Clearance phagocytosis by the retinal pigment epithelial during photoreceptor outer segment renewal: Molecular mechanisms and relation to retinal inflammation.

Mammalian photoreceptor outer segment renewal is a highly coordinated process that hinges on timed cell signaling between photoreceptor neurons and the adjacent retinal pigment epithelial (RPE). It is a strictly rhythmic, synchronized process that underlies in part circadian regulation. We highlight findings from recently developed methods that quantify distinct phases of outer segment renewal in retinal tissue. At light onset, outer segments expose the conserved "eat-me" signal phosphatidylserine exclusively at their distal, most aged tip. A coordinated two-receptor efferocytosis process follows, in which ligands bridge outer segment phosphatidylserine with the RPE receptors αvß5 integrin, inducing cytosolic signaling toward Rac1 and focal adhesion kinase/MERTK, and with MERTK directly, additionally inhibiting RhoA/ROCK and thus enabling F-actin dynamics favoring outer segment fragment engulfment. Photoreceptors and RPE persist for life with each RPE cell in the eye servicing dozens of overlying photoreceptors. Thus, RPE cells phagocytose more often and process more material than any other cell type. Mutant mice with impaired outer segment renewal largely retain functional photoreceptors and retinal integrity. However, when anti-inflammatory signaling in the RPE via MERTK or the related TYRO3 is lacking, catastrophic inflammation leads to immune cell infiltration that swiftly destroys the retina causing blindness.

Lens differentiation is controlled by the balance between PDGF and FGF signaling.

How multiple receptor tyrosine kinases coordinate cell fate determination is yet to be elucidated. We show here that the receptor for platelet-derived growth factor (PDGF) signaling recruits the p85 subunit of Phosphoinositide 3-kinase (PI3K) to regulate mammalian lens development. Activation of PI3K signaling not only prevents B-cell lymphoma 2 (BCL2)-Associated X (Bax)- and BCL2 Antagonist/Killer (Bak)-mediated apoptosis but also promotes Notch signaling to prevent premature cell differentiation. Reducing PI3K activity destabilizes the Notch intracellular domain, while the constitutive activation of Notch reverses the PI3K deficiency phenotype. In contrast, fibroblast growth factor receptors (FGFRs) recruit Fibroblast Growth Factor Receptor Substrate 2 (Frs2) and Rous sarcoma oncogene (Src) Homology Phosphatase 2 (Shp2) to activate Mitogen-Activated Protein Kinase (MAPK) signaling, which induces the Notch ligand Jagged 1 (Jag1) and promotes cell differentiation. Inactivation of Shp2 restored the proper timing of differentiation in the p85 mutant lens, demonstrating the antagonistic interaction between FGF-induced MAPK and PDGF-induced PI3K signaling. By selective activation of PI3K and MAPK, PDGF and FGF cooperate with and oppose each other to balance progenitor cell maintenance and differentiation.

Drug repurposing strategies in the development of potential antifungal agents.

The morbidity and mortality caused by invasive fungal infections are increasing across the globe due to developments in transplant surgery, the use of immunosuppressive agents, and the emergence of drug-resistant fungal strains, which has led to a challenge in terms of treatment due to the limitations of three classes of drugs. Hence, it is imperative to establish effective strategies to identify and design new antifungal drugs. Drug repurposing is a potential way of expanding the application of existing drugs. Recently, various existing drugs have been shown to be useful in the prevention and treatment of invasive fungi. In this review, we summarize the currently used antifungal agents. In addition, the most up-to-date information on the effectiveness of existing drugs with antifungal activity is discussed. Moreover, the antifungal mechanisms of existing drugs are highlighted. These data will provide valuable knowledge to stimulate further investigation and clinical application in this field. KEY POINTS: • Conventional antifungal agents have limitations due to the occurrence of drug-resistant strains. • Non-antifungal drugs act as antifungal agents in various ways toward different targets. • Non-antifungal drugs with antifungal activity are demonstrated as effective antifungal strategies.

