Nikon Eclipse E600

Pontodrilus litoralis is a cosmopolitan littoral earthworm known to exhibit bioluminescence. Recently, a congeneric species Pontodrilus longissimus from Thailand was described. These species are sympatric but their burrowing depths on Thai beaches are different. In this study, we examined the in vivo and in vitro bioluminescence properties of P. longissimus and P. litoralis. Mechanical stimulation induced in vivo luminescence in P. litoralis, as reported previously, but not in P. longissimus. In vitro cross-reaction tests between these species revealed the absence of luciferin and luciferase activities in P. longissimus. P. litoralis had strong fluorescence in a coelomic fluid that matches to the spectral maximum of its bioluminescence, but P. longissimus did not. These results suggest that P. longissimus does not have luminescence ability due to the lack of all bioluminescent components, luciferin, luciferase, and light emitter, despite its close relationship to the luminous P. litoralis. The presence of both luminous and non-luminous species in a single genus is uncommon, and our present findings will shed insight on the possible functions of bioluminescence in the earthworm, such as avoiding predation by littoral earwigs.

Teerapong Seesamut, Daichi Yano, Jose Paitio, Ikuhiko Kin, Somsak Panha, Yuichi Oba. 2020.

BackgroundWidely used in recent years, mesenchymal stem cells (MSCs) expressing enhanced green fluorescent protein (eGFP) can be tracked during migration to injury sites, while also supporting tri-lineage differentiation. However, the relationship between the expression of green fluorescence and the magnitude of osteogenic differentiation is not clearly defined. Despite increasing use of eGFP-MSCs derived from the transgenic pigs and non-viral eGFP plasmid transfected MSCs in recent years, it remains unclear which cells are suitable for tracking during osteogenic differentiation, and whether the transfected plasmid alters osteogenic potential.MethodsWe compared the expression of green fluorescence and the magnitude of osteogenic differentiation between eGFP MSCs from a transgenic pig (group 1) and non-virally transfected eGFP-MSCs using transIT^®-2020 (group 2). Non-transfected MSCs were used as control group (group 3). We also use a scaffold to compare the osteogenic induction environments created by 2-D monolayer cultures and 3-D cultures, respectivelyResultsIn the monolayer culture, flow cytometry from day 7 to day 28 showed that the percentage of green fluorescent cells in groups 1 and 2 were 99.6% and 59.7% of total cell counts, respectively. Quantification showed that eGFP expression peaked on day 7, decreased after day 14, and plateaued to day 28 in group 1 and group 2. Significant aggregation of eGFP over bone-like nodules was appreciated in group 1. In 3-D culture, eGFP expression increased from day 7 to day 28 in both groups, and was higher in group 1 than in group 2 at each time point. Osteogenic profiles and immunohistochemistry showed more significant osteogenic activity in group 1 and group 3 than in group 2. ConclusionsThe expression of eGFP in the test groups did not significantly change after osteogenic induction. However, quantification data was different in monolayer and 3-D cultures due to spatial limitations, differing extracellular environments, and heterogeneous cell morphology and methods of division. Osteogenic profiles and immunohistochemistry data confirmed that osteogenic potential did not change in transgenic pig-derived MSCs. However osteogenic potential decreased in pig MSCs (pig MSCs) treated with the transfection reagent, likely from related toxicity.

Ming-Kai Hsieh, Chia-Jung Wu, Chi-Yun Wang, Tsung-Ting Tsai, Chi-Chien Niu, Po-Liang Lai, Shinn-Chih Wu. 2020.

Paracrine signaling in the tissue microenvironment is a central mediator of morphogenesis, and modeling this dynamic intercellular activity in vitro is critical to understanding normal and abnormal development. For example, Sonic Hedgehog (Shh) signaling is a conserved mechanism involved in multiple developmental processes and strongly linked to human birth defects including orofacial clefts of the lip and palate. SHH ligand produced, processed, and secreted from the epithelial ectoderm is shuttled through the extracellular matrix where it binds mesenchymal receptors, establishing a gradient of transcriptional response that drives orofacial morphogenesis. In humans, complex interactions of genetic predispositions and environmental insults acting on diverse molecular targets are thought to underlie orofacial cleft etiology. Consequently, there is a need for tractable in vitro approaches that model this complex cellular and environmental interplay and are sensitive to disruption across the multistep signaling cascade. We developed a microplate-based device that supports an epithelium directly overlaid onto an extracellular matrix-embedded mesenchyme, mimicking the basic tissue architecture of developing orofacial tissues. SHH ligand produced from the epithelium generated a gradient of SHH-driven transcription in the adjacent mesenchyme, recapitulating the gradient of pathway activity observed in vivo. Shh pathway activation was antagonized by small molecule inhibitors of epithelial secretory, extracellular matrix transport, and mesenchymal sensing targets, supporting the use of this approach in high-content chemical screening of the complete Shh pathway. Together, these findings demonstrate a novel and practical microphysiological model with broad utility for investigating epithelial-mesenchymal interactions and environmental signaling disruptions in development.

