SFTPB in serum extracellular vesicles as a biomarker of progressive pulmonary fibrosis.

Progressive pulmonary fibrosis (PPF), defined as the worsening of various interstitial lung diseases (ILDs), currently lacks useful biomarkers. To identify novel biomarkers for early detection of patients at risk of PPF, we performed a proteomic analysis of serum extracellular vesicles (EVs). Notably, the identified candidate biomarkers were enriched for lung-derived proteins participating in fibrosis-related pathways. Among them, pulmonary surfactant-associated protein B (SFTPB) in serum EVs could predict ILD progression better than the known biomarkers, serum KL-6 and SP-D, and it was identified as an independent prognostic factor from ILD-gender-age-physiology index. Subsequently, the utility of SFTPB for predicting ILD progression was evaluated further in 2 cohorts using serum EVs and serum, respectively, suggesting that SFTPB in serum EVs but not in serum was helpful. Among SFTPB forms, pro-SFTPB levels were increased in both serum EVs and lungs of patients with PPF compared with those of the control. Consistently, in a mouse model, the levels of pro-SFTPB, primarily originating from alveolar epithelial type 2 cells, were increased similarly in serum EVs and lungs, reflecting pro-fibrotic changes in the lungs, as supported by single-cell RNA sequencing. SFTPB, especially its pro-form, in serum EVs could serve as a biomarker for predicting ILD progression.

In situ hybridization analysis of odorant receptor expression in the olfactory organ of the pig-nosed turtle Carettochelys insculpta.

The turtle olfactory organ consists of upper (UCE) and lower (LCE) chamber epithelium, which send axons to the ventral and dorsal portions of the olfactory bulbs, respectively. Generally, the UCE is associated with glands and contains ciliated olfactory receptor neurons (ORNs), while the LCE is devoid of glands and contains microvillous ORNs. However, the olfactory organ of the pig-nosed turtle Carettochelys insculpta appears to be a single olfactory system morphologically: there are no associated glands; ciliated ORNs are distributed throughout the olfactory organ; and the olfactory bulb is not divided into ventral and dorsal portions. In this study, we analyzed the expression of odorant receptors (ORs), the major olfactory receptors in turtles, in the pig-nosed turtle olfactory organ, via in situ hybridization. Of 690 ORs, 375 were classified as class I and 315 as class II. Some class II ORs were expressed predominantly in the posterior dorsomedial walls of the nasal cavity, while other class II ORs and all class I ORs examined were expressed in the remaining region. These results suggest that the pig-nosed turtle olfactory organ can be divided into two regions according to the expression of ORs.

Modulation of Context-Dependent Spatiotemporal Patterns within Packets of Spiking Activity.

Recent experiments have shown that stereotypical spatiotemporal patterns occur during brief packets of spiking activity in the cortex, and it has been suggested that top-down inputs can modulate these patterns according to the context. We propose a simple model that may explain important features of these experimental observations and is analytically tractable. The key mechanism underlying this model is that context-dependent top-down inputs can modulate the effective connection strengths between neurons because of short-term synaptic depression. As a result, the degree of synchrony and, in turn, the spatiotemporal patterns of spiking activity that occur during packets are modulated by the top-down inputs. This is shown using an analytical framework, based on avalanche dynamics, that allows calculating the probability that a given neuron spikes during a packet and numerical simulations. Finally, we show that the spatiotemporal patterns that replay previously experienced sequential stimuli and their binding with their corresponding context can be learned because of spike-timing-dependent plasticity.

Individuality embedded in the isolation calls of captive beluga whales (Delphinapterus leucas).

