| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Tarvainen, Ilari, et al.
(författare)
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Identification of phthalate mixture exposure targets in the human and mouse ovary in vitro
- 2023
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Ingår i: Reproductive Toxicology. - : Elsevier. - 0890-6238 .- 1873-1708. ; 119
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Tidskriftsartikel (refereegranskat)abstract
- Chemical health risk assessment is based on single chemicals, but humans and wildlife are exposed to extensive mixtures of industrial substances and pharmaceuticals. Such exposures are life-long and correlate with multiple morbidities, including infertility. How combinatorial effects of chemicals should be handled in hazard charac-terization and risk assessment are open questions. Further, test systems are missing for several relevant health outcomes including reproductive health and fertility in women. Here, our aim was to screen multiple ovarian cell models for phthalate induced effects to identify biomarkers of exposure. We used an epidemiological cohort study to define different phthalate mixtures for in vitro testing. The mixtures were then tested in five cell models representing ovarian granulosa or stromal cells, namely COV434, KGN, primary human granulosa cells, primary mouse granulosa cells, and primary human ovarian stromal cells. Exposures at epidemiologically relevant levels did not markedly elicit cytotoxicity or affect steroidogenesis in short 24-hour exposure. However, significant effects on gene expression were identified by RNA-sequencing. Altogether, the exposures changed the expression of 124 genes on the average (9-479 genes per exposure) in human cell models, without obvious concentration or mixture-dependent effects on gene numbers. The mixtures stimulated distinct changes in different cell models. Despite differences, our analyses suggest commonalities in responses towards phthalates, which forms a starting point for follow-up studies on identification and validation of candidate biomarkers that could be developed to novel assays for regulatory testing or even into clinical tests.
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| 3. |
- Chen, Tianyi, et al.
(författare)
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Maternal exposure to PM2.5/BC during pregnancy predisposes children to allergic rhinitis which varies by regions and exclusive breastfeeding
- 2022
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Ingår i: Environment International. - : Elsevier. - 0160-4120 .- 1873-6750. ; 165
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundIncreasing prevalence of childhood allergic rhinitis(AR) needs a deeper understanding on the potential adverse effects of early life exposure to air pollution.ObjectivesThe main aim was to evaluate the effects of maternal exposure to PM2.5 and chemical constituents during pregnancy on preschool children’s AR, and further to explore the modification effects of regions and exclusive breastfeeding.MethodsA multi-center population-based study was performed in 6 cities from 3 regions of China in 2011–2012. Maternal exposure to ambient PM2.5 and main chemical constituents(BC, OM, SO42−, NO3−, NH4+) during pregnancy was assessed and a longitudinal prospective analysis was applied on preschool children’s AR. The modification effects of regions and exclusive breastfeeding were investigated.ResultsA total of 8.8% and 9.8% of children reported doctor-diagnosed allergic rhinitis(DDAR) and current hay fever, respectively, and 48.6% had less than 6 months of exclusive breastfeeding. The means of PM2.5 during pregnancy were 52.7 μg/m3, 70.3 μg/m3 and 76.4 μg/m3 in the east, north and central south of China, respectively. Multilevel log-binomial model regression showed that each interquartile range(IQR) increase of PM2.5 during pregnancy was associated with an average increase in prevalence ratio (PR) of DDAR by 1.43(95% confidence interval(CI): 1.11, 1.84) and current hay fever by 1.79(95% CI: 1.26, 2.55), respectively. Among chemical constituents, black carbon (BC) had the strongest associations. Across 3 regions, the eastern cities had the highest associations, followed by those in the central south and the north. For those equal to or longer than 6 months of exclusive breastfeeding, the associations were significantly reduced.ConclusionsChildren in east of China had the highest risks of developing AR per unit increase of maternal exposure to PM2.5 during pregnancy, especially BC constituent. Remarkable decline was found in association with an increase in breastfeeding for ≥6 months, in particular in east of China.
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| 4. |
- Chong, Hui, et al.
