Compromised Viability Research Articles

Hybrid nanoparticle-mediated simultaneous ROS scavenging and STING activation improve the antitumor immunity of in situ vaccines.

In situ vaccine (ISV) is a versatile and personalized local immunotherapeutic strategy. However, the compromised viability and function of dendritic cells (DCs) in a tumor microenvironment (TME) largely limit the therapeutic efficacy. We designed a hybrid nanoparticle-based ISV, which accomplished superior cancer immunotherapy via simultaneously scavenging reactive oxygen species (ROS) and activating the stimulator of interferon genes (STING) pathway in DCs. This ISV was constructed by encapsulating a chemodrug, SN38, into diselenide bond-bridged organosilica nanoparticles, followed by coating with a Mn2+-based metal phenolic network. We show that this ISV can activate the STING pathway through Mn2+ and SN38 comediated signaling and simultaneously scavenge preexisting H2O2 in the TME and Mn2+-catalyzed •OH by leveraging the antioxidant property of diselenide and polyphenol. This ISV effectively activated DCs and protected them from oxidative damage, leading to remarkable downstream T cell activation and systemic antitumor immunity. This work highlights a nanoparticle design that manipulates DCs in the TME for improving the ISV.

Construction nanobiotechnology approach for performance enhancement of microbially induced biomineralization (MIB) using a biopolymer encapsulated spore-based system.

The integration of green construction practices within the built environment has been significantly advanced by biotechnological innovations, among which microbially induced biomineralization (MIB), predominantly facilitated by various strains of spore-forming bacilli, emerges as a pivotal mechanism for the self-healing of concrete. However, the practical deployment of this technology faces challenges, notably the compromised viability of bacterial spores due to germination triggered by severe shear stress during concrete mixing. To address this limitation, a water-insoluble polymer (extracellular polymeric substance) produced by Cellulomonas flavigena was utilized to encapsulate and protect the spores. The encapsulation process was rigorously verified through physicochemical methodologies, including X-ray diffraction (XRD) analysis, which revealed alterations in the interlayer spacings of the extracellular polymeric substance (EPS) structure during the encapsulation process, indicating successful EPS coating of the spores. Furthermore, a proof of concept for the enhanced biomineralization capacity of EPS-coated spores was demonstrated. Standard analytical techniques confirmed the precipitation of calcite and vaterite among other minerals, underscoring the effectiveness of this novel approach. This breakthrough paves the way for the development of innovative, sustainable bioconcrete applications, aligning with broader environmental objectives and advancing the field of green construction technology.IMPORTANCEDevelopment of bioconcrete with self-healing capability through MIB constitutes an important sustainable construction biotechnology approach for restoration and repair of built environment. Like every promising technology, MIB also suffers from certain shortcomings in terms of compromised viability of the microbial cells after premature germination of the spores on exposure to shear stress caused during concrete mixing. In this study, these challenges were adequately addressed by successfully providing a protective coating of indigenously extracted EPS to the bacterial spores and elucidating the interactive mechanisms between them. The results showed stable encapsulation of the spores while providing mechanistic insights of the encapsulation phenomenon. The data also showed enhanced rate of biomineralization by encapsulated microbes when subjected to stress conditions.

Mitochondrial viability in neurogenic bladder urothelium after sigmoidocolocystoplasty. Implications for persistent vesicoureteral reflux.

We investigated whether inflammatory cell infiltration (ICI), fibrosis, and mitochondrial viability of the neurogenic bladder urothelium are involved in the mechanism of persistent vesicoureteral reflux (VUR) after sigmoidocolocystoplasty (SCP). Bladder biopsies obtained 1994-2023 from 62 neurogenic bladder patients were examined by hematoxylin and eosin for ICI, Masson's trichrome for fibrosis, and immunofluorescence for urothelial growth differentiation factor 15 (GDF15; a mitochondrial stress-responsive cytokine) (positive/negative) and heat shock protein 60 (HSP60; a mitochondrial matrix marker) (strong ≥ 50%/weak≤ 50%) expression. GDF15 + /weak HSP60 indicated compromised mitochondrial viability. Cystometry measured neobladder compliance/capacity. Mean ages (years) at SCP and bladder biopsies were 9.4 ± 4.6 and 14.2 ± 7.1, respectively. VUR was present in 38/62 patients (51 ureters) at SCP and resolved with SCP alone in 4/38 patients, with SCP and ureteroneocystostomy in 17/38, and persisted in 17/38. Fibrosis was significantly denser in GDF15 + (n = 24)/weak HSP60 (n = 31) compared with GDF15- (n = 38)/strong HSP60 (n = 31) (p < 0.001 and p < 0.01, respectively). Differences in ICI were significant for GDF15 + vs. GDF15- (p < 0.05) but not for HSP60. Patients with VUR after SCP had higher incidence of GDF15 + /weak HSP60 compared with cases without VUR (p < 0.05 and p < 0.001, respectively). Viability of mitochondria appears to be compromised with possible etiologic implications for VUR persisting after SCP.

