Due to the technical challenges of large-scale microscopy and analysis, to date only limited knowledge has been made available about axon morphometry (diameter, shape, myelin thickness, volume fraction), thereby limiting our understanding of neuronal microstructure and slowing down research on neurodegenerative pathologies. This study addresses this knowledge gap by establishing a state-of-the-art acquisition and analysis framework for mapping axon morphometry, and providing the first comprehensive mapping of axon morphometry in the human spinal cord. We dissected, fixed and stained a human spinal cord with osmium tetroxide, and used a scanning electron microscope to image the entirety of 23 axial slices, covering C1 to L5 spinal levels. An automatic method based on deep learning was then used to segment each axon and myelin sheath to produce maps of axon morphometry. These maps were then registered to a standard spinal cord magnetic resonance imaging (MRI) template. Between 500,000 (lumbar) and 1 million (cervical) myelinated axons were segmented at each level of this human spinal cord. Morphometric features show a large disparity between tracts, but high right-left symmetry. Our results suggest a modality-based organization of the dorsal column in the human, as it has been observed in the rat. The generated axon morphometry template is publicly available at https://osf.io/8k7jr/ and could be used as a reference for quantitative MRI studies. The proposed framework for axon morphometry mapping could be extended to other parts of the central or peripheral nervous system that exhibit coherently-oriented axons.

Liver dysfunction during sepsis is an independent risk factor leading to increased mortality rates. Specifically, dysregulation of hepatic biotransformation capacity, especially of the cytochrome P450 (CYP) system, represents an important distress factor during host response. The activity of the conserved stress enzyme sphingomyelin phosphodiesterase 1 (SMPD1) has been shown to be elevated in sepsis patients, allowing for risk stratification. Therefore, the aim of the present study was to investigate whether SMPD1 activity has an impact on expression and activity of different hepatic CYP enzymes using an animal model of polymicrobial sepsis. Polymicrobial sepsis was induced in SMPD1 wild-type and heterozygous mice and hepatic ceramide content as well as CYP mRNA, protein expression and enzyme activities were assessed at two different time points, at 24 h, representing the acute phase, and at 28 days, representing the post-acute phase of host response. In the acute phase of sepsis, SMPD1+/+ mice showed an increased hepatic C16- as well as C18-ceramide content. In addition, a downregulation of CYP expression and activities was detected. In SMPD1+/- mice, however, no noticeable changes of ceramide content and CYP expression and activities during sepsis could be observed. After 28 days, CYP expression and activities were normalized again in all study groups, whereas mRNA expression remained downregulated in SMPD+/+ animals. In conclusion, partial genetic inhibition of SMPD1 stabilizes hepatic ceramide content and improves hepatic monooxygenase function in the acute phase of polymicrobial sepsis. Since we were also able to show that the functional inhibitor of SMPD1, desipramine, ameliorates downregulation of CYP mRNA expression and activities in the acute phase of sepsis in wild-type mice, SMPD1 might be an interesting pharmacological target, which should be further investigated.

Platinum is the most employed electrocatalyst for the reactions taking place in energy converters, such as the oxygen reduction reaction in proton exchange membrane fuel cells, despite being a very low abundant element in the earth's crust and thus extremely expensive. The search for more active electrocatalysts with ultra-low Pt loading is thus a very active field of investigation. Here, surface-limited redox replacement (SLRR) that utilizes the monolayer-limited nature of underpotential deposition (UPD) was used to prepare ultrathin deposits of Pt, using Te as sacrificial metal. Cyclic voltammetry and anodic potentiodynamic scanning experiments have been performed to determine the optimal deposition conditions. Physicochemical and electrochemical characterization of the deposited Pt was carried out. The deposit comprises a series of contiguous Pt islands that form along the grain interfaces of the Au substrate. The electrochemical surface area (ECSA) of the Pt deposit obtained after 5 replacements, estimated to be 18 m²/g, is in agreement with the ECSA of extended surface catalysts on flat surfaces.