Brian P. Johnson, Ross A. Vitek, Molly M. Morgan, Dustin M. Fink, Tyler G. Beames, Peter G. Geiger, David J. Beebe, Robert J. Lipinski. Frontiers in Cell and Developmental Biology, 9, 2021.

Simultaneous or functional hermaphrodites possessing both ovary and testis at the same time are good materials for studying sexual development. However, previous research on sex determination and differentiation was mainly conducted in gonochoristic species and studies on simultaneous hermaphrodites are still limited. In this study, we conducted a combined morphological, endocrine and molecular study on the gonadal development of a hermaphroditic scallop Argopecten irradians aged 2–10 month old. Morphological analysis showed that sex differentiation occurred at 6 months of age. By examining the dynamic changes of progesterone, testosterone and estradiol, we found testosterone and estradiol were significantly different between the ovaries and testes almost throughout the whole process, suggesting the two hormones may be involved in scallop sex differentiation. In addition, we identified two critical sex-related genes FoxL2 and Dmrt1L, and investigated their spatiotemporal expression patterns. Results showed that FoxL2 and Dmrt1L were female- and male-biased, respectively, and mainly localized in the germ cells and follicular cells, indicating their feasibility as molecular markers for early identification of sex. Further analysis on the changes of FoxL2 and Dmrt1L expression in juveniles showed that significant sexual dimorphic expression of FoxL2 occurred at 2 months of age, earlier than that of Dmrt1L. Moreover, FoxL2 expression was significantly correlated with estradiol/testosterone ratio (E₂/T). All these results indicated that molecular sex differentiation occurs earlier than morphological sex differentiation, and FoxL2 may be a key driver that functions through regulating sex steroid hormones in the scallop. This study will deepen our understanding of the molecular mechanism underlying sex differentiation and development in spiralians.

Huilan Wei, Wanru Li, Tian Liu, Yajuan Li, Liangjie Liu, Ya Shu, Lijing Zhang, Shi Wang, Qiang Xing, Lingling Zhang, Zhenmin Bao. Frontiers in Cell and Developmental Biology, 9, 2021.

Osseointegration is a well-established concept used in applications including the percutaneous Bone-Anchored Hearing System (BAHS) and auricular rehabilitation. To date, few retrieved implants have been described. A systematic review including cases where percutaneous bone-anchored implants inserted in the temporal bone were retrieved and analyzed was performed. We also present the case of a patient who received a BAHS for mixed hearing loss. After the initial surgery, several episodes of soft tissue inflammation accompanied by pain were observed, leading to elective abutment removal 14 months post-surgery. Two years post-implantation, the implant was removed due to pain and subjected to a multiscale and multimodal analysis: microbial DNA using molecular fingerprinting, gene expression using quantitative real-time polymerase chain reaction (qPCR), X-ray microcomputed tomography (micro-CT), histology, histomorphometry, backscattered scanning electron microscopy (BSE-SEM), Raman spectroscopy, and fluorescence in situ hybridization (FISH). Evidence of osseointegration was provided via micro-CT, histology, BSE-SEM, and Raman spectroscopy. Polymicrobial colonization in the periabutment area and on the implant, including that with Staphylococcus aureus and Staphylococcus epidermidis, was determined using a molecular analysis via a 16S-23S rDNA interspace [IS]-region-based profiling method (IS-Pro). The histology suggested bacterial colonization in the skin and in the peri-implant bone. FISH confirmed the localization of S. aureus and coagulase-negative staphylococci in the skin. Ten articles (54 implants, 47 patients) met the inclusion criteria for the literature search. The analyzed samples were either BAHS (35 implants) or bone-anchored aural epitheses (19 implants) in situ between 2 weeks and 8 years. The main reasons for elective removal were nonuse/changes in treatment, pain, or skin reactions. Most samples were evaluated using histology, demonstrating osseointegration, but with the absence of bone under the implants’ proximal flange. Taken together, the literature and this case report show clear evidence of osseointegration, despite prominent complications. Nevertheless, despite implant osseointegration, chronic pain related to the BAHS may be associated with a chronic bacterial infection and raised inflammatory response in the absence of macroscopic signs of infection. It is suggested that a multimodal analysis of peri-implant health provides possibilities for device improvements and to guide diagnostic and therapeutic strategies to alleviate the impact of complications.

