Nonpermissive Research Articles

The Fungal Protein Mes1 Is Required for Morphogenesis and Virulence in the Dimorphic Phytopathogen Ustilago maydis.

Polarized growth is a defining property of filamentous fungi, which plays an important role in different aspects of their biology, including virulence. However, little information is available about the determinants of cell surface organization and their role in polarized growth. The fungal protein MesA was identified in a genetic screen in Aspergillus nidulans and is involved in the stabilization of the polarity axes, but it has no evident role in budding yeast. In this work, I present evidence that in the dimorphic fungal phytopathogen Ustilago maydis MesA/Mes1 is involved in cell wall stability and polarized growth. mes1 mutants were more sensitive to drugs provoking cell wall stress, and they displayed a temperature-sensitive phenotype. Actin cytoskeleton was disorganized in a mes1 mutant, suggesting that there is a connection between Mes1, the actin cytoskeleton and polarized morphogenesis. The septin ring was also absent from the bud tip, but not the bud neck. Deletion of mes1 provoked defects in endocytosis and vacuolar organization in the cells. Mes1 was essential for strong polarized growth in the hyphal form, but it was dispensable during low or moderate polarized growth in the yeast form in U. maydis at a permissive temperature. Consistently, mes1 mutants showed delayed mating and they were avirulent.

Evaluation of Recombinant Live-Attenuated Respiratory Syncytial Virus (RSV) Vaccines RSV/ΔNS2/Δ1313/I1314L and RSV/276 in RSV-Seronegative Children.

This United States-based study compared 2 candidate vaccines: RSV/ΔNS2/Δ1313/I1314L, attenuated by NS2 gene-deletion and temperature-sensitivity mutation in the polymerase gene; and RSV/276, attenuated by M2-2 deletion. RSV-seronegative children aged 6-24 months received RSV/ΔNS2/Δ1313/I1314L (106 plaque-forming units [PFU]), RSV/276 (105 PFU), or placebo intranasally. Participants were monitored for vaccine shedding, reactogenicity, and RSV serum antibodies, and followed over the subsequent RSV season. Enrollment occurred September 2017 to October 2019. During 28 days postinoculation, upper respiratory illness and/or fever occurred in 64% of RSV/ΔNS2/Δ1313/I1314L, 84% of RSV/276, and 58% of placebo recipients. Symptoms were generally mild. Cough was more common in RSV/276 recipients than RSV/ΔNS2/Δ1313/I1314L (48% vs 12%; P = .012) or placebo recipients (17%; P = .084). There were no lower respiratory illness or serious adverse events. Eighty-eight and 96% of RSV/ΔNS2/Δ1313/I1314L and RSV/276 recipients were infected with vaccine (shed vaccine and/or had ≥4-fold rises in RSV antibodies). Serum RSV-neutralizing titers and anti-RSV F IgG titers increased ≥4-fold in 60% and 92% of RSV/ΔNS2/Δ1313/I1314L and RSV/276 vaccinees, respectively. Exposure to community RSV during the subsequent winter was associated with strong anamnestic RSV-antibody responses. Both vaccines had excellent infectivity and were well tolerated. RSV/276 induced an excess of mild cough. Both vaccines were immunogenic and primed for strong anamnestic responses. NCT03227029 and NCT03422237.

Design, synthesis and biological evaluation of biphenyl-benzamides as potent FtsZ inhibitors

The rapid emergence of antibiotic resistance has become a prevalent threat to public health, thereby development of new antibacterial agents having novel mechanisms of action is in an urgent need. Targeting at the cytoskeletal cell division protein filamenting temperature-sensitive mutant Z (FtsZ) has been validated as an effective and promising approach for antibacterial drug discovery. In this study, a series of novel biphenyl-benzamides as FtsZ inhibitors has been rationally designed, synthesized and evaluated for their antibacterial activities against various Gram-positive bacteria strains. In particular, the most promising compound 30 exhibited excellent antibacterial activities, especially against four different Bacillus subtilis strains, with an MIC range of 0.008 μg/mL to 0.063 μg/mL. Moreover, compound 30 also showed good pharmaceutical properties with low cytotoxicity (CC50 > 20 μg/mL), excellent human metabolic stability (T1/2 = 111.98 min), moderate pharmacokinetics (T1/2 = 2.26 h, F = 61.2%) and in vivo efficacy, which can be identified as a promising FtsZ inhibitor worthy of further profiling.

