Nucleoside diphosphate kinases (NDPK/NME/Nm23) are enzymes composed of subunits NME1/NDPK A and NME2/NDPK B, responsible for the maintenance of the cellular (d)NTP pool and involved in other cellular processes, such as metastasis suppression and DNA damage repair. Although eukaryotic NDPKs are active only as hexamers, it is unclear whether other NME functions require the hexameric form, and how the isoenzyme composition varies in different cellular compartments. To examine the effect of DNA damage on intracellular localization of NME1 and NME2 and the composition of NME oligomers in the nucleus and the cytoplasm, we used live-cell imaging and the FRET/FLIM technique. We showed that exogenous NME1 and NME2 proteins co-localize in the cytoplasm of non-irradiated cells, and move simultaneously to the nucleus after gamma irradiation. The FRET/FLIM experiments imply that, after DNA damage, there is a slight shift in the homomer/heteromer balance between the nucleus and the cytoplasm. Collectively, our results indicate that, after irradiation, NME1 and NME2 engage in mutual functions in the nucleus, possibly performing specific functions in their homomeric states. Finally, we demonstrated that fluorophores fused to the N-termini of NME polypeptides produce the largest FRET effect and thus recommend this orientation for use in similar studies.

The process of fracture healing consists of an inflammatory reaction and cartilage and bone tissue reconstruction. The inflammatory cytokine interleukin-1β (IL-1β) signal is an important major factor in fracture healing, whereas its relevance to retinoid receptor (an RAR inverse agonist, which promotes endochondral bone formation) remains unclear. Herein, we investigated the expressions of IL-1β and retinoic acid receptor gamma (RARγ) in a rat fracture model and the effects of IL-1β in the presence of one of several RAR inverse agonists on chondrocytes. An immunohistochemical analysis revealed that IL-1β and RARγ were expressed in chondrocytes at the fracture site in the rat ribs on day 7 post-fracture. In chondrogenic ATDC5 cells, IL-1β decreases the levels of aggrecan and type II collagen but significantly increased the metalloproteinase-13 (Mmp13) mRNA by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. An RAR inverse agonist (AGN194310) inhibited IL-1β-stimulated Mmp13 and Ccn2 mRNA in a dose-dependent manner. Phosphorylated extracellular signal regulated-kinases (pERK1/2) and p-p38 mitogen-activated protein kinase (MAPK) were increased time-dependently by IL-1β treatment, and the IL-1β-induced p-p38 MAPK was inhibited by AGN194310. Experimental p38 inhibition led to a drop in the IL-1β-stimulated expressions of Mmp13 and Ccn2 mRNA. MMP13, CCN2, and p-p38 MAPK were expressed in hypertrophic chondrocytes near the invaded vascular endothelial cells. As a whole, these results point to role of the IL-1β via p38 MAPK as important signaling in the regulation of the endochondral bone formation in fracture healing, and to the actions of RAR inverse agonists as potentially relevant modulators of this process.

Under stressful conditions, the early larvae of C. elegans enter dauer diapause, a non-aging period, driven by the seemingly opposite influence of ascaroside pheromones (ASCRs) and steroid hormone dafachronic acids (DAs). However, the molecular basis of how these small molecules engage in competitive crosstalk in coordination with insulin/IGF-1 signaling (IIS) remains elusive. Here we report a novel transcriptional regulatory pathway that seems to operate between the ASCR and DA biosynthesis under ad libitum (AL) feeding conditions or bacterial deprivation (BD). Although expression of the ASCR and DA biosynthetic genes reciprocally inhibit each other, ironically and interestingly, such dietary cue-mediated modulation requires the presence of the competitors. Under BD, induction of ASCR biosynthetic gene expression required DA, while ASCR suppresses the expression of the DA biosynthetic gene daf-36. The negative regulation of DA by ASCR was IIS-dependent, whereas daf-36 regulation appeared to be independent of IIS. These observations suggest that the presence of ASCR determines the IIS-dependency of DA gene expression regardless of dietary conditions. Thus, our work defines a molecular basis for a novel reciprocal gene regulation of pheromones and hormones to cope with stressful conditions during development and aging.