Martin L. Johansson, Tim G.A. Calon, Omar Omar, Furqan A. Shah, Margarita Trobos, Peter Thomsen, Robert J. Stokroos, Anders Palmquist. Frontiers in Cellular and Infection Microbiology, 11, 2021.

Obesity cardiomyopathy increases the risk of heart failure and death. Obesity is curable, leading to the restoration of the heart phenotype, but it is not clear if there are any after-effects of obesity present after weight loss. We characterize the proteomic landscape of obesity cardiomyopathy with an evaluation of whether the cardiac phenotype is still shaped after weight loss. Cardiomyopathy was validated by cardiac hypertrophy, fibrosis, oversized myocytes, and mTOR upregulation in a rat model of cafeteria diet-induced developmental obesity. By global proteomic techniques (LC-MS/MS) a plethora of molecular changes was observed in the heart and circulation of obese animals, suggesting abnormal utilization of metabolic substrates. This was confirmed by increased levels of cardiac ACSL-1, a key enzyme for fatty acid degradation and decreased GLUT-1, a glucose transporter in obese rats. Calorie restriction and weight loss led to the normalization of the heart’s size, but fibrosis was still excessive. The proteomic compositions of cardiac tissue and plasma were different after weight loss as compared to control. In addition to morphological consequences, obesity cardiomyopathy involves many proteomic changes. Weight loss provides for a partial repair of the heart’s architecture, but the trace of fibrotic deposition and proteomic alterations may occur.

Arkadiusz D. Liśkiewicz, Łukasz Marczak, Katarzyna Bogus, Daniela Liśkiewicz, Marta Przybyła, Joanna Lewin-Kowalik. Frontiers in Endocrinology, 12, 2021.

Normal aging is characterized by declines in processing speed, learning, memory, and executive function even in the absence of neurodegenerative diseases such as Alzheimer's Disease (AD). In normal aging monkeys and humans, neuronal loss does not account for cognitive impairment. Instead, loss of white matter volume and an accumulation of myelin sheath pathology begins in middle age and is associated with cognitive decline. It is unknown what causes this myelin pathology, but it likely involves increased neuroinflammation in white matter and failures in oligodendrocyte function (maturation and repair). In frontal white matter tracts vulnerable to myelin damage, microglia become chronically reactive and secrete harmful pro-inflammatory cytokines. Despite being in a phagocytic state, these microglia are ineffective at phagocytosing accruing myelin debris, which directly inhibits myelin sheath repair. Here, we asked whether reported age-related increases in pro-inflammatory markers were accompanied by an adaptive immune response involving T cells. We quantified T cells with immunohistochemistry in the brains of 34 cognitively characterized monkeys and found an age-related increase in perivascular T cells that surround CNS vasculature. We found a surprising age-related increase in T cells that infiltrate the white matter parenchyma. In the cingulum bundle the percentage of these parenchymal T cells increased with age relative to those in the perivascular space. In contrast, infiltrating T cells were rarely found in surrounding gray matter regions. We assessed whether T cell infiltration correlated with fibrinogen extravasation from the vasculature as a measure of BBB leakiness and found no correlation, suggesting that T cell infiltration is not a result of passive extravasation. Importantly, the density of T cells in the cingulum bundle correlated with microglial reactivity and with cognitive impairment. This is the first demonstration that T cell infiltration of white matter is associated with cognitive decline in the normal aging monkey.

Katelyn V. Batterman, Payton E. Cabrera, Tara L. Moore, Douglas L. Rosene. Frontiers in Immunology, 12, 2021.

Different types and inclusion levels of palm oil were incorporated in the enriched diets of L-type rotifer, Brachionus plicatilis, and fed to Asian seabass (Lates calcarifer) larvae. The dietary fish oil was replaced with either 50 or 75% of crude palm oil, CPO (CPO50, CPO75) and refined bleached deodorized palm olein, RPO (RPO50, RPO75). The enriched diet containing 100% fish oil (FO100) was used as the experimental control. Triplicate groups of the fish larvae of initial length 2.72 ± 0.14 mm were fed with enriched rotifer for 15 days. In general, palm oil-based enriched diets performed better than the control diet (FO100). Specifically, final mean body weight (31.3 ± 9.2 mg), final mean total length (11.5 ± 1.6 mm), SGR (29.0 ± 1.4%/day) and WG (7,769.4 ± 1,510.8%) of Asian seabass larvae fed RPO75 were significantly higher (P < 0.05) compared to those fed the other palm oil-based diet and FO100. The rotifer enriched with palm oil significantly affected the body proximate composition and fatty acid profiles of the fed larvae. The present study suggests that RPO and CPO can be considered as a good alternative dietary lipid for enrichment of rotifer to positively influence the nutritional requirements of the Asian seabass larvae and support their survival and growth.