Live Imaging of LysoTracker-Labelled Phagolysosomes Tracks Diurnal Phagocytosis of Photoreceptor Outer Segment Fragments in Rat RPE Tissue Ex Vivo.

Renewal of rod photoreceptor outer segments in the mammalian eye involves synchronized diurnal shedding after light onset of spent distal outer segment fragments (POS) linked to swift clearance of shed POS from the subretinal space by the adjacent retinal pigment epithelium (RPE). Engulfed POS phagosomes in RPE cells mature to acidified phagolysosomes, which accomplish enzymatic degradation of POS macromolecules. Here, we used an acidophilic fluorophore LysoTracker to label acidic organelles in freshly dissected, live rat RPE tissue flat mounts. We observed that all RPE cells imaged contained numerous acidified POS phagolysosomes whose abundance per cell was dramatically increased 2 h after light onset as compared to either 1 h before or 4 h after light onset. Lack of organelles of similar diameter (of 1-2 µm) in phagocytosis-defective mutant RCS rat RPE confirmed that LysoTracker live imaging detected POS phagolysosomes. Lack of increase in lysosomal membrane protein LAMP-1 in RPE/choroid during the diurnal phagocytic burst suggests that formation of POS phagolysosomes in RPE in situ may not involve extra lysosome membrane biogenesis. Taken together, we report a new imaging approach that directly detects POS phagosome acidification and allows rapid tracking and quantification of POS phagocytosis by live RPE -tissue ex situ.

Clinicopathological characteristics of placental chorioangioma: A clinicopathological study of 30 cases.

OBJECTIVE: The objective is to explore clinicopathological characteristics, diagnosis, differential diagnoses, treatment, and prognoses of placental chorioangioma (PCA). MATERIALS AND METHODS: The pathological data of 30 cases of PCA were collected; the color Doppler ultrasound, Down's screening, fetal survival, and pathological characteristics were observed; and the literature was reviewed. RESULTS: Of the 30 patients, the ages ranged from 20 to 38 years, with an average of 29.6 years. Pregnancy comorbidity occurred in 14 patients; intrauterine fetal death occurred in 4; the gross appearance of the tumor: a reddish-brown nodule, slightly round, 0.5-8 cm in diameter, can be seen on the cut surface of the placenta Pregnancy comorbidity occurred in 14 patients and intrauterine fetal death in 4. On sectioning the placenta, tumors grossly appeared as reddish-brown nodules, slightly round and ranging in diameter from 0.5 to 8 cm. Microscopically, the tumor has small, densely packed capillaries with fibrous connective tissue in the stroma. There were 10 cases with high risk of Down's syndrome screening, and the immunophenotype CD34 (+) and Ki-67 proliferation index were less than 10%. CONCLUSIONS: PCA is rare and may be misdiagnosed as malignant tumor, which may be related to pregnancy comorbidity and high risk of Down's screening, so improving the understanding of PCA can provide the basis for clinical diagnosis and intervention. PCA is a rare tumor which may be misdiagnosed as a malignancy. It may be related to pregnancy comorbidity and high risk of Down's screening. Improving the understanding of PCA could provide the basis for clinical diagnosis and intervention.

Placental mesenchymal dysplasia: A case report and review of literature.

ABSTRACT: Placental mesenchymal dysplasia is a rare disorder of the placenta with only a few reported cases. A case of interstitial dysplasia of the placenta was reported. The diagnosis and differential diagnosis were made by gross observation, microscopic findings, and immunohistochemistry.

Riboflavin Targets the Cellular Metabolic and Ribosomal Pathways of Candida albicans In Vitro and Exhibits Efficacy against Oropharyngeal Candidiasis.

