ResearchPad - collagens https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Collagen methionine sulfoxide and glucuronidine/LW-1 are markers of coronary artery disease in long-term survivors with type 1 diabetes. The Dialong study]]> https://www.researchpad.co/article/elastic_article_13877 Type 1 diabetes is a risk factor for coronary heart disease. The underlying mechanism behind the accelerated atherosclerosis formation is not fully understood but may be related to the formation of oxidation products and advanced glycation end-products (AGEs). We aimed to examine the associations between the collagen oxidation product methionine sulfoxide; the collagen AGEs methylglyoxal hydroimidazolone (MG-H1), glucosepane, pentosidine, glucuronidine/LW-1; and serum receptors for AGE (RAGE) with measures of coronary artery disease in patients with long-term type 1 diabetes.MethodsIn this cross-sectional study, 99 participants with type 1 diabetes of ≥ 45-year duration and 63 controls without diabetes had either established coronary heart disease (CHD) or underwent Computed Tomography Coronary Angiography (CTCA) measuring total, calcified and soft/mixed plaque volume. Skin collagen methionine sulfoxide and AGEs were measured by liquid chromatography-mass spectrometry and serum sRAGE/esRAGE by ELISA.ResultsIn the diabetes group, low levels of methionine sulfoxide (adjusted for age, sex and mean HbA1c) were associated with normal coronary arteries, OR 0.48 (95% CI 0.27–0.88). Glucuronidine/LW-1 was associated with established CHD, OR 2.0 (1.16–3.49). MG-H1 and glucuronidine/LW-1 correlated with calcified plaque volume (r = 0.23–0.28, p<0.05), while pentosidine correlated with soft/mixed plaque volume (r = 0.29, p = 0.008), also in the adjusted analysis.ConclusionsLow levels of collagen-bound methionine sulfoxide were associated with normal coronary arteries while glucuronidine/LW-1 was positively associated with established CHD in long-term type 1 diabetes, suggesting a role for metabolic and oxidative stress in the formation of atherosclerosis in diabetes. ]]> <![CDATA[Assessing the effects of intratendinous genipin injections: Mechanical augmentation and spatial distribution in an ex vivo degenerative tendon model]]> https://www.researchpad.co/article/N537496c4-2c39-41e6-9ca3-002a318b88b6

Background

Tendinopathy is a common musculoskeletal disorder and current treatment options show limited success. Genipin is an effective collagen crosslinker with low cytotoxicity and a promising therapeutic strategy for stabilizing an intratendinous lesion.

Purpose

This study examined the mechanical effect and delivery of intratendinous genipin injection in healthy and degenerated tendons.

Study design

Controlled laboratory study

Methods

Bovine superficial digital flexor tendons were randomized into four groups: Healthy control (N = 25), healthy genipin (N = 25), degenerated control (N = 45) and degenerated genipin (N = 45). Degeneration was induced by Collagenase D injection. After 24h, degenerated tendons were subsequently injected with either 0.2ml of 80mM genipin or buffer only. 24h post-treatment, samples were cyclically loaded for 500 cycles and then ramp loaded to failure. Fluorescence and absorption assays were performed to analyze genipin crosslink distribution and estimate tissue concentration after injection.

Results

Compared to controls, genipin treatment increased ultimate force by 19% in degenerated tendons (median control 530 N vs. 633 N; p = 0.0078). No significant differences in mechanical properties were observed in healthy tendons, while degenerated tendons showed a significant difference in ultimate stress (+23%, p = 0.049), stiffness (+27%, p = 0.037), work to failure (+42%, p = 0.009), and relative stress relaxation (-11%, p < 0.001) after genipin injection. Fluorescence and absorption were significantly higher in genipin treated tendons compared to control groups. A higher degree of crosslinking (+45%, p < 0.001) and a more localized distribution were observed in the treated healthy compared to degenerated tendons, with higher genipin tissue concentrations in healthy (7.9 mM) than in degenerated tissue (2.3 mM).

Conclusion

Using an ex-vivo tendinopathy model, intratendinous genipin injections recovered mechanical strength to the level of healthy tendons. Measured by genipin tissue distribution, injection is an effective method for local delivery.

Clinical relevance

This study provides a proof of concept for the use of intratendinous genipin injection in the treatment of tendinopathy. The results demonstrate that a degenerated tendon can be mechanically augmented by a clinically viable method of local genipin delivery. This warrants further in vivo studies towards the development of a clinically applicable treatment based on genipin.