Iatrogenic bilocular femoral artery pseudoaneurysm in a patient with end-stage renal disease undergoing regular hemodialysis: An unexpected occurrence after a common procedure and surgical cure

Introduction: Pseudoaneurysms of the femoral artery can develop as an uncommon complication following penetrating trauma or arterial catheterization for cardiovascular procedures or hemodialysis. It is commonly diagnosed with color duplex imaging and usually a single pseudoaneurysm is present. Case Report: We present a 62-year-old woman with end-stage renal disease who developed a very rare bilocular pseudoaneurysm of the femoral artery caused by inadvertent puncture of the artery during elective hemodialysis which remained uneventful and undiagnosed until the pseudoaneurysm was itself punctured by the dialysis catheter iatrogenically and another large connecting pseudoaneurysm was formed above the initial one. Typical unilocular pseudoaneurysm presents as Yin-Yang sign on color duplex imaging but here bidirectional turbulent flow was seen as double Yin-Yang sign suggestive of bilocular pseudoaneurysm mimicking the shape of a snowman, hence Yin-Yang Snowman sign. The patient presented with signs and symptoms of rapid progression of the pseudoaneurysm resulting in femoral nerve compression with compromised skin viability. Therefore, surgical management was planned and the bilocular pseudoaneurysms were excised with repair of the femoral artery and then covered with vascularized rectus femoris muscle flap. Conclusion: An arterial pseudoaneurysm should be included in the differential diagnosis for a groin mass after femoral catheterization or other vascular procedures. A non-invasive imaging approach using color flow duplex sonography can be used with confidence and safety. But in complicated cases or cases with impending complications, surgical exploration and excision remains the mainstay of treatment.

How do local banks respond to natural disasters?

The increasing frequency and intensity of catastrophic natural disasters have the potential to stress and imperil banks to the point of compromised viability or even bankruptcy. Using data of approximately 907 domestic/local banks and Spatial Hazard Events and Losses Database for the United States during the period 2010–2019, we explore how natural disasters impact bank stability. Our main findings support the aforementioned hypothesis that natural disasters decrease bank stability because total deposit and equity (capital) become more volatile and the bank is prone to increased lending margins, as well as a provision of loan loss. Thus, banks lose their competitiveness, ROA deteriorates, and Z-score becomes lower. Strong corporate governance and healthy financial strategy, nevertheless, assist bank recovery in the aftermath of these weather extreme events. Last but not least, we find a non-linear relationship between natural disasters and bank stability and posit the role of indemnity paid out from the Federal insurance programme (after natural hazards) in the high-damage group.

Stability of the nitroxide biradical AMUPol in intact and lysed mammalian cells

Dynamic Nuclear Polarization (DNP) enhanced solid state NMR increases experimental sensitivity, potentially enabling detection of biomolecules at their physiological concentrations. The sensitivity of DNP experiments is due to the transfer of polarization from electron spins of free radicals to the nuclear spins of interest. Here, we investigate the reduction of AMUPol in both lysed and intact HEK293 cells. We find that nitroxide radicals are reduced with first order reduction kinetics by cell lysates at a rate of ∼ 12% of the added nitroxide radical concentration per hour. We also found that electroporation delivered a consistent amount of AMUPol to intact cells and that nitroxide radicals are reduced just slightly more rapidly (∼15% per hour) by intact cells than by cell lysates. The two nitroxide radicals of AMUPol are reduced independently and this leads to considerable accumulation of the DNP-silent monoradical form of AMUPol, particularly in preparations of intact cells where nearly half of the AMUPol is already reduced to the DNP silent monoradical form at the earliest experimental time points. This confirms that the loss of the DNP-active biradical form of AMUPol is faster than the nitroxide reduction rate. Finally, we investigate the effect of adding N-ethyl maleimide, a well-known inhibitor of thiol (-SH) group-based reduction of nitroxide biradicals in cells, on AMUPol reduction, cellular viability, and DNP performance. Although pre-treatment of cells with NEM effectively inhibited the reduction of AMUPol, exposure to NEM compromised cellular viability and, surprisingly, did not improve DNP performance. Collectively, these results indicate that, currently, the most effective strategy to obtain high DNP enhancements for DNP-assisted in-cell NMR is to minimize room temperature contact times with cellular constituents and suggest that the development of bio-resistant polarization agents for DNP could considerably increase the sensitivity of DNP-assisted in-cell NMR experiments.