Potent neuroprotective effects of photobiomodulation with 670 nm red light (RL) have been demonstrated in several models of retinal disease. RL improves mitochondrial metabolism, reduces retinal inflammation and oxidative cell stress, showing its ability to enhance visual function. However, the current knowledge is limited to the main hypothesis that the respiratory chain complex IV, cytochrome c oxidase, serves as the primary target of RL. Here, we demonstrate a comprehensive cellular, molecular, and functional characterization of neuroprotective effects of 670 nm RL and 810 nm near-infrared light (NIRL) on blue light damaged murine primary photoreceptors. We show that respiratory chain complexes I and II are additional PBM targets, besides complex IV, leading to enhanced mitochondrial energy metabolism. Accordingly, our study identified mitochondria related RL- and NIRL-triggered defense mechanisms promoting photoreceptor neuroprotection. The observed improvement of mitochondrial and extramitochondrial respiration in both inner and outer segments is linked with reduced oxidative stress including its cellular consequences and reduced mitochondria-induced apoptosis. Analysis of regulatory mechanisms using gene expression analysis identified upregulation α-crystallins that indicate enhanced production of proteins with protective functions that point to the rescued mitochondrial function. The results support the hypothesis that energy metabolism is a major target for retinal light therapy.

A previous study indicated that mutations in the transmembrane protease serine 3 (TMPRSS3) gene, which encodes a transmembrane serine protease, cause nonsyndromic hearing loss (NSHL). This was the first description of a serine protease involved in hearing loss (HL). In Taiwan, however, data on the TMPRSS3 gene's association with NSHL is still insufficient. In this study, we described 10 mutations of TMPRSS3 genes found in 14 patients after screening 230 children with NSHL. The prevalence of the TMPRSS3 mutation appeared to be 6.09% (14/230). Of the 10 mutations, three were missense mutations: c.239G>A (p.R80H), c.551T>C (p.L184S), and 1253C>T (p.A418V); three were silent mutations, and four were mutations in introns. To determine the functional importance of TMPRSS3 mutations, we constructed plasmids carrying TMPRSS3 mutations of p.R80H, p.L184S, and p.A418V. TMPRSS3 function can be examined by secretory genetic assay for site-specific proteolysis (sGASP) and Xenopus oocyte expression system. Our results showed that p.R80H, p.L184S, and p.A418V TMPRSS3 mutations gave ratios of 19.4%, 13.2%, and 27.6%, respectively, via the sGASP system. Moreover, these three TMPRSS3 mutations failed to activate the epithelial sodium channel (ENaC) in the Xenopus oocyte expression system. These results indicate that the p.R80H, p.L184S, and p.A418V missense mutations of TMPRSS3 resulted in greatly diminishing the proteolytic activity of TMPRSS3. Our study provides information for understanding the importance of TMPRSS3 in the NSHL of Taiwanese children and provides a novel molecular explanation for the role of TMPRSS3 in HL.

casper has been a widely used transparent mutant of zebrafish. It possesses a combined loss of reflective iridophores and light-absorbing melanophores, which gives rise to its almost transparent trunk throughout larval and adult stages. Nevertheless, genomic causal mutations of this transparent phenotype are poorly defined. To identify the potential genetic basis of this fascinating morphological phenotype, we constructed genome maps by performing genome sequencing of 28 zebrafish individuals including wild-type AB strain, roy orbison (roy), and casper mutants. A total of 4.3 million high-quality and high-confidence homozygous single nucleotide polymorphisms (SNPs) were detected in the present study. We also identified a 6.0-Mb linkage disequilibrium block specifically in both roy and casper that was composed of 39 functional genes, of which the mpv17 gene was potentially involved in the regulation of iridophore formation and maintenance. This is the first report of high-confidence genomic mutations in the mpv17 gene of roy and casper that potentially leads to defective splicing as one major molecular clue for the iridophore loss. Additionally, comparative transcriptomic analyses of skin tissues from the AB, roy and casper groups revealed detailed transcriptional changes of several core genes that may be involved in melanophore and iridophore degeneration. In summary, our updated genome and transcriptome sequencing of the casper and roy mutants provides novel genetic clues for the iridophore loss. These new genomic variation maps will offer a solid genetic basis for expanding the zebrafish mutant database and in-depth investigation into pigmentation of animals.

