Delivering therapeutics to the central nervous system (CNS) is difficult because of the blood–brain barrier (BBB). Therapeutic delivery across the tight junctions of the BBB can be achieved through various endogenous transportation mechanisms. Receptor-mediated transcytosis (RMT) is one of the most widely investigated and used methods. Drugs can hijack RMT by expressing specific ligands that bind to receptors mediating transcytosis, such as the transferrin receptor (TfR), low-density lipoprotein receptor (LDLR), and insulin receptor (INSR). Cell-penetrating peptides and viral components originating from neurotropic viruses can also be utilized for the efficient BBB crossing of therapeutics. Exosomes, or small extracellular vesicles, have gained attention as natural nanoparticles for treating CNS diseases, owing to their potential for natural BBB crossing and broad surface engineering capability. RMT-mediated transport of exosomes expressing ligands such as LDLR-targeting apolipoprotein B has shown promising results. Although surface-modified exosomes possessing brain targetability have shown enhanced CNS delivery in preclinical studies, the successful development of clinically approved exosome therapeutics for CNS diseases requires the establishment of quantitative and qualitative methods for monitoring exosomal delivery to the brain parenchyma in vivo as well as elucidation of the mechanisms underlying the BBB crossing of surface-modified exosomes. The central nervous system (CNS) is one of the most in-demand areas for the development of new therapeutics owing to the increasing occurrence rate of neurodegenerative disorders. However, it remains the most difficult area for drug development because of the blood–brain barrier (BBB), which prevents most of the currently developed drugs from entering the brain parenchyma. The BBB functions as a tight barrier to protect the CNS from potential neurotoxic substances, and regulates the selective transport of specific molecules and nutrients to maintain CNS homeostasis. Water molecules and small ions cross brain capillaries through channels, and small molecules under 500 Da can cross the BBB via passive diffusion . HoweverAmong the novel therapeutics, exosomes have recently gained attention because of their role as therapeutic vehicles for delivering various active pharmaceutical ingredients to the brain. Exosomes, or small extracellular vesicles (EVs), are a subtype of EVs defined as single-membrane lipid bilayer vesicles generated by vesicle budding into endosomes that mature into multivesicular bodies or by direct vesicle budding from the plasma membrane . DiffereNoninvasive delivery of therapeutics to the CNS can be achieved by hijacking endogenous transport pathways, such as receptor-mediated transcytosis (RMT) and adsorptive-mediated transcytosis , Table 1Transcytosis is the vesicular crossing of macromolecules from one side of the cell membrane to another . RMT is The transferrin receptor (TfR) is a widely used and validated receptor for the RMT-mediated BBB crossing of therapeutics. Transferrin is an iron-binding glycoprotein that delivers iron to cells by binding TfR. Although TfR is a ubiquitously expressed receptor, proteomics analysis confirmed that TfR is one of the highest expressed receptors that induces transcytosis in mice  and humaThe low-density lipoprotein receptor (LDLR) family is mainly responsible for the endocytosis of low-density lipoproteins (LDLs), such as apolipoprotein B (ApoB) and apolipoprotein E (ApoE). Each LDL particle contains a single apolipoprotein surrounded by fat molecules, such as cholesterol, phospholipids, and triglycerides, which mediate the delivery of these fatty acids into cells in need. LDLR is not only a ubiquitously expressed receptor, but is also widely expressed in the brain, rendering it an efficient transporter of therapeutics. The conjugation of ApoB- and ApoE-derived peptides to proteins, such as lysosomal enzymes, has been demonstrated to successfully transport proteins across the BBB ,26,27,28Insulin receptor (INSR) is widely expressed in various tissues. A recent study comparing the expression levels of various receptors mediating transcytosis showed that only INSR was overexpressed in human brain microvessels compared to brain parenchymal and peripheral tissues . A similCD98 heavy chain (CD98hc), also known as 4F2 antigen, is a heterodimer membrane protein consisting of a type 2-glycosylated 80-kDa heavy chain linked to a 37-kDa light chain by disulfide bonds ,54. CD98Glucose transporter 1 (GLUT1), also known as SLC2A1, is a glucose transporter highly expressed on both the apical and basolateral membranes of BCECs . The humCell-penetrating peptides (CPPs), or protein transduction domains, are a family of short peptides (<30 amino acids) that can induce the translocation of biologically active macromolecules across cell membranes without interacting with specific receptors ,16. AlthNeurotropic viruses can cross the BBB and invade the brain parenchyma, which prompted the investigation of viruses or viral components as transporters for the brain delivery of therapeutics. For instance, peptides derived from rabies virus glycoprotein (RVG) exhibit efficient penetration through the BBB and target neurons . AlthougUnmodified exosomes from various cell types show <1% delivery to the brain after systemic injection ,78, implTransport across the BBB is enhanced under specific pathological conditions. In mice exhibiting brain inflammation, macrophage-derived exosomes showed over three-fold increased delivery to the brain compared to those in normal mice . EnhanceCD9 as a control or CD9/LEL170-ApoB, in which ApoB was inserted between the 170–171 amino acid of CD9, to generate ApoB-expressing exosomes. Control exosomes (CD9 exosomes) or ApoB-expressing exosomes (CD9-ApoB exosomes) were isolated and purified from the supernatants of transfected Expi293F cells. To characterize the tissue distribution of the injected exosomes, we labeled CD9 exosomes with DiO and CD9-ApoB exosomes with DiD lipophilic fluorescent dyes, which were injected intravenously into mice. A laser scanning intravital confocal microscope was used to visualize the labeled exosomes in the cerebral cortex of mice by implanting a cranial window. We observed the accumulation of CD9-ApoB exosomes in the cortical blood vessels compared to CD9 exosomes, which were not detected in the vessels  peptide, which binds to integrin αhemistry . Click chemistry ,96,97,98hemistry .Exosomes are gaining attention because of their potential as next-generation nanoparticles for treating CNS diseases owing to their potential for natural BBB crossing and broad surface-engineering capability. Various technologies to efficiently incorporate drugs and active pharmaceutical ingredients into exosomes are being actively developed ,99,100.


One organoid line, UK1254, was predicted to be resistant to carboplatin based on its EC50 value (50.2 µM) being above clinically achievable Cmax. UK1254 had a significantly shorter PFS than the rest of the subjects (p = 0.0253) and was treated as a platinum-resistant recurrence. Subsequent gene expression analysis revealed extensively interconnected, differentially expressed pathways related to NF-kB, cellular differentiation (PRDM6 activation), and the linkage of B-cell receptor signaling to the PI3K–Akt signaling pathway (PI3KAP1 activation). This study demonstrates that patient-derived tumor organoids can be developed from patients at the time of primary or interval debulking surgery and may be used to predict clinical platinum sensitivity status or to investigate drivers of carboplatin resistance.The development of patient-derived tumor organoids (TOs) from an epithelial ovarian cancer tumor obtained at the time of primary or interval debulking surgery has the potential to play an important role in precision medicine. Here, we utilized TOs to test front-line chemotherapy sensitivity and to investigate genomic drivers of carboplatin resistance. We developed six high-grade, serous epithelial ovarian cancer tumor organoid lines from tissue obtained during debulking surgery (two neoadjuvant-carboplatin-exposed and four chemo-naïve). Each organoid line was screened for sensitivity to carboplatin at four different doses . Cell viability curves and resultant EC Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. In 2021, it is estimated that 21,410 women will be diagnosed with ovarian cancer and that it will be responsible for 13,770 deaths in the U.S. [The current standard of care treatment for advanced stage disease includes surgical debulking, with the goal of removing all macroscopic disease (R0 cytoreduction), in combination with platinum-based neoadjuvant or adjuvant chemotherapy . DespiteDespite the dramatic differences in outcomes between platinum-sensitive, -resistant, and -refractory groups, there is no validated method to predict clinical response to platinum-based chemotherapy, and all individuals receive the same up-front therapy. Chemosensitivity assays using patient-derived tumor cells have been increasingly explored to satisfy this unmet clinical need. In fact, some National Comprehensive Cancer Network (NCCN) centers employ the use of chemosensitivity assays to guide management in the face of recurrence when there are multiple equivalent chemotherapy options available . Early iHere, we developed and validated six patient-derived epithelial ovarian cancer TO lines that were subsequently screened for sensitivity to front line standard of care chemotherapeutic agents. TO genetic sequencing was used to identify genomic determinants of carboplatin resistance. Our primary objective was to assess the ability of TOs to predict clinical outcomes to initial chemotherapy. Secondary objectives included the identification of an integrated genomic signature of platinum resistance in EOC.Women with suspected or