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<![CDATA[A mass sacrifice of children and camelids at the Huanchaquito-Las Llamas site, Moche Valley, Peru]]> https://www.researchpad.co/article/5c897756d5eed0c4847d2a4c

Here we report the results of excavation and interdisciplinary study of the largest child and camelid sacrifice known from the New World. Stratigraphy, associated artifacts, and radiocarbon dating indicate that it was a single mass killing of more than 140 children and over 200 camelids directed by the Chimú state, c. AD 1450. Preliminary DNA analysis indicates that both boys and girls were chosen for sacrifice. Variability in forms of cranial modification (head shaping) and stable isotope analysis of carbon and nitrogen suggest that the children were a heterogeneous sample drawn from multiple regions and ethnic groups throughout the Chimú state. The Huanchaquito-Las Llamas mass sacrifice opens a new window on a previously unknown sacrificial ritual from fifteenth century northern coastal Peru. While the motivation for such a massive sacrifice is a subject for further research, there is archaeological evidence that it was associated with a climatic event (heavy rainfall and flooding) that could have impacted the economic, political and ideological stability of one of the most powerful states in the New World during the fifteenth century A.D.

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<![CDATA[Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs]]> https://www.researchpad.co/article/5c6c7580d5eed0c4843cfe25

Adipose-derived stem cells (ASCs) are multipotent and immune-privileged mesenchymal cells, making them ideal candidates for therapeutic purposes to manage tendon disorders. Providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices. To this aim we investigated the in vitro tenogenic differentiation potential of ASCs using a chemically defined serum-free medium (SF) or a xenogenic-free human pooled platelet lysate medium (hPL) suitable for cell therapy and both supplemented with CTGF, TGFβ-3, BMP-12 and ascorbic acid (AA) soluble factors. Human ASCs were isolated from 4 healthy donors and they were inducted to differentiate until 14 days in both hPL and SF tenogenic media (hPL-TENO and SF-TENO). Cell viability and immunophenotype profile were analysed to evaluate mesenchymal stem cell (MSC) characteristics in both xenogenic-free media. Moreover, the expression of stemness and tendon-related markers upon cell differentiation by RT-PCR, protein staining and cytofluorimetric analysis were also performed. Our results showed the two xenogenic-free media well support cell viability of ASCs and maintain their MSC nature as demonstrated by their typical immunophenototype profile and by the expression of NANOG, OCT4 and Ki67 genes. Moreover, both hPL-TENO and SF-TENO expressed significant high levels of the tendon-related genes SCX, COL1A1, COL3A1, COMP, MMP3 and MMP13 already at early time points in comparison to the respective controls. Significant up-regulations in scleraxis, collagen and tenomodulin proteins were also demonstrated at in both differentiated SF and hPL ASCs. In conclusion, we demonstrated firstly the feasibility of both serum and xenogenic-free media tested to culture ASCs moving forward the GMP-compliant approaches for clinical scale expansion of human MSCs needed for therapeutical application of stem cells. Moreover, a combination of CTGF, BMP-12, TGFβ3 and AA factors strongly and rapidly induce human ASCs to differentiate into tenocyte-like cells.

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<![CDATA[Mechanisms underpinning the permanent muscle damage induced by snake venom metalloprotease]]> https://www.researchpad.co/article/5c59febed5eed0c484135375

Snakebite is a major neglected tropical health issue that affects over 5 million people worldwide resulting in around 1.8 million envenomations and 100,000 deaths each year. Snakebite envenomation also causes innumerable morbidities, specifically loss of limbs as a result of excessive tissue/muscle damage. Snake venom metalloproteases (SVMPs) are a predominant component of viper venoms, and are involved in the degradation of basement membrane proteins (particularly collagen) surrounding the tissues around the bite site. Although their collagenolytic properties have been established, the molecular mechanisms through which SVMPs induce permanent muscle damage are poorly understood. Here, we demonstrate the purification and characterisation of an SVMP from a viper (Crotalus atrox) venom. Mass spectrometry analysis confirmed that this protein is most likely to be a group III metalloprotease (showing high similarity to VAP2A) and has been referred to as CAMP (Crotalus atrox metalloprotease). CAMP displays both collagenolytic and fibrinogenolytic activities and inhibits CRP-XL-induced platelet aggregation. To determine its effects on muscle damage, CAMP was administered into the tibialis anterior muscle of mice and its actions were compared with cardiotoxin I (a three-finger toxin) from an elapid snake (Naja pallida) venom. Extensive immunohistochemistry analyses revealed that CAMP significantly damages skeletal muscles by attacking the collagen scaffold and other important basement membrane proteins, and prevents their regeneration through disrupting the functions of satellite cells. In contrast, cardiotoxin I destroys skeletal muscle by damaging the plasma membrane, but does not impact regeneration due to its inability to affect the extracellular matrix. Overall, this study provides novel insights into the mechanisms through which SVMPs induce permanent muscle damage.

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<![CDATA[The tumor suppressor p53 can promote collective cellular migration]]> https://www.researchpad.co/article/5c5df35cd5eed0c4845811ac

Loss of function of the tumor suppressor p53 is known to increase the rate of migration of cells transiting the narrow pores of the traditional Boyden chamber assay. Here by contrast we investigate how p53 impacts the rate of cellular migration within a 2D confluent cell layer and a 3D collagen-embedded multicellular spheroid. We use two human carcinoma cell lines, the bladder carcinoma EJ and the colorectal carcinoma HCT116. In the confluent 2-D cell layer, for both EJ and HCT cells the migratory speeds and effective diffusion coefficients for the p53 null cells were significantly smaller than in p53-expressing cells. Compared to p53 expressers, p53-null cells exhibited more organized cortical actin rings together with reduced front-rear cell polarity. Furthermore, loss of p53 caused cells to exert smaller traction forces upon their substrates, and reduced formation of cryptic lamellipodia. In the 3D multicellular spheroid, loss of p53 consistently reduced collective cellular migration into surrounding collagen matrix. As regards the role of p53 in cellular migration, extrapolation from the Boyden chamber assay to other cellular microenvironments is seen to be fraught even in terms of the sign of the effect. Together, these paradoxical results show that the effects of p53 on cellular migration are context-dependent.

