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1.
Front Neurosci ; 18: 1346610, 2024.
Article in English | MEDLINE | ID: mdl-38638695

ABSTRACT

Introduction: The remarkable diversity observed in the structure and development of the molluscan nervous system raises intriguing questions regarding the molecular mechanisms underlying neurogenesis in Mollusca. The expression of SoxB family transcription factors plays a pivotal role in neuronal development, thereby offering valuable insights into the strategies of neurogenesis. Methods: In this study, we conducted gene expression analysis focusing on SoxB-family transcription factors during early neurogenesis in the gastropod Lymnaea stagnalis. We employed a combination of hybridization chain reaction in situ hybridization (HCR-ISH), immunocytochemistry, confocal microscopy, and cell proliferation assays to investigate the spatial and temporal expression patterns of LsSoxB1 and LsSoxB2 from the gastrula stage to hatching, with particular attention to the formation of central ring ganglia. Results: Our investigation reveals that LsSoxB1 demonstrates expanded ectodermal expression from the gastrula to the hatching stage, whereas expression of LsSoxB2 in the ectoderm ceases by the veliger stage. LsSoxB1 is expressed in the ectoderm of the head, foot, and visceral complex, as well as in forming ganglia and sensory cells. Conversely, LsSoxB2 is mostly restricted to the subepithelial layer and forming ganglia cells during metamorphosis. Proliferation assays indicate a uniform distribution of dividing cells in the ectoderm across all developmental stages, suggesting the absence of distinct neurogenic zones with increased proliferation in gastropods. Discussion: Our findings reveal a spatially and temporally extended pattern of SoxB1 expression in a gastropod representative compared to other lophotrochozoan species. This prolonged and widespread expression of SoxB genes may be interpreted as a form of transcriptional neoteny, representing a preadaptation to prolonged neurogenesis. Consequently, it could contribute to the diversification of nervous systems in gastropods and lead to an increase in the complexity of the central nervous system in Mollusca.

2.
Biol Reprod ; 110(2): 365-376, 2024 Feb 10.
Article in English | MEDLINE | ID: mdl-37971359

ABSTRACT

The implementation of live imaging in reproductive research is crucial for studying the physiological dynamics. Sperm transport is a highly dynamic process regulated by tubular contractions and luminal flows within the male reproductive tract. However, due to the lack of imaging techniques to capture these dynamics in vivo, there is little information on the physiological and biomechanical regulation of sperm transport through the male reproductive tract. Here, we present a functional in vivo imaging approach using optical coherence tomography, enabling live, label-free, depth-resolved, three-dimensional, high-resolution visualization of the mouse testis and epididymis. With this approach, we spatiotemporally captured tubular contractility in mouse testis and epididymis, as well as microstructures of these reproductive organs. Our findings demonstrated that the contraction frequency varies significantly depending on the epididymal regions, suggesting the spatial regulation of epididymal contractility. Furthermore, we implemented quantitative measurements of the contraction wave and luminal transport through the epididymal duct, revealing the physiological dynamics within the male reproductive tract. The results show that the contraction wave propagates along the epididymal duct and the wave propagation velocity was estimated in vivo. In conclusion, this is the first study to develop in vivo dynamic volumetric imaging of the male reproductive tract, which allows for quantitative analysis of the dynamics associated with sperm transport. This study sets a platform for various studies investigating normal and abnormal male reproductive physiology as well as the pharmacological and environmental effects on reproductive functions in mouse models, ultimately contributing to a comprehensive understanding of male reproductive disorders.


Subject(s)
Epididymis , Testis , Mice , Animals , Male , Epididymis/diagnostic imaging , Epididymis/physiology , Testis/diagnostic imaging , Tomography, Optical Coherence , Semen , Spermatozoa
3.
Actas urol. esp ; 47(10): 661-667, Dic. 2023. tab
Article in English, Spanish | IBECS | ID: ibc-228317