(författare)
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Organo-ptii complexes for potent photodynamic inactivation of multi-drug resistant bacteria and the influence of configuration
- 2024
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Ingår i: Advanced Science. - : John Wiley & Sons. - 2198-3844. ; 11:14
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Tidskriftsartikel (refereegranskat)abstract
- PtII based organometallic photosensitizers (PSs) have emerged as novel potent photodynamic inactivation (PDI) reagents through their enhanced intersystem crossing (ISC) processes. Currently, few PtII PSs have been investigated as antibacterial materials, with relatively poor performances reported and with structure-activity relationships not well described. Herein, a pair of configurational isomers are reported of Bis-BODIPY (4,4-difluoro-boradizaindacene) embedded PtII PSs. The cis-isomer (cis-BBP) displayed enhanced 1O2 generation and better bacterial membrane anchoring capability as compared to the trans-isomer (trans-BBP). The effective PDI concentrations (efficiency > 99.9%) for cis-BBP in Acinetobacter baumannii (multi-drug resistant (MDR)) and Staphylococcus aureus are 400 nM (12 J cm−2) and 100 nM (18 J cm−2), respectively; corresponding concentrations and light doses for trans-BBP in the two bacteria are 2.50 µM (30 J cm−2) and 1.50 µM (18 J cm−2), respectively. The 50% and 90% minimum inhibitory concentration (MIC50 and MIC90) ratio of trans-BBP to cis-BBP is 22.22 and 24.02 in A. baumannii (MDR); 21.29 and 22.36 in methicillin resistant S. aureus (MRSA), respectively. Furthermore, cis-BBP displays superior in vivo antibacterial performance, with acceptable dark and photoinduced cytotoxicity. These results demonstrate cis-BBP is a robust light-assisted antibacterial reagent at sub-micromolecular concentrations. More importantly, configuration of PtII PSs should be an important issue to be considered in further PDI reagents design.
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| 5. |
- Dou, Tianyi, et al.
(författare)
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Nanoscale Structural Characterization of Individual Viral Particles Using Atomic Force Microscope Infrared (AFM-IR) and Tip-Enhanced Raman Spectroscopy (TERS)
- 2020
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 92:16, s. 11297-11304
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Tidskriftsartikel (refereegranskat)abstract
- Viruses are infections species that infect a large spectrum of living systems. Although displaying a wide variety of shapes and sizes, they are all composed of nucleic acid encapsulated into a protein capsid. After virions enter the host cell, they replicate to produce multiple copies of themselves. They then lyse the host, releasing virions to infect new cells. High proliferation rate of viruses is the underlying cause of their fast transmission among living species. Although many viruses are harmless, some of them are responsible for severe diseases such as AIDS, viral hepatitis and flu. Traditionally, electron microscopy is used to identify and characterize viruses. This approach is time and labor consuming, which is problematic upon pandemic proliferation of previously unknown viruses. Herein, we demonstrate a novel diagnosis approach for label-free identification and structural characterization of individual viruses that is based on a combination of nanoscale Raman and Infrared spectroscopy. Using atomic force microscopy infrared spectroscopy (AFM-IR), we were able to probe structural organization of the virions of herpes simplex type 1 viruses and bacteriophage MS2. We also showed that tip enhanced Raman spectroscopy could be used to reveal protein secondary structure and amino acid composition of the virus surface. Our results show that AFM-IR and TERS provide different but complimentary information about the structure of complex biological specimens. This structural information can be used for fast and reliable identification of viruses. This nanoscale bimodal imaging approach can be also used to investigate the origin of viral polymorphism and study mechanisms of virion assembly.
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| 6. |
- Fang, Jianping, et al.
(författare)
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Enzyme overexpression - an exercise toward understanding regulation of heparan sulfate biosynthesis
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Biosynthesis of heparan sulfate (HS) involves conversion of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) units catalyzed by glucuronyl C5-epimerase (Hsepi). IdoA units are the favored substrate for 2-O-sulfotransferase (2OST). We used HEK293 cells as a model to investigate the effects of overexpression of these enzymes on HS structure. Overexpression of Hsepi alone resulted in an unexpected increase in HS chain length. A Hsepi point-mutant (Y168A), devoid of catalytic activity, failed to affect chain length. Moreover, the effect of Hsepi overexpression on HS chain length was abolished by simultaneous overexpression of 2OST. These findings raise novel aspects on regulation of HS biosynthesis. We propose a hypothetical enzyme-binding protein (EBP) with distinct, specific and partly overlapping binding sites, the interactions of which will determine levels of enzymes available to the biosynthetic process.
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| 7. |
- Li, Tianyi, et al.