Design of a US28 ORF Deletion Virus in a Temperature-Sensitive Cytomegalovirus Strain Fails to Promote Lytic Replication in Hematopoietic Cells.

Human cytomegalovirus (CMV) is a ubiquitous pathogen that latently resides in hematopoietic cells. Latently infected individuals with dysfunctional immune systems often experience CMV reactivation, which can cause devastating disease and mortality. While factors dictating the balance between latency and reactivation are not completely understood, CMV US28 is required for maintaining latent infection, and viral mutants that alter US28 function result in a lytic-like, rather than latent, infection in hematopoietic cells. In turn, viral lytic factors alter the host cell, making it challenging to characterize the US28-specific changes in the cellular milieu. To circumvent this, we generated a temperature-sensitive TB40/E recombinant virus, TB40/EgfpC510G (tsC510G), into which we engineered an amino acid change at position 510 (C510G) of IE2, as previously described in the CMV Towne strain. Using tsC510G, we then deleted the US28 ORF, termed tsC510G-US28Δ. Consistent with previous findings, tsC510G-US28Δ fails to undergo latency in Kasumi-3 cells at the permissive temperature. However, parallel cultures maintained at the non-permissive temperature showed a significant reduction in infectious center frequency, as measured by limiting dilution assay. Thus, we generated a new US28 mutant virus for use as a tool to study US28-specific changes in latently infected hematopoietic cells in the absence of induced lytic replication.

FtsZ at mid-cell is essential in Escherichia coli until the late stage of constriction.

There has been recent debate as to the source of constriction force during cell division. FtsZ can generate a constriction force on tubular membranes in vitro, suggesting it may generate the constriction force in vivo. However, another study showed that mutants of FtsZ did not affect the rate of constriction, whereas mutants of the PG assembly did, suggesting that PG assembly may push the constriction from the outside. Supporting this model, two groups found that cells that have initiated constriction can complete septation while the Z ring is poisoned with the FtsZ targeting antibiotic PC190723. PC19 arrests treadmilling but leaves FtsZ in place. We sought to determine if a fully assembled Z ring is necessary during constriction. To do this, we used a temperature-sensitive FtsZ mutant, FtsZ84. FtsZ84 supports cell division at 30 °C, but it disassembles from the Z ring within 1 min upon a temperature jump to 42 °C. Following the temperature jump we found that cells in early constriction stop constricting. Cells that had progressed to the later stage of division finished constriction without a Z ring. These results show that in Escherichia coli, an assembled Z ring is essential for constriction except in the final stage, contradicting the simplest interpretation of previous studies using PC19.

HLH-1 Modulates Muscle Proteostasis During Caenorhabditis elegans Larval Development.

Muscle proteostasis is shaped by the myogenic transcription factor MyoD which regulates the expression of chaperones during muscle differentiation. Whether MyoD can also modulate chaperone expression in terminally differentiated muscle cells remains open. Here we utilized a temperature-sensitive (ts) conditional knockdown nonsense mutation in MyoD ortholog in C. elegans, HLH-1, to ask whether MyoD plays a role in maintaining muscle proteostasis post myogenesis. We showed that hlh-1 is expressed during larval development and that hlh-1 knockdown at the first, second, or third larval stages resulted in severe defects in motility and muscle organization. Motility defects and myofilament organization were rescued when the clearance of hlh-1(ts) mRNA was inhibited, and hlh-1 mRNA levels were restored. Moreover, hlh-1 knockdown modulated the expression of chaperones with putative HLH-1 binding sites in their promoters, supporting HLH-1 role in muscle maintenance during larval development. Finally, mild disruption of hlh-1 expression during development resulted in earlier dysregulation of muscle maintenance and function during adulthood. We propose that the differentiation transcription factor, HLH-1, contributes to muscle maintenance and regulates cell-specific chaperone expression post differentiation. HLH-1 may thus impact muscle proteostasis and potentially the onset and manifestation of sarcopenia.