ABSTRACT

Objetivo Evaluar si la tasa libre de litiasis afecta a los resultados del estudio metabólico en pacientes con alto riesgo de litiasis recidivante tras tratamiento completo mediante ureteroscopia. Pacientes y métodos Un total de 78 pacientes sometidos a cirugía retrógrada intrarrenal (CRIR) para el tratamiento de litiasis fueron incluidos en este estudio. Cuatro semanas después del tratamiento, los casos se dividieron en dos grupos en base a los resultados de la tomografía computarizada sin contraste (TCSC). Los casos del grupo 1 (n=54) presentaban una tasa libre de litiasis del 100% y los del grupo 2 (n=24) presentaban litiasis residuales en el riñón. Cuatro semanas después de la ureteroscopia flexible (URF) se realizó un análisis completo de orina de 24h a todos los pacientes de ambos grupos, para detectar los factores de riesgo implicados en la litogénesis. Los resultados del estudio metabólico (orina de 24h y suero) se compararon entre los dos grupos. Resultados La evaluación preoperatoria en orina y suero de los factores de riesgo asociados a la formación de cálculos no reveló diferencias estadísticas entre los dos grupos. El análisis comparativo de los factores de riesgo implicados en la formación de la litiasis mediante pruebas de orina de 24h tampoco reveló diferencias estadísticamente significativas entre los resultados preoperatorios y postoperatorios en los casos del grupo 2 con cálculos residuales. Tampoco se observaron diferencias significativas entre las medias de las variables séricas preoperatorias y postoperatorias de ambos grupos. Conclusiones Según nuestros resultados, y dada la similitud de los hallazgos obtenidos en los estudios metabólicos de los casos con y sin litiasis residual, la tasa libre de litiasis puede no constituir un factor imprescindible para la realización del estudio metabólico detallado (suero y orina de 24h) tras las intervenciones endourológicas para la extracción de los cálculos renales. (AU)


Objective To evaluate the impact of stone free status on the outcomes of metabolic evaluation in recurrent stone formers after ureteroscopic stone removal. Patients and methods A total of 78 patients undergoing retrograde intrarenal surgery (RIRS) for renal stones were included and cases were divided into two groups after 4 weeks based on the NCCT findings. While cases in the Group 1 (n=54) was completely stone free, cases in Group 2 (n=24) had residual fragments in the kidney. A full 24-hour urine analysis for relevant stone forming risk factors has been performed after 4 weeks following the fURS procedures in all patients of both groups. Outcomes of metabolic evaluation (24-hour urine and serum) have been comparatively evaluated in both groups. Results Evaluation of the preoperative serum and urine stone forming risk factors revelaed no statistical difference in both groups. Comparative evaluation of the 24-hour urinary stone forming risk factors also revealed no statistically significant difference between preoperative and postoperative findings in cases of Group 2 with residual stones. Last but not least, no significant difference was observed between the mean preoperative and postoperative serum variables between two groups. Conclusions Our results show that in the light of the similar metabolic evaluation outcomes obtained in cases with and without residual fragments, ‘stone free status’ may not be an essential factor to perform a detailed metabolic evaluation (24-hour urine analysis and serum parameters) after endourological stone removal procedures. (AU)


Subject(s)
Humans , Male , Female , Lithiasis/surgery , Lithiasis/therapy , Nephrolithiasis/surgery , Nephrolithiasis/therapy , Ureteroscopy/rehabilitation , Metabolic Flux Analysis
4.
Genome Biol Evol ; 15(10)2023 Oct 06.
Article in English | MEDLINE | ID: mdl-37708413

ABSTRACT

The sleeping chironomid Polypedilum vanderplanki is capable of anhydrobiosis, a striking example of adaptation to extreme desiccation. Tolerance to complete desiccation in this species is associated with emergence of multiple paralogs of protective genes. One of the gene families highly expressed under anhydrobiosis and involved in this process is protein-L-isoaspartate (D-aspartate) O-methyltransferases (PIMTs). Recently, another closely related midge was discovered, Polypedilum pembai, which is able not only to tolerate desiccation but also to survive multiple desiccation-rehydration cycles. To investigate the evolution of anhydrobiosis in these species, we sequenced and assembled the genome of P. pembai and compared it with P. vanderplanki and also performed a population genomics analysis of several populations of P. vanderplanki and one population of P. pembai. We observe positive selection and radical changes in the genetic architecture of the PIMT locus between the two species, including its amplification in the P. pembai lineage. In particular, PIMT-4, the most highly expressed of these PIMTs, is present in six copies in the P. pembai; these copies differ in expression profiles, suggesting possible sub- or neofunctionalization. The nucleotide diversity of the genomic region carrying these new genes is decreased in P. pembai, but not in the orthologous region carrying the ancestral gene in P. vanderplanki, providing evidence for a selective sweep associated with postduplication adaptation in the former. Overall, our results suggest an extensive relatively recent and likely ongoing adaptation of the mechanisms of anhydrobiosis.