Oropharyngeal candidiasis (OPC), which has a high incidence in immunocompromised and denture stomatitis patients, is commonly caused by Candida albicans infection and in some cases develops into disseminated candidiasis throughout the throat and esophagus, resulting in high mortality. New drugs are needed to combat OPC because of the limited treatment options currently available and increasing resistance to existing drugs. Here, we confirmed that riboflavin (RF), a cofactor of flavin adenine mononucleotide and flavin adenine dinucleotide, has broad-spectrum anti-Candida activity. The formation of C. albicans hyphae and biofilm was inhibited by RF. Mechanistically, RF disrupted membrane and cell wall integrity, as well as promoting reactive oxygen species and pyruvate accumulation. Furthermore, RF targeted multiple essential pathways via functional disruption of thiamine and RF metabolic pathways, central carbon metabolism, and ribosome metabolism. Similar to the results in vitro, the inhibitory effect of RF on C. albicans hyphae was confirmed in a mouse model of OPC. Moreover, after 5 consecutive days of intraperitoneal injection, RF exhibited therapeutic efficacy, as demonstrated by phenotype investigation, the fungal burden, and histopathological analysis. These findings revealed that RF exerts a multifaceted anti-Candida effect and has potential benefits in the treatment of OPC. IMPORTANCE Candida species are common pathogens in fungal infections, causing mucosal infection and invasive infection in immunodeficient patients. Given the limited classes of drugs and resistance to these drugs, new antifungal agents need to be developed. Drug repurposing is a potential method for antifungal drug development. This study demonstrated that riboflavin (RF) exhibited broad-spectrum anti-Candida activity. RF affected multiple targets involving the membrane and cell wall integrity, the accumulation of reactive oxygen species and pyruvate, and the altered metabolic pathways in C. albicans. Moreover, RF exhibited efficacy in the treatment of C. albicans in an oropharyngeal candidiasis mouse model. Taken together, the antifungal activity and the promising clinical application of RF were highlighted.

Glomus tumor of the crissum: A case report and review of literature.

Glomus tumor is a rare mesenchymal neoplasm arising from the modified smooth muscle cells of the glomus body. Primary crissum glomus tumor is extremely rare without any published in the literature. In this article, we report the first case of primary crissum glomus tumor in an 80-year-old man with recurrent anal pain for 8 years, increased pain for 1 year. Rectal MRI for inflammatory lesions (sinus tract). Microscopic examination showed the tumor cells were arranged in sheets and nests, surrounding blood vessels and nerve bundles. At high magnification, the neoplastic cells show regular round shape with light eosinophilic and translucent cytoplasm. The cell boundary is clear, the nucleus is round and located in the center. The stroma of the tumor shows hyaline degeneration. Immunohistochemically, the tumor cells were positive for smooth muscle actin, h-caldesmon, Calponin, synaptophysin, Collagen IV and CD34, but completely negative for HMB45, S100, EMA, desmin, CgA and CD56. The histologic features and immunohistochemical profile supported a diagnosis of primary crissum glomus tumor. The patient was asymptomatic and disease free after the procedure.

Antifungal Activity of Sodium New Houttuyfonate Against Aspergillus fumigatus in vitro and in vivo.

Aspergillus fumigatus is an important pathogen causing invasive aspergillosis, which is associated with high morbidity and mortality in immunocompromised people. However, the treatment of A. fumigatus infection is a growing challenge, owing to the limited availability antifungal agents and the continual emergence of drug-resistant strains. Drug repurposing is a potential strategy to solve this current problem. Sodium new houttuyfonate (SNH), derived from houttuynin, extracted from Houttuynia cordata, has anti-bacterial and anti-Candida albicans effects. However, whether it has anti-A. fumigatus activity had not been reported. In this study, the antifungal properties of SNH against A. fumigatus, including the standard strain AF293, itraconazole resistant clinical strains, and voriconazole resistant clinical strains, were evaluated in vitro and in vivo. Moreover, the potential mechanism of SNH was characterized. SNH exhibited significant fungicidal activity toward various A. fumigatus strains. SNH also inhibited fungal growth, sporulation, conidial germination and pigment formation, and biofilm formation. Further investigations revealed that SNH interfered with the A. fumigatus cell steroid synthesis pathway, as indicated by transcriptomic and quantitative real-time polymerase chain reaction analyses, and inhibited ergosterol synthesis, as indicated by cell membrane stress assays and ergosterol quantification. Moreover, daily gastric gavage of SNH significantly decreased the fungal burden in mice with disseminated infection (kidney, liver, and lung) and local tissue damage. In addition, the application of SNH downregulated the production of IL-6 and IL-17A. Together, these findings provided the first confirmation that SNH may be a promising antifungal agent for the treatment of A. fumigatus infection.

