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Abstract In the Japanese hagfish Eptatretus burgeri, 16 chromosomes (eliminated [E]-chromosomes) have been lost in somatic cells (2n = 36), which is equivalent to approx. 21% of the genomic DNA in germ cells (2n = 52). At least seven of the 12 eliminated repetitive DNA families isolated in eight hagfish species were selectively amplified in the germline genome of this species. One of them, EEEb1 (eliminated element of E. burgeri 1) is exclusively localized on all E-chromosomes. Herein, we identified four novel eliminated repetitive DNA families (named EEEb3–6) through PCR amplification and suppressive subtractive hybridization (SSH) combined with Southern-blot hybridization. EEEb3 was mosaic for 5S rDNA and SINE elements. EEEb4 was GC-rich repeats and has one pair of direct and inverted repeats, whereas EEEb5 and EEEb6 were AT-rich repeats with one pair and two pairs of sub-repeats, respectively. Interestingly, all repeat classes except EEEb3 were transcribed in the testes, although no open reading frames (ORF) were identified. We conducted fluorescence in situ hybridization (FISH) to examine the chromosomal localizations of EEEb3–6 and EEEb2, which was previously isolated from the germline genome of E. burgeri. All sequences were only found on all EEEb1-positive E-chromosomes. Copy number estimation of the repeated elements by slot-blot hybridization revealed that (i) the EEEb1–6 family members occupied 39.9% of the total eliminated DNA, and (ii) a small number of repeats were retained in somatic cells, suggesting that there is incomplete elimination of the repeated elements. These results provide new insights into the mechanisms involved in the chromosome elimination and the evolution of E-chromosomes.

Amyloid fibrils of β2-microglobulin (β2m) can cause dialysis-related amyloidosis. Here, the authors show that a decrease in serum albumin levels in long-term dialysis deteriorates the inhibitory effects of serum milieux on supersaturation-limited amyloid formation of β2m, suggesting that macromolecular crowding protects the onset of amyloidosis.

Much effort has been devoted to elucidate mechanisms of amyloid fibril formation using various kinds of additives, such as salts, metals, detergents, and biopolymers. Here, we review the effects of additives with a focus on polyphosphate (polyP) on amyloid fibril formation of β2-microglobulin (β2m) and α-synuclein (αSyn). PolyP, consisting of up to 1,000 phosphoanhydride bond-linked phosphate monomers, is one of the most ancient, enigmatic, and negatively charged molecules in biology. Amyloid fibril formation of both β2m and αSyn could be accelerated by counter anion-binding and preferential hydration at relatively lower and higher concentrations of polyP, respectively, depending on the chain length of polyP. These bimodal concentration-dependent effects were also observed in salt- and heparin-induced amyloid fibril formation, indicating the generality of bimodal effects. We also address the effects of detergents, alcohols, and isoelectric point precipitation on amyloid fibril formation, in comparison with the effects of salts. Because polyP is present all around us, from cellular components to food additives, clarifying its effects and consequent biological roles will be important to further advance our understanding of amyloid fibrils. This review article is an extended version of the Japanese article, Linking Protein Folding to Amyloid Formation, published in SEIBUTSU BUTSURI Vol. 61, p. 358–365 (2021).

Abstract Parkinson’s disease is a neurodegenerative disease characterized by the formation of neuronal inclusions of α-synuclein in patient brains. As the disease progresses, toxic α-synuclein aggregates transmit throughout the nervous system. No effective disease-modifying therapy has been established, and preventing α-synuclein aggregation is thought to be one of the most promising approaches to ameliorate the disease. In this study, we performed a two-step screening using the thioflavin T assay and a cell-based assay to identify α-synuclein aggregation inhibitors. The first screening, thioflavin T assay, allowed the identification of 30 molecules, among a total of 1262 FDA-approved small compounds, which showed inhibitory effects on α-synuclein fibrilization. In the second screening, a cell-based aggregation assay, seven out of these 30 candidates were found to prevent α-synuclein aggregation without causing substantial toxicity. Of the seven final candidates, tannic acid was the most promising compound. The robustness of our screening method was validated by a primary neuronal cell model and a Caenorhabditis elegans model, which demonstrated the effect of tannic acid against α-synuclein aggregation. In conclusion, our two-step screening system is a powerful method for the identification of α-synuclein aggregation inhibitors, and tannic acid is a promising candidate as a disease-modifying drug for Parkinson’s disease.