Natural products play vital roles against infectious diseases since ancient times and most drugs in use today are derived from natural sources. Worldwide, multi-drug resistance becomes a massive threat to the society with increasing mortality. Hence, it is very crucial to identify alternate strategies to control these 'super bugs'. Pseudomonas aeruginosa is an opportunistic pathogen reported to be resistant to a large number of critically important antibiotics. Quorum sensing (QS) is a cell-cell communication mechanism, regulates the biofilm formation and virulence factors that endow pathogenesis in various bacteria including P. aeruginosa. In this study, we identified and evaluated quorum sensing inhibitors (QSIs) from plant-based natural products against P. aeruginosa. In silico studies revealed that catechin-7-xyloside (C7X), sappanol and butein were capable of interacting with LasR, a LuxR-type quorum sensing regulator of P. aeruginosa. In vitro assays suggested that these QSIs significantly reduced the biofilm formation, pyocyanin, elastase, and rhamnolipid without influencing the growth. Especially, butein reduced the biofilm formation up to 72.45% at 100 µM concentration while C7X and sappanol inhibited the biofilm up to 66% and 54.26% respectively. Microscale thermophoresis analysis revealed that C7X had potential interaction with LasR (KD = 933±369 nM) and thermal shift assay further confirmed the biomolecular interactions. These results suggested that QSIs are able to substantially obstruct the P. aeruginosa QS. Since LuxR-type transcriptional regulator homologues are present in numerous bacterial species, these QSIs may be developed as broad spectrum anti-infectives in the future.

Cell-penetrating peptides might have great potential for enhancing the therapeutic effect of drug molecules against such dangerous pathogens as Mycobacterium tuberculosis (Mtb), which causes a major health problem worldwide. A set of cationic cell-penetration peptides with various hydrophobicity were selected and synthesized as drug carrier of isoniazid (INH), a first-line antibacterial agent against tuberculosis. Molecular interactions between the peptides and their INH-conjugates with cell-membrane-forming lipid layers composed of DPPC and mycolic acid (a characteristic component of Mtb cell wall) were evaluated, using the Langmuir balance technique. Secondary structure of the INH conjugates was analyzed and compared to that of the native peptides by circular dichroism spectroscopic experiments performed in aqueous and membrane mimetic environment. A correlation was found between the conjugation induced conformational and membrane affinity changes of the INH-peptide conjugates. The degree and mode of interaction were also characterized by AFM imaging of penetrated lipid layers. In vitro biological evaluation was performed with Penetratin and Transportan conjugates. Results showed similar internalization rate into EBC-1 human squamous cell carcinoma, but markedly different subcellular localization and activity on intracellular Mtb.

Vitamin D is associated with cardiovascular health through activating the vitamin D receptor that targets genes related to cardiovascular disease (CVD). The human cardiac microvascular endothelial cells (HCMECs) were used to develop mechanically and TGF-β1-induced fibrosis models, and the rat was used as the isoproterenol (ISO)-induced fibrosis model. The rats were injected with ISO for the first five days, followed by vitamin D injection for the consecutive three weeks before being sacrificed on the fourth week. Results showed that mechanical stretching reduced endothelial cell marker CD31 and VE-cadherin protein expressions, as well as increased α-smooth muscle actin (α-SMA) and fibronectin (FN). The transforming growth factor-β1 (TGF-β1) reduced CD31, and increased α-SMA and FN protein expression levels. Vitamin D presence led to higher protein expression of CD31, and lower protein expressions of α-SMA and FN compared to the control in the TGF-β1-induced fibrosis model. Additionally, protein expression of VE-cadherin was increased and fibroblast-specific protein-1 (FSP1) was decreased after vitamin D treatment in the ISO-induced fibrosis rat. In conclusion, vitamin D slightly inhibited fibrosis development in cell and animal models. Based on this study, the beneficial effect of vitamin D may be insignificant; however, further investigation of vitamin D's effect in the long-term is required in the future.