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<![CDATA[Connective tissue growth factor mediates transforming growth factor β-induced collagen expression in human endometrial stromal cells]]> https://www.researchpad.co/article/5c59feb0d5eed0c4841352f2

Background

Adenomyosis is a medical condition defined by the abnormal presence of endometrial tissue within the myometrium, in which fibrosis occurs with new collagen deposition and myofibroblast differentiation. In this study, the effect of several mediators and growth factors on collagen expression was investigated on human endometrial stromal cells (fibroblasts) derived from adenomyotic endometrium.

Experimental approach

RT-PCR, Western blot analysis, pharmacological interventions and siRNA interference were applied to primary cultured human endometrial stromal cells (fibroblasts). Immunohistochemistry was used to analyze protein expression in adenomyotic endometrium tissue specimens.

Results

Of the tested mediators, transforming growth factor β1 (TGFβ1) and its isoforms were effective to induce collagen and connective tissue growth factor (CTGF) expression. Collagen and CTGF induction by TGFβ1 could be reduced by the inhibitors targeting DNA transcription, protein translation, and Smad2/3 signaling. Interestingly, TGFβ1 induced Smad2/3 phosphorylation and CTGF mRNA expression, but not collagen mRNA expression, suggesting that TGFβ1 mediates collagen expression through CTGF induction and Smad2/3 activation. In parallel, TGFβ1 and CTGF also induced expression of heat shock protein (HSP) 47, a protein required for the synthesis of several types of collagens. However, only CTGF siRNA knockdown, could compromise TGFβ1-induced collagen expression. Finally, the immunohistochemistry revealed vimentin- and α-SMA-positive staining for (myo)fibroblasts, TGFβ1, collagen, and CTGF in the subepithelial stroma region of human adenomyotic endometria.

Conclusion and implications

We reveal here that TGFβ1, collagen, and CTGF are expressed in the stroma of adenomyotic endometria and demonstrate that TGFβ1 can induce collagen production in endometrium-derived fibroblasts through cellular Smad2/3-dependent signaling pathway and CTGF expression, suggesting that endometrial TGFβ may take part in the pathogenesis of adenomyosis and ectopic endometrium may participate in uterine adenomyosis.

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<![CDATA[Medieval mummies of Zeleny Yar burial ground in the Arctic Zone of Western Siberia]]> https://www.researchpad.co/article/5c79afdad5eed0c4841e3874

Notwithstanding the pioneering achievements of studies on arctic mummies in Siberia, there are insufficient data for any comprehensive understanding of the bio-cultural details of medieval people living in the region. In the Western Siberian arctic, permafrost mummies have been found in 12th to 13th century graves located in the Zeleny Yar (Z-Y) burial ground (66°19'4.54"С; 67°21'13.54"В). In 2013–2016, we were fortunate to be able to excavate that cemetery, locating a total of 47 burials, including cases of mummification. Some of these mummies had been wrapped in a multi-layered birch-bark cocoon. After removal of the cocoon, we conducted interdisciplinary studies using various scientific techniques. Gross anatomical examination and CT radiography showed that the internal organs were still well preserved inside the body cavities. Under light and electron microscopy, the histological findings were very similar to those for naturally mummified specimens discovered in other countries. Ancient DNA analysis showed that the Z-Y mummies’ mtDNA haplotypes belong to five different haplogroups, namely U5a (#34), H3ao (#53), D (#67–1), U4b1b1 (#67–2), and D4j8 (#68), which distinguish them for their unique combination of Western- and Eastern Siberia-specific mtDNA haplogroups. Our interdisciplinary study obtained fundamental information that will form the foundation of successful future investigations on medieval mummies found in the Western Siberian arctic.