INTRODUCTION: Species with fission-fusion social systems tend to exchange individualized contact calls to maintain group cohesion. Signature whistles by bottlenose dolphins are unique compared to the contact calls of other non-human animals in that they include identity information independent of voice cues. Further, dolphins copy the signatures of conspecifics and use them to label specific individuals. Increasing our knowledge of the contact calls of other cetaceans that have a fluid social structure may thus help us better understand the evolutionary and adaptive significance of all forms of individually distinctive calls. It was recently reported that one type of broadband pulsed sounds (PS1), rather than whistles, may function as individualized contact calls in captive belugas. The objective of this study was to assess the function and individual distinctiveness of PS1 calls in an isolation context. Recordings were made from five captive belugas, including both sexes and various ages. RESULTS: PS1 was the predominant call type (38 % in total) out of five broader sound categories. One sub-adult and three adults had individually distinctive and stereotyped pulse repetition pattern in PS1; one calf showed no clear stereotyped pulse repetition pattern. While visual inspection of the PS1 power spectra uncovered no apparent individual specificity, statistical analyses revealed that both temporal and spectral parameters had inter-individual differences and that there was greater inter-individual than intra-individual variability. Discriminant function analysis based on five temporal and spectral parameters classified PS1 calls into individuals with an overall correct classification rate of 80.5 %, and the most informative parameter was the average Inter-pulse interval, followed by peak frequency. CONCLUSION: These results suggest that belugas use individually distinctive contact calls in an isolation context. If belugas encode signature information in PS1 calls, as seen in bottlenose dolphins, the pulse repetition pattern may be the carrier, as it is individually stereotyped and appears to require vocal development. This idea is supported by the finding that the average inter-pulse interval is the most powerful discriminator in discriminant analysis. Playback experiments will elucidate which parameters are perceived as individual characteristics, and whether one of the parameters functions as a signature.

[Duloxetine for chronic pain management: pharmacology and clinical use].

The purpose of this article is to provide an overview of some of the important information related to safety and tolerability of duloxetine. Duloxetine, a potent reuptake inhibitor of serotonin and noradrenaline, is effective for the treatment of major depressive disorder, anxiety disorder, and painful diabetic neuropathy (PDN). Duloxetine is safe and well-tolerated across indications, with few reported serious side effects. Common adverse events are consistent with the pharmacology of the molecule and are mainly referable to the gastrointestinal and the nervous systems. Duloxetine should not be used in combination with CYP 1A2 inhibitors or nonselective, irreversible monoamine oxidase inhibitors. Duloxetine has a generally favorable side effect profile and dosing is simple. Nausea is the most common side effect, but it occurs less frequently if treatment is initiated at 30 mg . day-1 and titrated after one week to 60 mg . day-1, an efficacious dosage at which pain relief can occur within one week. Clinical trials have demonstrated the analgesic efficacy of duloxetine for PDN and fibromyalgia in addition to improvements in quality-of-life measurements. Furthermore trials for osteoarthritis, headache, and the pain associated with Parkinson disease may provide insight into alternative uses for duloxetine.

Variations in both TG1 and TG2 isozyme-specific in situ activities and protein expressions during mouse embryonic development.

Transglutaminase (TG) is a family of enzymes that catalyzes cross-linking reactions among proteins. Using fluorescent-labeled highly reactive substrate peptides, we recently developed a system to visualize isozyme-specific in situ enzymatic activity. In the present study, we investigated the in situ activities of TG1 (skin-type) and TG2 (tissue-type) using whole mouse sections of various embryonic developmental stages and neonates. In each case, we also successfully used immunostaining of identical whole mouse sections for protein expression after detection of enzymatic activities. In general, the enzymatic activity was correlated with TG protein expression. However, in some tissues, TG protein expression patterns, which were inconsistent with the enzymatic activities, suggested that inactive TGs were produced possibly by self cross-linking or other modifications. Our method allowed us to simultaneously observe developmental variations in both TG isozyme-specific activities and protein levels in mouse embryonic and neonate tissues.

Identification of a highly reactive substrate peptide for transglutaminase 6 and its use in detecting transglutaminase activity in the skin epidermis.