Transcriptome analysis of maize pollen grains under drought stress during flowering

Drought stress is detrimental to male reproduction in maize (Zea mays L.), largely through reducing the quantity and quality of pollen grains. However, transcriptional response of maize pollen grains to drought stress has not been well documented. We compared pollen gene expression for a maize hybrid (ZhongDan909) under well-watered and drought-stress conditions, based on RNA-Seq validated by quantitative real-time PCR analysis. Expression of 6424 genes and 1302 transcripts was altered in pollen grains of maize subjected to 7 days of drought during flowering. Gene Ontology annotations showed 308 differentially expressed genes, annotated and classified into 50 primary functional categories. Kyoto Encyclopedia of Genes and Genomes analyses revealed 44 differentially expressed genes in nine metabolic pathways. In relation to carbohydrate metabolism pathways, there was downregulation of a polygalacturonase gene, which could reduce cell wall lysis in early pollen germination, and an increase in callose synthase transcripts along with reduced cellulase transcripts. These altered gene expressions responsible for cell wall integrity may inhibit the initiation of pollen tube growth. The onset of tube growth could be further impeded by observed changes in gene expression that potentially influence hormone metabolism (including downregulation of AUXIN RESPONSE FACTOR 18 and EIN3-BINDING F-BOX), reduce mitochondrial function, and alter protein translation. Genes with potential roles in adaptation were also altered in their transcript levels. These included genes encoding the upregulated transcription factor ZmNF-YC2, and the downregulated ZmbHLH13, a negative regulator of jasmonic acid responses. The upregulated flavin enzyme gene DIHYDROLIPOYL DEHYDROGENASE 1, associated with increased levels of reactive oxygen species, is of interest in relating redox homeostasis to stress adaptation. Overall, the analyses identified a suite of genes involved in the development of pollen grains and tubes and responsive to drought stress. The findings enhance understanding of the gene networks underlying compromised pollen viability under drought stress.