Nik Siti Zaimah Safiin, Saleem Mustafa, Fui Fui Ching, Rossita Shapawi. Frontiers in Marine Science, 8, 2021.

Although Verrucomicrobia is widely distributed in the marine environment, their physiological or cellular properties are poorly characterized because of the lack of cultured representatives. Under the selective pressure of two antibiotics, ofloxacin and norfloxacin, a Gram-stain-negative, aerobic coccus with exopolysaccharide (EPS) production ability was isolated from the coastal sediment of Xiaoshi Island, Weihai, China. These antibiotics inhibited bacterial growth, giving rise to the relatively slow-growing Verrucomicrobia that formed colonies on the isolation plates. It may be an effective method for the isolation of Puniceicoccaceae. From the taxonomic data obtained in this study, the new marine isolate NFK12^T (=KCTC 72940^T = MCCC 1H00424^T) is proposed to be placed into a novel species within the genus Pelagicoccus for which the name Pelagicoccus enzymogenes sp. nov. is proposed. The EPS production of the strain NFK12^T and the related strains were investigated and the effect of EPS produced by the strain NFK12^T on the growth of other strains was examined. Besides, the effect of EPS on tolerance to ofloxacin and norfloxacin of the strain NFK12^T was studied by measuring the biomass of the strain NFK12^T. It was deduced that those strains that produced EPS tentatively protected themselves against the inhibitory effects of ofloxacin and norfloxacin.

Xi Feng, Ya Gong, Meng-Qi Ye, Zong-Jun Du. Frontiers in Marine Science, 8, 2021.

The contamination of soil with total petroleum hydrocarbons (TPH) may result in dramatic consequences and needs great attention, as soil rehabilitation would need more effort from a sustainability perspective. However, there is still no known general method since the remediation technology is strictly site-specific. Adaptive biological system dynamics can play a key role in understanding and addressing the potential of situ-specific biological combinations for soil pollutants removal. The potential worst-case of TPH contamination reflects soil affected by heavy industrial activities, such as oil refineries. Therefore, the experimental trial was conducted on a 2,000 m² area from a contaminated site located in northern Italy. We evaluated the remediation potential over time (270 days) assessing (i) the phytoremediation efficiency of two species of Poaceae (Festuca arundinacea Schreb. and Dactylis glomerata L.) and two species of Fabaceae (Medicago sativa L. and Lotus corniculatus L.) and (ii) the role of the indigenous bacteria flora and endo-mycorrhizae consortium addition in plant growth promotion. We also induced resistance to contamination stress in a field experiment. Thirty-three indigenous bacteria selected from the contaminated soils showed marked plant growth promotion. Moreover, functional metagenomics confirmed the metabolic capability of hydrocarbon-degrading microorganisms living in the polluted soil. Our data showed that soil enzymatic activities increased with hydrocarbon degradation rate after 60 days. Both Poaceae and Fabaceae resulted in remarkable remediation potential. Stress markers and antioxidant activity indicated that the selected plant species generally need some time to adapt to TPH stress. In conclusion, our evaluation implied both the rhizosphere effects and functional features of the plant and suggested that plants should (i) have marked tolerance to specific contaminants, (ii) be characterized by an extensive root system, and (iii) be susceptible to arbuscular mycorrhizal fungi (AMF) infection.

Daniela Zuzolo, Carmine Guarino, Maria Tartaglia, Rosaria Sciarrillo. Frontiers in Microbiology, 11, 2021.