In the Japanese hagfish, Eptatretus burgeri, approximately 21% of the genomic DNA in germ cells (2n = 52) consists of 16 chromosomes (eliminated [E]-chromosomes) that are eliminated from presumptive somatic cells (2n = 36). To uncover the eliminated genome (E-genome), we have identified 16 eliminated repetitive DNA families from eight hagfish species, with 11 of these repeats being selectively amplified in the germline genome of E. burgeri. Furthermore, we have demonstrated that six of these sequences, namely EEEb1-6, are exclusively localized on all 16 E-chromosomes. This has led to the hypothesis that the eight pairs of E-chromosomes are derived from one pair of ancestral chromosomes via multiple duplication events over a prolonged evolutionary period. NGS analysis has recently facilitated the re-assembly of two distinct draft genomes of E. burgeri, derived from the testis and liver. This advancement allows for the prediction of not only nonrepetitive eliminated sequences but also over 100 repetitive and eliminated sequences, accomplished through K-mer-based analysis. In this study, we report four novel eliminated repetitive DNA sequences (designated as EEEb7-10) and confirm the relative chromosomal localization of all eliminated repeats (EEEb1-10) by fluorescence in situ hybridization (FISH). With the exception of EEEb10, all sequences were exclusively detected on EEEb1-positive chromosomes. Surprisingly, EEEb10 was detected as an intense signal on EEEb1-positive chromosomes and as a scattered signal on other chromosomes in germ cells. The study further divided the eight pairs of E-chromosomes into six groups based on the signal distribution of each DNA family, and fiber-FISH experiments showed that the EEEb2-10 family was dispersed in the EEEb1-positive extended chromatin fiber. These findings provide new insights into the mechanisms underlying chromosome elimination and the evolution of E-chromosomes, supporting our previous hypothesis.

We hypothesized that maternal low or high nutrition would give unique effects to morphological and molecular dynamics in adipose tissue of fetus of fatty breed Wagyu (Japanese Black) cattle which produce highly marbled beef. This study aimed to determine the effects of maternal energy intake in Wagyu cows, during gestation on fetal adipose tissue development, histochemical properties, and gene and microRNA (miRNA) expression. Cows were allocated to one of two nutritional energy groups: 120% (HIGH) or 60% nutritional requirements of (LOW). Fetuses (n = 6 per treatment) were removed from pregnant cows by cesarean section at fetal age 260 ± 8 days and euthanized. Subcutaneous adipose tissue (SAT), thoracic cavity visceral adipose tissue (TVAT), and perirenal adipose tissue (PAT) were collected for analysis. In histochemical analysis, in SAT and PAT, HIGH fetuses had greater diameter of adipocytes than LOW fetuses (P

The supersaturation of a solution refers to a non-equilibrium phase in which the solution is trapped in a soluble state, even though the solute’s concentration is greater than its thermodynamic solubility. Upon breaking supersaturation, crystals form and the concentration of the solute decreases to its thermodynamic solubility. Soon after the discovery of the prion phenomena, it was recognized that prion disease transmission and propagation share some similarities with the process of crystallization. Subsequent studies exploring the structural and functional association between amyloid fibrils and amyloidoses solidified this paradigm. However, recent studies have not necessarily focused on supersaturation, possibly because of marked advancements in structural studies clarifying the atomic structures of amyloid fibrils. On the other hand, there is increasing evidence that supersaturation plays a critical role in the formation of amyloid fibrils and the onset of amyloidosis. Here, we review the recent evidence that supersaturation plays a role in linking unfolding/folding and amyloid fibril formation. We also introduce the HANABI (HANdai Amyloid Burst Inducer) system, which enables high-throughput analysis of amyloid fibril formation by the ultrasonication-triggered breakdown of supersaturation. In addition to structural studies, studies based on solubility and supersaturation are essential both to developing a comprehensive understanding of amyloid fibrils and their roles in amyloidosis, and to developing therapeutic strategies.

Amyloid fibrils have been an important subject as they are involved in the development of many amyloidoses and neurodegenerative diseases. The formation of amyloid fibrils is typically initiated by nucleation, whereas its exact mechanisms are largely unknown. With this situation, we have previously identified prefibrillar aggregates in the formation of insulin B chain amyloid fibrils, which have provided an insight into the mechanisms of protein assembly involved in nucleation. Here, we have investigated the formation of insulin B chain amyloid fibrils under different pH conditions to better understand amyloid nucleation mediated by prefibrillar aggregates. The B chain showed strong propensity to form amyloid fibrils over a wide pH range, and prefibrillar aggregates were formed under all examined conditions. In particular, different structures of amyloid fibrils were found at pH 5.2 and pH 8.7, making it possible to compare different pathways. Detailed investigations at pH 5.2 in comparison with those at pH 8.7 have suggested that the evolution of protofibril-like aggregates is a common mechanism. In addition, different processes of evolution of the prefibrillar aggregates have also been identified, suggesting that the nucleation processes diversify depending on the polymorphism of amyloid fibrils.