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<![CDATA[Effect of photobiomodulation and exercise on early remodeling of the Achilles tendon in streptozotocin-induced diabetic rats]]> https://www.researchpad.co/article/5c61e8d6d5eed0c48496f20d

The aim of this study was to compare the treatment effects of laser photobiomodulation (LPBM) therapy and aerobic exercise on the biomechanical properties, tissue morphology and the expression of tendon matrix molecules during early remodeling of Achilles tendon (AT) injury in diabetic rats. Animals were randomly assigned to five groups: injured non diabetic (I, n = 15), injured diabetic (ID, n = 15), injured diabetic plus LPBM (IDL, n = 16), injured diabetic plus aerobic exercise (IDE, n = 16) and injured diabetic plus aerobic exercise and LPBM (IDEAL, n = 17). Type 1 diabetes was induced via a single intravenous injection of Streptozotocin at a dose of 40 mg/kg. A partial tenotomy was performed in the right AT. LPBM was performed with an indium-gallium-aluminum-phosphide 660 nm 10 mW laser device (spot size 0.04 cm2, power density 250 mW/cm2, irradiation duration 16 s, energy 0.16 J, energy density 4 J/cm2) on alternate days for a total of 9 sessions over 3 weeks (total energy 1.44 J), using a stationary contact technique to a single point over the dorsal aspect of the AT. Moderate aerobic exercise was performed on a motorized treadmill (velocity 9 m/min for 60 minutes). At 3 weeks post-injury, biomechanical analyzes as well as assessment of fibroblast number and orientation were performed. Collagen 1 (Col1) and 3 (Col3) and matrix metalloproteinases (MMPs) -3 and 13 protein distributions were studied by immunohistochemistry; while Col1 and Col3 and MMP-2 and 9 gene expression were assessed by quantitative RT-PCR (qRT-PCR). IDEAL exhibited significant increases in several biomechanical parameters in comparison to the other groups. Moreover, IDEAL presented stronger Col1 immunoreactivity when compared to ID, and weaker Col3 immunoreactivity than IDE. Both IDL and IDEAL demonstrated weaker expression of MMP-3 in comparison to I, while IDL presented no expression of MMP-13 when compared to ID. ID, IDL and IDE showed an increased number of fibroblasts in comparison to I, while IDEAL decreased the number of these cells in comparison to ID and IDE. IDL and IDEAL groups exhibited decreased angular dispersion among the fibroblasts when compared to I. The gene expression results showed that IDE demonstrated a downregulation in Col1 mRNA expression in comparison to I and ID. IDEAL demonstrated upregulation of Col1 mRNA expression when compared to IDL or IDE alone and increased MMP-2 expression when compared to IDL and IDE. MMP-9 expression was upregulated in IDEAL when compared to I, IDL and IDE. Our results suggest a beneficial interaction of combining both treatment strategies i.e., aerobic exercise and LPBM, on the biomechanical properties, tissue morphology and the expression of matrix molecules in diabetic tendons.

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<![CDATA[A simplified flow cytometric method for detection of inherited platelet disorders—A comparison to the gold standard light transmission aggregometry]]> https://www.researchpad.co/article/5c52184bd5eed0c484797a80

Background

Flow cytometric platelet activation has emerged as an alternative diagnostic test for inherited platelet disorders. It is, however, labor intensive and few studies have directly compared the performance of flow cytometric platelet activation (PACT) to light transmission aggregometry (LTA). The aims of this study were 1/ to develop a simplified flow cytometric platelet activation assay using microtiter plates and 2/ to correlate the outcome to gold standard method LTA, and to clinical bleeding assessment tool scores (BAT score).

Methods

The PACT method was developed in microtiter plates using adenosine diphosphate (ADP), collagen-derived peptide (CRP-XL) and thrombin receptor activator for peptide 6 (TRAP-6) as agonists. Antibodies against GPIIb-IIIa activation epitope (PAC1), P-selectin (CD62P) and lysosome-associated membrane glycoprotein 3 (LAMP3; CD63) were used as platelet activation markers. Sixty-six patients referred to the coagulation unit for bleeding symptoms were included in this single-center observational study. Platelet activation was determined by PACT and LTA. The results of both methods were correlated to BAT score.

Results

A two-by-two analysis using Cohen’s kappa analysis gave moderate agreement between LTA and PACT (82%, kappa = 0.57), when PACT analysis with ADP and CRP-XL was compared to LTA. Using LTA as reference method, positive predictive value was 70% and negative predictive value was 87%. A substantial number of patients had high BAT score and normal LTA and PACT results. Patients with abnormal LTA or PACT results had higher BAT score than patients with normal results, but the difference was not significant.

Conclusions

The performance in microtiter plates simplified the PACT method and enabled analysis of more patients at the same time. Our results indicate that with modification of the current PACT assay, a higher negative predictive value can be obtained. Furthermore, with comparable result to LTA the PACT could be used as a screening assay for inherited platelet disorders.

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<![CDATA[Increased von Willebrand factor parameters in children with febrile seizures]]> https://www.researchpad.co/article/5c37b7a6d5eed0c4844907c3

Introduction

Primary blood coagulation and wound sealing are orchestrated by von Willebrand factor (VWF), a large multimeric glycoprotein. Upon release of arginine vasopressin (AVP), VWF containing high molecular weight multimers is secreted. By measuring copeptin, the C-terminal part of the AVP prohormone, we recently found strongly increased AVP levels in children with febrile seizures (FS) as compared to children with fever but without seizures. It is unknown if increased AVP levels in FS are of any biological function. Therefore, our a priori hypothesis was that children with FS have increased VWF parameters in parallel with higher AVP levels.