PSMB9 Mediates Resistance to Bortezomib in Multiple Myeloma

▪Introduction: Proteasomes are the chief degradation components of the ubiquitin proteasome system (UPS) and are comprised of several (α) structural and (ß) catalytic subunits. Targeting the ß5 subunit, with agents such bortezomib (Btz, 20S proteasome inhibitor, PI) has proven highly successful in MM pts. However, resistance to Btz (Btz-R) invariably ensues with disease evolution resulting in relapse and mortality. Our data highlight that in Btz-R cells, proteasome function is upregulated with increased reliance on other ß-catalytic subunits. We have uncovered a novel association between the ß5 subunit acting in concert with the ß1i proteasome subunit, to promote resistance to Btz. We hypothesized that targeting ß5 and ß1i together would be lethal to Btz-R MM cells.Methods: CD138+ cells from MM patients, Btz-sensitive (Btz-S) MM cell lines (OPM2, KMS11 and U266) and their isogenic Btz-R subclones were used in experiments. Fluorogenic peptide substrates were used to measure ß-subunit-specific enzymatic activity. Gene disruption was conducted using 2 different shRNA hairpins for PSMB5 (ß5) and PSMB9 (ß9). Affymetrix HT12v4 gene expression array and NanoString mRNA quantification were used for gene expression profiling (GEP), followed by qPCR for confirmation. Novel dual ß5-ß1i PI were synthesized and tested in vitro. Apoptosis was determined by annexin-V/PI staining and MTS/CellTiter Glo assay used to determine cell viability.Results: Btz IC50 was noted to be 284nM in Btz-R vs. 4nM in Btz-S cells. Sanger seq. revealed no mutations in PSMB5 indicating that Btz should effectively bind the ß5 subunit. ß-subunit chymotrypic activity in Btz-R vs. Btz-S cells was significantly increased (3.5 fold, p<0.001) but remained amenable to downregulation with Btz (as well as other PI). This suggested that ß5-subunit-independent mechanisms may account for increased proteasome function and resistance to Btz. GEP analysis revealed modulation of several genes; in particular, PSMB9 (ß1i) in Btz-R vs. Btz-S MM cells. Proteomic analysis confirmed protein/enzymatic upregulation of PSMB5/ß5 in conjunction with PSMB9/ß1i in primary cells from Btz-R MM patients and Btz-R MM cell lines. Clinical significance of PSMB9 upregulation was queried using the MMRF CoMMPass database (IA10), with analysis in pts. who received either Btz+dex or Len+Dex and did not respond to treatment. PSMB9 mRNA was significantly upregulated in Btz+Dex non-responders (n=42) vs. responders (n=92), whereas in Len+Dex pts. this difference was not significant. We hypothesized that downregulation of PSMB9 would resensitize Btz-R cells to Btz and noted a significant shift in IC50 (235nM to 108nM) in Btz-R-PSMB9 shRNA transfected cells. While ß1i knockdown alone was able to reduce Btz-R cell viability by ~25%, we posited that concurrent disruption of ß1i and ß5 would significantly reduce cell viability and proliferation. Targeted GEP in dual-ß5/ß1i knockdown Btz-R cells, showed >2-fold increase in CASP8, CASP9, CASP10, CDKN2A, CDKN1A , TNFRSF10B, FOXO4 and RNF43 (apoptosis genes) and decrease in CCB1, CCNA2, WNT5A, SKP2, CDC6 and FEN1 (cell cycle genes) vs. scramble-transfected controls. Indeed, both Btz-S and Btz-R MM cells demonstrated significantly compromised growth capacity and viability (Dual knockdown ß5/ß1i Btz-R cells: 0.32 million/mL vs. scramble Btz-R cells: 1.6 million/mL) (Fig 1). This prompted us to develop novel dual-ß5 and ß1i chemical inhibitors. Using a highly modified carbamate scaffold we developed 20 cmpds that display binding affinity for both ß5 and ß1i. In vitro screening of the top 5 cmpds showed >2 fold lower EC50 in Btz-R vs. Btz-S MM cell lines. 2 cmpds in particular showed ß1i and ß5 enzymatic inhibition of >50% at 1uM and 5uM, respectively and are being further optimized.Conclusions: We have found that upregulation of PSMB9/ß1i and PSMB5/ß5 is associated with resistance to Btz. These findings were confirmed in both MM cell lines as well as in primary MM cells from Btz-R pts. Moreover, increased PSMB9 mRNA expression in non-responding Btz+Dex pts. but not Len+dex MM pts. highlights this as a resistance mechanism unique to Btz. Direct genetic disruption or with novel dual-chemical inhibitors directed toward ß1i and ß5 induced lethality in Btz-R (as well as Btz-S) MM cells. Thus, our investigations point to development of agents, which inhibit ß1i and ß5-proteasome subunits to overcome resistance to Btz. [Display omitted] DisclosuresAilawadhi:Takeda: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pharmacyclics: Research Funding; Amgen: Consultancy, Honoraria.

Developmental kinetics and viability of bovine embryos produced in vitro with sex-sorted semen

The present study aimed to elucidate the developmental kinetics, growth potential, and viability of bovine embryos produced in vitro with sexed semen. Bovine oocytes were fertilized in vitro using unsorted and X-sorted semen from the same Holstein bulls, and the kinetics of in vitro development were continuously monitored for 10 d through time-lapse cinematography. The blastocyst formation rate was lower in the X-sorted group than in the unsorted group (P < 0.01), whereas the normal fertilization rate did not differ between groups. Morphokinetic evaluation revealed that the incidence of blastomere fusion during the first cleavage division, termed reverse cleavage, was higher in the X-sorted group (P < 0.01). Furthermore, embryos produced with X-sorted semen showed slower growth throughout the developmental period than embryos produced with unsorted semen (P < 0.01). The cell number of the trophectoderm and inner cell mass of blastocysts was reduced in the X-sorted group (P < 0.01). In embryos that developed to the blastocyst stage, the hatchability (P < 0.05), chromosomal normality (P < 0.01), and survivability after the conventional frozen-thawing process (P < 0.05) were reduced in the X-sorted group compared to that in the unsorted group, indicating a compromised viability of embryos derived from X-sorted semen. Taken together, the first cleavage dysmorphism, delayed embryo growth, and impaired viability of embryos developed to the blastocyst stage may explain the mechanism of reduced fertility in embryos derived from sexed semen. The kinetic evaluation of early embryo development and de-selection of embryos presenting the aberrant first cleavage would be valid for clinical application to produce sexed embryos with high implantation potential.

Perfluoroctanoic acid (PFOA) enhances NOTCH-signaling in an angiogenesis model of placental trophoblast cells.