5.
Actas Urol Esp (Engl Ed) ; 47(10): 661-667, 2023 Dec.
Article in English, Spanish | MEDLINE | ID: mdl-37355206

ABSTRACT

OBJECTIVE: To evaluate the impact of stone free status on the outcomes of metabolic evaluation in recurrent stone formers after ureteroscopic stone removal. PATIENTS AND METHODS: A total of 78 patients undergoing retrograde intrarenal surgery (RIRS) for renal stones were included and cases were divided into two groups after 4 weeks based on the NCCT findings. While cases in the Group 1 (n = 54) was completely stone free, cases in Group 2 (n = 24) had residual fragments in the kidney. A full 24-h urine analysis for relevant stone forming risk factors has been performed after 4 weeks following the fURS procedures in all patients of both groups. Outcomes of metabolic evaluation (24-h urine and serum) have been comparatively evaluated in both groups. RESULTS: Evaluation of the preoperative serum and urine stone forming risk factors revelaed no statistical difference in both groups. Comparative evaluation of the 24-h urinary stone forming risk factors also revealed no statistically significant difference between preoperative and postoperative findings in cases of Group 2 with residual stones. Last but not least, no significant difference was observed between the mean preoperative and postoperative serum variables between two groups. CONCLUSIONS: Our results show that in the light of the similar metabolic evaluation outcomes obtained in cases with and without residual fragments, 'stone free status' may not be an essential factor to perform a detailed metabolic evaluation (24-h urine analysis and serum parameters) after endourological stone removal procedures.


Subject(s)
Kidney Calculi , Lithotripsy , Humans , Treatment Outcome , Kidney Calculi/surgery , Kidney/surgery , Lithotripsy/methods , Postoperative Complications/epidemiology
6.
J Appl Physiol (1985) ; 134(5): 1256-1264, 2023 05 01.
Article in English | MEDLINE | ID: mdl-37055032

ABSTRACT

We aimed to explore the effect of the 3-day dry immersion, a model of physical unloading, on mitochondrial function, transcriptomic and proteomic profiles in a slow-twitch soleus muscle of six healthy females. We registered that a marked reduction (25-34%) in the ADP-stimulated respiration in permeabilized muscle fibers was not accompanied by a decrease in the content of mitochondrial enzymes (mass spectrometry-based quantitative proteomics), hence, it is related to the disruption in regulation of respiration. We detected a widespread change in the transcriptomic profile (RNA-seq) upon dry immersion. Downregulated mRNAs were strongly associated with mitochondrial function, as well as with lipid metabolism, glycolysis, insulin signaling, and various transporters. Despite the substantial transcriptomic response, we found no effect on the content of highly abundant proteins (sarcomeric, mitochondrial, chaperon, and extracellular matrix-related, etc.) that may be explained by long half-life of these proteins. We suggest that during short-term disuse the content of some regulatory (and usually low abundant) proteins such as cytokines, receptors, transporters, and transcription regulators is largely determined by their mRNA concentration. These mRNAs revealed in our work may serve as putative targets for future studies aimed at developing approaches for the prevention of muscle deconditioning induced by disuse.NEW & NOTEWORTHY Three-day dry immersion (a model of physical unloading) substantially changes the transcriptomic profile in the human soleus muscle, a muscle with predominantly slow-twitch fibers and strong postural function; despite this, we found no effect on the muscle proteome (highly abundant proteins). Dry immersion markedly reduces ADP-stimulated respiration; this decline is not accompanied by a decrease in the content of mitochondrial proteins/respiratory enzymes, indicating the disruption in regulation of cellular respiration.