High salt (HS) intake stimulates the production of marinobufagenin (MBG), an endogenous steroidal Na/K-ATPase ligand, which activates profibrotic signaling. HS is accompanied by a blood pressure (BP) increase in salt-sensitive hypertension, but not in normotensive animals. Here, we investigated whether HS stimulates MBG production and activates transforming growth factor-beta (TGF-β) profibrotic signaling in young normotensive rats, and whether these changes can be reversed by reducing salt to a normal salt (NS) level. Three-month old male Sprague⁻Dawley rats received NS for 4 and 8 weeks (0.5% NaCl; NS4 and NS8), or HS for 4 and 8 weeks (4% NaCl; HS4 and HS8), or HS for 4 weeks followed by NS for 4 weeks (HS4/NS4), n = 8/group. Systolic BP (SBP), pulse wave velocity (PWV), MBG excretion, aortic collagen 1α2, collagen 4α1 and TGF-β, Smad2, Smad3, Fli-1 mRNA, and total collagen abundance were measured at baseline (BL), and on weeks 4 and 8. Statistical analysis was performed using one-way ANOVA. SBP was not affected by HS (125 ± 5 and 126 ± 6 vs. 128 ± 7 mmHg, HS4 and HS8 vs. BL, p > 0.05). HS increased MBG (164 ± 19 vs. 103 ± 19 pmol/24 h/kg, HS4 vs. BL, p < 0.05) and PWV (3.7 ± 0.2 vs. 2.7 ± 0.2 m/s, HS4 vs. NS4, p < 0.05). HS8 was associated with a further increase in MBG and PWV, with an increase in aortic Col1a2 80%), Col4a1 (50%), Tgfb1 (30%), Smad2 (30%) and Smad3 (45%) mRNAs, and aortic wall collagen (180%) vs. NS8 (all p < 0.05). NS following HS downregulated HS-induced factors: in HS4/NS4, the MBG level was 91 ± 12 pmol/24 h/kg (twofold lower than HS8, p < 0.01), PWV was 3.7 ± 0.3 vs. 4.7 ± 0.2 m/s (HS4/NS4 vs. HS8, p < 0.05), aortic wall Tgfb1, Col1a2, Col4a1, Smad2, Smad3 mRNAs, and collagen abundance were reversed by salt reduction to the BL levels (p < 0.05). HS was associated with an activation of TGF-β signaling, aortic fibrosis and aortic stiffness accompanied by an MBG increase in the absence of SBP changes in young normotensive rats. The reduction of dietary salt following HS decreased MBG, PWV, aortic wall collagen and TGF-β. Thus, HS-induced aortic stiffness in normotensive animals occurred in the context of elevated MBG, which may activate SMAD-dependent TGF-β pro-fibrotic signaling. This data suggests that a decrease in salt consumption could help to restore aortic elasticity and diminish the risk of cardiovascular disease by reducing the production of the pro-fibrotic factor MBG.

Activation of nicotinic acetylcholine receptors containing α4 and β2 subunits (α4/β2* nAChRs) in the mammalian brain is necessary for nicotine reinforcement and addiction. We previously identified interactions between α4/β2* nAChRs and calcium/calmodulin-dependent protein kinase II (CaMKII) in mouse and human brain tissue. Following co-expression of α4/β2 nAChR subunits with CaMKII in HEK cells, mass spectrometry identified 8 phosphorylation sites in the α4 subunit. One of these sites and an additional site were identified when isolated α4/β2* nAChRs were dephosphorylated and subsequently incubated with CaMKII in vitro, while 3 phosphorylation sites were identified following incubation with protein kinase A (PKA) in vitro. We then isolated native α4/β2* nAChRs from mouse brain following acute or chronic exposure to nicotine. Two CaMKII sites identified in HEK cells were phosphorylated, and 1 PKA site was dephosphorylated following acute nicotine administration in vivo, whereas phosphorylation of the PKA site was increased back to baseline levels following repeated nicotine exposure. Significant changes in β2 nAChR subunit phosphorylation were not observed under these conditions, but 2 novel sites were identified on this subunit, 1 in HEK cells and 1 in vitro. These experiments identified putative CaMKII and PKA sites on α4/β2* nAChRs and novel nicotine-induced phosphorylation sites in mouse brain that can be explored for their consequences on receptor function.