Chloracidobacterium is the first and until now the sole genus in the phylum Acidobacteriota (formerly Acidobacteria) whose members perform chlorophyll-dependent phototrophy (i.e., chlorophototrophy). An axenic isolate of Chloracidobacterium thermophilum (strain B^T) was previously obtained by using the inferred genome sequence from an enrichment culture and diel metatranscriptomic profiling analyses in situ to direct adjustments to the growth medium and incubation conditions, and thereby a defined growth medium for Chloracidobacterium thermophilum was developed. These advances allowed eight additional strains of Chloracidobacterium spp. to be isolated from microbial mat samples collected from Mushroom Spring, Yellowstone National Park, United States, at temperatures of 41, 52, and 60°C; an axenic strain was also isolated from Rupite hot spring in Bulgaria. All isolates are obligately photoheterotrophic, microaerophilic, non-motile, thermophilic, rod-shaped bacteria. Chloracidobacterium spp. synthesize multiple types of (bacterio-)chlorophylls and have type-1 reaction centers like those of green sulfur bacteria. Light harvesting is accomplished by the bacteriochlorophyll a-binding, Fenna-Matthews-Olson protein and chlorosomes containing bacteriochlorophyll c. Their genomes are approximately 3.7 Mbp in size and comprise two circular chromosomes with sizes of approximately 2.7 Mbp and 1.0 Mbp. Comparative genomic studies and phenotypic properties indicate that the nine isolates represent three species within the genus Chloracidobacterium. In addition to C. thermophilum, the microbial mats at Mushroom Spring contain a second species, tentatively named Chloracidobacterium aggregatum, which grows as aggregates in liquid cultures. The Bulgarian isolate, tentatively named Chloracidobacterium validum, will be proposed as the type species of the genus, Chloracidobacterium. Additionally, Chloracidobacterium will be proposed as the type genus of a new family, Chloracidobacteriaceae, within the order Blastocatellales, the class Blastocatellia, and the phylum Acidobacteriota.

Mohit Kumar Saini, Aswathy Sebastian, Yoshiki Shirotori, Nathan T. Soulier, Amaya M. Garcia Costas, Daniela I. Drautz-Moses, Stephan C. Schuster, Istvan Albert, Shin Haruta, Satoshi Hanada, Vera Thiel, Marcus Tank, Donald A. Bryant. Frontiers in Microbiology, 12, 2021.

Serotonin (5-HT) is member of a family of indolamine molecules that participate in a wide variety of biological processes. Despite its important role in the regulation of local blood systems, little is known about the physiological function of 5-HT in reproductive organs, its functional implications, and its role in the reproduction of mammals. In the present work, we evaluated the localization and distribution of 5-HT (using histochemical analysis of indolamines) and different components of the serotoninergic system in rat testes. We detected local synthesis and degradation through immunofluorescence and western blot analyses against the TPH1, MAO_A, 5-HT_T, and VMAT1 serotonin transporters. We also identified the localization and distribution of the 5-HT_1B, 5-HT_2A, and 5-HT_3Areceptors. RT-PCR results showed the presence of the Tph1, Maoa, Slc6a4, and Htr3a genes in testes and in the brain stem (Tph1 was used as a negative control). High-performance liquid chromatography was used to determine the presence of 5-HT and the activity of tryptophan hydroxylase in testes homogenatesin vitro. Our observations suggest that TPH1 activity and local 5-HT synthesis befall in rat testes. We propose that 5-HT could participate in the regulation of testosterone synthesis and in the spermatogenesis process via local serotoninergic system. However, more studies are needed before concluding that rat testes, or those of other mammals, contain an active form of tryptophan hydroxylase and produce 5-HT.

Francisco Jiménez-Trejo, Isabel Coronado-Mares, Cristian Arriaga-Canon, Luis A. Herrera, Bladimir Roque-Ramírez, Margarita Chávez-Saldaña, Julio Rojas-Castañeda, Marco Cerbón, Rosa M. Vigueras-Villaseñor. Frontiers in Neuroanatomy, 14, 2021.

Extracts from Holothuria scabra (HS) have been shown to possess anti-inflammation, anti-oxidant and anti-cancer activities. More recently, it was shown to have neuroprotective potential in Caenorhabditis elegans PD model. Here, we assessed whether HS has neuroprotective and neurorestorative effects on dopaminergic neurons in both mouse and cellular models of PD. We found that both pre-treatment and post-treatment with HS improved motor deficits in PD mouse model induced with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as determined by grid walk test. This was likely mediated by HS protective and restorative effects on maintaining the numbers of dopaminergic neurons and fibers in both substantia nigra pars compacta (SNpc) and striatum. In a cellular model of PD, HS significantly attenuated 1-methyl-4-phenylpyridinium (MPP⁺)-induced apoptosis of DAergic-like neurons differentiated from SH-SY5Y cells by enhancing the expression of Bcl-2, suppressing the expression of cleaved Caspase 3 and preventing depolarization of mitochondrial membrane. In addition, HS could stimulate the expression of tyrosine hydroxylase (TH) and suppressed the formation of α-synuclein protein. Taken together, our in vivo and in vitro findings suggested that HS is an attractive candidate for the neuroprotection rather than neurorestoration in PD.

Kunwadee Noonong, Prasert Sobhon, Morakot Sroyraya, Kulathida Chaithirayanon. Frontiers in Neuroscience, 14, 2020.