Subject(s)
Immersion , Transcriptome , Female , Humans , Proteomics , Muscle, Skeletal/metabolism , Mitochondria/metabolism , Muscle Fibers, Slow-Twitch/metabolism
7.
Soft Matter ; 19(13): 2430-2437, 2023 Mar 29.
Article in English | MEDLINE | ID: mdl-36930054

ABSTRACT

Fibrin and its modifications, particularly those with functionalized polyethylene glycol (PEG), remain highly attractive as a biomaterial in drug delivery and regenerative medicine. Despite the extensive knowledge of fibrinogenesis, there is little information on the processes occurring after its modification. Previously, we found structural differences between native fibrin and its conjugates with PEG that allows us to hypothesize that a combination of methods such as terahertz (THz) pulsed spectroscopy and rheology may contribute to the characterization of gelation and reveal the effect of PEG on the polymerization dynamics. Compared to native fibrin, PEGylated fibrins had a homogenously soft surface; PEGylation also led to a significant decrease in the gelation time: from 42.75 min for native fibrin to 31.26 min and 35.09 min for 5 : 1 and 10 : 1 PEGylated fibrin, respectively. It is worth noting that THz pulsed spectroscopy makes it possible to reliably investigate only the polymerization process itself, while it does not allow us to observe statistically significant differences between the distinct PEGylated fibrin gels. The polymerization time constant of native fibrin measured by THz pulsed spectroscopy was 14.4 ± 2.8 min. However, it could not be calculated for PEGylated fibrin because the structural changes were too rapid. These results, together with those previously reported, led us to speculate that PEG-fibrin conjugates formed homogenously distributed highly water-shelled aggregates without bundling compared to native fibrin, ensuring rapid gelation and stabilization of the system without increasing its complexity.


Subject(s)
Fibrin , Polyethylene Glycols , Polyethylene Glycols/chemistry , Fibrin/chemistry , Polymerization , Biocompatible Materials/chemistry , Regenerative Medicine
8.
J Exp Zool B Mol Dev Evol ; 340(1): 34-55, 2023 01.
Article in English | MEDLINE | ID: mdl-35438249

ABSTRACT

Organization and functioning of immune system remain unevenly studied in different taxa of lophotrochozoan animals. We analyzed transcriptomic data on coelomocytes of the lugworm Arenicola marina (Linnaeus, 1758; Annelida, Polychaeta) to gain insights into the molecular mechanisms involved in polychaete immunity. Coelomocytes are specialized motile cells populating coelomic fluid of annelids, responsible for cellular defense reactions and providing humoral immune factors. The transcriptome was enriched with immune-related transcripts by challenging the cells in vitro with lipopolysaccharides of Escherichia coli and Zymosan from Saccharomyces cerevisiae. Our analysis revealed a multifaceted and complex internal defense system of the lugworm. A. marina possesses orthologs of proto-complement-like factors: six thioester-containing proteins, a complement-like receptor, and a MASP-related serine protease (MReM2). A. marina coelomocytes employ pattern-recognition receptors to detect pathogens and regulate immune responses. Among them, there are 18 Toll-like receptors and various putative lectin-like proteins with evolutionary conserved and taxa-specific domains. C-type lectins and a novel family of Gal-binding and CUB domains containing receptors were the most abundant in the transcriptome. The array of pore-forming proteins in the coelomocytes was surprisingly reduced compared to that of other invertebrate species. We characterized a set of conserved proteins metabolizing reactive oxygen species and nitric oxide and expanded the arsenal of potential antimicrobial peptides. Phenoloxidase activity in immune cells of lugworm is mediated only by laccase enzyme. The described repertoire of immune-associated molecules provides valuable candidates for further functional and comparative research on the immunity of annelids.


Subject(s)
Annelida , Polychaeta , Animals , Polychaeta/genetics , Transcriptome , Gene Expression Profiling , Invertebrates
9.
Hum Genomics ; 16(1): 24, 2022 07 22.
Article in English | MEDLINE | ID: mdl-35869513