Polyunsaturated fatty acids (PUFAs) play a significant role in the modulation and prevention of various diseases, and hence are attracting increasing attention from the biotech industry. Thraustochytrids are marine heterokonts that exhibit robust growth rates, high PUFA content, and more specifically, a large percentage of omega-3 fatty acids like docosahexaenoic acid (DHA). Recently, ionic liquids (ILs) have been shown to improve the efficiency of organic solvent extraction of oils from wet oleaginous yeast and microalgae under mild conditions. Two ILs, the imidazolium 1-ethyl-3-methylimidazolium ethylsulfate [C₂mim][EtSO₄] IL and the phosphonium (tetrabutylphosphonium propanoate [P4444][Prop]) IL were assessed for their ability to facilitate extraction of PUFA-containing lipids from a Thraustochytrium sp. (T18) through efficient cell wall disruption. The oil extracted after IL pretreatment was further characterized with respect to fatty acid methyl ester (FAME) composition, while the effects of process parameters, such as the ratio of ionic liquid to co-solvent, the mass ratio of microalgae to the mixture of ionic liquid, and type of co-solvent were also investigated for both ILs. The results indicate that these ILs can disrupt the cells of Thraustochytrium sp. when mixed with a co-solvent (methanol), and facilitated the recovery of oils over a large degree of dewatered Thraustochytrium biomass (0⁻77.2 wt% water) in a short period of time (60 min) at ambient temperature, hence demonstrating a water compatible, low-energy, lipid recovery method. The lipid recovery was not affected by repeated usage of recycled ILs (tested up to five times).

Estrogen receptors promote target gene transcription when they form a dimer, in which two identical (homodimer) or different (heterodimer) proteins are bound to each other. In hormone-dependent cancers, hormone receptor dimerization plays pivotal roles, not only in the pathogenesis or development of the tumors, but also in the development of therapeutic resistance. Protein⁻protein interactions (PPIs), including dimerization and complex formation, have been also well-known to be required for proteins to exert their functions. The methods which could detect PPIs are genetic engineering (i.e., resonance energy transfer) and/or antibody technology (i.e., co-immunoprecipitation) using cultured cells. In addition, visualization of the target proteins in tissues can be performed using antigen⁻antibody reactions, as in immunohistochemistry. Furthermore, development of microscopic techniques (i.e., electron microscopy and confocal laser microscopy) has made it possible to visualize intracellular and/or intranuclear organelles. We have recently reported the visualization of estrogen receptor dimers in breast cancer tissues by using the in situ proximity ligation assay (PLA). PLA was developed along the lines of antibody technology development, and this assay has made it possible to visualize PPIs in archival tissue specimens. Localization of PPI in organelles has also become possible using super-resolution microscopes exceeding the resolution limit of conventional microscopes. Therefore, in this review, we summarize the methodologies used for studying PPIs in both cells and tissues, and review the recently reported studies on PPIs of hormones.

Coffee is a beverage widely consumed in the world. The coffee species most commercialized worldwide are Arabica (Coffea arabica) and Robusta (Coffea canephora). Roasted coffee beans are the most used, but coffee leaves are also consumed as infusion in several countries for traditional medicinal purposes. They contain several interesting phenolic antioxidant compounds mainly belonging to chlorogenic acids (CGAs). In the present work, a liquid chromatography-electrochemical detection (LC-EC) method was developed for the determination of three main chlorogenic acid isomers, namely 3-, 4-, and 5-caffeoylquinic acids (CQA), in coffee leaves aqueous extracts. Samples from eight coffee species, namely; Coffea arabica, Coffea canephora, Coffea liberica, Coffea humilis, Coffea mannii, Coffea charrieriana, Coffea anthonyi, and Coffea liberica var. liberica, were grown and collected in tropical greenhouses. Linearity of the calibration graphs was observed in the range from the limit of quantification to 1.0 × 10-5 M, with R² equal to 99.9% in all cases. High sensitivity was achieved with a limit of detection of 1.0 × 10-8 M for 3-CQA and 5-CQA (i.e., 3.5 µg/L) and 2.0 × 10-8 M for 4-CQA (i.e., 7.1 µg/L). The chromatographic profile of the samples harvested for each Coffea species was studied comparatively. Obtained raw data were pretreated for baseline variations and shifts in retention times between the chromatographic profiles. Principal Component Analysis (PCA) was applied to the pretreated data. According to the results, three clusters of Coffea species were found. In the water sample extracts, 5-CQA appeared to be the major isomer, and some species contained a very low amount of CQAs. Fluctuations were observed depending on the Coffea species and harvesting period. Significant differences between January and July were noticed regarding CQAs content. The species with the best CQAs/caffeine ratio was identified. The LC-EC data were validated by liquid chromatography-high resolution mass spectrometry (LC-HRMS).