β2-microglobulin (β2m), the light chain of the MHC-I complex, is associated with dialysis-related amyloidosis (DRA). Recently, a hereditary systemic amyloidosis was discovered, caused by a naturally occurring D76N β2m variant, which showed a structure remarkably similar to the wild-type (WT) protein, albeit with decreased thermodynamic stability and increased amyloidogenicity. Here, we investigated the role of the D76N mutation in the amyloid formation of β2m by point mutations affecting the Asp76-Lys41 ion-pair of WT β2m and the charge cluster on Asp38. Using a variety of biophysical techniques, we investigated the conformational stability and partial unfolding of the native state of the variants, as well as their amyloidogenic propensity and the stability of amyloid fibrils under various conditions. Furthermore, we studied the intermolecular interactions of WT and mutant proteins with various binding partners that might have in vivo relevance. We found that, relative to WT β2m, the exceptional amyloidogenicity of the pathogenic D76N β2m variant is realized by the deleterious synergy of diverse effects of destabilized native structure, higher sensitivity to negatively charged amphiphilic molecules (e.g., lipids) and polyphosphate, more effective fibril nucleation, higher conformational stability of fibrils, and elevated affinity for extracellular components, including extracellular matrix proteins.

For agricultural purposes, the drainage and deforestation of Southeast Asian peatland resulted in high greenhouse gases’ (GHGs, e.g., CO2, N2O and CH4) emission. A peatland regenerating initiative, by rewetting and vegetation restoration, reflects evidence of subsequent forest recovery. In this study, we compared GHG emissions from three Malaysian tropical peatland systems under the following different land-use conditions: (i) drained oil palm plantation (OP), (ii) rewetting-restored forest (RF) and (iii) undrained natural forest (NF). Biweekly temporal measurements of CO2, CH4 and N2O fluxes were conducted using a closed-chamber method from July 2017 to December 2018, along with the continuous measurement of environmental variables and a one-time measurement of the soil physicochemical properties. The biweekly emission data were integrated to provide cumulative fluxes using the trapezoidal rule. Our results indicated that the changes in environmental conditions resulting from draining (OP) or rewetting historically drained peatland (RF) affected CH4 and N2O emissions more than CO2 emissions. The cumulative CH4 emission was significantly higher in the forested sites (RF and NF), which was linked to their significantly higher water table (WT) level (p < 0.05). Similarly, the high cumulative CO2 emission trends at the RF and OP sites indicated that the RF rewetting-restored peatland system continued to have high decomposition rates despite having a significantly higher WT than the OP (p < 0.05). The highest cumulative N2O emission at the drained-fertilized OP and rewetting-restored RF sites was linked to the available substrates for high decomposition (low C/N ratio) together with soil organic matter mineralization that provided inorganic nitrogen (N), enabling ideal conditions for microbial mediated N2O emissions. Overall, the measured peat properties did not vary significantly among the different land uses. However, the lower C/N ratio at the OP and the RF sites indicated higher decomposition rates in the drained and historically drained peat than the undrained natural peat (NF), which was associated with high cumulative CO2 and N2O emissions in our study.

Measurements of greenhouse gas 􀀉GHG􀀊 emissions from paddy fields can often include flux measurement errors due to either instrument errors or unfavorable weather. Therefore, data post-processing, including the gap-filling process, is required to improve data quality and quantify the GHG flux budget. This study applied machine learning 􀀉ML􀀊 techniques with polynomial and multivariate polynomial regression models for gap-filling methane 􀀉CH4􀀊 and carbon dioxide 􀀉CO2􀀊 fluxes from closed chamber 􀀉CC􀀊 method measurements and compared results with mean diurnal variation 􀀉MDV􀀊 and look-up table 􀀉LUT􀀊 techniques. The most influential factors affecting methane emissions in the paddy field were used for input variables in the models: air temperature, soil temperature, soil redox potential, soil water content, solar radiation, and days after transplanting. The models' performances were compared using mean absolute error 􀀉MAE􀀊 and root mean square error 􀀉RMSE􀀊. The results showed that MAE and RMSE for gap-filling CH4 fluxes were 1.299-2.984 and 2.499-4.981 mg CH4 m-2 h-1, respectively. Also, the multivariate polynomial regression models performed better for gap-filling CH4 fluxes 􀀉RMSE = 2.499 mg CH4 m-2 h-1􀀊 than the polynomial regression models, MDV 􀀉RMSE = 3.210 mg CH4 m-2 h-1􀀊, and LUT 􀀉RMSE = 3.339 mg CH4 m-2 h-1􀀊 techniques. The MAE and RMSE for gap-filling CO2 fluxes were 0.282-0.949 and 0.435-1.078 g CO2 m-2 h-1, respectively. The ML techniques with polynomial regression using solar radiation 􀀉RMSE = 0.435 g CO2 m-2 h-1􀀊 and multivariate models 􀀉RMSE = 0.445 g CO2 m-2 h-1􀀊 perform better on gap-filling CO2 fluxes than MDV 􀀉RMSE = 0.544 g CO2 m-2 h-1􀀊, and LUT 􀀉RMSE = 0.553 g CO2 m-2 h-1􀀊 techniques. The gap-filling using the multivariate polynomial regression models used in this study improved the reliability of the diurnal variation in GHG fluxes. Therefore, ML techniques could be a proper alternative for gap-filling GHG fluxes. [ABSTRACT FROM AUTHOR]