ABSTRACT

BACKGROUND: More than half of human protein-coding genes have an alternative transcription start site (TSS). We aimed to investigate the contribution of alternative TSSs to the acute-stress-induced transcriptome response in human tissue (skeletal muscle) using the cap analysis of gene expression approach. TSSs were examined at baseline and during recovery after acute stress (a cycling exercise). RESULTS: We identified 44,680 CAGE TSS clusters (including 3764 first defined) belonging to 12,268 genes and annotated for the first time 290 TSSs belonging to 163 genes. The transcriptome dynamically changes during the first hours after acute stress; the change in the expression of 10% of genes was associated with the activation of alternative TSSs, indicating differential TSSs usage. The majority of the alternative TSSs do not increase proteome complexity suggesting that the function of thousands of alternative TSSs is associated with the fine regulation of mRNA isoform expression from a gene due to the transcription factor-specific activation of various alternative TSSs. We identified individual muscle promoter regions for each TSS using muscle open chromatin data (ATAC-seq and DNase-seq). Then, using the positional weight matrix approach we predicted time course activation of "classic" transcription factors involved in response of skeletal muscle to contractile activity, as well as diversity of less/un-investigated factors. CONCLUSIONS: Transcriptome response induced by acute stress related to activation of the alternative TSSs indicates that differential TSSs usage is an essential mechanism of fine regulation of gene response to stress stimulus. A comprehensive resource of accurate TSSs and individual promoter regions for each TSS in muscle was created. This resource together with the positional weight matrix approach can be used to accurate prediction of TFs in any gene(s) of interest involved in the response to various stimuli, interventions or pathological conditions in human skeletal muscle.


Subject(s)
Gene Expression Regulation , Transcriptome , Humans , Muscle, Skeletal , Promoter Regions, Genetic/genetics , Transcription Initiation Site , Transcriptome/genetics
10.
Cancers (Basel) ; 15(1)2022 Dec 26.
Article in English | MEDLINE | ID: mdl-36612140

ABSTRACT

Diffuse gliomas continue to be an important problem in neuro-oncology. To solve it, studies have considered the issues of molecular pathogenesis from the intratumoral heterogeneity point. Here, we carried out a comparative dynamic analysis of the different cell populations' content in diffuse gliomas of different molecular profiles and grades, considering the cell populations' functional properties and the relationship with patient survival, using flow cytometry, immunofluorescence, multiparametric fluorescent in situ hybridization, polymerase chain reaction, and cultural methods. It was shown that an increase in the IDH-mutant astrocytomas and oligodendrogliomas malignancy is accompanied by an increase in stem cells' proportion and mesenchymal cell populations' appearance arising from oligodendrocyte-progenitor-like cells with cell plasticity and cells' hypoxia response programs' activation. In glioblastomas, malignancy increase is accompanied by an increase in both stem and definitive cells with mesenchymal differentiation, while proneuronal glioma stem cells are the most likely the source of mesenchymal glioma stem cells, which, in hypoxic conditions, further give rise to mesenchymal-like cells. Clinical confirmation was a mesenchymal-like cell and mesenchymal glioma stem cell number, and the hypoxic and plastic molecular programs' activation degree had a significant effect on relapse-free and overall survival. In general, we built a multi-vector model of diffuse gliomas' pathogenetic tracing up to the practical plane.

11.
Membranes (Basel) ; 11(10)2021 Oct 01.
Article in English | MEDLINE | ID: mdl-34677528

ABSTRACT

Lipid transfer proteins (LTPs) participate in many important physiological processes in plants, including adaptation to stressors, e.g., salinity. Here we address the mechanism of this protective action of LTPs by studying the interaction between LTPs and abscisic acid (ABA, a "stress" hormone) and their mutual participation in suberin deposition in root endodermis of salt-stressed pea plants. Using immunohistochemistry we show for the first time NaCl induced accumulation of LTPs and ABA in the cell walls of phloem paralleled by suberin deposition in the endoderm region of pea roots. Unlike LTPs which were found localized around phloem cells, ABA was also present within phloem cells. In addition, ABA treatment resulted in both LTP and ABA accumulation in phloem cells and promoted root suberization. These results suggested the importance of NaCl-induced accumulation of ABA in increasing the abundance of LTPs and of suberin. Using molecular modeling and fluorescence spectroscopy we confirmed the ability of different plant LTPs, including pea Ps-LTP1, to bind ABA. We therefore hypothesize an involvement of plant LTPs in ABA transport (unloading from phloem) as part of the salinity adaptation mechanism.