histologically confirmed epithelial ovarian cancer with a plan to undergo cytoreductive surgery were eligible for study inclusion. All patients who were potentially eligible were approached for enrollment by trained clinical research staff during their pre-operative clinic visit, which occurred 1–4 weeks before the scheduled debulking surgery. Written informed consent was obtained from all subjects, and they were enrolled in the Total Cancer Care Protocol: A Lifetime Partnership with Patients Who Have or May be at Risk of Cancer . Clinical outcome data were prospectively collected, deidentified, and correlated with TO chemosensitivity assay and genomic data by an honest broker. Disease assessments were performed per routine clinical practice by the treating provider to assess progression-free survival (PFS). Patient outcomes were followed until all patients demonstrated clinical evidence of recurrence or progression as defined by the RECIST version 1.1 criteria . The stu® Growth Factor Reduced Basement Membrane Matrix (Corning) in vitro using factor-defined media [® matrix with trypsin-EDTA/TrypLE followed by gentle mechanical dissociation. Once ovarian cancer TOs were established, the TOs were fixed, and then representative sections were H&E stained and compared with a primary tumor by a board-certified pathologist.Fresh ovarian tumor tissue was obtained from patients at the time of debulking surgery, dissociated into a single ovarian cancer cell suspension, and established in Matrigeled media ,15. TOs 50 values were determined. Established TOs were enzymatically dissociated into single cells and plated in 384-well plates. The cells were cultured for 72 h prior to the administration of carboplatin at five different doses . After culturing for an additional 72 h, organoids were incubated with Hoechst nuclear counterstain and imaged on a spinning disc confocal high content imager. After imaging was completed, viability was measured by with an MTS assay (Promega). Raw data were generated in triplicate, and the average cell viability for each drug concentration was determined after normalizing values to untreated negative controls. Cell viability curves were generated, and ECThe Tempus xT next generation targeted oncology sequencing assay was utilized to perform a gene mutation and expression analyses for all six generated TO cell lines. TO total nucleic acid was extracted and digested by proteinase K. RNA was purified from the total nucleic acid by DNase-I digestion. DNA and RNA sequencing was performed as previously described . BrieflyFor somatic mutation analysis, an oncoplot was generated based on the maftools  package 50 value to the clinically achievable plasma concentration of carboplatin. Resistant cell lines were defined as having a carboplatin EC50 above the plasma Cmax of carboplatin [50 within achievable plasma concentrations. One sample t-test was used to compare carboplatin-resistant and pooled carboplatin-sensitive EC50 values using GraphPad Prism 8. The Kaplan–Meier method was used to estimate PFS curves for platinum-sensitive and -resistant patients. The PFS curves were compared via the log-rank test using R 4.0.3. A p < 0.05 was considered as statistically significant.The classification of each TO cell line as carboplatin-sensitive or -resistant was based on the comparison of the carboplatin ECboplatin . SensitiThe tumor samples used to generate the TO lines were derived from a relatively homogenous population. All subjects had histologically proven advanced-stage epithelial ovarian or fallopian tube cancer. Histologic subtype was exclusively high-grade serous. Primary disease sites were localized to the ovary (75%) and fallopian tubes (25%). One TO line (UK1393) was generated from a metastatic implant in the omentum, but all others were developed from the primary site of disease . The treatment courses of the study subjects were also relatively homogenous. All participants were treated with a platinum and taxane doublet. One subject’s taxane therapy (UK1236) was switched from paclitaxel to abraxane due to allergic reaction. Most subjects were chemo-naïve (66.7%) at the time of debulking surgery. However, two subjects (33.3%) were exposed to three cycles of neoadjuvant chemotherapy before interval debulking surgery. Optimal cytoreduction was achieved in 50% of patients, and all other debulking surgeries achieved <0.5 cm of residual disease. Most patients did not receive maintenance therapy. However, one patient received olaparib, and another was enrolled in a Gynecologic Oncology Group (GOG) clinic trial studying the effects of the PARP inhibitor rucaparib and immunotherapy agent nivolumab . It is uHistologic concordance between each ovarian cancer TO cell line and its respective primary ovarian cancer tumor sample was confirmed. After the establishment of the TO cell line, a sample of it was formalin-fixed and stained using hematoxylin and eosin (H&E) . Primary50 values were determined for all generated TO lines  and was the highest of all TO line EC50 values. Conversely, all other TO lines were determined to be sensitive to carboplatin, with a significantly lower pooled cell viability EC50 mean value (p = 0.018). All carboplatin-sensitive TO cell lines demonstrated EC50 values within the range of achievable plasma concentrations  . UK1254  = 0.025 . ClinicaA limited mutation analysis was performed for all generated TO lines using the Tempus xT gene panel. Genes that were mutated in multiple cell lines and a selection of DNA repair genes were specifically interrogated to explore similarities in mutation profiles between TO cell lines for all enrolled subjects . As expeWe then performed a gene expression analysis using the RNA sequencing data in Qiagen’s IPA to evaluate differences in expression and pathways to better understand the mechanism of carboplatin resistance. All subjects had TO RNA sequencing data available. A total of 71 genes were significantly differentially expressed  between the carboplatin-resistant and carboplatin-sensitive TOs after appropriate thresholds were applied . Genes tThe top upregulated and downregulated differentially expressed genes comparing the carboplatin-resistant group to the carboplatin-sensitive group are displayed in Next, an in depth network analysis used the RNA sequencing data to determine cell-specific pathways impacted by carboplatin resistance. Notably, leukocyte extravasation signaling, GP6 signaling, cardiac hypertrophy signaling, and PI3K signaling in B lymphocytes were predicted to be increasingly activated in the carboplatin-resistant TO, as demonstrated by pathways represented with shades of orange. Conversely, the CD40 signaling, HER-2 signaling, and MSP-RON signaling in macrophages were suspected to have decreased activation in the carboplatin-resistant phenotype and are represented in shades of blue A. Pathwap-value). The color of the boxes correlates with the z-score, with the intensity of blue representing z ≤ 0 and the intensity of orange representing z ≥ 0. Pathways related to organismal injury and abnormalities, cancer, gastrointestinal disease, and reproductive system disease predominated. This suggests that carboplatin resistance is partly mediated by the alteration of injury-associated biological mechanisms and well-established cancer-related pathways.To better assess the clinical applicability of the gene expression analysis, we converted the pathway analysis to a heatmap with analysis by disease and organ system A. The lep-values with expression fold changes ≥ 0. We focused on the most significantly altered gene network  when unThe main strength of our study was that we were able to successfully correlate TO chemosensitivity assay results with clinical PFS despite only including six subjects in the analysis and that we identified genomic predictors of response. These results contrast prior ovarian cancer tumor organoid publications that report in vitro sensitivity to antineoplastics but fail to include correlation with clinical outcome and genomic predictors of resistance. Genomic predictors of platinum resistance identified at the initial surgery have the potential to guide subsequent clinical management with the advantages of convenience and speed over organoid sensitivity testing ,45,46,47The early stratification of patients into carboplatin-sensitive and -resistant cohorts, before clinical recurrence, may help delineate who should receive maintenance therapy with bevacizumab or a biosimilar. Furthermore, the combined use of TO chemosensitivity assay results and genomic markers of carboplatin resistance into a predictive scoring system of recurrence may provide a basis for additional cycles of cytotoxic chemotherapy beyond the traditional six. We envision that if the methodologies utilized here are applied to a larger cohort, we could develop a novel epithelial ovarian cancer predictive scoring system. The Oncotype DX test is a similar system that is currently the standard of care for adjuvant chemotherapy stratification in early stage, ER+, HER2/neu-negative breast cancer, and intermediate-risk prostate cancer. The development of an accurate scoring system that predicts an individual’s front line PFS has the potential to change the standard of care for high-grade serous ovarian cancer treatment and improve outcomes for thousands of patients every year. Tumor organoid (TO) development from chemo-naïve and neoadjuvant-chemotherapy-exposed epithelial ovarian cancer patients is both feasible and potentially predictive of clinical response to front line therapy. An integrated TO mutation and gene expression analysis can be utilized to investigate the molecular mechanisms of carboplatin resistance. The combination of these methods may provide the basis for development of a predictive recurrence scoring system that can be utilized to tailor maintenance and additional adjuvant therapy to individual patient needs.