Acute RhoA/Rho Kinase Inhibition Is Sufficient to Restore Phagocytic Capacity to Retinal Pigment Epithelium Lacking the Engulfment Receptor MerTK.

The diurnal phagocytosis of spent photoreceptor outer segment fragments (POS) by retinal pigment epithelial (RPE) cells is essential for visual function. POS internalization by RPE cells requires the assembly of F-actin phagocytic cups beneath surface-tethered POS and Mer tyrosine kinase (MerTK) signaling. The activation of the Rho family GTPase Rac1 is necessary for phagocytic cup formation, and Rac1 is activated normally in MerTK-deficient RPE. We show here that mutant RPE lacking MerTK and wild-type RPE deprived of MerTK ligand both fail to form phagocytic cups regardless of Rac1 activation. However, in wild-type RPE in vivo, a decrease in RhoA activity coincides with the daily phagocytosis burst, while RhoA activity in MerTK-deficient RPE is constant. Elevating RhoA activity blocks phagocytic cup formation and phagocytosis by wild-type RPE. Conversely, inhibiting RhoA effector Rho kinases (ROCKs) rescues both F-actin assembly and POS internalization of primary RPE if MerTK or its ligand are lacking. Most strikingly, acute ROCK inhibition is sufficient to induce the formation and acidification of endogenous POS phagosomes by MerTK-deficient RPE ex vivo. Altogether, RhoA pathway inactivation is a necessary and sufficient downstream effect of MerTK phagocytic signaling such that the acute manipulation of cytosolic ROCK activity suffices to restore phagocytic capacity to MerTK-deficient RPE.

Phase transition specified by a binary code patterns the vertebrate eye cup.

The developing vertebrate eye cup is partitioned into the neural retina (NR), the retinal pigmented epithelium (RPE), and the ciliary margin (CM). By single-cell analysis, we showed that fibroblast growth factor (FGF) signaling regulates the CM in its stem cell­like property of self-renewal, differentiation, and survival, which is balanced by an evolutionarily conserved Wnt signaling gradient. FGF promotes Wnt signaling in the CM by stabilizing ß-catenin in a GSK3ß-independent manner. While Wnt signaling converts the NR to either the CM or the RPE depending on FGF signaling, FGF transforms the RPE to the NR or CM dependent on Wnt activity. The default fate of the eye cup is the NR, but synergistic FGF and Wnt signaling promotes CM formation both in vivo and in human retinal organoid. Our study reveals that the vertebrate eye develops through phase transition determined by a combinatorial code of FGF and Wnt signaling.

Smooth muscle tumor of uncertain malignant potential (STUMP): a clinicopathologic analysis of 26 cases.

BACKGROUND: To investigate the clinicopathologic features, differential diagnosis, and factors associated with recurrence in patients with smooth muscle tumors of uncertain malignant potential (STUMP). METHODS: The clinical and pathologic data of STUMP patients diagnosed in Mindong Hospital of Ningde City from 2017 to 2018 were collected and slides reviewed, the high-frequency color Doppler ultrasound and pathological characteristics were observed, and the literature was reviewed. RESULTS: All the STUMP diagnoses were confirmed by slide review. The age of onset was 23-61 years (mean 42.96 years). The main clinical symptoms were leiomyoma of uterus, prolonged menstruation, and increased menstruation. Color Doppler ultrasonography showed hypoechoic uterine wall nodules. The mean follow-up time was 62.9 months (range: 13-96 months). CONCLUSIONS: Smooth muscle tumors of undetermined malignant potential (STUMP) in the uterus are one of the rare gynecologic neoplasms. Although not malignant, they should be considered as low malignant potential tumors because they occasionally recur. Six of 13 recurrent tumors recurred in the years following hysterectomy with preservation. These six recurrent tumors are the only ones that had a strong immune response to p16 and p53. In support of early observation, these markers may help predict STUMP behavior. Patients diagnosed with STUMP should be monitored over time.