Exposure to perfluoroalkyl substances (PFAS) was found to be associated with several pathological endpoints, including high cholesterol levels, specific defective functions of the immune system and reduced birth weight. While environmental PFAS have been recognized as threats for public health, surprisingly little is known about the underlying mechanisms of toxicity. We hypothesized that some of the observed vascular and developmental effects of environmental PFAS may share a common molecular pathway. At elevated levels of exposure to PFAS, a reduction in mean birth weight of newborns has been observed in combination with a high incidence rate of preeclampsia. As both, preeclampsia and reduced birth weight are consequences of an inadequate placental vascularization, we hypothesized that the adaptation of placental vasculature may get compromised by PFAS. We analyzed pseudo-vascular network formation and protein expression in the HTR8/SVneo cell line, an embryonic trophoblast cell type that is able to form vessel-like vascular networks in 3D-matrices, similar to endothelial cells. PFOA (perfluoroctanoic acid), but not PFOS (perfuoroctanesulfonic acid), induced morphological changes in the vascular 3D-network structure, without indications of compromised cellular viability. Incubation with PFOA reduced cellular sprouting and elongated isolated stalks in pseudo-vascular networks, while a γ-secretase inhibitor BMS-906024 induced directional opposite effects. We found a PFOA-induced increase in NOTCH intracellular domain (NICD) abundance in HTR8/SVneo, indicating that PFOA enhances NOTCH-signaling in this cell type. Enhancement of NOTCH-pathway by PFOA may be a key to understand the mode of action of PFAS, as this pathway is critically involved in many confirmed physiological/toxicological symptoms associated with PFAS exposure.

Molecular changes associated with spinal cord aging.

Age-related muscle weakness and loss of muscle mass (sarcopenia) is a universal problem in the elderly. Our previous studies indicate that alpha motor neurons (α-MNs) play a critical role in this process. The goal of the current study is to uncover changes in the aging spinal cord that contribute to loss of innervation and the downstream degenerative processes that occur in skeletal muscle. The number of α-MNs is decreased in the spinal cord of wildtype mice during aging, beginning in middle age and reaching a 41% loss by 27months of age. There is evidence for age-related loss of myelin and mild inflammation, including astrocyte and microglia activation and an increase in levels of sICAM-1. We identified changes in metabolites consistent with compromised neuronal viability, such as reduced levels of N-acetyl-aspartate. Cleaved caspase-3 is more abundant in spinal cord from old mice, suggesting that apoptosis contributes to neuronal loss. RNA-seq analysis revealed changes in the expression of a number of genes in spinal cord from old mice, in particular genes encoding extracellular matrix components (ECM) and a 172-fold increase in MMP-12 expression. Furthermore, blood-spinal cord barrier (BSCB) permeability is increased in old mice, which may contribute to alterations in spinal cord homeostasis and exacerbate neuronal distress. Together, these data show for the first time that the spinal cord undergoes significant changes during aging, including progressive α-MNs loss that is associated with low-grade inflammation, apoptosis, changes in ECM, myelination, and vascular permeability.

Improving pressure ulcer risk identification: a pilot project by ambulance staff

Background: A quality improvement initiative was designed to identify patients at risk of compromised tissue viability before they were admitted to hospital. Paramedics were educated to better identify patients with pressure ulcers or pressure damage, or those at risk of compromised tissue viability, and these patients were fitted with a pressure ulcer alert bracelet so that emergency department staff could identify them. Aims: The aims of the current initiative were to educate paramedics to better identify patients with pressure ulcers or those at risk of compromised tissue viability to emergency department staff, and fit them with a pressure ulcer alert bracelet to highlight them to emergency department staff so they would receive prompt intervention. Methods: A plan, do, study, act improvement methodology was used, and data from a 3-month period were retrospectively analysed. Patients identified as being at risk of compromised tissue viability were flagged as requiring assessment via a pressure ulcer risk assessment tool to enable prevention. Results: Paramedics identified 130 at-risk patients (aged 23–100 years), and data from 127 patients were analysed. Most at-risk patients fitted with pressure ulcer alert bracelets were aged 70 years or over, and there was an even female/male division. More than half (53%) of patients were found to have a pressure ulcer and alerted to emergency department staff. More than one in four (27%) patients who were identified as being at risk of pressure ulcers lived in nursing or residential homes, and 43% lived alone or in warden-controlled accommodation. Conclusions: Paramedics effectively identified potential risk factors for pressure ulcer development, indicating a need for immediate intervention. This study gives insight into how pressure ulcer risk assessment using an alert bracelet may be used in paramedic practice in emergency department handovers. Success depends on hospital staff acting upon paramedic recommendation.

Chlamydomonas CHT7 Is Required for an Effective Quiescent State by Regulating Nutrient-Responsive Cell Cycle Gene Expression.