The pathogenicity of the common phytopathogenic bacterium Pseudomonas syringae toward Caenorhabditis elegans has been recently demonstrated. However, the major virulence factors involved in this interaction remain unknown. In this study, we investigated the nematocidal activity of P. syringae against C. elegans under iron-sufficient/limited conditions, primarily focusing on the role of the ferric chelator pyoverdine in a P. syringae-C. elegans liquid-based pathogenicity model. Prediction-based analysis of pyoverdine-encoding genes in the genome of the wild-type P. syringae strain MB03 revealed that the genes are located in one large cluster. Two non-ribosomal peptide synthetase genes (pvdD and pvdJ) were disrupted via a Rec/TE recombination system, resulting in mutant strains with abrogated pyoverdine production and attenuated virulence against C. elegans. When used alone, pure pyoverdine also showed nematocidal activity. The role of iron used alone or with pyoverdine was further investigated in mutant and MB03-based bioassays. The results indicated that pyoverdine in P. syringae MB03 is a robust virulence factor that promotes the killing of C. elegans. We speculate that pyoverdine functions as a virulence determinant by capturing environmentally available iron for host bacterial cells, by limiting its availability for C. elegans worms, and by regulating and/or activating other intracellular virulence factors that ultimately kills C. elegans worms.

Tobacco (Nicotiana tabacum), is a world's major non-food agricultural crop widely cultivated for its economic value. Among several color change associated biological processes, plastid pigment metabolism is of trivial importance in postharvest plant organs during curing and storage. However, the molecular mechanisms involved in carotenoid and chlorophyll metabolism, as well as color change in tobacco leaves during curing, need further elaboration. Here, proteomic analysis at different curing stages (0 h, 48 h, 72 h) was performed in tobacco cv. Bi'na1 with an aim to investigate the molecular mechanisms of pigment metabolism in tobacco leaves as revealed by the iTRAQ proteomic approach. Our results displayed significant differences in leaf color parameters and ultrastructural fingerprints that indicate an acceleration of chloroplast disintegration and promotion of pigment degradation in tobacco leaves due to curing. In total, 5931 proteins were identified, of which 923 (450 up-regulated, 452 down-regulated, and 21 common) differentially expressed proteins (DEPs) were obtained from tobacco leaves. To elucidate the molecular mechanisms of pigment metabolism and color change, 19 DEPs involved in carotenoid metabolism and 12 DEPs related to chlorophyll metabolism were screened. The results exhibited the complex regulation of DEPs in carotenoid metabolism, a negative regulation in chlorophyll biosynthesis, and a positive regulation in chlorophyll breakdown, which delayed the degradation of xanthophylls and accelerated the breakdown of chlorophylls, promoting the formation of yellow color during curing. Particularly, the up-regulation of the chlorophyllase-1-like isoform X2 was the key protein regulatory mechanism responsible for chlorophyll metabolism and color change. The expression pattern of 8 genes was consistent with the iTRAQ data. These results not only provide new insights into pigment metabolism and color change underlying the postharvest physiological regulatory networks in plants, but also a broader perspective, which prompts us to pay attention to further screen key proteins in tobacco leaves during curing.

Identification of disease-associated autoantibodies is of high importance. Their assessment could complement current diagnostic modalities and assist the clinical management of patients. We aimed at developing and validating high-throughput protein microarrays able to screen patients' sera to determine disease-specific autoantibody-signatures for pancreatic cancer (PDAC), chronic pancreatitis (CP), autoimmune pancreatitis and their subtypes (AIP-1 and AIP-2). In-house manufactured microarrays were used for autoantibody-profiling of IgG-enriched preoperative sera from PDAC-, CP-, AIP-1-, AIP-2-, other gastrointestinal disease (GID) patients and healthy controls. As a top-down strategy, three different fluorescence detection-based protein-microarrays were used: large with 6400, intermediate with 345, and small with 36 full-length human recombinant proteins. Large-scale analysis revealed 89 PDAC, 98 CP and 104 AIP immunogenic antigens. Narrowing the selection to 29 autoantigens using pooled sera first and individual sera afterwards allowed a discrimination of CP and AIP from PDAC. For validation, predictive models based on the identified antigens were generated which enabled discrimination between PDAC and AIP-1 or AIP-2 yielded high AUC values of 0.940 and 0.925, respectively. A new repertoire of autoantigens was identified and their assembly as a multiplex test will provide a fast and cost-effective tool for differential diagnosis of pancreatic diseases with high clinical relevance.