Dairy, as a major component of a high protein diet, is a critical dietary source of branched chain amino acids (BCAA), which are biomarkers of health and diseases. While BCAA are known to be key stimulators of protein synthesis, elevated circulatory BCAA is an independent risk factor for type 2 diabetes mellitus. This study examined the impact of altered dairy intake on plasma BCAA and their potential relationship to insulin sensitivity. Healthy adults (n = 102) were randomized to receive dietary advice to reduce, maintain, or increase habitual dairy intake for 1 month. Food intake was recorded with food frequency questionnaires. Self-reported protein intake from dairy was reported to be reduced (-14.6 ± 3.0 g/day), maintained (-4.0 ± 2.0 g/day) or increased (+13.8 ± 4.1 g/day) according to group allocation. No significant alterations in circulating free amino acids (AA), including BCAA, were measured. Insulin sensitivity, as assessed by homeostatic model assessment-insulin resistance (HOMA-IR), was also unaltered. A significant change in dairy protein intake showed no significant effect on fasting circulatory BCAA and insulin sensitivity in healthy populations.

Sustained endoplasmic reticular stress (ERS) is implicated in aggressive metastasis of cancer cells and increased tumor cell proliferation. Cancer cells activate the unfolded protein response (UPR), which aids in cellular survival and adaptation to harsh conditions. Inhibition of apoptosis, in contrast, is a mechanism adopted by cancer cells with the help of the inhibitor of an apoptosis (IAP) class of proteins such as Survivin to evade cell death and gain a proliferative advantage. In this study, we aimed to reveal the interrelation between ERS and Survivin. We initially verified the expression of Survivin in Winnie (a mouse model of chronic ERS) colon tissues by using immunohistochemistry (IHC) and immunofluorescence (IF) in comparison with wild type Blk6 mice. Additionally, we isolated the goblet cells and determined the expression of Survivin by IF and protein validation. Tunicamycin was utilized at a concentration of 10 µg/mL to induce ERS in the LS174T cell line and the gene expression of the ERS markers was measured. This was followed by determination of inflammatory cytokines. Inhibition of ERS was carried out by 4Phenyl Butyric acid (4PBA) at a concentration of 10 mM to assess whether there was a reciprocation effect. The downstream cell death assays including caspase 3/7, Annexin V, and poly(ADP-ribose) polymerase (PARP) cleavage were evaluated in the presence of ERS and absence of ERS, which was followed by a proliferative assay (EdU click) with and without ERS. Correspondingly, we inhibited Survivin by YM155 at a concentration of 100 nM and observed the succeeding ERS markers and inflammatory markers. We also verified the caspase 3/7 assay. Our results demonstrate that ERS inhibition not only significantly reduced the UPR genes (Grp78, ATF6, PERKandXBP1) along with Survivin but also downregulated the inflammatory markers such as IL8, IL4, and IL6, which suggests a positive correlation between ERS and the inhibition of apoptosis. Furthermore, we provided evidence that ERS inhibition promoted apoptosis in LS174T cells and shortened the proliferation rate. Moreover, Survivin inhibition by YM155 led to a comparable effect as that of ERS inhibition, which includes attenuation of ERS genes and inflammatory markers as well as the promotion of programmed cell death via the caspase 3/7 pathway. Together, our results propose the interrelation between ERS and inhibition of apoptosis assigning a molecular and therapeutic target for cancer treatment.