In angiosperms, double fertilization requires pollen tubes to transport non-motile sperm to distant egg cells housed in a specialized female structure known as the pistil, mediating the ultimate fusion between male and female gametes. During this journey, the pollen tube encounters numerous physical barriers that must be mechanically circumvented, including the penetration of the stigmatic papillae, style, transmitting tract, and synergid cells as well as the ultimate fusion of sperm cells to the egg or central cell. Additionally, the pollen tube must maintain structural integrity in these compact environments, while responding to positional guidance cues that lead the pollen tube to its destination. Here, we discuss the nature of these physical barriers as well as efforts to genetically and cellularly identify the factors that allow pollen tubes to successfully, specifically, and quickly circumnavigate them. In angiosperms, the productive interaction between male and female gametes resulting in the formation of a new embryo and surrounding endosperm tissue is known as double fertilization. In contrast to many animal systems, sperm cells in higher plants are non-motile and thus rely on a specialized structure associated with the male gametophyte, known as the pollen tube, to move sperm cells to the egg. Additionally, the female reproductive tissue surrounding the egg cell is structurally complex and inaccessible to the male gamete. Therefore, the pollen tube must invade these tissues, circumvent numerous physical barriers, and discharge sperm cells to complete the fertilization process. Communication between pollen and pistil tissues is essential to mediate these successful fertilization events.The pollen grain initially interacts with specialized finger-like cells known as stigmatic papillae, and if this interaction is productive, the pollen grain will be hydrated by the stigmatic papillae cells and subsequently germinate to form a pollen tube . The polBrassicaceae species with dry-type stigmas.Along the pollen tube’s trajectory, the female tissue imposes numerous physical barriers , includiPollen tubes grow both in vitro and in vivo via a specialized process called tip growth in which the tip of the pollen tube elongates in a polarized fashion ,5,6. PolD-galacturonic acid residues that can be modified with acetyl or methyl esters [Plant cell walls are complex polysaccharide-rich extracellular matrices that commonly consist of three functionally distinct but interconnected polysaccharide networks: cellulose, neutral hemicelluloses, and acidic pectins ,8. Pollel esters . Methyl l esters ,12. Thisl esters ,10.GAUT genes (GAUT13 and GAUT14) are highly expressed in pollen tubes. Analysis of gaut13 and gaut14 loss-of-function single mutants did not reveal a substantial impact on pollen tube function, but double gaut13/gaut14 mutants produced pollen tubes that germinate but fail to elongate and additionally exhibited tip rupture under both in vitro and in vivo conditions [gaut13/gaut14 double mutants are devoid of de-methyl esterified HG in the pollen tube shank, but retain methyl esterified HG at the pollen tube tip. These observations indicate that GAUT13 and GAUT14 redundantly participate in the synthesis of pollen tube HG, and further indicate that this HG deposition is required for pollen tube elongation.HG synthesis requires the activity of homogalacturonan:galacturonosyltransferases (GAUTs) ,14. In Anditions . These epagr mutants produced defective pollen tubes that were unable to elongate under in vitro conditions and did not transmit in self-fertilization assays [RG-I and RG-II biosynthesis also appear to be critical for pollen tube elongation, although the biosynthesis of these polysaccharides is relatively poorly understood. RG-I is structurally composed of a backbone that consists of a repeating rhamnose α-(1→4)-galacturonic acid α-(1→2)-disaccharide . This ban assays . These oRG-II is an exceptionally complex polysaccharide that exhibits structural conservation across the green lineage . This poInvestigating pollen tube growth through the pistil tissue would be an ideal way to study physical barriers associated with this process. Although various methodologies are used to realize this goal ,28, thisCamellia japonica pollen tubes with a diameter ranging from 13–21 µm were germinated and directed to navigate a series of microgaps ranging from 17 to 10 µm. The pollen tubes were imaged during their growth trajectories, and several outcomes were observed, including successful navigation of the narrow opening, pollen tube bursting, or stalling. The frequency of these outcomes was linked to the ratio of opening size versus pollen tube diameter with smaller microgap sizes being associated with bursting or stalling. Similar work on Torenia fournieri pollen tubes resulted in similar conclusions [TipChip , a microclusions , demonstOne of the first barriers encountered by the pollen grain and subsequently formed pollen tube are the stigmatic papillae cells A. The poCDEF1 transcripts are highly expressed in pollen during initial interactions with the stigmatic papillae, but cdef1 null mutant pollen are capable of normal fertilization, suggesting that additional cutin degrading factors may be important for pollen tube entry through the cuticle.While the identity of plant cutinases has remained somewhat enigmatic, an Arabidopsis GDSL family esterase known as CUTIN DESTRUCTING FACTOR1 (CDEF1) caused substantial cutin degradation in transgenic plants over-expressing this enzyme . IntriguKATANIN mutants, or in response to the microtubule depolymerizing drug oryzalin. The loss of CMT anisotropy generally led to loss of cellulose microfibril anisotropy and caused pollen tube coiling around the papillar cell, suggesting that cellulose microfibrils deposited in an anisotropic orientation contribute to pollen tube guidance through the papillar cell wall. The authors further demonstrated by Atomic Force Microscopy (AFM) that ktn1-5 loss-of-function mutants had cell walls that were softer than wild-type papillar cells at the same developmental stage, indicating that wall stiffness surprisingly plays a role in efficient pollen tube growth through the papillar cell. These observations highlight the importance of both the cuticle and cell wall in pollen tube invasion at the papillar cell, but also indicate that the processes that actuate this invasive behavior are largely unclear.Once the pollen tube circumvents the papillar cell cuticle, it must rapidly elongate through the space between layers of primary and secondary cell wall . AlthougTIR-NBS-LRR)-type receptor family proteins appear to be enriched in this transcript subset, and these enzymes were recently described as NAD cleaving enzymes that promote cell death [NUCLEOREDOXIN1 (NRX1) and XIPOTL, that significantly impact pollen tube guidance through the pistil.After penetrating through a papillar cell, the pollen tube must begin to elongate through the apoplastic spaces of tightly associated cells within the style . During ll death . AdditioNRX1 encodes for a protein containing thioredoxin and C1-like domains [NRX1 in lipid signaling. However, NRX1 is a cytoplasmic and nuclear-localized protein that possesses disulfide bond reductase activity. Therefore, this protein may be less likely to play a role in plant lipid metabolism and more likely to contribute to protein folding of critical pollen tube targets, based on the observation that nrx1 mutants display male-specific segregation distortion [XIPOTL encodes for one of three Arabidopsis S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferases (PEAMT), which are required for phosphatidylcholine synthesis. Phosphatidylcholine is a major membrane phospholipid and precursor of phosphatidic acid, an important lipid signaling molecule [NRX1 and XIPOTL display abnormal growth patterns compared to wild-type pollen tubes, such as approaching but not entering the ovule micropyle, growing toward the chalazal end instead of the micropylar end of the ovule, and multiple pollen tubes being attracted to the same ovule [NRX1 and XIPOTL are necessary for accurate ovule targeting, but the mechanistic basis for these observations remains unresolved. domains . As C1 dstortion . XIPOTL molecule . Mutantsme ovule . OverallAnother elegant approach to elucidate pollen tube factors that contribute to enhanced growth within the pistil made use of high-throughput RNA-sequencing and automated SNP detection in fertilization assays between multiple ecotypes. This approach allowed for delineating gene expression changes in crosses between Cape Verde Island-0 (Cvi) and Columbia (Col-0) ecotype reproductive tissues. Upon evaluation of the differentially expressed genes using RNA-seq analysis, 277 genes were determined to be expressed specifically from the pollen tube and 6838 genes were expressed specifically from the pistil tissue . This hiO-FUCOSYLTRANSFERASE1 (AtOFT1) gene confers pollen tubes the ability to penetrate the stigma-style interface [AtOFT1 transcripts are expressed in pollen tubes and increase in abundance upon penetration through the stigma and style. oft1 mutant pollen tubes were indistinguishable from wild-type when grown in vitro, but oft1 mutant pollen tubes penetrated the stigma-style interface much slower compared to Col-0 pollen tube controls, resulting in an approximate 2000-fold decrease in oft1 pollen transmission and a nearly 10-fold reduction in seed set. The stigma-style interface proved to be a significant barrier for oft1, as excision of the stigma-style interface [oft1 mutant pollen to fertilize ovules at a much higher frequency and produce progeny [Despite these high-quality transcriptome studies, very few mutants have been isolated that are specifically compromised in their ability to penetrate the stigma-style interface. While there must be a continuum between mutants that impact growth in the style versus growth in the transmitting tract, we will define the former class of mutants as having specific impacts in the stigma-style interface as evidenced by reduced pollen tube penetration in SIV experiments. The Arabidopsis nterface . AtOFT1 nterface  from the progeny .OFT-like genes that all bear amino acid sequence similarity to metazoan Protein O-FucosylTransferases (POFTs). POFTs utilize GDP-fucose as a sugar nucleotide donor to transfer L-fucose onto serine and threonine residues of target proteins which typically contain Epidermal Growth Factor (EGF) or Thrombospondin Repeat (TSR) domains in metazoan systems, and these post-translational fucosylation events often confer critical regulatory functions. For example, in the Notch signaling cascade, mono-O-fucosylation of the Notch extracellular domain potentiates interactions with cognate Notch ligands, such as Serrate, Delta, and Jagged [O-fucosylation events ultimately communicate mechanical information between cells to regulate cell adhesion and cell fate [AtOFT1 remain to be elucidated. However, it is proposed that oft1 mutant pollen tubes might fail to penetrate the stigma-style interface due to their inability to degrade the pistil cell wall extracellular matrix or that AtOFT1 might post-translationally modify other proteins that serve as receptors for receiving positional guidance cues from the stigma, style, and transmitting tract [The Arabidopsis genome contains 39 putative d Jagged ,50,51,52ell fate . Substrang tract . It is ang tract ,56.VACUOLAR PROTEIN SORTING41 (VPS41) gene encodes a novel factor that facilitates control of pollen-stigma interaction. vps41 loss-of-function mutants display a phenotype of disrupted stigma/style penetration, resulting in male gametophyte sterility. VPS41 localizes to pollen tube late endosomes and tonoplasts and interacts with RAB GTPases  that is responsible for approximately 80% of the PME activity in pollen tubes. The vgd1 mutants produce pollen tubes that successfully penetrate through stigmatic papillae cells but fail to rapidly elongate at the stigma-style interface [Additional work highlights the importance of regulated pectin metabolism in pollen tube stigma/style penetration. nterface . BecauseAfter penetrating the stigma-style interface, the pollen tube continues growing through the transmitting tract (TT). It is important to note that the pollen tube must balance two important functions during this time. First, the pollen tube must elongate over a substantial distance and successfully navigate toward an unfertilized ovule. Second, the pollen tube is literally built to burst, but it must not burst prematurely. Thus, positional guidance