GATA-type transcription factor MrNsdD regulates dimorphic transition, conidiation, virulence and microsclerotium formation in the entomopathogenic fungus Metarhizium rileyi.

The GATA-type sexual development transcription factor NsdD has been implicated in virulence, secondary metabolism and asexual development in filamentous fungi. However, little is known about its function in the yeast-to-hypha transition and in microsclerotium formation. In the current study, the orthologous NsdD gene MrNsdD in the entomopathogenic fungus Metarhizium rileyi was characterized. Transcriptional analysis indicated that MrNsdD was involved in yeast-to-hypha transition, conidiation and microsclerotium formation. After targeted deletion of MrNsdD, dimorphic transition, conidiation, fungal virulence and microsclerotium formation were all impaired. Compared with the wild-type strain, the ΔMrNsdD mutants were hypersensitive to thermal stress. Furthermore, transcriptome sequencing analysis revealed that MrNsdD regulated a distinct signalling pathway in M. rileyi during the yeast-to-hypha transition or microsclerotium formation, but exhibited overlapping regulation of genes during the two distinct developmental stages. Taken together, characterization of the MrNsdD targets in this study will aid in the dissection of the molecular mechanisms of dimorphic transition and microsclerotium development.

A Novel Quantitative Index of Meibomian Gland Dysfunction, the Meibomian Gland Tortuosity.

Purpose: To quantitatively measure meibomian gland (MG) tortuosity in meibomian gland dysfunction (MGD) patients and normal controls and to observe the efficacy of evaluating MG tortuosity for the diagnosis of MGD. Methods: This cross-sectional study enrolled 32 obstructive MGD patients and 28 normal volunteers. Clinical assessments were performed, including symptom questionnaires, tear meniscus height, tear break-up time (TBUT), corneal fluorescein staining, lid margin abnormality, MG expressibility, and meibography. The meibomian gland tortuosity and meibomian gland density were measured by VIA software. Results: The mean age of the patients in the MGD group was 33.28 ± 9.28 years, and that of the normal controls was 25.25 ± 11.19 years. The average tortuosity of all MGs in the MGD patients was significantly larger than in the normal controls (P< 0.05). We further stratified the MGD patients into symptomatic MGD and asymptomatic groups. The average tortuosity of all MGs and of the central eight MGs was significantly higher in the symptomatic MGD patients than in the asymptomatic MGD patients (P< 0.05). Significant linear correlations were found between MG tortuosity and the lid margin score, meiboscore, meibum expressibility score, and TBUT (P< 0.05). When the diagnosis of obstructive MGD was based on the tortuosity of the central eight MGs of both eyelids, the sensitivity and specificity were 100% and 100%, respectively. Conclusions: MG tortuosity is an effective index to delineate MG morphology and to diagnose MGD, especially for the diagnosis of early-stage MGD. Translation Relevance: Calculating tortuosity quantitatively may play an important role in the diagnosis of MGD.

[High expression of activated CD4+ memory T cells and CD8+ T cells and low expression of M0 macrophage are associated with better clinical prognosis in bladder cancer patients].

Objective To study the infiltration pattern of bladder cancer immune cells and explore the relationship between immune cells and tumor prognosis. Methods The bladder cancer transcript data and corresponding clinical data were downloaded from The Cancer Genome Atlas (TCGA). CIBERSORT software deconvolution method was used to calculate the proportions of 22 kinds of immune cells. R software was used to analyze the immune cell infiltration pattern in each clinical stage. The relationship between each immune cell and prognosis was analyzed by Kaplan-Meier survival. Results A total of 433 cases of gene transcript data were downloaded from the TCGA database, including 414 cases of bladder cancer tissues and 19 normal tissues. After the data were corrected, the proportions of immune cells were calculated using CIBERSORT software. Screening was performed, and 146 cases of bladder cancer tissue and 4 cases of normal bladder tissue were obtained. Activated CD4+ memory T cells and CD8+ T cells had the lowest expression and M0 macrophages had the highest expression in clinical stage IV of bladder cancer. The bladder cancer patients with high expression of activated CD4+ memory T cells, CD8+ T cells and low expression of M0 macrophages had beneficial prognosis. Conclusion The bladder cancer patients with high expression of activated CD4+ memory T cells, CD8+ T cells and low expression of M0 macrophages might have better clinical prognosis.

