthesis) in biological triplicates, from an obligate apomictic and a full sexual genotype. De novo assemblies with Trinity yielded 103,699 and 100,114 transcripts for the apomictic and sexual samples respectively. A global comparative analysis involving reads from all developmental stages revealed 19,352 differentially expressed sense transcripts, of which 13,205 (68%) and 6147 (32%) were up- and down-regulated in apomictic samples with respect to the sexual ones. Interestingly, 100 differentially expressed antisense transcripts were detected, 55 (55%) of them up- and 45 (45%) down-regulated in apomictic libraries. A stage-by-stage comparative analysis showed a higher number of differentially expressed candidates due to heterochronicity discrimination: the highest number of differential sense transcripts was detected at premeiosis (23,651), followed by meiosis (22,830), postmeiosis (19,100), and anthesis (17,962), while the highest number of differential antisense transcripts were detected at anthesis (495), followed by postmeiosis (164), meiosis (120) and premeiosis (115). Members of the AP2, ARF, MYB and WRKY transcription factor families, as well as the auxin, jasmonate and cytokinin plant hormone families appeared broadly deregulated. Moreover, the chronological expression profile of several well-characterized apomixis controllers was examined in detail. Conclusions This work provides a quantitative sense/antisense gene expression catalogue covering several subsequent reproductive developmental stages from premeiosis to anthesis for apomictic and sexual P. notatum, with potential to reveal heterochronic expression between reproductive types and discover sense/antisense mediated regulation. We detected a contrasting transcriptional and hormonal control in apomixis and sexuality as well as specific sense/antisense modulation occurring at the onset of parthenogenesis.

Quorum sensing (QS) is a bacterial communication mechanism in which secreted signaling molecules impact population function and gene expression. QS-like phenomena have been reported in eukaryotes with largely unknown contributing molecules, functions, and mechanisms. We identify Qsp1, a secreted peptide, as a central signaling molecule that regulates virulence in the fungal pathogen Cryptococcus neoformans. QSP1 is a direct target of three transcription factors required for virulence, and qsp1Δ mutants exhibit attenuated infection, slowed tissue accumulation, and greater control by primary macrophages. Qsp1 mediates autoregulatory signaling that modulates secreted protease activity and promotes cell wall function at high cell densities. Peptide production requires release from a secreted precursor, proQsp1, by a cell-associated protease, Pqp1. Qsp1 sensing requires an oligopeptide transporter, Opt1, and remarkably, cytoplasmic expression of mature Qsp1 complements multiple phenotypes of qsp1Δ. Thus, C. neoformans produces an autoregulatory peptide that matures extracellularly but functions intracellularly to regulate virulence.

Abstract Background Apomixis is considered an evolutionary deviation of the sexual reproductive pathway leading to the generation of clonal maternal progenies by seeds. Recent evidence from model and non-model species suggested that this trait could be modulated by epigenetic mechanisms involving small RNAs (sRNAs). Here we profiled floral sRNAs originated from apomictic and sexual Paspalum notatum genotypes in order to identify molecular pathways under epigenetic control that might be involved in the transition from sexuality to agamospermy. Results The mining of genes participating in sRNA-directed pathways from floral Paspalum transcriptomic resources showed these routes are functional during reproductive development, with several members differentially expressed in apomictic and sexual plants. Triplicate floral sRNA libraries derived from apomictic and a sexual genotypes were characterized by using high-throughput sequencing technology. EdgeR was apply to compare the number of sRNA reads between sexual and apomictic libraries that map over all Paspalum floral transcripts. A total of 1525 transcripts showed differential sRNA representation, including genes related to meiosis, plant hormone signaling, biomolecules transport, transcription control and cell cycle. Survey for miRNA precursors on transcriptome and genome references allowed the discovery of 124 entities, including 40 conserved and 8 novel ones. Fifty-six clusters were differentially represented in apomictic and sexual plants. All differentially expressed miRNAs were up-regulated in apomictic libraries but miR2275, which showed different family members with opposed representation. Examination of predicted miRNAs targets detected 374 potential candidates. Considering sRNA, miRNAs and target surveys together, 14 genes previously described as related with auxin metabolism, transport and signaling were detected, including AMINO ACID/AUXIN PERMEASE 15, IAA-AMIDO SYNTHETASE GH3–8, IAA30, miR160, miR167, miR164, miR319, ARF2, ARF8, ARF10, ARF12, AFB2, PROLIFERATING CELL FACTOR 6 and NITRATE TRANSPORTER 1.1. Conclusions This work provides a comprehensive survey of the sRNA differential representation in flowers of sexual and apomictic Paspalum notatum plants. An integration of the small RNA profiling data presented here and previous transcriptomic information suggests that sRNA-mediated regulation of auxin pathways is pivotal in promoting apomixis. These results will underlie future functional characterization of the molecular components mediating the switch from sexuality to apomixis.