cues in the transmitting tract must encourage the pollen tube to elongate toward ovules and actively prevent premature pollen tube rupture.ANXUR (ANX) 1 and 2 are pollen expressed Catharanthus roseus receptor-like kinase (CrRLK)-type proteins that coordinate proper rupturing time and release of the two sperm nuclei into the ovule. Interestingly, anx1 or anx2 mutant pollen tubes display premature rupturing in vitro. Similarly, double anx1/anx2 mutant pollen tubes prematurely rupture in the transmitting tract or style tissue when applied to pistils [Several recent studies have elegantly highlighted the complex mechanical signaling mechanisms that prevent premature pollen tube bursting in the transmitting tract. The  pistils . As pollanx1/anx2 mutants were observed in mutants of BUDDHA’S PAPER SEAL (BUPS) 1 and 2, a second pair of CrRLK-type receptor-like kinases [RAPID ALKALINIZATION FACTORS (RALF) 4 and 19 [LORELEI-LIKE GENE (LLG) 2 and 3 [anx1/anx2 phenotypes. These initial observations led multiple groups to demonstrate that LLG, ANX, and BUPS proteins form a multi-protein receptor complex that perceives RALF4 and 19 peptides that are secreted from the pollen tube and actively prevent the pollen tube from bursting. RALF peptides are also secreted by ovules, and the ovule-secreted RALF34 is capable of displacing RALF4 or 19 from the ANX-BUPS-LLG receptor complex in vitro, leading to rapid and quantitative pollen tube bursting [Phenotypes that are similar to  kinases ,61. Addi4 and 19 , as well 2 and 3 ,63, recabursting . These obursting , suggestbups mutant pollen tubes could be compressed while growing through small microfluidic channels similar to TipChip described above, but that bups pollen tubes ruptured shortly after exiting the narrow channel [bups mutation, suggesting that ROP1-mediated exocytosis is likely important for the mechanotransduction associated with pollen tube exit from the stigma-style interface, and that this process may be regulated by physical interactions between the BUPS intracellular domain, interacting RopGEF factors, and ROP1.Despite these advances, many questions remain about the functions that are performed by this complex signaling system B. For ex channel . This obNicotiana tabacum, arabinogalactan proteins (AGPs) are a predominant class of extracellular matrix proteins found in the stylar transmitting tract [agp6; agp11 double mutants, suggesting a conserved role of AGPs in pollen tube growth and elongation [In addition to maintaining structural integrity, pollen tubes must follow long-range positional guidance cues in the transmitting tract C. In Nicng tract . AGPs arng tract . TTS proongation .POLLEN–PISTIL INCOMPATIBILITY 2 (POP2) mutant is compromised in the degradation of GABA due to a mutation in GABA transaminase, which leads to 20-fold higher levels of GABA in the transmitting tract [pop2 mutant. GABA is likely perceived by calcium-permeable ion channels at the pollen tube tip that stimulate periodic calcium influx that is associated with pollen tube growth [pop2 mutant, which leads to reduced growth of pollen tubes through the pistil and female sterility. These observations suggest that the GABA gradient established in the pistil may serve as a long-range positional guidance cue to help pollen tubes navigate through this structure.Genetic evidence also suggests a role for γ-amino-butyric acid (GABA) in pollen tube navigation through the transmitting tract. The Arabidopsis ng tract . GABA ste growth . Under nAs pollen tubes navigate the transmitting tract, they must also be directed to unfertilized ovules by positional guidance cues from the ovule. The recently identified LURE peptides play a large role in this portion of pollen–pistil interactions. LUREs are small defensin-like peptides secreted from synergid cells that attract pollen tubes alone in vitro or in the context of the ovule ,73. LUREAt an unfertilized ovule, the pollen tube must penetrate the synergid cells, rupture, and release sperm cells, which represents yet another unique cell penetration event . The ferFERONIA/SIRENE (FER/SRN) receptor-like kinase is expressed in synergid cells and localizes to the filiform apparatus to control pollen tube behavior during synergid cell penetration [fer mutants displayed female semi-sterility, pollen tube coiling within the synergid, and frequent polytubey [FER confers female gametophytes the ability to control pollen tube behavior following synergid cell penetration. The establishment of FER’s role in pollen tube signaling has allowed for subsequent elucidation of other effector proteins and members in its signaling pathway.The etration . Null feolytubey . Thus, Ffer mutant, including female sterility, polytubey, and pollen tube coiling in the synergid cell without pollen tube bursting [herk1/anj mutants [Mutations in the GPI-anchored protein LRE were isolated independently but displayed all of the hallmarks of the bursting . Similar mutants , indicat mutants . These oThe molecules that activate the FER-HERK1/ANJ-LRE complex and the mechanisms of downstream signaling leading to pollen tube rupture and blocking additional pollen tubes are less clear D. It is MYB97, 101, and 120 formed a closely related pollen tube-expressed subclade [myb97/101/120 triple mutants, revealing that only 48 genes were differentially expressed by these transcription factors, encompassing enriched functional categories of transporters, carbohydrate-active enzymes, and small secreted proteins. One of the small, secreted proteins identified is a cysteine-rich thionin that is specifically expressed in pollen tubes and requires the activity of all three MYB transcription factors for expression. As thionins are recognized for their ability to form transmembrane pores, it is proposed that this function might also aid in conferring the pollen tube its ability to burst in a controlled manner.The pollen tube also likely contributes to correctly-timed bursting within the synergid cell. Upon investigating which transcription factors display elevated expression during pollen development and pollen tube growth, subclade , leadingThe final physical barriers of double fertilization require that released sperm cells fuse separately with the egg and central cell to form the new embryo and endosperm tissue, respectively. Careful cytological analysis using sperm cell-localized photo-convertible fluorescent proteins suggest that sperm cells are equivalent and can fuse with either nuclei  and thatgex2 mutants exhibited male sterility that was ultimately linked to failed sperm fusion events, and, in an elegant assay, the authors of this study demonstrated that gex2 mutant sperm cells adhere to protoplasted egg cells less frequently than wild-type sperm [hap/gcs1 mutants were not compromised in egg-sperm cell interactions, suggesting that GEX2 plays an important role in this cellular interaction.GAMETE EXPRESSED 2 (GEX2) is a second plasma membrane-localized protein of unknown function that participates in cellular interactions between sperm and egg cells. The pe sperm . InteresDOMAIN OF UNKNOWN FUNCTION 679 Membrane Protein 9 (DMP) genes in Arabidopsis were found to be transcriptionally co-expressed with GEX2 and encode protein products that were likely expressed in sperm cells. Double dmp8/dmp9 mutants show various hallmarks of reduced male fertility, including reduced seed set and reduced pollen transmission, but these mutants also display the curious phenotype that they often produce aborted seeds with fertilized central cells, but unfertilized embryos [dmp8/dmp9 mutants were more often compromised in egg-sperm cell fusion events than in central cell-sperm fusions. These observations suggest that DMP proteins may contribute to cell fusion selectivity and that future work may reveal a role in other unique DMP family members toward mediating central cell-sperm fusion events.Recent work has shed light on how sperm cells select a fusion target. A small family of  embryos . FurtherThe information presented above suggests a model describing the unique functions of these proteins in overcoming the gamete fusion physical barrier. GEX2 seems to play a primary role in tethering cells together to facilitate membrane fusion, while HAP2/GCS1 mediates the formal process of membrane fusion. The recently identified DMP proteins may contribute to selectivity of the cell fusion events, and EC1 serves as a secreted signal that prepares the sperm cells for fusion prior to arriving at the egg or central cell. Despite this progress, it remains unclear whether proteins or other signals displayed on the egg or central cell have analogous or complementary activities that mediate cellular tethering, fusion, or cell fusion selectivity.Pollen tube penetration through the various cell types of the pistil requires the coordinated signaling of growth factors that facilitate pollen tube elongation and navigation ,72,75 asIt is quite fascinating that pollen tubes are capable of penetrating through extracellular spaces that are only a few hundred nanometers wide in vivo but lack this capacity in controlled in vitro settings. It is certainly clear that pollen tubes secrete an array of cell wall modifying enzymes that can degrade and capacitate cell wall material along the female reproductive tract . Howeveroft1 and vps41 mutants are similar  to many of the mutants that are impacted in this pathway, and their roles in regulating BUPS-ANX-LLG signaling should be investigated in greater detail. O-fucosylation events or other post-translational modifications might confer receptors or other proteins the ability to mechanically interact with their respective pistillar receptors to signal that navigation and penetration are taking place. This possible mechanical interaction could result in female tissue reorganizing or further cooperation between the pollen tube and the pistil.We have illustrated a wide array of signaling cascades and feedback mechanisms that pollen tubes utilize in order to successfully locate and fertilize an ovule. Recent work indicates that the BUPS-ANX-LLG receptor complex plays a critical and dynamic role during pollen–pistil interactions by maintaining pollen tube mechanical stability in the stigma/style interface and transmitting tract, while also facilitating bursting at the synergid. During mechanical signaling at the stigma-style interface, it is clear that ROP1-mediated polarized secretion participates in this process , but it Although a large body of recent work has highlighted mechanisms by which pollen tubes are attracted to ovules, growth attractants in the transmitting tract remain less clear. It will also be important to understand whether other factors secreted into the transmitting tract have long range guidance functions or serve to passivate the transmitting tract for easier pollen tube growth. Transcriptomic studies have attempted to delineate differences between pollen tube and pistillar cell transcriptomes, however, a large number of pistillar transcripts remain to be investigated. Thus, we also ask the questions: Are the pistillar cells receiving a signal from the pollen tube and facilitating its navigation? Or does this navigation depend on the pollen tube’s ability to navigate and penetrate independently of female tissue aid?As noted above, many questions remain unanswered regarding mechanical signaling at physical barriers during pollen–pistil interactions, with many of these mechanistic understanding gaps at the stigma-style interface or during growth in the transmitting tract. Perhaps these barriers could be better mechanistically defined by taking advantage of recent technological advances, which we highlight here. First, two-photon microscopy can provide a viable experimental platform to observe the behavior of live pollen tubes growing through pistil tissue . Thus, tInvestigating mechanical signaling in plant systems is quite difficult because the majority of cells in plant tissues adhere to one another though cell wall matrices. Pollen–pistil interactions represent a unique example of plant cell–cell interactions and cell–cell signaling that can be precisely controlled by the simple addition of pollen. While this system has vastly enhanced our understanding of mechanical signaling in the pistil and beyond, many questions remain to be resolved. Utilization of the approaches outlined above, as well as addressing some of these critical questions, will likely have important impacts on our understanding of pollen–pistil interactions, as well as broader questions associated with how plant cells interact to form the plant body.