12.
Cancers (Basel) ; 13(15)2021 Jul 30.
Article in English | MEDLINE | ID: mdl-34359733

ABSTRACT

Voltage-gated Na+ channels (VGSCs) are expressed widely in human carcinomas and play a significant role in promoting cellular invasiveness and metastasis. However, human tissue-based studies and clinical characterization are lacking. In several carcinomas, including colorectal cancer (CRCa), the predominant VGSC is the neonatal splice variant of Nav1.5 (nNav1.5). The present study was designed to determine the expression patterns and clinical relevance of nNav1.5 protein in human CRCa tissues from patients with available clinicopathological history. The immunohistochemistry was made possible by the use of a polyclonal antibody (NESOpAb) specific for nNav1.5. The analysis showed that, compared with normal mucosa, nNav1.5 expression occurred in CRCa samples (i) at levels that were significantly higher and (ii) with a pattern that was more delineated (i.e., apical/basal or mixed). A surprisingly high level of nNav1.5 protein expression also occurred in adenomas, but this was mainly intracellular and diffuse. nNav1.5 showed a statistically significant association with TNM stage, highest expression being associated with TNM IV and metastatic status. Interestingly, nNav1.5 expression co-occurred with other biomarkers associated with metastasis, including hERG1, KCa3.1, VEGF-A, Glut1, and EGFR. Finally, univariate analysis showed that nNav1.5 expression had an impact on progression-free survival. We conclude (i) that nNav1.5 could represent a novel clinical biomarker ('companion diagnostic') useful to better stratify CRCa patients and (ii) that since nNav1.5 expression is functional, it could form the basis of anti-metastatic therapies including in combination with standard treatments.

13.
Int J Mol Sci ; 22(16)2021 Aug 11.
Article in English | MEDLINE | ID: mdl-34445356

ABSTRACT

Ferritins comprise a conservative family of proteins found in all species and play an essential role in resistance to redox stress, immune response, and cell differentiation. Sponges (Porifera) are the oldest Metazoa that show unique plasticity and regenerative potential. Here, we characterize the ferritins of two cold-water sponges using proteomics, spectral microscopy, and bioinformatic analysis. The recently duplicated conservative HdF1a/b and atypical HdF2 genes were found in the Halisarca dujardini genome. Multiple related transcripts of HpF1 were identified in the Halichondria panicea transcriptome. Expression of HdF1a/b was much higher than that of HdF2 in all annual seasons and regulated differently during the sponge dissociation/reaggregation. The presence of the MRE and HRE motifs in the HdF1 and HdF2 promotor regions and the IRE motif in mRNAs of HdF1 and HpF indicates that sponge ferritins expression depends on the cellular iron and oxygen levels. The gel electrophoresis combined with specific staining and mass spectrometry confirmed the presence of ferric ions and ferritins in multi-subunit complexes. The 3D modeling predicts the iron-binding capacity of HdF1 and HpF1 at the ferroxidase center and the absence of iron-binding in atypical HdF2. Interestingly, atypical ferritins lacking iron-binding capacity were found in genomes of many invertebrate species. Their function deserves further research.


Subject(s)
Ferritins/genetics , Porifera/genetics , Animals , Conserved Sequence , Ferritins/chemistry , Ferritins/metabolism , Iron/metabolism , Metabolic Networks and Pathways/genetics , Models, Molecular , Phylogeny , Porifera/classification , Porifera/metabolism , Protein Domains/genetics , Sequence Analysis, DNA , Transcriptome/physiology
14.
Biology (Basel) ; 10(6)2021 Jun 20.
Article in English | MEDLINE | ID: mdl-34203013

ABSTRACT

The prevention of muscle atrophy carries with it clinical significance for the control of increased morbidity and mortality following physical inactivity. While major transcriptional events associated with muscle atrophy-recovery processes are the subject of active research on the gene level, the contribution of non-coding regulatory elements and alternative promoter usage is a major source for both the production of alternative protein products and new insights into the activity of transcription factors. We used the cap-analysis of gene expression (CAGE) to create a genome-wide atlas of promoter-level transcription in fast (m. EDL) and slow (m. soleus) muscles in rats that were subjected to hindlimb unloading and subsequent recovery. We found that the genetic regulation of the atrophy-recovery cycle in two types of muscle is mediated by different pathways, including a unique set of non-coding transcribed regulatory elements. We showed that the activation of "shadow" enhancers is tightly linked to specific stages of atrophy and recovery dynamics, with the largest number of specific regulatory elements being transcriptionally active in the muscles on the first day of recovery after a week of disuse. The developed comprehensive database of transcription of regulatory elements will further stimulate research on the gene regulation of muscle homeostasis in mammals.