Cilobradine (CIL, DK-AH269), an inhibitor of hyperpolarization-activated cation current (Ih), has been observed to possess pro-arrhythmic properties. Whether and how CIL is capable of perturbing different types of membrane ionic currents existing in electrically excitable cells, however, is incompletely understood. In this study, we intended to examine possible modifications by it or other structurally similar compounds of ionic currents in pituitary tumor (GH3) cells and in heart-derived H9c2 cells. The standard whole-cell voltage-clamp technique was performed to examine the effect of CIL on ionic currents. GH3-cell exposure to CIL suppressed the density of hyperpolarization-evoked Ih in a concentration-dependent manner with an effective IC50 of 3.38 μM. Apart from its increase in the activation time constant of Ih during long-lasting hyperpolarization, the presence of CIL (3 μM) distinctly shifted the steady-state activation curve of Ih triggered by a 2-s conditioning pulse to a hyperpolarizing direction by 10 mV. As the impedance-frequency relation of Ih was studied, its presence raised the impedance magnitude at the resonance frequency induced by chirp voltage. CIL also suppressed delayed-rectifier K+ current (IK(DR)) followed by the accelerated inactivation time course of this current, with effective IC50 (measured at late IK(DR)) or KD value of 3.54 or 3.77 μM, respectively. As the CIL concentration increased 1 to 3 μM, the inactivation curve of IK(DR) elicited by 1- or 10-s conditioning pulses was shifted to a hyperpolarizing potential by approximately 10 mV, and the recovery of IK(DR) inactivation during its presence was prolonged. The peak Na+ current (INa) during brief depolarization was resistant to being sensitive to the presence of CIL, yet to be either decreased by subsequent addition of A-803467 or enhanced by that of tefluthrin. In cardiac H9c2 cells, unlike the CIL effect, the addition of either ivabradine or zatebradine mildly led to a lowering in IK(DR) amplitude with no conceivable change in the inactivation time course of the current. Taken together, the compound like CIL, which was tailored to block hyperpolarization-activated cation (HCN) channels effectively, was also capable of altering the amplitude and gating of IK(DR), thereby influencing the functional activities of electrically excitable cells, such as GH3 cells.

Antxr1/Tem8 is highly expressed in tumor endothelial cells and is a receptor for anthrax toxin. Mutation of Antxr1 causes GAPO syndrome, which is characterized by growth retardation, alopecia, pseudo-anodontia, and optic atrophy. However, the mechanism underlying the growth retardation remains to be clarified. Runx2 is essential for osteoblast differentiation and chondrocyte maturation and regulates chondrocyte proliferation through Ihh induction. In the search of Runx2 target genes in chondrocytes, we found that Antxr1 expression is upregulated by Runx2. Antxr1 was highly expressed in cartilaginous tissues and was directly regulated by Runx2. In skeletal development, the process of endochondral ossification proceeded similarly in wild-type and Antxr1-/- mice. However, the limbs of Antxr1-/- mice were shorter than those of wild-type mice from embryonic day 16.5 due to the reduced chondrocyte proliferation. Chondrocyte-specific Antxr1 transgenic mice exhibited shortened limbs, although the process of endochondral ossification proceeded as in wild-type mice. BrdU-uptake and apoptosis were both increased in chondrocytes, and the apoptosis-high regions were mineralized. These findings indicated that Antxr1, of which the expression is regulated by Runx2, plays an important role in chondrocyte proliferation and that overexpression of Antxr1 causes chondrocyte apoptosis accompanied by matrix mineralization.