Mycobacterium tuberculosis (Mtb) infection is the marked heterogeneity that exists, spanning lesion type differences to microenvironment changes as infection progresses. A mechanistic understanding of how this heterogeneity affects Mtb growth and treatment efficacy necessitates single bacterium level studies in the context of intact host tissue architecture; however, such an evaluation has been technically challenging. Here, we exploit fluorescent reporter Mtb strains and the C3HeB/FeJ murine model in an integrated imaging approach to study microenvironment heterogeneity within a single lesion in situ, and analyze how these differences relate to non-uniformity in Mtb replication state, activity, and drug efficacy. We show that the pH and chloride environments differ spatially even within a single caseous necrotic lesion, with increased acidity and chloride levels in the lesion cuff versus core. Strikingly, a higher percentage of Mtb in the lesion core versus cuff were in an actively replicating state, and correspondingly active in transcription/translation. Finally, examination of three first-line anti-tubercular drugs showed that isoniazid efficacy was conspicuously poor against Mtb in the lesion cuff. Our study reveals spatial relationships of intra-lesion heterogeneity, sheds light on important considerations in anti-tubercular treatment strategies, and establishes a foundational framework for Mtb infection heterogeneity analysis at the single bacterium level in situ.A hallmark of  Mycobacterium tuberculosis (Mtb), is non-uniformity in bacterial physiology and disease progression. This heterogeneity is thought to be a major reason for the need for prolonged treatment, and understanding what and how facets of the infection differ within a host or single lesion is thus vital for the development of improved therapeutic strategies. However, elucidation and direct demonstration of bacterial non-uniformity during whole animal infection, in the context of intact tissue architecture, has been extremely challenging and consequently lacking. We show here using an integrated imaging approach that Mtb experiences different pH and chloride microenvironments depending on its sublocation within a structured lesion, which strikingly correlates with Mtb replication status and transcriptional/translational activity. We further find that these spatial differences impact critically on drug efficacy, with the first-line anti-tubercular drug isoniazid demonstrating conspicuously poor efficacy against Mtb residing in the structured lesion cuff, versus the bacteria present in the lesion core. Our study reveals how several aspects of non-uniformity in Mtb infection biology are related to spatial location within a structured lesion, and lays the groundwork for analysis of Mtb infection heterogeneity at the single bacterium level in intact tissue.A critical aspect of tuberculosis disease, caused by the bacterium  Mycobacterium tuberculosis (Mtb) is significantly impeded by the marked heterogeneity of the infection across multiple levels, including non-uniformity in local microenvironments  -GFP replication reporter to determine the replication status of Mtb in sublocations within a lesion. In this reporter strain, the Mtb SSB protein is translationally fused to GFP, driven by the native acterium ,26. Stririum [-] , with a The replication state of Mtb can differentially impact drug efficacy and is implicated as a major contributor to the difficulties of successfully treating Mtb infection, as well as to the necessity for a prolonged treatment time course –31. Yet in situ. A dual fluorescent system where one fluorophore is placed under the control of an inducible promoter and a second spectrally distinct fluorophore is expressed constitutively has been utilized in differentiating transcriptionally active versus non-active Mtb in cultured macrophages  is additionally present even within a lesion sublocation. The local pH and [Cl-] environment experienced by intracellular Mtb within the lesion cuff is expected to be driven by the actions of both the host and the bacterium, given the decrease in pH and increase in [Cl-] that occur during macrophage phagosome maturation [+-ATPase [-] in the local environment still observed even within the population of Mtb residing in the necrotic core was not correlated with specific sub-regions of the core , and whether these differences have active drivers will require further investigation.Despite the consensus on the importance of heterogeneity on Mtb infection progression and treatment outcome, population-level readouts such as bacterial load and host cytokine levels continue to be the primary means by which these outcomes are measured. Our establishment here of an integrated imaging approach for d caseum ,10, by pturation , and the+-ATPase . The varWe anticipate that other vital environmental signals such as nitric oxide, which is actively produced by host immune cells, are likely to also exhibit intra-lesion heterogeneity. In addition, pimonidazole-based histological labeling has indicated the presence of hypoxia in the cuff of caseous necrotic lesions ,47. Reduin vivo and in situ comparison of the replication state of Mtb present in the caseous necrotic core versus in macrophages in the lesion cuff, with a higher percentage of Mtb in the lesion core versus cuff found to be in an actively replicating state. While this fits with the less acidic pH/lower [Cl-] microenvironment of the lesion core versus cuff, the result is intriguing in the context of ex vivo experiments with caseum Mtb from a rabbit infection model that reported decreased replication state of the bacteria [in situ and in vivo context, and not against bacteria in broth or in vitro macrophage infection conditions. Indeed, the percentage of Mtb residing in the necrotic lesion core undergoing active DNA replication is lower than what is observed for bacteria grown in broth (76–80% for Mtb in broth) [ex vivo caseum study utilized caseum extracted from lesions in rabbits that had been infected for 12 to 16 weeks [in vivo infections, to delineate if the conditions within the caseous necrotic core change with time and infection state. Additional analyses expanding on the reporters utilized to encompass those that report on Mtb metabolism, such as cholesterol utilization [Excitingly, utilization of the SSB-GFP replication reporter enabled, to our knowledge, the first bacteria . A key dn broth) . Our stun broth) . In cont16 weeks . It willlization , will fu606’::mKO-tetON, smyc’::mCherry reporter provided a method for analyzing bacterial transcriptional/translational activity in vivo and in situ. While we posit that the lack of mKO signal seen from bacteria in the very central core of the caseous necrotic lesion arises from impedance of dox penetration into the lesion core, it will be interesting in future studies to directly examine dox penetration into caseous necrotic lesions with fine spatial resolution, using methods such as MALDI-IMS or fluorescent dox derivatives. A previous study utilizing laser capture microdissection from Mtb-infected rabbits had reported that dox concentrations in the caseum were comparable to those from the cellular region surrounding the necrotic core, but did not examine for possible differences in dox levels between the peripheral regions of the necrotic core versus the very central region [606’::mKO-tetON, smyc’::mCherry reporter Mtb can be observed will be an additional method for probing dox lesion penetration characteristics.Complementary to the SSB-GFP replication reporter, the Pl region ,54. Thesl region ). It is By elucidating and directly demonstrating the spatial non-uniformity in Mtb replication status and transcriptional/translational activity within a single lesion and its striking correlation to local differences in drug efficacy, this work sets a foundational framework for interrogation of: (i) the heterogeneity in diverse aspects of Mtb infection biology within and between lesions in a single host, via the use of various reporter Mtb strains ,52,56–58Staphylococcus aureus biology in abscesses and Yersinia pseudotuberculosis physiology and host response within microcolonies [Pseudomonas aeruginosa and Vibrio cholerae have also recently been elegantly demonstrated [in situ to other bacterial systems thus hold exciting potential for further revealing the impact of spatial heterogeneity during infection on pathogen growth and treatment success.Finally, the approaches and concepts established here are likely to also have broad applicability to other bacterial species, with the burgeoning appreciation of the importance of spatial heterogeneity in varied aspects of bacterial-host interactions for infection outcome of many pathogens. This spans spatial differences in colonies –62, to mcolonies ,64. Spatnstrated ,66. ExteAll animal procedures followed standards set by the National Institutes of Health “Guide for the Care and Use of Laboratory Animals”. Animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Tufts University (#B2021-139), in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care, the US Department of Agriculture, and the US Public Health Service guidelines. Light anesthesia during infection and oral gavage administration of drugs was via exposure to 2% isoflurane delivered by a vaporizer system. Euthanasia utilized carbon dioxide gas with regulated flow, consistent with American Veterinary Medical Association guidelines.smyc’::mCherry; rv2390c’::GFP, smyc’::mCherry; SSB-GFP, smyc’::mCherry) used for measuring differences in local microenvironment and replication status were in the Erdman background and have been previously described [606’::mKO-tetON, smyc’::mCherry