(författare)
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Persistent organic pollutants dysregulate energy homeostasis in human ovaries in vitro
- 2024
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Ingår i: Environment International. - : Elsevier. - 0160-4120 .- 1873-6750. ; 187
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Tidskriftsartikel (refereegranskat)abstract
- Exposure to persistent organic pollutants (POPs), such as dichlorodiphenyltrichloroethane (DDT) and polychlorinated biphenyls (PCBs), has historically been linked to population collapses in wildlife. Despite international regulations, these legacy chemicals are still currently detected in women of reproductive age, and their levels correlate with reduced ovarian reserve, longer time -to -pregnancy, and higher risk of infertility. However, the specific modes of action underlying these associations remain unclear. Here, we examined the effects of five commonly occurring POPs - hexachlorobenzene (HCB), p,p '-dichlorodiphenyldichloroethylene (DDE), 2,3,3 ' ,4,4 ' ,5-hexachlorobiphenyl (PCB156), 2,2 ' ,3,4,4 ' ,5,5 ' -heptachlorobiphenyl (PCB180), perfluorooctane sulfonate (PFOS) - and their mixture on human ovaries in vitro . We exposed human ovarian cancer cell lines COV434, KGN, and PA1 as well as primary ovarian cells for 24 h, and ovarian tissue containing unilaminar follicles for 6 days. RNA -sequencing of samples exposed to concentrations covering epidemiologically relevant levels revealed significant gene expression changes related to central energy metabolism in the exposed cells, indicating glycolysis, oxidative phosphorylation, fatty acid metabolism, and reactive oxygen species as potential shared targets of POP exposures in ovarian cells. Alpha-enolase ( ENO1 ), lactate dehydrogenase A ( LDHA ), cytochrome C oxidase subunit 4I1 ( COX4I1 ), ATP synthase F1 subunit alpha ( ATP5A ), and glutathione peroxidase 4 ( GPX4 ) were validated as targets through qPCR in additional cell culture experiments in KGN. In ovarian tissue cultures, we observed significant effects of exposure on follicle growth and atresia as well as protein expression. All POP exposures, except PCB180, decreased unilaminar follicle proportion and increased follicle atresia. Immunostaining confirmed altered expression of LDHA, ATP5A, and GPX4 in the exposed tissues. Moreover, POP exposures modified ATP production in KGN and tissue culture. In conclusion, our results demonstrate the disruption of cellular energy metabolism as a novel mode of action underlying POP -mediated interference of follicle growth in human ovaries.
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| 8. |
- Li, Tianyi
(författare)
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Targeting the ovary : mapping mechanisms to link endocrine-disrupting chemicals to female fertility
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In recent decades, growing attention has been paid to endocrine-disrupting chemicals (EDCs) as mounting evidence suggests that they can cause detrimental reproductive outcomes in animals and humans. Even though epidemiological studies show the link between EDC exposure and adverse reproductive outcomes such as sub-fertility, longer time-to-pregnancy, and decreased ovarian reserve in women, the underlying mechanisms are not fully elucidated. This is challenging for chemical regulation as we need mechanistic information to properly identify EDCs that may cause detrimental effects. In this thesis, we aim to map the mechanisms linking EDCs to female infertility, identify potential biomarkers of exposure using in vitro culture systems, and carry out cross-species comparisons using rat models. Paper I characterized the effects of in vitro culture alone on ovarian cortical tissue using transcriptomic profiling and investigated the feasibility of using this model to study the effects of exposure to EDCs on the human ovary. RNA sequencing (RNA-seq) revealed a marked change induced by tissue fragmentation and culture itself, including changes in energy metabolism (i.e., glycolysis). Follicles were activated to grow during the culture, which could be explained by the disruption of the Hippo signaling, and partially, by upregulation of the glycolysis pathway. Papers II-III investigated the effects of selected chemicals (pharmaceuticals: DES, KTZ; persistent organic pollutants (POPs): HCB, DDE, PCB156, PCB180, PFOS, and their mixture) on ovarian cells and tissue in culture. All exposures affected follicle growth in culture. Additionally, exposure to POPs resulted in increased follicle atresia in in vitro culture. Using transcriptomic profiling, we found disruption of lipid biology and energy homeostasis as well as increased oxidative stress as potential novel mechanisms connecting chemical exposures to disrupted folliculogenesis. Furthermore, we identified stearoyl-CoA desaturase (SCD) and 7-dehydrocholesterol reductase (DHCR7) as potential biomarkers of chemical exposure. Papers IV-V investigated the reproductive outcomes induced by DES and KTZ exposure in rats and explored the changes in endpoint sensitivity during pubertal and adult exposure. Moreover, we also developed and assessed the feasibility of using surface photo counting (SPC) as a fast tool to prioritize chemical exposure groups for further histological evaluation. In general, we found that high-dose exposure to DES and KTZ disrupted folliculogenesis in rats. When comparing endpoint sensitivity between different exposure periods, our results suggested that no profound differences can be observed, although pubertal exposure allowed the inclusion of vaginal opening as a sensitive endpoint to estrogenic chemicals. In addition, we showed that the quantification results obtained from the SPC method were significantly correlated with that of traditional histological assessment. Therefore, SPC could be used as a complementary method to prioritize groups for histology analysis. In summary, in vitro ovarian tissue culture can be used to study the impact of chemicals on follicle survival and growth, and underlying mechanisms. Utilizing this model, we found that exposure to the selected chemicals affected folliculogenesis, through disruption of their energy metabolism and increased oxidative stress. Similarly, we showed that high-dose DES and KTZ exposure disrupted follicle growth in rats, but not in low- and middle-dose groups. This suggests that the investigated endpoints in the in vivo study were not sensitive enough. It advocates the need for a sensitive and human-relevant assay for the screening of EDCs present in the global market. The identified common signatures and biomarkers might be used as a base for the future development of such screening assays after validation. Even though this is a small step, we are moving towards the future of a chemical-safe world.