Essential roles of phosphatidylinositol 4-phosphate phosphatases Sac1p and Sjl3p in yeast autophagosome formation

Autophagy is regulated by phosphoinositides. We have previously shown that phosphatidylinositol 4-phosphate (PtdIns(4)P) is localized in the autophagosomal membrane. Additionally, in yeast cells, phosphatidylinositol 4-kinases Pik1p and Stt4p play important roles in the formation of the autophagosome and its fusion with the vacuole, respectively. In this study, we analyzed the primary role of PtdIns(4)P phosphatases in yeast autophagy. The PtdIns(4)P labeling densities in the membranes of the vacuoles, mitochondria, nucleus, endoplasmic reticulum, and plasma membrane dramatically increased in the phosphatase deletion mutants sac1∆ and sjl3∆, and the temperature-sensitive mutant sac1ts/sjl3∆ at the restrictive temperature. GFP-Atg8 processing assay indicated defective autophagy in the sac1∆ and sac1ts/sjl3∆ mutants. In contrast to the localization of PtdIns(4)P in the luminal leaflet of autophagosomal membranes in the wild-type yeast, PtdIns(4)P was localized in both the luminal and cytoplasmic leaflets of the autophagosomal membranes in the sac1∆ strain. In addition, the number of autophagic bodies in the vacuole significantly decreased in the sac1∆ strain, although autophagosomes were present in the cytoplasm. In the sac1ts/sjl3∆ strain, the number of autophagosomes in the cytoplasm dramatically decreased at the restrictive temperature. Considering that the numbers of autophagosomes and autophagic bodies in the sjl3∆ strain were comparable to those in the wild-type yeast, we found that the autophagosome could not be formed when PtdIns(4)P phosphatase activities of both Sac1p and Sjl3p were diminished. Together, these results indicate that the turnover of PtdIns(4)P by phosphatases is essential for autophagosome biogenesis.

Temperature Influences on Powdery Mildew Susceptibility and Development in the Hop Cultivar Cascade.

The hop cultivar 'Cascade' possesses partial resistance to powdery mildew (Podosphaera macularis) that can be overcome by recently emerged, virulent isolates of the fungus. Given that hop is a long-lived perennial and that brewers still demand Cascade, there is a need to better understand factors that influence the development of powdery mildew on this cultivar. Growth chamber experiments were conducted to quantify the effect of constant, transient, and fluctuating temperature on Cascade before, concurrent to, and after inoculation as contrasted with another powdery mildew-susceptible cultivar, 'Symphony'. Exposure of plants to supraoptimal temperature (26 and 32°C) before inoculation led to more rapid onset of ontogenic resistance in intermediately aged leaves in Cascade as compared with Symphony. Cascade was overall less susceptible to powdery mildew when exposed to constant temperature ranging from 18 to 32°C directly after inoculation. However, cultivar also interacted with temperature such that proportionately fewer and smaller colonies developed on Cascade than Symphony at supraoptimal yet permissive temperatures for disease. When plants were inoculated and then exposed to high temperature, colonies became progressively more tolerant to temperatures of 26 to 30°C with increasing time from inoculation to exposure, as moderated by cultivar, the specific temperature, and their interaction. Subjecting plants to simulated diurnal temperature regimes at the time of inoculation or 24 h later indicated Cascade and Symphony responded proportionately similarly on days predicted to be marginally unfavorable or marginally favorable for powdery mildew, although Cascade was quantitatively less susceptible than Symphony. In sum, this research indicates that Cascade is overall less susceptible to powdery mildew than Symphony, and supraoptimal temperature before, concurrent to, or after infection may interact differentially to moderate disease risk in Cascade. Therefore, cultivar-specific risk assessments for powdery mildew appear warranted.