Etv transcription factors functionally diverge from their upstream FGF signaling in lens development.

The signal regulated transcription factors (SRTFs) control the ultimate transcriptional output of signaling pathways. Here, we examined a family of FGF-induced SRTFs - Etv1, Etv 4, and Etv 5 - in murine lens development. Contrary to FGF receptor mutants that displayed loss of ERK signaling and defective cell differentiation, Etv deficiency augmented ERK phosphorylation without disrupting the normal lens fiber gene expression. Instead, the transitional zone for lens differentiation was shifted anteriorly as a result of reduced Jag1-Notch signaling. We also showed that Etv proteins suppresses mTOR activity by promoting Tsc2 expression, which is necessary for the nuclei clearance in mature lens. These results revealed the functional divergence between Etv and FGF in lens development, demonstrating that these SRTFs can operate outside the confine of their upstream signaling.

Many cells contain proteins known as signal-induced transcription factors, which are poised to receive messages from the environment and then react by activating genes required for the cell to respond appropriately. It is commonly thought that these transcription factors faithfully follow the instructions they receive from the external signal: for instance, if the message was to encourage the cell to grow, the transcription factors would switch on growth-related genes. As the eyes of mice and other mammals develop, a signal known as FGF is required for certain cells to specialize into lens fiber cells: these long, thin, transparent cells form the bulk of the lens, the structure that allows focused vision. Previous studies suggest that FGF activates three transcription factors known as Etv1, Etv4 and Etv5, but their precise roles in the development of the lens has remained unclear. Here, Garg, Hannan, Wang et al. confirm that FGF signaling does indeed activate all three proteins. However, mutant mice that lacked Etv1, Etv4 and Etv5 still created lens fiber cells, suggesting that the transcription factors are largely unnecessary for lens fiber cells formation. Instead, the Etv proteins participated in a cascade of molecular events involving a protein called Notch; as a result, if the transcription factors were absent, the lens fiber cells formed prematurely. In addition, deactivating Etv1, Etv4 and Etv5 also promoted the activity of a protein which interfered with the removal of internal cell compartments, a process required for lens fiber cells to mature properly. These findings reveal that the roles of Etv1, Etv4 and Etv5 deviate from and even oppose FGF signaling in the lenses of mice. Transcription factors control the ultimate fate of a cell, and there is therefore increased interest in targeting them for therapy. The work by Garg, Hannan, Wang et al. reveals an unexpected complexity in how these proteins respond to upstream signals, highlighting the importance of further dissecting these relationships.

[Evaluation of immunohistochemistry staining and cytologic diagnosis by using cell block sections prepared with effusion fluid cytology specimens].

OBJECTIVE: To study the values of immunohistochemistry staining and cytological diagnosis by using cell block sections prepared with the effusion fluid cytology specimens. METHODS: Ninety-nine effusion cytology specimens with the diagnoses of reactive mesothelial hyperplasia, atypical cells and metastatic carcinoma were enrolled into the study. The cytospin preparations/smears, cell block sections and immunohistochemical study were performed and correlated with the clinical findings and follow-up data. RESULTS: Amongst the 99 cases studied, the percentage with positive diagnosis using cytospin preparations/smears was 68.7% (68/99). The percentages with negative and equivocal diagnoses were 16.2% (16/99) and 15.1% (15/99), respectively. As for cell block sections, the percentages were 71.7% (71/99), 16.2% (16/99) and 12.1% (12/99), respectively. On the other hands, the percentages became 76.8% (76/99), 20.2% (20/99) and 3.0% (3/99), respectively, when coupled with immunohistochemical findings. The overall percentages of positive, negative and equivocal diagnoses were 77.8% (77/99), 17.2% (17/99) and 5.0% (5/99), respectively, upon clinicopathologic correlation. The difference between cytospin preparations/smears and cell block sections was not statistically significant (P > 0.05). When coupled with immunohistochemical findings or clinicopathologic correlation, the difference in rates of equivocal diagnosis however carried statistical significance (P < 0.05). The false-negative rate of immunohistochemical study applied on cell block sections was 1.0% (1/99). CONCLUSIONS: Immunohistochemistry, when applied on cell block sections, is useful in delineation of the primary origins of the tumor cells in effusion fluid cytology specimens. Combination of morphologic examination, immunohistochemical findings and clinicopathologic correlation can further improve the rate of positive diagnosis.