Methods

We conducted a prospective, cross-sectional study of children aged between 6 months and 5 years. Children that presented at our emergency department with fever or a recent FS (within four hours) were evaluated to be included to the study. We measured serum copeptin and VWF parameters, including analyses of VWF:Antigen (WVF:Ag), VWF:collagen binding activity (VWF:CB) and VWF multimers in children with FS, febrile infections without seizures and additionally, in a non-febrile control group.

Results

We included 54 children in our study, 30 with FS, 10 in the febrile control group, and 14 in the non-febrile control group. Serum copeptin levels were significantly higher in children with FS (median [IQR] 24.73 pmol/l [13.65–68.65]) compared to the febrile control group (5.66 pmol/l [4.15–8.07], p = 0.002) and the non-febrile control group (4.78 pmol/l [3.33–5.3], p<0.001). VWF:CB levels were also significantly higher in children with FS (VWF:CB 2.29 U/ml [1.88–2.97]) as compared to the febrile (VWF:CB 1.41 U/ml [1.27–1.93], p = 0.048) and the non-febrile control group (VWF:CB 1.15 U/ml [0.98–1.21], p<0.001). VWF:Ag tended to be higher in children with FS compared to both control groups. Multivariate regression analysis revealed FS and copeptin as major determinants of VWF:CB.

Conclusions

Our results suggest that increased secretion of AVP in children with FS is associated with higher plasma levels of VWF parameters. Especially VWF:CB may serve as additional biomarker in the diagnosis of FS.

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<![CDATA[Dorset Pre-Inuit and Beothuk foodways in Newfoundland, ca. AD 500-1829]]> https://www.researchpad.co/article/5c3d0114d5eed0c4840381ce

Archaeological research on the Canadian island of Newfoundland increasingly demonstrates that the island’s subarctic climate and paucity of terrestrial food resources did not restrict past Pre-Inuit (Dorset) and Native American (Beothuk) hunter-gatherer populations to a single subsistence pattern. This study first sought to characterize hunter-gatherer diets over the past 1500 years; and second, to assess the impact of European colonization on Beothuk lifeways by comparing the bone chemistry of Beothuk skeletal remains before and after the intensification of European settlement in the early 18th century. We employed radiocarbon dating and stable carbon and nitrogen isotope ratio analysis of bulk bone collagen from both Dorset (n = 9) and Beothuk (n = 13) cultures, including a naturally mummified 17th century Beothuk individual. Carbon and nitrogen isotope analysis of 108 faunal samples from Dorset and Beothuk archaeological sites around the island were used as a dietary baseline for the humans. We combined our results with previously published isotope data and radiocarbon dates from Dorset (n = 12) and Beothuk (n = 18) individuals and conducted a palaeodietary analysis using Bayesian modelling, cluster analysis and comparative statistical tests. Dorset diets featured more marine protein than those of the Beothuk, and the diets of Beothuk after the 18th century featured less high trophic level marine protein than those of individuals predating the 18th century. Despite inhabiting the same island, Dorset and Beothuk cultures employed markedly different dietary strategies, consistent with interpretations of other archaeological data. Significantly, European colonization had a profound effect on Beothuk lifeways, as in response to the increasing European presence on the coast, the Beothuk relied more extensively on the limited resources of the island’s boreal forests and rivers.

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<![CDATA[Small molecule inhibition of IRE1α kinase/RNase has anti-fibrotic effects in the lung]]> https://www.researchpad.co/article/5c3fa5c3d5eed0c484ca83f8

Endoplasmic reticulum stress (ER stress) has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a disease of progressive fibrosis and respiratory failure. ER stress activates a signaling pathway called the unfolded protein response (UPR) that either restores homeostasis or promotes apoptosis. The bifunctional kinase/RNase IRE1α is a UPR sensor/effector that promotes apoptosis if ER stress remains high and irremediable (i.e., a “terminal” UPR). Using multiple small molecule inhibitors against IRE1α, we show that ER stress-induced apoptosis of murine alveolar epithelial cells can be mitigated in vitro. In vivo, we show that bleomycin exposure to murine lungs causes early ER stress to activate IRE1α and the terminal UPR prior to development of pulmonary fibrosis. Small-molecule IRE1α kinase-inhibiting RNase attenuators (KIRAs) that we developed were used to evaluate the contribution of IRE1α activation to bleomycin-induced pulmonary fibrosis. One such KIRA—KIRA7—provided systemically to mice at the time of bleomycin exposure decreases terminal UPR signaling and prevents lung fibrosis. Administration of KIRA7 14 days after bleomycin exposure even promoted the reversal of established fibrosis. Finally, we show that KIRA8, a nanomolar-potent, monoselective KIRA compound derived from a completely different scaffold than KIRA7, likewise promoted reversal of established fibrosis. These results demonstrate that IRE1α may be a promising target in pulmonary fibrosis and that kinase inhibitors of IRE1α may eventually be developed into efficacious anti-fibrotic drugs.