Characterization and application of a novel immortalized murine thick ascending limb (imTAL) cell line to investigate the role of myosin II motor proteins Myh9 and Myh10 in TAL cargo transport

Conditional genetic inactivation of Myh9and Myh10, the genes which encode the heavy chains of nonmuscle myosin IIA and IIB, respectively, in adult murine renal epithelium leads to aberrant trafficking of the thick ascending limb‐specific GPI‐anchored glycoprotein uromodulin (UMOD) and its subcellular accumulation in cells of the thick ascending limb of Henle’s loop (Otterpohl et al. 2020). A novel immortalized murine thick ascending limb (imTAL) cell line harboring doxycycline inducible Myh9and Myh10conditional knockout (cKO) capability was established in our lab to facilitate long term in vitrostudies. We utilized biotinylated anti‐uromodulin antibody to isolate cells from collagenase digested Myh9&10cKO whole mouse kidneys. Characterization of these cells has revealed endogenous expression of TAL‐specific protein UMOD and NKCC2. RT‐qPCR detected mRNA expression of TAL specific proteins. We generated imTAL cells stably expressing GFP‐tagged UMOD using retroviral transduction. GFP‐UMOD expressing cells underwent 72‐hour doxycycline treatment and, along with untreated control cells, were fixed and analyzed via confocal microscope. While GFP‐UMOD is enriched along the plasma membrane in control cells, we observe accumulation of GFP‐UMOD within vesicular structures upon doxycycline‐induced loss of Myh9 and Myh10 proteins. We also utilized imTAL cells stably expressing an GFP‐tagged thermosensitive variant of vesicular stomatitis virus glycoprotein (VSV‐GtsO45‐GFP), which has been widely employed in studying the secretory pathway. The temperature sensitive nature of VSV‐GtsO45 enabled us to perform pulse‐chase type experiments, following its anterograde transport through the ER, Golgi, and ultimately to the plasma membrane. Following incubation at the restrictive temperature (40°C) overnight, cells were moved to permissive temperature (32°C) and followed until 45 minutes. VSV‐GtsO45‐GFP in control cells undergoes ER export immediately (5 minutes) after incubation at the permissive temperature and is transported along the secretory pathway, arriving at the plasma membrane 30‐40 minutes later. Inactivation of Myh9and Myh10 results in sequestration of VSV‐GtsO45‐GFP in perinuclear vesicular and tubular structures. Immunolocalization studies are being performed to identify the vesicular compartment(s) in which VSV‐GtsO45‐GFP is sequestered in the absence of Myh9 and Myh10 proteins. imTAL cells stably expressing NKCC2‐GFP are also being analyzed to assess the anterograde transport of NKCC2 in doxycycline‐treated cells. Taken together, we have established a novel, immortalized, inducible, Myh9&10 cKO murine TAL cell line to enable long term in vitro studies and have expanded the molecular toolbox to elucidate the mechanisms underlying NM2 motor mediated TAL cargo transport.

SUMO Regulation of Bir1 in Mitotic Progression

Organisms utilize many mechanisms in order to preserve their genomic integrity when faced with DNA damage or chromosomal instability. One such mechanism is protein sumoylation, in which E1, E2, and E3 ligases covalently link small ubiquitin‐like modifier (SUMO) peptides or chains to target proteins. The goal of this research is to examine the contribution of Bir1 sumoylation to genome stability in Saccharomyces cerevisiae. Bir1 is the target of one of the three S. cerevisiae E3 SUMO ligases, Mms21, and is a subunit of the chromosomal passenger complex (CPC). The CPC mediates the spindle assembly checkpoint (SAC) to establish chromosome bi‐orientation during mitosis. Therefore, it has previously been hypothesized that the sumoylation of Bir1 influences mitotic progression. In order to explore this hypothesis, BIR1 was genetically modified to mimic conditions in which Bir1 is constantly sumoylated (BIR1‐SuON) or cannot be sumoylated (BIR1‐SuOFF). The BIR1‐SuOFF mutants appeared to be more resistant to DNA damage caused by methyl methanesulfonate (MMS) and ultraviolet radiation than the BIR1‐SuON mutant, as well as the control strain (BIR1‐SuC). BIR1‐SuOff mutants phenocopied the temperature sensitive mutant of Ipl1, ipl1‐321, suggesting that mitotic checkpoint regulation may be affected in BIR1‐SuOff mutants. However, cell cycle analyses of BIR1‐SuOff mutants revealed that mitotic checkpoint activation was comparable to the control strain, at least in response to treatment with nocodazole, an agent that depolymerizes microtubules. We will further characterize the status of mitotic checkpoint activation in BIR1 mutants by examining Pds1 degradation and cell cycle analyses in the presence of other genotoxic agents.