Mammalian transglutaminases (TGs) are a family of enzymes that catalyze the formation of covalent crosslinks between glutamine and lysine residues in proteins. These catalytic reactions play roles in several essential biological processes, including blood coagulation, skin formation, and stabilization of the extracellular matrix. Among the members of this family, factor XIII and TGs 1-5 have been characterized well, but very little is known about the novel members TG6 and TG7. Recently, however, autoantibodies against TG6 were found in a patient with gluten ataxia, a disease caused by enzymatically modified gluten-derived peptides in neuronal cells. To characterize the possible physiological functions of TG6, in this study we screened a phage-displayed random peptide library to find highly reactive glutamine donor substrate peptides. From several candidate peptides, one sequence, designated Y25, appeared to have the highest reactivity. The Y25 sequence also has apparent isozyme specificity when evaluated by incorporation of the labeled glutamine acceptor substrate as a fusion protein with glutathione-S-transferase. Also, the sequence retained high reactivity as well as the isozyme specificity in the peptide form. Analyses with the biotin-labeled and fluorescence-labeled peptides showed TG6 to be an active enzyme and react to specific substrates in the skin, which is consistent with the results of the expression pattern of its transcripts.

Transglutaminase 2 and Factor XIII catalyze distinct substrates in differentiating osteoblastic cell line: utility of highly reactive substrate peptides.

Differentiated osteoblastic cell line, MC3T3-E1 expresses transglutaminase 2 (TG2) and Factor XIII (FXIII). In previous studies, we identified isozyme-specific and highly reactive glutamine-donor substrate peptides (pepF11KA and pepT26) for each isozyme. Using these peptides, we compared the reaction products with lysine-donor substrates for each isozyme in differentiating MC3T3-E1 cells. By this analysis, distinct substrates for the activated TG2 and FXIII were detected in cultured cellular extract. Possible substrates that incorporated biotin-labeled peptides were further purified using streptavidin-affinity chromatography. Several isozyme-specific substrates were identified by mass spectrometry analysis of the purified fractions. These analyses also indicate the benefit of the substrate peptides for obtaining distinct substrates in a reaction mixture where two isozymes co-exist.

Vena cava and aortic smooth muscle cells express transglutaminases 1 and 4 in addition to transglutaminase 2.

Transglutaminase (TG) function facilitates several vascular processes and diseases. Although many of these TG-dependent vascular processes have been ascribed to the function of TG2, TG2 knockout mice have a mild vascular phenotype. We hypothesized that TGs besides TG2 exist and function in the vasculature. Biotin-pentylamide incorporation, a measure of general TG activity, was similar in wild-type and TG2 knockout mouse aortae, and the general TG inhibitor cystamine reduced biotin-pentylamine incorporation to a greater extent than the TG2-specific inhibitor Z-DON, indicating the presence of other functional TGs. Additionally, 5-hydroxytryptamine-induced aortic contraction, a TG-activity-dependent process, was decreased to a greater extent by general TG inhibitors vs. Z-DON (maximum contraction: cystamine = abolished, monodansylcadaverine = 28.6 ± 14.9%, and Z-DON = 60.2 ± 15.2% vehicle), providing evidence for the importance of TG2-independent activity in the vasculature. TG1, TG2, TG4, and Factor XIII (FXIII) mRNA in rat aortae and vena cavae was detected by RT-PCR. Western analysis detected TG1 and TG4, but not FXIII, in rat aortae and vena cavae and in TG2 knockout and wild-type mouse aortae. Immunostaining confirmed the presence of TG1, TG2, and TG4 in rat aortae and vena cavae, notably in smooth muscle cells; FXIII was absent. K5 and T26, FITC-labeled peptide substrates specific for active TG1 and TG2, respectively, were incorporated into rat aortae and vena cavae and wild-type, but not TG2 knockout, mouse aortae. These studies demonstrate that TG2-independent TG activity exists in the vasculature and that TG1 and TG4 are expressed in vascular tissues.