reporter has also been previously described [Reporter Mtb strains  containing 0.05% Tween 80, under light anesthesia with 2% isoflurane. Mice were sacrificed at 2, 6, 7, or 8 weeks post-infection, and the lungs fixed overnight in 4% paraformaldehyde (PFA) in PBS, before transfer and storage in PBS prior to analysis. For histology imaging, lung lobes were paraffin-embedded and processed for standard hematoxylin and eosin staining (Tufts Comparative Pathology Services), with images captured using a Nikon Eclipse E400 microscope equipped with a SPOT Insight color digital camera.6–8 week old female C3HeB/FeJ wild type mice  were intranasally infected with 10606’::mKO-tetON, smyc’::mCherry reporter strain infection, infections were allowed to establish for one or six weeks, before provision of the mice with water containing 5% sucrose ± 1 mg/ml doxycycline, with one additional water change during the one week treatment period. For the drug treatment infections, C3HeB/FeJ wild type mice were infected with the SSB-GFP, smyc’::mCherry reporter and infection allowed to establish for six weeks, before commencement of treatment with 10 mg/kg isoniazid (INH), 150 mg/kg pyrazinamide (PZA), or 10 mg/kg rifampicin (RIF) via oral gavage five times a week for 2 weeks (in a 200 μl volume). Control infected mice were mock-treated with sterile water. All drugs were prepared weekly, and all oral gavage treatments were carried out under light anesthesia with 2% isoflurane. INH and PZA working solutions were prepared directly in sterile water. RIF working solution was prepared by diluting 50 mg/ml RIF stock in DMSO to 10 mg/kg RIF + 5% DMSO in sterile water.For the PFixed lung lobes were embedded in 4% agarose in PBS and 250 μm sections obtained with a Leica VT1000S vibratome . Stainin606’::mKO-tetON, smyc’::mCherry reporter strain, high magnification and resolution images for reporter signal quantification for Mtb residing in the lesion core were taken both from the central core and the more peripheral regions of the core. Regions were selected based only on bacteria presence, and without regard for reporter signal. In each case, 3–6 images were taken, each with a 40x oil immersion objective at 2x digital zoom. In the case of the P606’::mKO-tetON, smyc’::mCherry reporter strain infection, as indicated in the Results section, the lack of apparent doxycycline penetration into the central core precluded analysis of the reporter signal of the bacteria residing there, and thus in this case, high magnification and resolution images for reporter signal quantification of Mtb in the lesion core were taken only from the more peripheral regions of the core. Images for reporter quantification of Mtb residing in the lesion cuff were taken from areas containing bacteria in the CD68-positive region ringing the lesion core. As with the images from the core, regions were selected based only on bacteria presence, and without regard for reporter signal. To further ensure robustness of data, at least 5 lesions across multiple mice were analyzed.For broad xy-plane imaging, the Leica LAS X Navigator module was used to obtain multiple overlapping images that were then automatically merged together. High resolution images for reporter quantification were 10 μm in depth, reconstructed into 3D using Volocity software  from images taken at 0.5 μm z-steps. For all experiments except for those with the Prv2390c’::GFP reporter, the volume of each bacterium was measured via the mCherry channel, and the corresponding total GFP signal for that given object (bacterium) simultaneously measured. The settings for the GFP channel were maintained across samples to allow for comparison of values. Statistical analysis was performed using a Mann-Whitney test for comparison of reporter signal in bacteria present in the cuff versus core region in each lesion. A multiple t-test with a Holm-Sidak correction was used for statistical analysis of the binned data of reporter signal in the cuff versus core across all lesions. The P606’::mKO-tetON, smyc’::mCherry reporter strain was quantified in the same manner as for the rv2390c’::GFP reporter. For the SSB-GFP reporter, individual bacteria were identified via the mCherry channel and the number of bacteria with SSB-GFP puncta determined. Numbers of bacteria quantified in each case are indicated in the figure legends. Statistical analysis was performed using a Wilcoxon matched-pairs signed rank test for comparison of lesion cuff versus core values, and with a Mann-Whitney statistical test for comparing drug to mock treatment in a lesion sublocation.Quantification of reporter signal was carried out essentially as previously described using Volocity software ,25,67. IS1 FigC3HeB/FeJ mice were infected with Mtb for 6 or 8 weeks, before sacrifice and lung homogenates plated for colony forming unit determination. Each point represents a single mouse. Horizontal lines indicate the median.(TIF)Click here for additional data file.


Injury to the peripheral or central nervous systems often results in extensive loss of motor and sensory function that can greatly diminish quality of life. In both cases, macrophage infiltration into the injury site plays an integral role in the host tissue inflammatory response. In particular, the temporally related transition of macrophage phenotype between the M1/M2 inflammatory/repair states is critical for successful tissue repair. In recent years, biomaterial implants have emerged as a novel approach to bridge lesion sites and provide a growth-inductive environment for regenerating axons. This has more recently seen these two areas of research increasingly intersecting in the creation of ‘immune-modulatory’ biomaterials. These synthetic or naturally derived materials are fabricated to drive macrophages towards a pro-repair phenotype. This review considers the macrophage-mediated inflammatory events that occur following nervous tissue injury and outlines the latest developments in biomaterial-based strategies to influence macrophage phenotype and enhance repair. Macrophages are innate immune cells and play a critical role in modulating injury and potential repair in the peripheral nervous system (PNS) and the central nervous system (CNS). Together with the neuronal-specific immune cells, the microglia, macrophages can undergo phenotypic shifts into either a pro-inflammatory M1) or a range pro-repair M2) sub-types  [1]. Phe or a ran sub-typeThe PNS has a remarkable capacity for repair. Damaged axons are capable of spontaneous, supported regeneration following injury, and can restore functional motor and sensory connections to distal targets depending on the defect length. A series of orchestrated events occur to mend the affected axons and recover neural functions after a traumatic peripheral nerve injury (PNI). This repair process is divided into three sequential stages encompassing 1. Wallerian degeneration, 2. axonal regrowth and 3. restoration of nerve function . Wallerian degeneration is mediated by an initial innate-immune response after PNI. This involves the activation, migration, proliferation and differentiation of Schwann cells , fibroblasts and macrophages to degrade damaged axons and prepare the environment for regrowth . FirstlyOver the course of nerve repair in the proximal and distal injured nerve segments PNS, the Schwann cell population change their phenotype to progenitor-like Schwann cells and start overexpressing pro-inflammatory cytokines and chemokines to initiate macrophage activity. At the same time, the blood–nerve barrier, mainly composed of endothelial cells , breaks At approximately 7–10 days post-injury, the axonal regrowth phase starts . ProgeniIn instances where there is nerve transection, a tissue bridge of macrophages, neutrophils, fibroblasts, endothelial cells, fibronectin and elastin must be built between the gap. This cellular bridge serves as a ‘travel path’ for Schwann cells and axons ,20. At tIn the final phase, which can take approximately 20 days , the inf2 < 10 mmHg) and necrotic . I. I187]. In addition to chemically crosslinking the biomaterial, creating a biomaterial for electrochemical stimulation can also modulate macrophage behavior in the peripheral nerve. Graphene oxide (GO) has recently gained interest due to its bio-adaptability and electroconductivity, which are necessary to facilitate the transmission of action potentials ,193. SevNanodiamonds (NDs) represent a non-graphene carbon-based approach to chemically manipulating macrophage behaviours in PNI. Contrarily to graphene, NDs are not electrically conductive, but still have an excellent biocompatibility . In addiTherapeutic treatment with other biological compounds revealed another promising approach for PNI inflammation . MiconazAn additional approach can be the use of biological factors such as cytokines. For this reason, several studies proposed their use to regulate macrophages polarization after PNI. Mokarram et al. found that the local delivery of IFN-γ or IL-4 within a polymeric guidance channel is effective in inducing macrophage polarization towards the M1 phenotype and M2 phenotype, respectively. In this case, the delivery approach was performed by filling a polysulfone tube with agarose hydrogel mixed with the cytokines . Similar+ cells in vivo. However, given that SKPSCs also express high levels of IL-6, their use in combination with anti-IL-6 treatment can be beneficial to avoid detrimental effects associated to this pro-inflammatory cytokine  (PHPMA)) based hydrogels for brain  and SCI Other synthetic polymers, however, have been reported to reduce overall macrophage numbers in the lesion site. PLLA multi-channel conduits fabricated with a ladder-like porous channel