An Evaluation of the Stability of Splicing Protein Dib1 Through Circular Dichroism Spectroscopy

Pre‐messenger RNA (pre‐mRNA) splicing is the process in which introns, noncoding regions of DNA, are removed and exons, coding regions of DNA, are ligated to produce a mature mRNA transcript. This process is mediated by the spliceosome, a dynamic macromolecular complex composed of five small nuclear ribonucleoproteins and other protein factors. At the catalytic center of the splicing machinery is the evolutionarily conserved yeast protein, Dib1, composed of 143 amino acids essential for splicing and cell viability. Dib1 and its human ortholog, hDim1, have demonstrated peptidase activity, specifically autocleavage of the last thirteen and fourteen amino acids, respectively, of their carboxy‐terminal tails. The mechanism and function of this autocleavage activity of the carboxy‐terminal tail is uncharacterized.Previous yeast growth assays comparing mutants to wildtype Dib1 have established that deletion of fifteen and sixteen amino acids off the carboxy‐terminal tail results in temperature‐sensitivity of yeast cell growth at 37ºC. Since Dib1 and splicing are essential for cell viability, the temperature‐sensitive mutants suggest that truncations to the carboxy‐terminal tail of Dib1 may result in defective splicing. Thus, the carboxy‐terminal tail may play a direct role in splicing or destabilize splicing complexes. To address these two possible hypotheses, we are investigating the effects of the carboxy‐terminal truncations on Dib1 stability. Protein purification of wild type and a series of truncated Dib1 mutants were performed. Circular dichroism spectroscopy employing increasing concentrations of guanidinium hydrochloride, a protein denaturant, was used to determine the stability of Dib1 and its truncated mutants. Overall, our findings show that Dib1 stability is directly linked to the length of the tail region and lead us to speculate that the tail is critical for stabilization of the spliceosomal complex.

Uncovering early thyroid hormone signalling events through temperature-mediated activation of molecular memory in the cultured bullfrog tadpole tail fin

Thyroid hormone (TH) is a critical signalling molecule for all vertebrate organisms, playing a crucial role in postembryonic development. The best-studied mechanism of TH response is through modulating gene expression, however TH’s involvement in coordinating the early steps in the TH signal transduction pathway is still poorly understood. The American bullfrog, Rana [Lithobates] catesbeiana, is a useful model to study these early responses as tadpole post-embryonic development in the form of metamorphosis of the tadpole into a frog can be experimentally induced by TH exposure. The rate of TH-induced metamorphosis can be modulated by temperature where sufficiently cold temperatures (5 °C) completely halt precocious metamorphosis. Interestingly, when premetamorphic tadpoles exposed to exogenous THs at 5 °C are shifted to permissive temperatures (24 °C), their metamorphic rate exceeds that of TH-exposed tadpoles at the permissive temperature. This suggests that a molecular memory of TH exposure is retained at 5 °C even after THs are cleared at this low temperature. However, the molecular memory machinery is poorly understood. Herein we use RNA-seq analysis to identify potential components of the molecular memory in cultured tail fin that allows for the recapitulation of the molecular memory phenomenon. Eighty-one gene transcripts were TH-responsive at 5 °C compared to matched controls indicating that the molecular memory is more complex than previously thought. Many of these transcripts encode transcription factors including thyroid hormone-induced B/Zip, thibz, and a novel krüppel-like factor family member, klfX. Actinomycin D and cycloheximide treatment had no effect on their TH induction suggesting that a change in transcription or translation is not required. Rather a change in RNA stability may be a possible mechanism contributing to the molecular memory. The ability to manipulate temperature and TH response in cultured organs provide an exciting opportunity to further elucidate the early TH signalling mechanisms during postembryonic development.