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<![CDATA[Increased water intake reduces long-term renal and cardiovascular disease progression in experimental polycystic kidney disease]]> https://www.researchpad.co/article/5c3667b9d5eed0c4841a61de

Polycystic kidney disease (PKD) is the most common inherited cause of kidney failure and currently has limited treatment options. Increasing water intake reduces renal cyst growth in the pck rat (a genetic ortholog of autosomal recessive PKD) but it is not clear if this beneficial effect is present in other models of PKD. In this study, we tested the hypothesis that high water intake (HWI) reduces the progression of cystic renal disease in Lewis polycystic kidney (LPK) rats (a genetic ortholog of human nephronophthisis-9). Groups of female and male LPK (n = 8–10 per group) and Lewis (n = 4 per group) rats received water ad libitum supplemented with or without 5% glucose [to simulate HWI or normal water intake (NWI) respectively] from postnatal weeks 3 to 16. Water intake increased ~1.3-fold in the LPK+HWI group compared to LPK+NWI rats between weeks 3 to 10 but the differences were not significant at later timepoints. In LPK rats, HWI reduced the increases in the kidney to body weight ratio by 54% at week 10 and by 42% at week 16 compared to NWI (both p<0.01). The reduction in kidney enlargement was accompanied by decreases in the percentage renal cyst area, percentage renal interstitial collagen and proteinuria (all p<0.05). At week 16, HWI reduced systolic blood pressure and the heart to body to weight ratio by 16% and 21% respectively in males LPK rats (both p<0.01). In conclusion, a modest increase in water intake during the early phase of disease was sufficient to attenuate renal cystic disease in LPK rats, with secondary benefits on hypertension and cardiovascular disease. These data provide further preclinical evidence that increased water intake is a potential intervention in cystic renal diseases.

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<![CDATA[Bioprinted liver provides early insight into the role of Kupffer cells in TGF-β1 and methotrexate-induced fibrogenesis]]> https://www.researchpad.co/article/5c3667efd5eed0c4841a69e6

Hepatic fibrosis develops from a series of complex interactions among resident and recruited cells making it a challenge to replicate using standard in vitro approaches. While studies have demonstrated the importance of macrophages in fibrogenesis, the role of Kupffer cells (KCs) in modulating the initial response remains elusive. Previous work demonstrated utility of 3D bioprinted liver to recapitulate basic fibrogenic features following treatment with fibrosis-associated agents. In the present study, culture conditions were modified to recapitulate a gradual accumulation of collagen within the tissues over an extended exposure timeframe. Under these conditions, KCs were added to the model to examine their impact on the injury/fibrogenic response following cytokine and drug stimuli. A 28-day exposure to 10 ng/mL TGF-β1 and 0.209 μM methotrexate (MTX) resulted in sustained LDH release which was attenuated when KCs were incorporated in the model. Assessment of miR-122 confirmed early hepatocyte injury in response to TGF-β1 that appeared delayed in the presence of KCs, whereas MTX-induced increases in miR-122 were observed when KCs were incorporated in the model. Although the collagen responses were mild under the conditions tested to mimic early fibrotic injury, a global reduction in cytokines was observed in the KC-modified tissue model following treatment. Furthermore, gene expression profiling suggests KCs have a significant impact on baseline tissue function over time and an important modulatory role dependent on the context of injury. Although the number of differentially expressed genes across treatments was comparable, pathway enrichment suggests distinct, KC- and time-dependent changes in the transcriptome for each agent. As such, the incorporation of KCs and impact on baseline tissue homeostasis may be important in recapitulating temporal dynamics of the fibrogenic response to different agents.

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<![CDATA[Suppression of angiotensin II-induced pathological changes in heart and kidney by the caveolin-1 scaffolding domain peptide]]> https://www.researchpad.co/article/5c269747d5eed0c48470f1b0

Dysregulation of the renin-angiotensin system leads to systemic hypertension and maladaptive fibrosis in various organs. We showed recently that myocardial fibrosis and the loss of cardiac function in mice with transverse aortic constriction (TAC) could be averted by treatment with the caveolin-1 scaffolding domain (CSD) peptide. Here, we used angiotensin II (AngII) infusion (2.1 mg/kg/day for 2 wk) in mice as a second model to confirm and extend our observations on the beneficial effects of CSD on heart and kidney disease. AngII caused cardiac hypertrophy (increased heart weight to body weight ratio (HW/BW) and cardiomyocyte cross-sectional area); fibrosis in heart and kidney (increased levels of collagen I and heat shock protein-47 (HSP47)); and vascular leakage (increased levels of IgG in heart and kidney). Echocardiograms of AngII-infused mice showed increased left ventricular posterior wall thickness (pWTh) and isovolumic relaxation time (IVRT), and decreased ejection fraction (EF), stroke volume (SV), and cardiac output (CO). CSD treatment (i.p. injections, 50 μg/mouse/day) of AngII-infused mice significantly suppressed all of these pathological changes in fibrosis, hypertrophy, vascular leakage, and ventricular function. AngII infusion increased β1 and β3 integrin levels and activated Pyk2 in both heart and kidney. These changes were also suppressed by CSD. Finally, bone marrow cell (BMC) isolated from AngII-infused mice showed hyper-migration toward SDF1. When AngII-infused mice were treated with CSD, BMC migration was reduced to the basal level observed in cells from control mice. Importantly, CSD did not affect the AngII-induced increase in blood pressure (BP), indicating that the beneficial effects of CSD were not mediated via normalization of BP. These results strongly indicate that CSD suppresses AngII-induced pathological changes in mice, suggesting that CSD can be developed as a treatment for patients with hypertension and pressure overload-induced heart failure.