COMPROMISED HYDROLYSIS OF TRIACYLGLYCEROLS7 (CHT7) in Chlamydomonas (Chlamydomonas reinhardtii) was previously shown to affect the transcription of a subset of genes during nitrogen (N)-replete growth and following N refeeding. Here, we show that an extensive derepression of genes involved in DNA metabolism and cell cycle-related processes, as well as downregulation of genes encoding oxidoreductases and nutrient transporters, occurs in the cht7 mutant during N deprivation. Cellular mutant phenotypes are consistent with the observed transcriptome misregulation, as cht7 cells fail to properly arrest growth, nuclear replication, and cell division following N deprivation. Reduction in cht7 colony formation following N refeeding is explained by its compromised viability during N deprivation and by the occurrence of abortive divisions during N refeeding. Surprisingly, the largely unstructured C-terminal half of CHT7 with predicted protein binding domains, but not the canonical CXC DNA binding domain, is essential for the ability of CHT7 to form stable complexes and reverse the cellular phenotypes and transcription levels in the cht7 mutant. Hence, although lacking the presumed DNA binding domain, CHT7 modulates the expression of cell cycle genes in response to N availability, which is essential for establishing an effective quiescent state and the coordinated resumption of growth following N refeeding.

Snapshot Multispectral Imaging Is Not Inferior to SPY Laser Fluorescence Imaging When Predicting Murine Flap Necrosis.

Objective assessment of tissue viability is critical to improve outcomes of cosmetic and reconstructive procedures. A widely used method to predict tissue viability is indocyanine green angiography. The authors present an alternative method that determines the relative proportions of oxyhemoglobin to deoxyhemoglobin through multispectral reflectance imaging. This affordable, hand-held device is noninvasive and may be used in clinic settings. The authors hypothesize that multispectral reflectance imaging is not inferior to indocyanine green angiography in predicting flap necrosis in the murine model. Reverse McFarlane skin flaps measuring 10 × 3 cm were raised on 300- to 400-g male Sprague-Dawley rats. Indocyanine green angiography and multispectral reflectance imaging was performed before surgery, immediately after surgery, and 30 minutes after surgery. Clinical outcome images acquired 72 hours after surgery were evaluated by three independent plastic surgeons. Objective data obtained immediately after surgery were compared to postsurgical clinical outcomes to determine which method more accurately predicted flap necrosis. Nine reverse McFarlane skin flaps were evaluated 72 hours after flap elevation. Data analysis demonstrated that the 95 percent confidence intervals for the sensitivity of postoperative multispectral reflectance imaging and indocyanine green angiography imaging to predict 72-hour tissue viability at a fixed specificity of 90 percent for predicting tissue necrosis were 86.3 to 91.0 and 79.1 to 86.9, respectively. In this experimental animal model, multispectral reflectance imaging does not appear to be inferior to indocyanine green angiography in detecting compromised tissue viability. With the advantages of noninvasiveness, portability, affordability, and lack of disposables, multispectral reflectance imaging has an exciting potential for widespread use in cosmetic and reconstructive procedures.

Abstract A133: Role of CXCR2 in the acquisition of pan-resistant phenotype in high grade serous ovarian cancer cells