This study aims to locate defects in polymer electrolyte fuel cells (PEFCs) caused by electrolyte breakage, electrical contact failure, contamination of electrical insulators, or the like in an easy and instantaneous manner by a noninvasive method. Specifically, the density of the magnetic flux generated around the PEFC during power generation is measured, and then value of the electric current in the electrodes of the PEFC is estimated from the magnetic flux density through inverse problem analysis using sparse modeling. Since the estimated current values are affected by the variables used in the inverse problem analysis, the procedure for determining the variables was first discussed using a simulated fuel cell that imitated the current flow inside the fuel cell. Then, the authors tried to detect a defect of 10 mm x 10 mm inside the fuel cell containing one layer of MEA (Membrane Electrode Assembly) with electrode area of 50 mm x 50 mm according to the above determination procedure. Each estimated current at the three characteristic defect locations was 0.00 A, and the estimated current values at other locations were higher than 0.08 A. From this current distribution, the location of the defect was able to be clearly identified. Particularly, it became possible to detect defects at the central part of the electrode, which was impossible with the Tikhonov regularization method.

Complement component C1q is a protein complex of the innate immune system with well-characterized binding partners that constitutes part of the classical complement pathway. In addition, C1q was recently described in the central nervous system as having a role in synapse elimination both in the healthy brain and in neurodegenerative diseases. However, the molecular mechanism of C1q-associated synapse phagocytosis is still unclear. Here, we designed monomer and multimer protein constructs, which comprised the globular interaction recognition parts of mouse C1q (globular part of C1q [gC1q]) as single-chain molecules (sc-gC1q proteins) lacking the collagen-like effector region. These molecules, which can competitively inhibit the function of C1q, were expressed in an Escherichia coli expression system, and their structure and capabilities to bind known complement pathway activators were validated by mass spectrometry, analytical size-exclusion chromatography, analytical ultracentrifugation, CD spectroscopy, and ELISA. We further characterized the interactions between these molecules and immunoglobulins and neuronal pentraxins using surface plasmon resonance spectroscopy. We demonstrated that sc-gC1qs potently inhibited the function of C1q. Furthermore, these sc-gC1qs competed with C1q in binding to the embryonal neuronal cell membrane. We conclude that the application of sc-gC1qs can reveal neuronal localization and functions of C1q in assays in vivo and might serve as a basis for engineering inhibitors for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Amyloid fibrils, crystal-like fibrillar aggregates of proteins associated with various amyloidoses, have the potential to propagate via a prion-like mechanism. Among known methodologies to dissolve preformed amyloid fibrils, acid treatment has been used with the expectation that the acids will degrade amyloid fibrils similar to acid inactivation of protein functions. Contrary to our expectation, treatment with strong acids, such as HCl or H2SO4, of β2-microglobulin (β2m) or insulin actually promoted amyloid fibril formation, proportionally to the concentration of acid used. A similar promotion was observed at pH 2.0 upon the addition of salts, such as NaCl or Na2SO4. Although trichloroacetic acid, another strong acid, promoted amyloid fibril formation of β2m, formic acid, a weak acid, did not, suggesting the dominant role of anions in promoting fibril formation of this protein. Comparison of the effects of acids and salts confirmed the critical role of anions, indicating that strong acids likely induce amyloid fibril formation via an anion-binding mechanism. The results suggest that although the addition of strong acids decreases pH, it is not useful for degrading amyloid fibrils, but rather induces or stabilizes amyloid fibrils via an anion-binding mechanism. [ABSTRACT FROM AUTHOR]