Sexuality and apomixis are interconnected plant reproductive routes possibly behaving as polyphenic traits under the influence of the environment. In the subtropical grass Paspalum notatum, one of the controllers of apospory, a main component of gametophytic apomixis reproduction, is TRIMETHYLGUANOSINE SYNTHASE 1 (TGS1), a multifunctional gene previously associated with RNA cleavage regulation (including mRNA splicing as well as rRNA and miRNA processing), transcriptional modulation and the establishment of heterochromatin. In particular, the downregulation of TGS1 induces a sexuality decline and the emergence of aposporous-like embryo sacs. The present work was aimed at identifying TGS1 target RNAs expressed during reproductive development of Paspalum notatum. First, we mined available RNA databases originated from spikelets of sexual and apomictic plants, which naturally display a contrasting TGS1 representation, to identify differentially expressed mRNA splice variants and miRNAs. Then, the role of TGS1 in the generation of these particular molecules was investigated in antisense tgs1 sexual lines. We found that CHLOROPHYLL A-B BINDING PROTEIN 1B-21 (LHC Ib-21, a component of the chloroplast light harvesting complex), QUI-GON JINN (QGJ, encoding a MAP3K previously associated with apomixis) and miR2275 (a meiotic 24-nt phasi-RNAs producer) are directly or indirectly targeted by TGS1. Our results point to a coordinated control exercised by signal transduction and siRNA machineries to induce the transition from sexuality to apomixis.

The capacity for apomixis in Paspalum notatum is controlled by a single-dominant genomic region, which shows strong synteny to a portion of rice chromosome 12 long arm. The locus LOC_Os12g40890, encoding the Auxin/Indole-3-Acetic Acid (Aux/IAA) family member OsIAA30, is located in this rice genomic segment. The objectives of this work were to identify transcripts coding for Aux/IAA proteins expressed in reproductive tissues of P. notatum, detect the OsIAA30 putative ortholog and analyze its temporal and spatial expression pattern in reproductive organs of sexual and apomictic plants. Thirty-three transcripts coding for AUX/IAA proteins were identified. Predicted protein alignment and phylogenetic analysis detected a highly similar sequence to OsIAA30 (named as PnIAA30) present in both sexual and apomictic samples. The expression assays of PnIAA30 showed a significant down-regulation in apomictic spikelets compared to sexual ones at the stages of anthesis and post-anthesis, representation levels negatively correlated with apospory expressivity and different localizations in sexual and apomictic ovules. Several PnIAA30 predicted interactors also appeared differentially regulated in the sexual and apomictic floral transcriptomes. Our results showed that an auxin-response repressor similar to OsIAA30 is down-regulated in apomictic spikelets of P. notatum and suggests a contrasting regulation of auxin signaling during sexual and asexual seed formation.

Simple Summary: Liver cancer is one of the most difficult solid tumors to treat and is responsible for one-third of cancer-related deaths worldwide. In particular, the quest for effective therapeutic strategies for hepatocellular carcinoma, which often arises from a chronic inflammatory background, remains an open challenge. In this review, we aim to provide an overview of the current therapeutic options available, focusing on recent advances in targeted therapies and the pursuit of emerging potential targets. Liver cancer, predominantly hepatocellular carcinoma (HCC), globally ranks sixth in incidence and third in cancer-related deaths. HCC risk factors include non-viral hepatitis, alcohol abuse, environmental exposures, and genetic factors. No specific genetic alterations are unequivocally linked to HCC tumorigenesis. Current standard therapies include surgical options, systemic chemotherapy, and kinase inhibitors, like sorafenib and regorafenib. Immunotherapy, targeting immune checkpoints, represents a promising avenue. FDA-approved checkpoint inhibitors, such as atezolizumab and pembrolizumab, show efficacy, and combination therapies enhance clinical responses. Despite this, the treatment of hepatocellular carcinoma (HCC) remains a challenge, as the complex tumor ecosystem and the immunosuppressive microenvironment associated with it hamper the efficacy of the available therapeutic approaches. This review explores current and advanced approaches to treat HCC, considering both known and new potential targets, especially derived from proteomic analysis, which is today considered as the most promising approach. Exploring novel strategies, this review discusses antibody drug conjugates (ADCs), chimeric antigen receptor T-cell therapy (CAR-T), and engineered antibodies. It then reports a systematic analysis of the main ligand/receptor pairs and molecular pathways reported to be overexpressed in tumor cells, highlighting their potential and limitations. Finally, it discusses TGFβ, one of the most promising targets of the HCC microenvironment. [ABSTRACT FROM AUTHOR]