In situ detection of active transglutaminases for keratinocyte type (TGase 1) and tissue type (TGase 2) using fluorescence-labeled highly reactive substrate peptides.

Transglutaminase is a calcium-dependent enzyme that posttranslationally modifies proteins by cross-linking between glutamine and lysine residues or attachment of a primary amine to specific polypeptide-bound glutamine residues. Eight isozymes play essential roles in various mammalian biological processes. The authors have recently identified 12­amino acid preferred substrate peptide sequences that are highly reactive and act in an isozyme-specific manner. In this study, a rapid, isozyme-specific, and sensitive detection of active keratinocyte type (TGase 1) and tissue type (TGase 2) was successful using fluorescence-labeled peptides. This procedure involved using whole-body sections of a mouse to extensively analyze the tissue distribution of both enzymes that revealed clearly distinct patterns. Strong active TGase 1 was observed in epithelial tissues such as tongue, developing teeth, forestomach, and skin epidermis. Significantly active TGase 2 was observed in various types of tissues as predicted and at particularly higher levels in the intestinal mucosa, muscle membrane, and whole veins in the liver. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Identification of a preferred substrate peptide for transglutaminase 3 and detection of in situ activity in skin and hair follicles.

Transglutaminases (TGases) are a family of enzymes that catalyze cross-linking reactions between proteins. During epidermal differentiation, these enzymatic reactions are essential for formation of the cornified envelope, which consists of cross-linked structural proteins. Two main transglutaminases isoforms, epidermal-type (TGase 3) and keratinocyte-type (TGase 1), are cooperatively involved in this process of differentiating keratinocytes. Information regarding their substrate preference is of great importance to determine the functional role of these isozymes and clarify their possible co-operative action. Thus far, we have identified highly reactive peptide sequences specifically recognized by TGases isozymes such as TGase 1, TGase 2 (tissue-type isozyme) and the blood coagulation isozyme, Factor XIII. In this study, several substrate peptide sequences for human TGase 3 were screened from a phage-displayed peptide library. The preferred substrate sequences for TGase 3 were selected and evaluated as fusion proteins with mutated glutathione S-transferase. From these studies, a highly reactive and isozyme-specific sequence (E51) was identified. Furthermore, this sequence was found to be a prominent substrate in the peptide form and was suitable for detection of in situ TGase 3 activity in the mouse epidermis. TGase 3 enzymatic activity was detected in the layers of differentiating keratinocytes and hair follicles with patterns distinct from those of TGase 1. Our findings provide new information on the specific distribution of TGase 3 and constitute a useful tool to clarify its functional role in the epidermis.

Soft and hard keratin expression in Epstein-Barr-virus-associated gastric carcinoma.

BACKGROUND: Epstein-Barr-virus-associated gastric carcinoma (EBVaGC) is a distinct subset of gastric carcinoma (GC). The expressions of cytokeratins (CK)7, 8, 18, 19 and 20 and truncated basic hair keratin 1 (hHb1-deltaN) were investigated in GC to clarify the characteristics of EBVaGC. MATERIALS AND METHODS: For immunohistochemical evaluation, 173 GC tissues were examined and 31 GC tissues and 5 GC cell lines were used for quantitative real-time RT-PCR (qrt-RT-PCR) and RT-PCR. RESULTS: EBVaGC showed significantly lower immunohistochemical positivity of CK7 (-/(+/-)/+/+ +/+ + +; 27/15/4/1/1) compared to EBV-negative GC (12/29/27/44/13), even after stratification by histological types. The qrt-RT-PCR test demonstrated decreased amounts of CK7, 18 and 19 mRNAs in EBVaGC. Two among 5 GC cell lines showed a decrease of CK7 mRNA level after recombinant EBV infection. hHb1-deltaN expression was not specific to EBVaGC. CONCLUSION: Abnormalities of CK7, 18 and 19 expressions, especially a decreased amount of CK7 expression, are characteristics of EBV-associated epithelial malignancies and might be important in carcinogenesis.