To understand functions of biomolecules such as proteins, not only structures but their conformational change and kinetics need to be characterized, but its atomistic details are hard to obtain both experimentally and computationally. Here, we review our recent computational studies using novel enhanced sampling techniques for conformational sampling of biomolecules and calculations of their kinetics. For efficiently characterizing the free energy landscape of a biomolecule, we introduce the multiscale enhanced sampling method, which uses a combined system of atomistic and coarse-grained models. Based on the idea of Hamiltonian replica exchange, we can recover the statistical properties of the atomistic model without any biases. We next introduce the string method as a path search method to calculate the minimum free energy pathways along a multidimensional curve in high dimensional space. Finally we introduce novel methods to calculate kinetics of biomolecules based on the ideas of path sampling: one is the Onsager⁻Machlup action method, and the other is the weighted ensemble method. Some applications of the above methods to biomolecular systems are also discussed and illustrated.

A series of novel bivalent β-carboline derivatives were designed and synthesized, and in vitro cytotoxicity, cell apoptosis, and DNA-binding affinity were evaluated. The cytotoxic results demonstrated that most bivalent β-carboline derivatives exhibited stronger cytotoxicity than the corresponding monomer against the five selected tumor cell lines (A549, SGC-7901, Hela, SMMC-7721, and MCF-7), indicating that the dimerization at the C³ position could enhance the antitumor activity of β-carbolines. Among the derivatives tested, 4B, 6i, 4D, and 6u displayed considerable cytotoxicity against A549 cell line. Furthermore, 4B, 6i, 4D, and 6u induced cell apoptosis in a dose-dependent manner, and caused cell cycle arrest at the S and G2/M phases. Moreover, the levels of cytochrome C in mitochondria, and the expressions of bcl-2 protein, decreased after treatment with β-carbolines, which indicated that 6i and 6u could induce mitochondria-mediated apoptosis. In addition, the results of UV-visible spectral, thermal denaturation, and molecular docking studies revealed that 4B, 6i, 4D, and 6u could bind to DNA mainly by intercalation.

Ogura cytoplasmic male sterility (CMS) contributes considerably to hybrid seed production in Brassica crops. To detect the key protein species and pathways involved in Ogura-CMS, we analysed the proteome of the cabbage Ogura-CMS line CMS01-20 and its corresponding maintainer line F01-20 using the isobaric tags for the relative and absolute quantitation (iTRAQ) approach. In total, 162 differential abundance protein species (DAPs) were identified between the two lines, of which 92 were down-accumulated and 70 were up-accumulated in CMS01-20. For energy metabolism in the mitochondrion, eight DAPs involved in oxidative phosphorylation were down-accumulated in CMS01-20, whereas in the tricarboxylic acid (TCA) cycle, five DAPs were up-accumulated, which may compensate for the decreased respiration capacity and may be associated with the elevated O2 consumption rate in Ogura-CMS plants. Other key protein species and pathways involved in pollen wall assembly and programmed cell death (PCD) were also identified as being male-sterility related. Transcriptome profiling revealed 3247 differentially expressed genes between the CMS line and the fertile line. In a conjoint analysis of the proteome and transcriptome data, 30 and 9 protein species/genes showed the same and opposite accumulation patterns, respectively. Nine noteworthy genes involved in sporopollenin synthesis, callose wall degeneration, and oxidative phosphorylation were presumably associated with the processes leading to male sterility, and their expression levels were validated by qRT-PCR analysis. This study will improve our understanding of the protein species involved in pollen development and the molecular mechanisms underlying Ogura-CMS.