The Tc964 protein was initially identified by its presence in the interactome associated with the LYT1 mRNAs, which code for a virulence factor of Trypanosoma cruzi. Tc964 is annotated in the T. cruzi genome as a hypothetical protein. According to phylogenetic analysis, the protein is conserved in the different genera of the Trypanosomatidae family; however, recognizable orthologues were not identified in other groups of organisms. Therefore, as a first step, an in-depth molecular characterization of the Tc946 protein was carried out. Based on structural predictions and molecular dynamics studies, the Tc964 protein would belong to a particular class of GTPases. Subcellular fractionation analysis indicated that Tc964 is a nucleocytoplasmic protein. Additionally, the protein was expressed as a recombinant protein in order to analyze its antigenicity with sera from Chagas disease (CD) patients. Tc964 was found to be antigenic, and B-cell epitopes were mapped by the use of synthetic peptides. In parallel, the Leishmania major homologue (Lm964) was also expressed as recombinant protein and used for a preliminary evaluation of antigen cross-reactivity in CD patients. Interestingly, Tc964 was recognized by sera from Chronic CD (CCD) patients at different stages of disease severity, but no reactivity against this protein was observed when sera from Colombian patients with cutaneous leishmaniasis were analyzed. Therefore, Tc964 would be adequate for CD diagnosis in areas where both infections (CD and leishmaniasis) coexist, even though additional assays using larger collections of sera are needed in order to confirm its usefulness for differential serodiagnosis.

Human pluripotent stem cells have the potential to change the way in which human diseases are cured. Clinical-grade human embryonic stem cells and human induced pluripotent stem cells have to be created according to current good manufacturing practices and regulations. Quality and safety must be of the highest importance when humans' lives are at stake. With the rising number of clinical trials, there is a need for a consensus on hPSCs characterization. Here, we summarize mandatory and 'for information only' characterization methods with release criteria for the establishment of clinical-grade hPSC lines.

Mothers with a history of gestational diabetes mellitus (GDM) have an increased risk of developing diabetes in the future and a lifelong cardiovascular risk. Postpartal expression profile of cardiovascular/cerebrovascular disease associated microRNAs was assessed 3-11 years after the delivery in whole peripheral blood of young and middle-aged mothers with a prior exposure to GDM with the aim to identify a high-risk group of mothers at risk of later development of diabetes mellitus and cardiovascular/cerebrovascular diseases who would benefit from implementation of early primary prevention strategies and long-term follow-up. The hypothesis of the assessment of cardiovascular risk in women was based on the knowledge that a series of microRNAs play a role in the pathogenesis of diabetes mellitus and cardiovascular/cerebrovascular diseases. Abnormal expression profile of multiple microRNAs was found in women with a prior exposure to GDM (miR-1-3p, miR-16-5p, miR-17-5p, miR-20a-5p, miR-20b-5p, miR-21-5p, miR-23a-3p, miR-24-3p, miR-26a-5p, miR-29a-3p, miR-100-5p, miR-103a-3p, miR-125b-5p, miR-126-3p, miR-130b-3p, miR-133a-3p, miR-143-3p, miR-145-5p, miR-146a-5p, miR-181a-5p, miR-195-5p, miR-199a-5p, miR-221-3p, miR-342-3p, miR-499a-5p, and-miR-574-3p). Postpartal combined screening of miR-1-3p, miR-16-5p, miR-17-5p, miR-20b-5p, miR-21-5p, miR-23a-3p, miR-26a-5p, miR-29a-3p, miR-103a-3p, miR-133a-3p, miR-146a-5p, miR-181a-5p, miR-195-5p, miR-199a-5p, miR-221-3p, and miR-499a-5p showed the highest accuracy for the identification of mothers with a prior exposure to GDM at a higher risk of later development of cardiovascular/cerebrovascular diseases (AUC 0.900, p 0.001, sensitivity 77.48%, specificity 93.26%, cut off >0.611270413). It was able to identify 77.48% mothers with an increased cardiovascular risk at 10.0% FPR. Any of changes in epigenome (upregulation of miR-16-5p, miR-17-5p, miR-29a-3p, and miR-195-5p) that were induced by GDM-complicated pregnancy are long-acting and may predispose mothers affected with GDM to later development of diabetes mellitus and cardiovascular/cerebrovascular diseases. In addition, novel epigenetic changes (upregulation of serious of microRNAs) appeared in a proportion of women that were exposed to GDM throughout the postpartal life. Likewise, a previous occurrence of either GH, PE, and/or FGR, as well as a previous occurrence of GDM, is associated with the upregulation of miR-1-3p, miR-17-5p, miR-20a-5p, miR-20b-5p, miR-29a-3p, miR-100-5p, miR-125b-5p, miR-126-3p, miR-130b-3p, miR-133a-3p, miR-143-3p, miR-145-5p, miR-146a-5p, miR-181a-5p, miR-199a-5p, miR-221-3p, and miR-499a-5p. On the other hand, upregulation of miR-16-5p, miR-21-5p, miR-23a-3p, miR-24-3p, miR-26a-5p, miR-103a-3p, miR-195-5p, miR-342-3p, and miR-574-3p represents a unique feature of aberrant expression profile of women with a prior exposure to GDM. Screening of particular microRNAs may stratify a high-risk group of mothers with a history of GDM who might benefit from implementation of early primary prevention strategies.