Abstract Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy. Indeed, EOC epidemiological scenario is partially due to its late diagnosis, when most of tumor cells have acquired metastatic and chemoresistant phenotype. Despite the initial satisfactory response of EOC cells to platinum/taxanes-based therapy, chemoresistance emerges in an uncontrolled pace, specifically when the patient carries a high-grade serous adenocarcinoma (HGS-AC). Therefore, EOC clinic urges for novel treatment possibilities, aiming the reversal of chemoresistance, the improvement of patients’ quality of life, and their survival rates. cDNA microarray data from our group demonstrated that chemokines secreted to the tumor microenvironment, as CXCL2, IL-6, and IL-8 likely play a crucial role in EOC cells chemoresistance. Thereafter, the present study focused on the role of CXCL2, which binds to its receptor CXCR2 in the membrane of target cells. Nonetheless its function in HGS-AC is unclear. Our study model was based in two HGS-AC lineages: chemosensitive A2780, and its pan-resistant derived ACRP (IC50 to cisplatin 7.3uM vs. 26,56uM, p&amp;lt;0.001; to doxuribicin 0.07uM vs. 1.5uM, p&amp;lt;0.0001, and to paclitaxel 0.22uM vs. 1.7uM, p&amp;lt;0.0001, respectively). CXCR2 was knockdown (KD), and its expression was decreased to 0.6-fold as compared to control cells (p= 0.0015). As compared to the control experimental condition (100%), CXCR2 KD HGS-AC cells described compromised cellular proliferation and metabolic viability (BrdU and Presto blue/SRB assays: A2780 21% vs. ACRP 23%, p&amp;lt;0.0001), and lower cell viability (clonogenic assay: A2780 70% vs. ACRP 49%, p&amp;lt;0.0001). Although angiogenesis does not seem to influence our observations, chicken embryo chorioallantoic membrane assay revealed that CXCR2 KD ACRP-induced tumors shrunk by 3.3-fold in comparison the control condition. Intriguingly, similar results were not observed when cells were treated with the pharmacological inhibitor of CXCR2. We lately discovered that the receptor was anomalously expressed in the cells nucleus, thus justifying our observations while opening novel avenues for future investigations in the field. We, then, argued whether CXCR2 KD would modulate carcinogenic classic signaling pathways. We observed a biological tendency of less P-AKT (0.5- fold, p=0.09), but not P-ERK (0.3-fold, p=0.7) when compared to control. We also investigated the involvement of EMT in our studied phenomena. Our findings demonstrated significative decline in the expression of SLUG (0.9-fold vs. control, p=0.022) and SNAIL (1.1-fold vs. control, p=0.005) in CXCR2 KD ACRP cells. Finally, in silico analysis including over 200 HGS-AC cases revealed that overexpression of CXCR2 is robustly correlated to the worse disease prognosis and patients’ overall survival rate, specially in late stage disease (p=0.035). Altogether, our results present innovative strategy to fight pan-chemoresistant HGS-AC by silencing anomalously overexpressed CXCR2. Citation Format: Taciane Henriques, Diandra dos Santos, Isabella Guimaraes, Nayara Tessarollo, Paulo Lyra, Patricia Mesquisa, Diana Padua, Ana Luisa Amaral, Luisa Pereira, Bruno Cavadas, Ian Silva, Raquel Almeida, Leticia Rangel. Role of CXCR2 in the acquisition of pan-resistant phenotype in high grade serous ovarian cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A133. doi:10.1158/1535-7163.TARG-19-A133

Growth potential of bovine embryos presenting abnormal cleavage observed through time lapse cinematography

Time-lapse monitoring (TLM) has emerged as a novel technology for the continuous and noninvasive evaluation of embryos. TLM has revealed the prevalence of specific dysmorphisms such as abnormal development during the early-cleavage stage of embryos. However, little information is available on the prevalence and consequences of abnormal cleavage in bovine embryos. Hence, this study aimed to investigate growth potential of bovine embryos presenting abnormal cleavage, such as reverse cleavage (RC), direct cleavage (DC), and irregular and unsmooth ruffling of the oolema membrane (ruffling). Bovine embryos derived through in vitro fertilization (IVF) were cultured in the microwell culture dishes, and the kinetics of in vitro development were observed through TLM at 20-min intervals for 10 d. Approximately 36% of embryos that developed into a blastocyst presented abnormal cleavage. Morphokinetic evaluations revealed that RC, DC, and ruffling embryos showed slower development compared to embryos with normal cleavage (P < 0.01). Embryos with RC and DC, but not ruffling, revealed impaired hatchability (P < 0.05) with increased collapses of the blastocyst cavity until hatching (P < 0.0001). Moreover, the RC and DC embryos presented increased chromosomal aneuploidy (P < 0.05). These results suggest a compromised viability of embryos with RC and DC. This is the first report that clarified the effect of abnormal cleavage on the morphokinetics and growth potential of bovine IVF embryos. Results indicate that the kinetic evaluation of bovine embryos using the time-lapse imaging system will be beneficial for selecting embryos with a high viability.

Elimination of Sestrin 2 Compromises Viability in Extracellular Matrix-Detached SKOV3 Ovarian Cancer Cells

Epithelial ovarian carcinoma (EOC) is considered the deadliest gynecological cancer, largely due to the fact that it is often diagnosed once the cancer has already metastasized, thus making the disease more difficult to treat. Throughout metastasis, ovarian epithelial cancer cells must overcome many feats, including surviving in extracellular matrix (ECM) detachment. ECM-detached cancer cells must evade a number of insults, including increased intracellular reactive oxygen species (ROS). Recent evidence suggests ECM-detached cancer cells rely on antioxidant enzymes to combat these increasing levels of ROS to promote survival; however, the specific antioxidant enzymes involved in this process have yet to be fully elucidated. Sestrin 2 (SESN2) is a multi-functional protein that has been found to be instrumental in many different signaling pathways; notably, it has been recognized to play a critical role in eliminating ROS. Here, we show that SESN2 plays a unique role in maintaining the viability of ECM-detached metastatic ovarian epithelial cancer cells, and elimination of this critical protein results in compromised viability. Thus, these data identify SESN2 as a potentially interesting therapeutic target for treating this deadly metastatic disease.