Canine babesiosis and ehrlichiosis are important tick-borne infections in South Africa. Many South African general veterinary practitioners perceive co-infection with Ehrlichia spp. as a common occurrence in dogs with babesiosis. Studies about the prevalence of co-infection in South African dogs are lacking. This retrospective study aimed to determine the prevalence of Ehrlichia co-infection in dogs with babesiosis. Additionally, the predicative value of specific haematological variables for co-infection was evaluated. The study population consisted of 205 dogs diagnosed with canine babesiosis presented to the Onderstepoort Veterinary Academic Hospital (OVAH) in 2006 and between 2011 and 2013. The Babesia-infected dogs were grouped based on presence or absence of an Ehrlichia spp. co-infection. Ehrlichia spp. co-infection was confirmed using polymerase chain reaction. Positive and negative predictive values (PPVs and NPVs) of leukopenia or thrombocytopenia for co-infection were also calculated. The prevalence of Babesiaspp. and Ehrlichia spp. co-infection in this cohort of dogs was 2%. In the babesiosis dogs, the PPV of leukopenia for co-infection with Ehrlichia spp. was 1.3%, and the NPV 97.4%. Similarly, the PPV and NPVs of thrombocytopenia for co-infection were 2.1% and 100%, respectively. Co-infection with Ehrlichia spp. was a rare occurrence in dogs with babesiosis presented to the OVAH. Normal leukocyte or platelet counts confidently ruled out the presence of concurrent ehrlichiosis in this cohort of dogs. However, the diagnosis of Ehrlichia co-infection based on the presence of thrombocytopenia or leukopenia would have been associated with false positive results in more than 97.4% of cases.

In recent years, caesarean section rates continue to evoke worldwide concern because of their steady increase, lack of consensus on the appropriate caesarean section rate and the associated short- and long-term risks. This study sought to identify the rate of caesarean section and associated factors in two districts in rural southern Ghana.

High-flow nasal cannula (HFNC) can be used as an initial support strategy for patients with acute respiratory failure (ARF) and after extubation. However, no clear evidence exists to support or oppose HFNC use in clinical practice. We summarized the effects of HFNC, compared to conventional oxygen therapy (COT) and noninvasive ventilation (NIV), on important outcomes including treatment failure and intubation/reintubation rates in adult patients with ARF and after extubation.

Measles (MEV) and mumps virus (MUV) are enveloped, non-segmented, negative single stranded RNA viruses of the family Paramyxoviridae, and are the cause of measles and mumps, respectively, both preventable by vaccination. Aside from proteins coded by the viral genome, viruses are considered to contain host cell proteins (HCPs). The presence of extracellular vesicles (ECVs), which are often co-purified with viruses due to their similarity in size, density and composition, also contributes to HCPs detected in virus preparations, and this has often been neglected. The aim was to identify which virus-coded proteins are present in MEV and MUV virions, and to try to detect which HCPs, if any, are incorporated inside the virions or adsorbed on their outer surface, and which are more likely to be a contamination from co-purified ECVs.

Nuclear genes or family-based mitochondrial screening have been the focus of genetic studies into essential hypertension. Studies into the role of mitochondria in sporadic Chinese hypertensives are lacking. The objective of the study was to explore the relationship between mitochondrial DNA (mtDNA) variations and the development of maternally inherited essential hypertension (MIEH) in China.

TAR DNA binding protein 43 (TDP-43) is the main disease protein in most patients with amyotrophic lateral sclerosis (ALS) and about 50% of patients with frontotemporal dementia (FTD). TDP-43 pathology is not restricted to patients with missense mutations in TARDBP, the gene encoding TDP-43, but also occurs in ALS/FTD patients without known genetic cause or in patients with various other ALS/FTD gene mutations. Mutations in progranulin (GRN), which result in a reduction of ~ 50% of progranulin protein (PGRN) levels, cause FTD with TDP-43 pathology. How loss of PGRN leads to TDP-43 pathology and whether or not PGRN expression protects against TDP-43-induced neurodegeneration is not yet clear.

Although tamoxifen is a highly effective drug for treating estrogen receptor-positive (ER+) breast cancer, nearly all patients with metastasis with initially responsive tumors eventually relapse, and die from acquired drug resistance. Unfortunately, few molecular mediators of tamoxifen resistance have been described. Here, we describe AFF3 (AF4/FMR2 family member 3), which encodes a nuclear protein with transactivation potential that confers tamoxifen resistance and enables estrogen-independent growth.