Exploration of the inhibitory mechanism of PC190723 on FtsZ protein by molecular dynamics simulation

Bacterial resistance caused by widespread use and abuse of antibiotics is threatening human health, and the development of new antibacterial agents with novel antibacterial targets has become urgent. Filamenting temperature-sensitive mutant Z (FtsZ), as a key protein in bacterial division, has received extensive attention. PC190723 exhibits an outstanding antibacterial activity by producing potent inhibitory ability on FtsZ protein, but its influence on the conformation of FtsZ protein at the molecular level is still unclear. In this study, we explored the effect of PC190723 on the conformation and function of FtsZ protein through molecular dynamics (MD) simulation and post-analysis. The results showed that PC190723 increased the high-affinity conformational stability of FtsZ protein, which disrupts the normal assembly of the Z-ring. In particular, the interactions of residues S8-sheet (VAL260-GLY266) increased in the FtsZPC190723 system, which may be the reason for promotes the formation of protofilament. In brief, the mechanism of PC190723 inhibiting FtsZ protein was explained at the molecular level by MD simulation, which provides new ideas for the identification of new FtsZ inhibitors as antibacterial agents.

Cross-scale analysis of temperature compensation in the cyanobacterial circadian clock system

Circadian clock proteins often reveal temperature-compensatory responses that counteract temperature influences to keep their enzymatic activities constant over a physiological range of temperature. This temperature-compensating ability at the reaction level is likely crucial for circadian clock systems, to which the clock proteins are incorporated, to achieve the system-level temperature compensation of the oscillation frequency. Nevertheless, temperature compensation is yet a puzzling phenomenon, since side chains that make up the clock proteins fluctuate more frequently due to greater thermal energy at higher temperature. Here, we investigated temperature influences on the dynamics of KaiC, a temperature-compensated enzyme (ATPase) that hydrolyzes ATP into ADP in the cyanobacterial circadian clock system, using quasielastic neutron scattering. The frequency of picosecond to sub-nanosecond incoherent local motions in KaiC was accelerated by a factor of only 1.2 by increasing the temperature by 10 °C. This temperature insensitivity of the local motions was not necessarily unique to KaiC, but confirmed also for a series of temperature-sensitive mutants of KaiC and proteins other than clock-related proteins. Rather, the dynamics associated with the temperature-compensatory nature of the reaction- and system-level was found in global diffusional motions, which was suggested to regulate the temperature dependence of ATPase activity and dephosphorylation process presumably through changes in the hexamer conformation of KaiC. The spatiotemporal scale at which cross-scale causality of the temperature sensitivity is established is finite, and extends down to picosecond to sub-nanosecond dynamics only in a very limited part of KaiC, not in its entire part.

Repurposing antiparasitic antimonials to noncovalently rescue temperature-sensitive p53 mutations.

The tumor suppressor p53 is inactivated by over hundreds of heterogenous mutations in cancer. Here, we purposefully selected phenotypically reversible temperature-sensitive (TS) p53 mutations for pharmacological rescue with thermostability as the compound-screening readout. This rational screening identified antiparasitic drug potassium antimony tartrate (PAT) as an agent that can thermostabilize the representative TS mutant p53-V272M via noncovalent binding. PAT met the three basic criteria for a targeted drug: availability of a co-crystal structure, compatible structure-activity relationship, and intracellular target specificity, consequently exhibiting antitumor activity in a xenograft mouse model. At the antimony dose in clinical antiparasitic therapy, PAT effectively and specifically rescued p53-V272M in patient-derived primary leukemia cells in single-cell RNA sequencing. Further scanning of 815 frequent p53-missense mutations identified 65 potential PAT-treatable mutations, most of which were temperature sensitive. These results lay the groundwork for repurposing noncovalent antiparasitic antimonials for precisely treating cancers with the 65 p53 mutations.

Single amino acid substitutions in hydrophobic cores at a head-coiled coil junction region of cohesin facilitate its release of DNA during anaphase.