Regulation of pollen lipid body biogenesis by MAP kinases and downstream WRKY transcription factors in Arabidopsis.

Signaling pathways that control the activities in non-photosynthetic plastids, important sites of plant metabolism, are largely unknown. Previously, we demonstrated that WRKY2 and WRKY34 transcription factors play an essential role in pollen development downstream of mitogen-activated protein kinase 3 (MPK3) and MPK6 in Arabidopsis. Here, we report that GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSLOCATOR 1 (GPT1) is a key target gene of WRKY2/WRKY34. GPT1 transports glucose-6-phosphate (Glc6P) into plastids for starch and/or fatty acid biosynthesis depending on the plant species. Loss of function of WRKY2/WRKY34 results in reduced GPT1 expression, and concomitantly, reduced accumulation of lipid bodies in mature pollen, which leads to compromised pollen viability, germination, pollen tube growth, and male transmission in Arabidopsis. Pollen-specific overexpression of GPT1 rescues the pollen defects of wrky2 wrky34 double mutant. Furthermore, gain-of-function activation of MPK3/MPK6 enhances GPT1 expression; whereas GPT1 expression is reduced in mkk4 mkk5 double mutant. Together, this study revealed a cytoplasmic/nuclear signaling pathway capable of coordinating the metabolic activities in plastids. High-level expression of GPT1 at late stages of pollen development drives Glc6P from cytosol into plastids, where Glc6P is used for fatty acid biosynthesis, an important step of lipid body biogenesis. The accumulation of lipid bodies during pollen maturation is essential to pollen fitness and successful reproduction.

Monitoring the biomechanical and physiological effects of postural changes during leisure chair sitting

BackgroundIndividuals with limited mobility can spend prolonged periods in leisure chairs, increasing their risk of developing a seated acquired pressure ulcer. The present study aims to use objective measures of posture and tissue viability to identify the associated risks of leisure chair related pressure ulcers. MethodsHealthy participants (n = 13) were recruited to sit on a leisure chair with either a viscoelastic foam or air cushion. Participants were asked to adopt four different postures for a period of 10 min followed by a 10 min refractory period. Measurements at the leisure chair-participant interface included interface pressure, transcutaneous tissue gas tensions at the ischial tuberosities, accelerometer data collected from the sternum and subjective comfort levels. ResultsResults indicated that interface pressures remained consistent, with peak pressure index values of less than 60 mmHg across all conditions. A proportion of participants exhibited decreased oxygen tensions associated with increased carbon dioxide tensions during one or more test condition. This was particularly prevalent during the right lean posture on the air cushion (46%). In all cases, normal tissue viability was restored during standing. The accelerometer was able to detect significant changes (p < 0.05) in relative trunk angles during slump and right lean when compared to optimal sitting posture. ConclusionCommercially available leisure chairs have little evidence to support their pressure relieving properties. This study revealed that a proportion of healthy individuals demonstrated a compromised tissue viability in specific postures. Further research is required to assess the impact of these sitting conditions in vulnerable individuals.

Drp1-dependent mitochondrial fission mediates osteogenic dysfunction in inflammation through elevated production of reactive oxygen species.

Although previous studies have implicated pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), to be detrimental for osteogenic activity, the related regulatory mechanisms are not yet fully validated. Since mitochondria host several essential metabolic processes and play a pivotal role in cellular functions, whether and how mitochondrial function contributes to inflammation-induced bone destruction needs further exploration. Our findings revealed that TNF-α impaired osteoblast function, including decreased mRNA levels of osteogenic markers, suppressed ALP expression and activity, and compromised cellular viability. Moreover, increased reactive oxygen species (ROS)-mediated oxidative stress in the TNF-α-treated group enhanced excessive mitochondrial fragmentation and disrupted mitochondrial function. However, treatment with antioxidant N-acetyl cysteine (NAC) or mitochondrial division inhibitor Mdivi-1 protected the cells from these adverse phenomena. These findings provide new insights into the role of the Drp1-dependent mitochondrial pathway in the osteogenic dysfunction during inflammation, indicating that this pathway may be a target for the development of new therapeutic approaches for the prevention and treatment of inflammation-induced bone destruction.