15.
Genes (Basel) ; 11(12)2020 11 26.
Article in English | MEDLINE | ID: mdl-33256091

ABSTRACT

Synthetic promoters are vital for genetic engineering-based strategies for crop improvement, but effective methodologies for their creation and systematic testing are lacking. We report here on the comparative analysis of the promoters pro-SmAMP1 and pro-SmAMP2 from Stellaria media ANTIMICROBIAL PEPTIDE1 (AMP1) and ANTIMICROBIAL PEPTIDE2 (AMP2). These promoters are more effective than the well-known Cauliflower mosaic virus 35S promoter. Although these promoters share about 94% identity, the pro-SmAMP1 promoter demonstrated stronger transient expression of a reporter gene in Agrobacterium infiltration of Nicotiana benthamiana leaves, while the pro-SmAMP2 promoter was more effective for the selection of transgenic tobacco (Nicotiana tabacum) cells when driving a selectable marker. Using the cap analysis of gene expression method, we detected no differences in the structure of the transcription start sites for either promoter in transgenic plants. For both promoters, we used fine-scale deletion analysis to identify 160 bp-long sequences that retain the unique properties of each promoter. With the use of chimeric promoters and directed mutagenesis, we demonstrated that the superiority of the pro-SmAMP1 promoter for Agrobacterium-mediated infiltration is caused by the proline-inducible ACTCAT cis-element strictly positioned relative to the TATA box in the core promoter. Surprisingly, the ACTCAT cis-element not only activated but also suppressed the efficiency of the pro-SmAMP1 promoter under proline stress. The absence of the ACTCAT cis-element and CAANNNNATC motif (negative regulator) in the pro-SmAMP2 promoter provided a more constitutive gene expression profile and better selection of transgenic cells on selective medium. We created a new synthetic promoter that enjoys high effectiveness both in transient expression and in selection of transgenic cells. Intact promoters with differing properties and high degrees of sequence identity may thus be used as a basis for the creation of new synthetic promoters for precise and coordinated gene expression.


Subject(s)
Arabidopsis Proteins/genetics , Carboxypeptidases/genetics , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Promoter Regions, Genetic/genetics , Stellaria/genetics , Transgenes/genetics , Agrobacterium/genetics , Base Sequence , Caulimovirus/genetics , Gene Expression Regulation, Plant/genetics , Genes, Reporter/genetics , Plant Leaves/genetics , Plant Leaves/virology , Nicotiana/genetics , Nicotiana/virology , Transcription Initiation Site/physiology , Transcriptome/genetics
16.
J Biophotonics ; 13(12): e202000297, 2020 12.
Article in English | MEDLINE | ID: mdl-32881362

ABSTRACT

In this work, a thorough analysis of hyperosmotic agents for the immersion optical clearing (IOC) in terahertz (THz) range was performed. It was aimed at the selection of agents for the efficient enhancement of penetration depth of THz waves into biological tissues. Pulsed spectroscopy in the frequency range of 0.1 to 2.5 THz was applied for investigation of the optical properties of common IOC agents. Using the collimated transmission spectroscopy in visible range, binary diffusion coefficients of tissue water and agent in ex vivo rat brain tissue were measured. IOC agents were objectively compared using two-dimensional nomogram, accounting for their THz-wave absorption coefficients and binary diffusion coefficients. The results of this study demonstrate an interplay between the penetration depth enhancement and the diffusion rate and allow for pointing out glycerol as an optimal agent among the considered ones for particular applications in THz biophotonics.