wall alleviated the infiltration of  macrophages/microglia both inside and around the conduits, with the total number reducing much faster over time compared to the untreated control group . A similImidazole-poly(organophosphazenes) hydrogels implanted into the lesioned rat cord induced endothelial and fibroglial infiltration, which was accompanied by extensive macrophage infiltration. Four weeks post-injury, many were CD206+, indicating that most possessed an M2 phenotype . Hakim aSimilar to the findings for peripheral nerve graphs see , modificPore size has been shown to be an important feature for axonal repair in the CNS. Thomas and colleagues (2013) studied this effect after SCI using a variety of PLGA porosities, noting that porosities of 80%, corresponding to channel diameters of 230 µm, contained the largest numbers of regrowing axons , indicatThe stiffness of biomaterial scaffolds and hydrogels has a direct influence on macrophage and microglia behavior. In general, stiffnesses that mimic those of the spinal cord and brain (200–600 Pa)  induce sBiologically derived materials have been extensively used as CNS bridging materials due to their innate biocompatibility and bioactivity, biodegradability, and low antigenicity, and because the exhibit similar soft properties as their target tissues . They arInjectable formulations of acellular peripheral nerve tissue have been developed that form hydrogels once in situ and more efficiently fill the lesion cavity of the injured cord and facilitate cellular transplant efficacy , encouraInjectable extracellular matrix (ECM) derived hydrogels have also been generated from porcine brain ,313, spiElsewhere, scaffolds composed of decellularized mammalian ECM have facilitated a constructive host response and diminished the number of M1 macrophages (CD86+/CD68+) around the implant and resulting fibrosis . FurtherSimilar to the results demonstrated in peripheral nerves, graphene oxide (GO) has also been shown to exert positive effects on macrophage phenotype after surgical insertion into the laterally hemisectioned rat spinal cord ,309. ED-Finally, self-assembling peptides (SAPs) comprise a family of biocompatible, biodegradable and easily modifiable short chain amino acid sequences, , capableDifferent organic and synthetic polymers have also been employed as injectable nanoparticle delivery vehicles and are capable of encapsulating, absorbing or conjugating different agents such as steroids (methylprednisolone), antibiotics (minocycline), and chemical (Chicago sky blue) cargoes in a controlled and high reproducible manner ,322 Tab. NanoparAcutely administered polycaprolactone nanoparticles loaded with the anti-inflammatory antibiotic minocycline injected directly into the compressed mouse spinal cord were readily taken up by activated microglia and macrophages . M1 macrUsing larger delivery vehicles to target SCI via the intravenous route was also explored by Saxena and colleagues (2015) and demonstrated the delivery of liposome particles carrying cargoes could be targeted to the injured cord. Liposomes containing the small molecule Chicago sky blue (CSB), a known macrophage migration inhibitory factor (MIF), was delivered 48 h after cord contusion injury . Using qPolymer scaffolds have also been used to carry cargoes that target macrophages and influence development of pro-reparative phenotypes ,310,311.Incorporating exogenous stem cells into implantable biomaterials has shown particular promise for CNS repair ,330,331 Biomaterial mediated delivery of MSCs exhibits trophic axon growth promoting properties ,335,336 Adult human bone marrow MSCs implanted into gelatin sponge scaffolds sheathed with a thin film of PLGA and inserted into the fully transected and excised (1.5 mm segment) rat spinal cord resulted in a three-fold decrease in CD68+ cells in the graft and a lower number of CD68+ cells surrounding the scaffold in the lesion site 1 week post-injury . HoweverNeural stem and progenitor cells (NSPCs) also exhibit immunomodulatory capabilities and a number of studies have demonstrated changes in macrophage responses after implantation ,344,345.NSPCs implanted in scaffolds such as methacrylamide chitosan (MAC) hydrogel protected by an outer chitosan conduit may modulate macrophage behavior, reduce ED-1+ cells in the lesion site and improve neuronal regrowth 8 weeks after hemisection injury . Of the Over the past two decades, the development of biomaterials to repair traumatic nervous system injury has increased in complexity as understanding of the multi-faceted nature of neuronal repair grows. In particular, the identification of the key role played by macrophages in the nerve micro-environment has given rise to the emerging field of immune-modulatory biomaterials.Attenuating the macrophage-mediated inflammatory response driven primarily by M1 macrophage phenotypes may have significant benefits, specifically, increasing axonal regrowth ,111. Of Furthermore, the ability to incorporate therapeutic immunomodulatory cargoes  has prompted the development of multi-component scaffolds/hydrogels. This offers the potential to develop temporally encoded sequential or sustained release of therapeutic agents that influence macrophage phenotype at different stages in the repair process. Although promising, there are several caveats for future work aimed at translation of controlled release strategies for macrophage modulation; namely, they will need to ensure the stability and reliability of any controlled release system. Any strategy that inadvertently exerts too rapid a change in macrophage number and/or polarization may prove counterproductive and impede the neuronal repair process. Immunomodulation is not the elimination of the M1 phenotype, but rather ensures the timely transition to M2 pro-repair phenotypes occurs at the right time and with sufficient effect to achieve a meaningful improvement in axonal regrowth. Care must be taken that introduced nucleic acids, proteins and cells do not result in their own immune reaction and induce or exacerbate chronic inflammation. Similarly, the degradation products or leachables of any particulate delivery system should not result in their own inflammatory reaction.On a more practical level, it is important that efforts are made for future studies to have clinical relevance. Current pre-clinical work almost always assumes biomaterial implantation immediately following PNS or CNS injury. However, in most cases, medical stabilization of the patient takes precedence, with surgical intervention usually occurring in the days to weeks after injury. Therefore, the biomaterial implant will encounter an entirely different cellular milieu with macrophage-mediated responses in transition from the initial inflammatory phase to the later proliferative and remodeling phases. For the future, it will be essential to verify that biomaterial implants retain immunomodulatory functionality at different stages of nervous tissue repair. An additional practical consideration is to ensure pre-clinical and clinical studies are comprehensive. There are known sex-based differences brought about by the anti-inflammatory effects on macrophages of estrogen, progesterone and allopregnanolone in PNS and CNS ,348. FaiRegarding CNS injuries, it has been shown that there are differences in terms of immune and inflammatory response between neonatal and adult brains. In particular, resident microglia are the main responding cells in adult injuries, whereas peripheral derived macrophages are predominant in injuries in developing brains . FurtherFundamentally, there remains a multitude of questions to be answered regarding the macrophage phenotype and its impact on neuronal regeneration. For example, do all M1 macrophages transition to the M2 phenotype of which they are capable? Furthermore, it will be necessary to determine within the injured tissue if all M2 subtypes develop from a common M2 progenitor or have a more direct activation pathway from an M1 subtype. Possessing a clearer understanding of macrophage activation pathways and where they develop in relation to repairing tissue structures will be key for future biomaterial designs. Evidence from several studies already indicates that biomaterial implants may affect the regional distribution of M1/M2 macrophages with greater numbers of M2 macrophages present within the internal structures of implants and with M1-type cells restricted to the periphery . These fThe pivotal role macrophages play in coordinating and regulating the inflammatory response to injury in the central and peripheral nervous systems has received much warranted attention over the past 20 years. It is now clear that macrophage-driven responses to acute injury and their synergistic interaction with glial cells at the site of injury are critical for host tissue driven inflammatory responses in both the CNS and PNS. Enhancing the necessary transition from the inflammation associated M1 phenotype to the tissue remodeling associated M2 phenotype, a key feature of peripheral nerve repair and one lacking from the CNS injury response, may drive a stronger regenerative response. Biomaterials offer considerable potential to help influence and enhance the M1/M2 transition in lesion sites through their composition and physiochemical properties, in addition to their ability to carry multiple different cargo types. Building on recent advances, coupled with careful tailoring of composite materials, internal architecture design and functionalization with various cargo types, will lead to future generations of biomaterial implants capable of exerting fine spatiotemporal control of macrophage behavior and drive better regenerative outcomes.