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<![CDATA[Coastal complexity: Ancient human diets inferred from Bayesian stable isotope mixing models and a primate analogue]]> https://www.researchpad.co/article/5c2544e3d5eed0c48442baa9

An extensive ecological literature applies stable isotope mixing models to derive quantitative dietary reconstructions from isotope ratios of consumer tissues. While this approach works well for some organisms, it is challenging for consumers with complex, varied diets, including humans; indeed, many archaeologists have avoided the use of mixing models because uncertainties in model outputs are sufficiently large that the findings are not helpful in understanding ancient lifeways. Here, we exploit an unparalleled opportunity to evaluate the feasibility of dietary quantification in a nutritionally and isotopically complex context on the Cape Peninsula, South Africa. Delta values (δ13C and δ15N) of 213 indigenous food samples enable us to characterise four food groups: terrestrial plants, terrestrial vertebrates, marine invertebrates and marine vertebrates. A recent study of baboons that consumed marine and terrestrial foods provides insight into the relationship between such foods and consumer tissue isotopes. We use this information to refine our interpretation of δ15N and especially δ13C in bone collagen from 35 archaeological hunter-gatherers, achieving better estimates of the relative importance of marine and terrestrial foods in the diet than has hitherto been possible. Based on Bayesian stable isotope mixing model (SIMM) outputs, we infer that the trophic enrichment factor (TEF) for δ13Cbone collagen in these coastal humans is closer to +3 than +5‰. In the most 13C- and 15N-rich individuals, 65–98% of bone collagen (95% credible intervals) derived from marine foods. Conversely, in 13C and 15N-poor individuals, 7–44% of bone collagen derived from marine foods. The uncertainties discussed here highlight the need for caution when implementing SIMMs in studies of consumers with complex diets. To our knowledge, this work constitutes the most detailed and most tightly constrained study of this problem to date.

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<![CDATA[Analysis of bone osteometry, mineralization, mechanical and histomorphometrical properties of tibiotarsus in broiler chickens demonstrates a influence of dietary chickpea seeds (Cicer arietinum L.) inclusion as a primary protein source]]> https://www.researchpad.co/article/5c196697d5eed0c484b52559

This study was focused on analyzing the effects of dietary inclusion of raw chickpea seed as a replacement of soybean meal as a primary protein source on bone structure in broiler chickens. Broiler chickens (n = 160) received in their diet either soybean meal (SBM) or raw chickpea seeds (CPS) as a primary protein source throughout the whole rearing period (n = 80 in each group). On the 42th day randomly selected chickens from each group (n = 8) were slaughtered. Collected tibiotarsus were subjected to examination of the biomechanical characteristics of bone mid-diaphysis, microstructure of the growth plate and articular cartilages; the analysis of mineral content and crystallinity of mineral phase, and the measurements of thermal stability of collagen in hyaline cartilage were also carried out. The inclusion of chickpea seeds resulted in increase of bone osteometric parameters (weight, length and mid-diaphysis cross-sectional area) and mechanical endurance (yield load, ultimate load, stiffness, Young modulus). However, when loads were adjusted to bone shape (yield and ultimate stress) both groups did not differ. Mineral density determined by means of densitometric measurements did not differ between groups, however the detailed analysis revealed the differences in the macro- and microelements composition. The results of FT-IR and XRD analyses showed no effect of diet type on mineral phase crystallinity and hydroxyapatite nanocrystallites size. In trabecular bone, the increase of real bone volume (BV/TV) and number of trabeculae was observed in the CPS group. Total thickness of articular cartilage was the same in both groups, save the transitional zone, which was thicker in the SBM group. The total thickness of the growth plate cartilage was significantly increased in the CPS group. The area of the most intense presence of proteoglycans was wider in the SBM group. The structural analysis of fibrous components of bone revealed the increase of fraction of thin, immature collagen content in articular cartilage, trabeculae and compact bone in the CPS group. The dietary inclusion of CPS affected the thermal stability of collagen, as decrease of net denaturation enthalpy was observed. This study showed a beneficial effect of CPS on the skeletal development, improving the overall bone development and the microarchitecture of cancellous bone. It suggests that CPS can be a promising replacement for SBM in broilers feeding in the aspect of animal welfare related to the development of the skeletal system.