Triple-negative tumor cells, a malignant subtype of breast cancer, lack a biologically targeted therapy. Given its DNA repair inhibiting properties, caffeine has been shown to enhance the effectiveness of specific tumor chemotherapies. In this work, we have investigated the effects of caffeine, cisplatin, and a combination of the two as potential treatments in energy metabolism for three cell lines, triple-negative breast cancer (MDA-MB-231), estrogen-receptor lacking breast cancer (MCF7) and breast epithelial cells (MCF10A) using a sensitive label-free approach, phasor-fluorescence lifetime imaging microscopy (phasor-FLIM). We found that solely using caffeine to treat MDA-MB-231 shifts their metabolism towards respiratory-chain phosphorylation with a lower ratio of free to bound NADH, and a similar trend is seen in MCF7. However, MDA-MB-231 cells shifted to a higher ratio of free to bound NADH when cisplatin was added. The combination of cisplatin and caffeine together reduced the survival rate for MDA-MD231 and shifted their energy metabolism to a higher fraction of bound NADH indicative of oxidative phosphorylation. The FLIM and viability results of MCF10A cells demonstrate that the treatments targeted cancer cells over the normal breast tissue. The identification of energy metabolism alteration could open up strategies of improving chemotherapy for malignant breast cancer.

Fat deposition, which influences pork production, meat quality and growth efficiency, is an economically important trait in pigs. Numerous studies have demonstrated that stearoyl-CoA desaturase (SCD), a key enzyme that catalyzes the conversion of saturated fatty acids into monounsaturated fatty acids, is associated with fatty acid composition in pigs. As SCD was observed to be significantly induced in 3T3-L1 preadipocytes differentiation, we hypothesized that it plays a role in porcine adipocyte differentiation and fat deposition. In this study, we revealed that SCD is highly expressed in adipose tissues from seven-day-old piglets, compared to its expression in tissues from four-month-old adult pigs. Moreover, we found that SCD and lipogenesis-related genes were induced significantly in differentiated porcine adipocytes. Using CRISPR/Cas9 technology, we generated SCD-/- porcine embryonic fibroblasts (PEFs) and found that the loss of SCD led to dramatically decreased transdifferentiation efficiency, as evidenced by the decreased expression of known lipid synthesis-related genes, lower levels of oil red O staining and significantly lower levels of triglyceride content. Our study demonstrates the critical role of SCD expression in porcine adipocyte differentiation and paves the way for identifying it as the promising candidate gene for less fat deposition in pigs.

Lentil (Lens culinaris Medikus) is an important source of protein for people in developing countries. Aphanomyces root rot (ARR) has emerged as one of the most devastating diseases affecting lentil production. In this study, we applied two complementary quantitative trait loci (QTL) analysis approaches to unravel the genetic architecture underlying this complex trait. A recombinant inbred line (RIL) population and an association mapping population were genotyped using genotyping by sequencing (GBS) to discover novel single nucleotide polymorphisms (SNPs). QTL mapping identified 19 QTL associated with ARR resistance, while association mapping detected 38 QTL and highlighted accumulation of favorable haplotypes in most of the resistant accessions. Seven QTL clusters were discovered on six chromosomes, and 15 putative genes were identified within the QTL clusters. To validate QTL mapping and genome-wide association study (GWAS) results, expression analysis of five selected genes was conducted on partially resistant and susceptible accessions. Three of the genes were differentially expressed at early stages of infection, two of which may be associated with ARR resistance. Our findings provide valuable insight into the genetic control of ARR, and genetic and genomic resources developed here can be used to accelerate development of lentil cultivars with high levels of partial resistance to ARR.