Cohesin holds sister chromatids together and is cleaved by separase/Cut1 to release DNA during the transition from mitotic metaphase to anaphase. The cohesin complex consists of heterodimeric structural maintenance of chromosomes (SMC) subunits (Psm1 and Psm3), which possess a head and a hinge, separated by long coiled coils. Non-SMC subunits (Rad21, Psc3 and Mis4) bind to the SMC heads. Kleisin/Rad21's N-terminal domain (Rad21-NTD) interacts with Psm3's head-coiled coil junction (Psm3-HCJ). Spontaneous mutations that rescued the cleavage defects in temperature-sensitive (ts) separase mutants were identified in the interaction interface, but the underlying mechanism is yet to be understood. Here, we performed site-directed random mutagenesis to introduce single amino acid substitutions in Psm3-HCJ and Rad21-NTD, and then identified 300 mutations that rescued the cohesin-releasing defects in a separase ts mutant. Mutational analysis indicated that the amino acids involved in hydrophobic cores (which may be in close contact) in Psm3-HCJ and Rad21-NTD are hotspots, since 80 mutations (approx. 27%) were mapped in these locations. Properties of these substitutions indicate that they destabilize the interaction between the Psm3 head and Rad21-NTD. Thus, they may facilitate sister chromatid separation in a cleavage-independent way through cohesin structural re-arrangement.

A Human Conditionally Immortalized Proximal Tubule Epithelial Cell Line as a Novel Model for Studying Senescence and Response to Senolytics.

Accumulating evidence suggests that senescence of kidney tubule epithelial cells leads to fibrosis. These cells secrete senescence-associated secretory phenotype (SASP) factors that are involved in diverse signaling pathways, influencing kidney fibrosis. Here, we investigated whether our previously established conditionally immortalized proximal tubule epithelial cell line overexpressing the organic anion transporter 1 (ciPTEC-OAT1) can be used as a valid in vitro model to study kidney senescence and senolytics response. CiPTEC-OAT1 proliferates rapidly at 33°C and exhibits a “senescence-like” arrest at 37°C, most likely due to suppression of SV40T expression and subsequent reactivation of the p53 and Rb pathways. To understand how permissive (33°C) and non-permissive (37°C) temperatures of the cell culture affect the senescence phenotype, we cultured ciPTEC-OAT1 for up to 12 days and evaluated the apoptosis and SASP markers. Day 0 in both groups is considered as the non-senescence group (control). Further, the potential of navitoclax, dasatinib, quercetin, and the combination of the latter two to clear senescent cells was evaluated. Maturation of ciPTEC-OAT1 at non-permissive temperature affected mRNA and protein levels of senescence markers. A remarkable upregulation in p21 gene expression was found in the non-permissive temperature group, whereas expression of Lamin B1 decreased significantly. SASP factors, including PAI-1A, IL-1β, CTGF, and IL-6 were upregulated, but no significant difference in Bcl-2 and Bcl-xl were found in the non-permissive temperature group. After culturing ciPTEC-OAT1 up to 12 days, cells in the non-permissive temperature group showed an upregulation in the apoptosis-associated proteins Bcl-2, BID, and Bax, and a downregulation in Mcl-1, Bad, Bak, and Bim at various time points. Further, Bcl-xl, Puma, Caspase 3, Caspase 7, and Caspase 9 showed initial upregulations followed by downregulations at later time points. The loss of Lamin B1, upregulation of SA-β-gal expression and increase in its activity, upregulation of p21 levels and downregulation of p53, along with the upregulation of SASP factors, confirmed that maturation at 37°C promotes senescence features. Finally, the senolytics response was evaluated by testing cell viability following exposure to senolytics, to which cells appeared dose-dependently sensitive. Navitoclax was most effective in eliminating senescent cells. In conclusion, culturing ciPTEC-OAT1 at 37°C induces a senescence phenotype characterized by increased expression of cell cycle arrest and anti-apoptosis markers, SASP factors, and responsiveness to senolytics treatment. Therefore, ciPTEC-OAT1 represents a valid model for studying kidney senescence by simply adjusting culture conditions.

Short-Term Exposure to Thermophilic Temperatures Facilitates CO Uptake by Thermophiles Maintained under Predominantly Mesophilic Conditions.