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| 9. |
- Liu, Yang, et al.
(författare)
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MiR-378a suppresses tenogenic differentiation and tendon repair by targeting at TGF-β2
- 2019
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Ingår i: Stem Cell Research and Therapy. - : Springer Science and Business Media LLC. - 1757-6512. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Tendons are a crucial component of the musculoskeletal system and responsible for transmission forces derived from muscle to bone. Patients with tendon injuries are often observed with decreased collagen production and matrix degeneration, and healing of tendon injuries remains a challenge as a result of limited understanding of tendon biology. Recent studies highlight the contribution of miR-378a on the regulation gene expression during tendon differentiation. Methods: We examined the tendon microstructure and tendon repair with using miR-378a knock-in transgenic mice, and the tendon-derived stem cells were also isolated from transgenic mice to study their tenogenic differentiation ability. Meanwhile, the expression levels of tenogenic markers were also examined in mouse tendon-derived stem cells transfected with miR-378a mimics during tenogenic differentiation. With using online prediction software and luciferase reporter assay, the binding target of miR-378a was also studied. Results: Our results indicated miR-378a impairs tenogenic differentiation and tendon repair by inhibition collagen and extracellular matrix production both in vitro and in vivo. We also demonstrated that miR-378a exert its inhibitory role during tenogenic differentiation through binding at TGFβ2 by luciferase reporter assay and western blot. Conclusions: Our investigation suggests that miR-378a could be considered as a new potential biomarker for tendon injury diagnosis or drug target for a possible therapeutic approach in future clinical practice.
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| 10. |
- Paucar, Martin, et al.
(författare)
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V374A KCND3 Pathogenic Variant Associated With Paroxysmal Ataxia Exacerbations
- 2021
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Ingår i: Neurology Genetics. - : American Academy of Neurology. - 2376-7839. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Objective: Ataxia channelopathies share common features such as slow motor progression and variable degrees of cognitive dysfunction. Mutations in potassium voltage-gated channel subfamily D member 3 (KCND3), encoding the K+ channel, Kv4.3, are associated with spinocerebellar ataxia (SCA) 19, allelic with SCA22. Mutations in potassium voltage-gated channel subfamily C member 3 (KCNC3), encoding another K+ channel, Kv3.3, cause SCA13. First, a comprehensive phenotype assessment was carried out in a family with autosomal dominant ataxia harboring 2 genetic variants in KCNC3 and KCND3. To evaluate the physiological impact of these variants on channel currents, in vitro studies were performed.Methods: Clinical and psychometric evaluations, neuroimaging, and genotyping of a family (mother and son) affected by ataxia were carried out. Heterozygous and homozygous Kv3.3 A671V and Kv4.3 V374A variants were evaluated in Xenopus laevis oocytes using 2-electrode voltage-clamp. The influence of Kv4 conductance on neuronal activity was investigated computationally using a Purkinje neuron model.Results: The main clinical findings were consistent with adult-onset ataxia with cognitive dysfunction and acetazolamide-responsive paroxysmal motor exacerbations in the index case. Despite cognitive deficits, fluorodeoxyglucose (FDG)-PET displayed hypometabolism mainly in the severely atrophic cerebellum. Genetic analyses revealed the new variant c.1121T>C (V374A) in KCND3 and c.2012T>C (A671V) in KCNC3. In vitro electrophysiology experiments on Xenopus oocytes demonstrated that the V374A mutant was nonfunctional when expressed on its own. Upon equal co-expression of wild-type (WT) and V374A channel subunits, Kv4.3 currents were significantly reduced in a dominant negative manner, without alterations of the gating properties of the channel. By contrast, Kv3.3 A671V, when expressed alone, exhibited moderately reduced currents compared with WT, with no effects on channel activation or inactivation. Immunohistochemistry demonstrated adequate cell membrane translocation of the Kv4.3 V374A variant, thus suggesting an impairment of channel function, rather than of expression. Computational modeling predicted an increased Purkinje neuron firing frequency upon reduced Kv4.3 conductance.Conclusions: Our findings suggest that Kv4.3 V374A is likely pathogenic and associated with paroxysmal ataxia exacerbations, a new trait for SCA19/22. The present FDG PET findings contrast with a previous study demonstrating widespread brain hypometabolism in SCA19/22.
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