Advanced Analysis of Photoreceptor Outer Segment Phagocytosis by RPE Cells in Culture.

Retinal pigment epithelial (RPE) cells are among the most actively phagocytic cells in nature. Primary RPE and stable RPE cell lines provide experimental model systems that possess the same phagocytic machinery as RPE in situ. Upon experimental challenge with isolated photoreceptor outer segment fragments (POS), these cells promptly and efficiently recognize, bind, internalize, and digest POS. Here, we describe experimental procedures to isolate POS from porcine eyes and to feed POS to RPE cells in culture. Furthermore, we provide experimental protocols to synchronize the POS binding and engulfment steps of phagocytosis. Finally, we describe three different and complementary methods to quantify total POS uptake by RPE cells and to discriminate surface-bound from engulfed POS.

blaNDM-5 carried by a hypervirulent Klebsiella pneumoniae with sequence type 29.

Background: A carbapenem-resistant hypermucoviscous Klebsiella pneumoniae isolate was recovered from human sputum. Methods: Whole genome sequencing of this isolate was carried out to reveal its clonal background, antimicrobial resistance determinants and virulence factors. Virulence assays were performed using wax moth larvae. The transfer of blaNDM-5 between bacterial strains was tested using conjugation. 59 genome assemblies of ST29 K. pneumoniae and 230 IncX3 plasmids regardless of the carriage of resistance gene were employed for phylogenetic analysis, respectively. Results: The strain carried a virulence plasmid pVir-SCNJ1 bearing the virulence gene rmpA and exhibited a high virulence in wax moth. This hypervirulent strain belongs to sequence type 29 and carries blaNDM-5, which is located on a conjugative plasmid, designated pNDM5-SCNJ1, belonging to type IncX3. pNDM5-SCNJ1 was fully sequenced and shows high similarity with pNDM_MGR194, except some deletion inside the ISAba125 region. Phylogenetic analysis of IncX3 plasmids revealed that although blaNDM-5 can be evolved from blaNDM-1 via point mutations within some IncX3 plasmids, most of blaNDM-5-carrying IncX3 plasmids probably have acquired blaNDM-5 in multiple events. Conclusions: In this study, we characterized a blaNDM-5-positive hypervirulent K. pneumoniae of sequence type 29 in China. Our results highlight the need for active surveillance on this lineage of carbapenem-resistant K. pneumoniae.

Crk proteins transduce FGF signaling to promote lens fiber cell elongation.

Specific cell shapes are fundamental to the organization and function of multicellular organisms. Fibroblast Growth Factor (FGF) signaling induces the elongation of lens fiber cells during vertebrate lens development. Nonetheless, exactly how this extracellular FGF signal is transmitted to the cytoskeletal network has previously not been determined. Here, we show that the Crk family of adaptor proteins, Crk and Crkl, are required for mouse lens morphogenesis but not differentiation. Genetic ablation and epistasis experiments demonstrated that Crk and Crkl play overlapping roles downstream of FGF signaling in order to regulate lens fiber cell elongation. Upon FGF stimulation, Crk proteins were found to interact with Frs2, Shp2 and Grb2. The loss of Crk proteins was partially compensated for by the activation of Ras and Rac signaling. These results reveal that Crk proteins are important partners of the Frs2/Shp2/Grb2 complex in mediating FGF signaling, specifically promoting cell shape changes.