Three phylogenetically and phenotypically distinct CO-oxidizing thermophiles (Alicyclobacillus macrosporangiidus CPP55 (Firmicutes), Meiothermus ruber PS4 (Deinococcus-Thermus) and Thermogemmatispora carboxidovorans PM5T (Chloroflexi)) and one CO-oxidizing mesophile (Paraburkholderia paradisi WAT (Betaproteobacteria)) isolated from volcanic soils were used to assess growth responses and CO uptake rates during incubations with constant temperatures (25 °C and 55 °C) and during multi-day incubations with a temperature regime that cycled between 20 °C and 55 °C on a diurnal basis (alternating mesophilic and thermophilic temperatures, AMTT). The results were used to test a conjecture that some thermophiles can survive in mesothermal habitats that experience occasional thermophilic temperatures. Meiothermus ruber PS4, which does not form spores, was able to grow and oxidize CO under all conditions, while the spore-forming Alicyclobacillus macrosporangiidus CPP55 grew and oxidized CO during the AMTT regime and at 55 °C, but was not active at 25 °C. Thermogemmatispora carboxidovorans PM5T, also a spore former, only grew at 55 °C but oxidized CO during AMTT and 55 °C incubations. In contrast, the non-sporing mesophile, Paraburkholderia paradisi WAT, was only able to grow and oxidize CO at 25 °C; growth and CO uptake ceased during the AMTT incubations after exposure to the initial round of thermophilic temperatures. Collectively, these results suggest that temporary, periodic exposure to permissive growth temperatures could help maintain populations of thermophiles in mesothermal habitats after deposition from the atmosphere or other sources.

Acridinium-conjugated aromatic heterocycles as highly potent FtsZ inhibitors: Design, synthesis, and biological evaluation.

The epidemic of multidrug resistance (MDR) is a serious threat to public health, and new classes of antibiotics with novel mechanisms of action are in critical need. We rationally designed and efficiently synthesized three series of new chemical entities with potential antibacterial activity targeting filamenting temperature-sensitive mutant Z (FtsZ). Evaluation of these compounds against a panel of Gram-positive bacteria including MDR and vancomycin-resistant Enterococcusstrains indicated that most compounds showed enhanced antibacterial efficacy, comparable or even superior to the reference drugs. The newly synthesized compounds proved to be substrates of the Escherichia coli efflux pump AcrB, thus affecting the activity. Their structure-activity relationshipswere summarized in detail. The most potent compound 10f quickly eliminated bacteria in a bactericidal mode, with low susceptibility to induce bacterial resistance. Further mechanistic studies with the BsFtsZ protein revealed that 10f functioned as an effective FtsZ inhibitor through altering the dynamics of FtsZ self-polymerization via a stimulatory mechanism, which leads to inhibition of cell division and cell death. Besides, 10f not only displayed no obvious cytotoxicity to mammalian cells but also had a high efficacy in a murine model of bacteremia in vivo. Regarded as a whole, our findings highlight 10f as a promising new FtsZ-targeting bactericidal agent.

Plasticity and conditional essentiality of modification enzymes for domain V of Escherichia coli 23S ribosomal RNA.

Escherichia coli rRNAs are post-transcriptionally modified at 36 positions but their modification enzymes are dispensable individually for growth, bringing into question their significance. However, a major growth defect was reported for deletion of the RlmE enzyme, which abolished a 2'O methylation near the peptidyl transferase center (PTC) of the 23S rRNA. Additionally, an adjacent 80-nt "critical region" around the PTC had to be modified to yield significant peptidyl transferase activity in vitro. Surprisingly, we discovered that an absence of just two rRNA modification enzymes is conditionally lethal (at 20°C): RlmE and RluC. At a permissive temperature (37°C), this double knockout was shown to abolish four modifications and be defective in ribosome assembly, though not more so than the RlmE single knockout. However, the double knockout exhibited an even lower rate of tripeptide synthesis than did the single knockout, suggesting an even more defective ribosomal translocation. A combination knockout of the five critical-region-modifying enzymes RluC, RlmKL, RlmN, RlmM, and RluE (not RlmE), which synthesize five of the seven critical-region modifications and 14 rRNA and tRNA modifications altogether, was viable (minor growth defect at 37°C, major at 20°C). This was surprising based on prior in vitro studies. This five-knockout combination had minimal effects on ribosome assembly and frameshifting at 37°C, but greater effects on ribosome assembly and in vitro peptidyl transferase activity at cooler temperatures. These results establish the conditional essentiality of bacterial rRNA modification enzymes and also reveal unexpected plasticity of modification of the PTC region in vivo.