Subject(s)
Glycerol , Immersion , Animals , Brain/diagnostic imaging , Diffusion , Rats , Water
17.
Biomolecules ; 10(9)2020 08 24.
Article in English | MEDLINE | ID: mdl-32847137

ABSTRACT

Water deficits inhibit plant growth and decrease crop productivity. Remedies are needed to counter this increasingly urgent problem in practical farming. One possible approach is to utilize rhizobacteria known to increase plant resistance to abiotic and other stresses. We therefore studied the effects of inoculating the culture medium of potato microplants grown in vitro with Azospirillum brasilense Sp245 or Ochrobactrum cytisi IPA7.2. Growth and hormone content of the plants were evaluated under stress-free conditions and under a water deficit imposed with polyethylene glycol (PEG 6000). Inoculation with either bacterium promoted the growth in terms of leaf mass accumulation. The effects were associated with increased concentrations of auxin and cytokinin hormones in the leaves and stems and with suppression of an increase in the leaf abscisic acid that PEG treatment otherwise promoted in the potato microplants. O. cytisi IPA7.2 had a greater growth-stimulating effect than A. brasilense Sp245 on stressed plants, while A. brasilense Sp245 was more effective in unstressed plants. The effects were likely to be the result of changes to the plant's hormonal balance brought about by the bacteria.


Subject(s)
Azospirillum brasilense/physiology , Ochrobactrum/physiology , Plant Growth Regulators/metabolism , Solanum tuberosum/metabolism , Solanum tuberosum/microbiology , Abscisic Acid/metabolism , Crop Production/methods , Crops, Agricultural/metabolism , Crops, Agricultural/microbiology , Cytokinins/metabolism , Droughts , Indoleacetic Acids/metabolism , Osmotic Pressure , Plant Leaves/metabolism , Plant Stems/metabolism , Polyethylene Glycols , Solanum tuberosum/growth & development
18.
Diagnostics (Basel) ; 10(5)2020 May 25.
Article in English | MEDLINE | ID: mdl-32466249

ABSTRACT

Parkinson's disease is the second most frequent neurodegenerative disease, representing a significant medical and socio-economic problem. Modern medicine still has no answer to the question of why Parkinson's disease develops and whether it is possible to develop an effective system of prevention. Therefore, active work is currently underway to find ways to assess the risks of the disease, as well as a means to extend the life of patients and improve its quality. Modern studies aim to create a method of assessing the risk of occurrence of Parkinson's disease (PD), to search for the specific ways of correction of biochemical disorders occurring in the prodromal stage of Parkinson's disease, and to personalize approaches to antiparkinsonian pharmacotherapy. In this review, we summarized all available clinically approved tests and techniques for PD diagnostics. Then, we reviewed major improvements and recent advancements in genomics, transcriptomics, and proteomics studies and application of metabolomics in PD research, and discussed the major metabolomics findings for diagnostics and therapy of the disease.

20.
PLoS One ; 15(2): e0228722, 2020.
Article in English | MEDLINE | ID: mdl-32084159

ABSTRACT

The ability to regulate oxygen consumption evolved in ancestral animals and is intrinsically linked to iron metabolism. The iron pathways have been intensively studied in mammals, whereas data on distant invertebrates are limited. Sea sponges represent the oldest animal phylum and have unique structural plasticity and capacity to reaggregate after complete dissociation. We studied iron metabolic factors and their expression during reaggregation in the White Sea cold-water sponges Halichondria panicea and Halisarca dujardini. De novo transcriptomes were assembled using RNA-Seq data, and evolutionary trends were analyzed with bioinformatic tools. Differential expression during reaggregation was studied for H. dujardini. Enzymes of the heme biosynthesis pathway and transport globins, neuroglobin (NGB) and androglobin (ADGB), were identified in sponges. The globins mutate at higher evolutionary rates than the heme synthesis enzymes. Highly conserved iron-regulatory protein 1 (IRP1) presumably interacts with the iron-responsive elements (IREs) found in mRNAs of ferritin (FTH1) and a putative transferrin receptor NAALAD2. The reaggregation process is accompanied by increased expression of IRP1, the antiapoptotic factor BCL2, the inflammation factor NFκB (p65), FTH1 and NGB, as well as by an increase in mitochondrial density. Our data indicate a complex mechanism of iron regulation in sponge structural plasticity and help to better understand general mechanisms of morphogenetic processes in multicellular species.


Subject(s)
Iron/metabolism , Porifera/metabolism , Animals , Computational Biology , Gene Expression Profiling , Iron-Regulatory Proteins/genetics , Iron-Regulatory Proteins/metabolism , Molecular Sequence Annotation , Phylogeny , Porifera/genetics , RNA-Seq
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