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<![CDATA[Feasibility of the annulus fibrosus repair with in situ gelating hydrogels – A biomechanical study]]> https://www.researchpad.co/article/5c12cf9ad5eed0c484914a3a

The surgical standard of care for lumbar discectomy leaves the annulus fibrosus (AF) defect unrepaired, despite considerable risk for a recurrent herniation. Identification of a viable defect repair strategy has until now been elusive. The scope of this ex vivo biomechanical study was to evaluate crosslinking hydrogels as potentially promising AF defect sealants, and provide a baseline for their use in combination with collagen scaffolds that restore disc volume. This study directly compared genipin crosslinked fibrin hydrogel (FibGen) as a promising preclinical candidate against a clinically available adhesive composed of glutaraldehyde and albumin (BioGlue). Forty-two bovine coccygeal functional spine units (FSU) were randomly allocated into four groups, namely untreated (control, n = 12), repaired with either one of the tested hydrogels (BioGlue, n = 12; FibGen, n = 12), or FibGen used in combination with a collagen hydrogel scaffold (FibGen+Scaffold, n = 6). All specimens underwent a moderate mechanical testing protocol in intact, injured and repaired states. After completion of the moderate testing protocol, the samples underwent a ramp-to-failure test. Lumbar discectomy destabilized the FSU as quantified by increased torsional range of motion (28.0° (19.1, 45.1) vs. 41.39° (27.3, 84.9), p<0.001), torsional neutral zone (3.1° (1.2, 7.7) vs. 4.8° (2.1, 12.1), Z = -3.49, p < 0.001), hysteresis(24.4 J (12.8, 76.0) vs. 27.6 J (16.4, 54.4), Z = -2.61, p = 0.009), with loss of both disc height (7.0 mm (5.0, 10.5) vs 6.1 mm (4.0, 9.3), Z = -5.16, p < 0.001) and torsional stiffness (0.76 Nmdeg-1 (0.38, 1.07) vs. 0.66 Nmdeg-1 (0.38, 0.97), Z = -3.98, p < 0.001). Most FibGen repaired AF endured the entire testing procedure whereas only a minority of BioGlue repaired AF and all FibGen+Scaffold repaired AF failed (6/10 vs. 3/12 vs. 0/6 respectively, p = 0.041). Both BioGlue and FibGen+Scaffold repaired AF partially restored disc height (0.47 mm (0.07, 2.41), p = 0.048 and 1.52 mm (0.41, 2.57), p = 0.021 respectively) compared to sham treatment (0.08 mm (-0.63, 0.88)) whereas FibGen-only repaired AF had no such effect (0.04 mm (-0.73, 1.13), U = 48.0, p = 1). The AF injury model demonstrated considerable change of FSU mechanics that could be partially restored by use of an AF sealant. While inclusion of a volumetric collagen scaffold led to repair failure, use of FibGen alone demonstrated clinically relevant promise for prevention of mechanical reherniation, outperforming an FDA approved sealant in this ex vivo test series.

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<![CDATA[Serum biomarkers of collagen turnover as potential diagnostic tools in diffuse systemic sclerosis: A cross-sectional study]]> https://www.researchpad.co/article/5c0ed760d5eed0c484f1400f

Background

Systemic sclerosis (SSc) is characterized by excessive fibrosis throughout the body. This leads to the release of extracellular matrix (ECM) fragments into circulation, where they may be quantified as biomarkers. The objectives were to investigate levels of ECM turnover biomarkers and the diagnostic power of these.

Methods

Diffuse SSc patients (n = 40) fulfilling the ACR/EULAR 2013 classification criteria and asymptomatic controls were included. Patients were divided into early (<2 years of symptoms; n = 20) and late (>10 years of symptoms; n = 20) diffuse SSc. Biomarkers of type I (C1M), III (C3A, C3M), IV (C4M), V (C5M) and VI (C6M) collagen degradation and type I (PRO-C1), II (PRO-C2), III (PRO-C3), IV (PRO-C4), V (PRO-C5) and VI (PRO-C6) collagen formation were measured in serum.

Repeated measures ANOVA was used to test for differences in biomarker levels and the area under the receiver operating characteristic curve (AUC) was used to investigate the ability of the biomarkers to separate groups.

Results

In early diffuse SSc, formation biomarkers of type III, IV, V and VI collagen were significantly increased compared to asymptomatic controls (p<0.0001). Moreover, in early diffuse SSc formation biomarkers of type III, V and VI collagen were significantly increased compared to late diffuse SSc (p = 0.0006, 0.003 and 0.004, respectively). Type I (p<0.0001), III (C3M: p = 0.001, and C3A: p = 0.02), IV (p<0.0001) and VI (p<0.0001) collagen degradation biomarkers significantly increased in early diffuse SSc compared to controls. C4M, C6M, PRO-C4, PRO-C5 and PRO-C6 had an AUC of >0.85 when assessing asymptomatic controls vs. diffuse SSc. Biomarkers of type VI collagen (PRO-C6 and C6M) turnover had the best separation with an AUC’s of >0.90.

Conclusion

Formation biomarkers of ECM turnover were shown to be significantly different between asymptomatic controls and diffuse SSc. This pilot study suggest that serological biomarkers of the ECM turnover is potentially applicable in SSc.

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