Complex oncological treatment can be associated with lymphatic vascular injury that is burdened by considerable morbidity. Lymphatic imaging and interventional techniques offer new minimally invasive treatment options. We report the case of a 59-year-old woman with an unusual lympho-veno-cutaneous fistula, diagnosed by magnetic resonance lymphangiography and treated by minimally invasive embolization therapy and venous recanalization. Lymphatic fistulas are rare, difficult to treat and potentially fatal complications of oncologic surgery or central venous catheterization. Lymphatic fistulas after surgery usually occur due to direct lymph vessel injury (e.g. of the thoracic duct) . In caseNew techniques to visualize lymph vessels and perform targeted minimally invasive treatment have been established . EspeciaWe report a case of a 59-year-old woman with cutaneous high-volume chylolymphatic discharge due to an unusual lympho-veno-cutaneous fistula without any direct lymph vessel injury, demonstrating the importance of selective lymphatic imaging to enable targeted therapy.A 59-year-old woman with advanced high-grade ovarian cancer presented with a high-volume chylolymphatic cutaneous fistula with a drainage volume of 1000–2000 ml chylous fluid (triglyceride levels > 500 mg/dl) per day from a left periclavicular wound after explantation of a pectoral port catheter system. Port explantation became necessary due to acute venous thrombosis of the left subclavian, internal jugular and brachiocephalic vein with oedema of the left arm.Initially conservative treatment was tried for 2 weeks with anticoagulation with heparin and parenteral nutrition at an outside hospital. However, drainage volume was unaffected and conservative therapy was ultimately unsuccessful with the patient developing progressing cachexia. Clinically the cause of lymphatic leakage remained unclear, as a post-operative lymph vessel injury was unlikely due to only superficial incision for port extraction. The patient was referred to our hospital for further treatment. We performed interstitial transpedal magnetic resonance lymphangiography (MRL). Transpedal MRL is described in detail elsewhere . In shorThe venous segment into which the thoracic duct drained was recanalized, but isolated from central and distal venous blood flow by persistent thrombosis of the brachiocephalic and axillary vein. The port catheter was previously inserted into this segment, resulting in lymph flow from the isolated venous segment through the established catheter tract into the port recess with subsequent cutaneous leakage Fig. . The cauInterventional treatment was planned with the intention of keeping central lymphatic drainage intact. Through a right femoral venous access, a 5F diagnostic catheter was used to pass the venous occlusion of the brachiocephalic vein. Additionally, the fistula was cannulated from the cutaneous porus using a microcatheter  and microwire . Digital-subtraction angiography confirmed MR-lymphangiographic findings. Central venous drainage was recanalized from by percutaneous transluminal angioplasty Fig.  and implLymphatic leakage ceased completely immediately after the intervention. There was no recurrence of leakage or post-interventional sequela during a follow-up of 4 weeks. After this period, the patient succumbed to her advanced stage ovarian cancer.This unusual case of an intractable lympho-veno-cutaneous fistula, initially thought to be due to post-surgical lymph vessel injury with high-volume chylolymphatic discharge, demonstrates the value of novel lymphatic imaging and interventional techniques. Although the incidence of post-surgical chylolymphatic leakage after head and neck surgery is low, the consequences of this complication such as malnutrition, infection and immunosuppression can be severe and life threatening . It is wConservative management is usually the first-line therapy of post-operative lymphatic fistulas . In thisIn conclusion, management of post-surgical chylolymphatic leakage remains challenging. Dedicated lymphatic imaging in combination with a growing range of interventional treatment options enable individualized and targeted treatment of these lymphatic pathologies.


Pseudomonas is a highly diverse genus that includes species that cause disease in both plants and animals. Recently, pathogenic pseudomonads from the Pseudomonas syringae and Pseudomonas fluorescens species complexes have caused significant outbreaks in several agronomically important crops in Turkey, including tomato, citrus, artichoke and melon. We characterized 169 pathogenic Pseudomonas strains associated with recent outbreaks in Turkey via multilocus sequence analysis and whole-genome sequencing, then used comparative and evolutionary genomics to characterize putative virulence mechanisms. Most of the isolates are closely related to other plant pathogens distributed among the primary phylogroups of P. syringae, although there are significant numbers of P. fluorescens isolates, which is a species better known as a rhizosphere-inhabiting plant-growth promoter. We found that all 39 citrus blast pathogens cluster in P. syringae phylogroup 2, although strains isolated from the same host do not cluster monophyletically, with lemon, mandarin orange and sweet orange isolates all being intermixed throughout the phylogroup. In contrast, 20 tomato pith pathogens are found in two independent lineages: one in the P. syringae secondary phylogroups, and the other from the P. fluorescens species complex. These divergent pith necrosis strains lack characteristic virulence factors like the canonical tripartite type III secretion system, large effector repertoires and the ability to synthesize multiple bacterial phytotoxins, suggesting they have alternative molecular mechanisms to cause disease. These findings highlight the complex nature of host specificity among plant pathogenic pseudomonads. All sequenced genomes from this study are available from GenBank through BioProject PRJNA680595 (accession numbers SAMN16885796–SAMN16885853).Pseudomonas syringae and Pseudomonas fluorescens species complexes include some of the most globally significant agricultural pathogens, and are driving a growing number of outbreaks on fruit and vegetable crops across Turkey. Here, we compare the genomes of bacterial strains driving several of these disease outbreaks. We show that distantly related strains can cause disease on the same crop, and that many pathogenic strains lack characteristic virulence factors like the type III secretion system and bacterial phytotoxins. Our results highlight the complex nature of host–pathogen interactions and suggest that even bacteria from the same species complex that cause disease on the same host will often do so by distinct virulence mechanisms.Plant pathogenic diseases often emerge without warning and can have devastating effects on global food security. However, the evolutionary origins and virulence mechanisms that drive agricultural outbreaks are usually unknown. Bacteria from the Pseudomonas is a highly complex genus that includes hundreds of described species, some of which cause devastating disease in both plants and animals . Pr. Pr34]. P. syringae strains [P. syringae  can be caused by distinct lineages of P. syringae, while phenotypically similar diseases on distinct, but closely related, hosts  can be caused by a single, closely related clade of pathogens. These observations raise interesting questions about the evolution of pathogen host specificity. For example, how frequently does convergent evolution onto the same host occur, and do these events typically entail minor variations of the same virulence mechanisms , or completely different virulence mechanisms? Do convergent virulence mechanisms target the same host immune complexes? Can we use these data to identify functionally similar groups of T3SEs or host immune complexes? Do all pathogen lineages have the potential to infect a broader array of hosts  or is there variation with some lineages being more specialized while others are more generalists? Is host generalism driven more by specific pathogen virulence factors or common host immune factors? What role do putatively non-specific virulence factors, such as toxins, play in host generalism? Unfortunately, addressing these questions will require both broad (i.e. from many different hosts) and deep (i.e. many isolates from the same population) sampling, which is currently not available.In this study, we analysed the population differentiation and virulence mechanisms of a diverse collection of Pseudomonas strains and is consistent with previous studies that have identified distinct lineages of isolates that cause similar disease symptoms on well-studied hosts like bean, tomato, cherry and kiwifruit [P. syringae phylogroups and, therefore, share a common ability to secrete T3SEs via the canonical T-PAI T3SS (Xanthomonas, is likely also a good framework for understanding host specificity convergence in P. syringae because of the diversity and functional redundancy of the major virulence factors in this species complex.The convergence of disease symptoms among distinct evolutionary lineages highlights the adaptive flexibility of virulence mechanisms employed by iwifruit . One posiwifruit . This isPAI T3SS . FurtherP. syringae T3SEs raises the interesting possibility that these T3SSs may contribute to virulence by secreting other virulence genes not traditionally associated with P. syringae pathogens. Further studies will be required to determine whether these strains harbour currently unknown effectors. While some common host-associated genes have been identified in comparative genomic studies of host range, it is rarely the case that genetic knock-out or knock-in experiments fully explain host-specificity [P. fluorescens complex, may enable the identification of these virulence factors via genome-wide association and predictive modelling analyses.Alternatively, molecular mechanisms independent of T3SE and phytotoxin repertoires may underlie the ability of divergent strains to cause the same disease. This seems more likely to be the case in tomato pith pathogens, which encompass a highly diverse collection of strains, do not harbour a canonical T-PAI T3SS, and secrete only a single phytotoxin. Interestingly, despite the fact that these strains do not harbour a canonical T-PAI T3SS, all of them do harbour either the S-PAI, F-PAI or C-PAI T3SS, which all have similar genetic architectures to the T-PAI T3SS. The presence of these T3SSs and the absence of homologues of the vast majority of cificity , illustrP. syringae strains. A common notion that has long been held in the P. syringae research community is that while the species complex as a whole has a broad host range, individual strains tend to be highly host-specific [Our observation of overlapping disease-causing lineages that do not cluster by host also raises a number of key questions related to the generalism of different specific . Recent specific . The citspecific . Whetherspecific , remainsP. syringae is ubiquitous as an epiphyte in a wide range of agricultural and non-agricultural settings [The emergence of recent outbreaks causing seedling blight of watermelon and leaf blight/necrosis of muskmelon in Turkey further suggest that naturally occurring host shifts are common. Both the watermelon seedling blight and muskmelon leaf necrosis pathogens arose in phylogroup 2, and were not accompanied by major changes to virulence repertoires. The propensity of phylogroup 2 strains to show weaker clustering by host of isolation may indicate that these strains have broader host ranges, which may be the result of their reduced reliance on potentially immune eliciting T3SEs for virulence . Becausesettings , an incrPseudomonas outbreaks in Southern Turkey continues to threaten the agricultural output in these regions [The frequency of severe  regions . However regions . We alsoClick here for additional data file.Click here for additional data file.