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Background The incidence of Achilles tendinopathy is high and underlying etiology as well as biochemical and morphological pathology associated with the disease is largely unknown. The aim of the present study was to describe biochemical and morphological differences in chronic Achilles tendinopathy. The expressions of growth factors, inflammatory mediators and tendon morphology were determined in both chronically diseased and healthy tendon parts. Methods Thirty Achilles tendinopathy patients were randomized to an expression-study ( n = 16) or a structural-study ( n = 14). Biopsies from two areas in the Achilles tendon were taken and structural parameters: fibril density, fibril size, volume fraction of cells and the nucleus/cytoplasm ratio of cells were determined. Further gene expressions of various genes were analyzed. Results Significantly smaller collagen fibrils and a higher volume fraction of cells were observed in the tendinopathic region of the tendon. Markers for collagen and its synthesis collagen 1, collagen 3, fibronectin, tenascin-c, transforming growth factor-β fibromodulin, and markers of collagen breakdown matrix metalloproteinase-2, matrix metalloproteinase-9 and metallopeptidase inhibitor-2 were significantly increased in the tendinopathic region. No altered expressions of markers for fibrillogenesis, inflammation or wound healing were observed. Conclusion The present study indicates that an increased expression of factors stimulating the turnover of connective tissue is present in the diseased part of tendinopathic tendons, associated with an increased number of cells in the injured area as well as an increased number of smaller and thinner fibrils in the diseased tendon region. As no fibrillogenesis, inflammation or wound healing could be detected, the present data supports the notion that tendinopathy is an ongoing degenerative process. Trial registration Current Controlled Trials ISRCTN20896880

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK-/- cardiomyocytes. Transmission electron microscopy of BK-/- ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK-/- permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK-/- hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK-/- hearts upon I/R injury in the absence of ischemic pre-conditioning (IP), but differed upon IP. While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK-/- hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations.

The exact location of the filtration barrier of the glomerular capillary wall, which consists of an endothelium, a basement membrane and a visceral epithelium, has not yet been determined. Apparent discrepancies between different investigators in the past could be explained if postmortem artifactual tissue changes, due to subnormal blood pressure or anoxia, have taken place in the endothelium before the tissue and tracers have been sufficiently fixed and immobilized by the fixative. To test this supposition, a new method of fixation, which includes a technique to maintain a physiological perfusion pressure and a fixative composed of an oxygen-carrying blood substitute fluid containing glutaraldehyde, was employed combined with contrast enhancement. New observations of the glomerular capillary wall revealed that filamentous plugs (about 90 nm in height) filled the capillary fenestrae and a filamentous surface coat about 60 nm thick covered the interfenestral domains of the endothelial cell. Based on these purely morphological data, we dare to suggest that the fenestral plugs are the primary site of the glomerular filtration barrier – albeit highly speculative, nevertheless a logical location – and consequently that the glomerular filtration process is a ‘tangential-flow’ as opposed to a ‘dead-end’ filtration process. A tangential-flow filtration would minimize ‘clogging’ and ‘concentration polarization’ in the ‘filter’.

A previously unknown ligament, the superficial anulus fibrosus ligament (SAFL), situated on the ventral part of the L5 intervertebral disc (ID) was observed and described from autopsy material. Twenty-eight cadaveric specimens from 12 black and 16 white persons aged 17-30 years were studied during routine forensic autopsies. The anterior longitudinal ligament was separated from the ID and the ventral part of the SAFL was visualized. The SAFL samples were removed, measured and studied with both conventional histology and examination by transmission electron microscopy. The SAFL was a completely separate ligament at the level of the L5-S1 ID situated between the anterior longitudinal ligament and the anulus fibrosus of the ID. The fibers of the ligament were vertically oriented. A difference in distance between the L5-S1 vertebral bodies ventrally was noted in the two groups studied (18.7 +/- 1.2 mm in the black vs. 15.2 +/- 1.0 mm in the white, p < 0.001), indicating a difference in the ventral thickness of the intervertebral disc. Also, there was a difference in the length (black: 17.7 +/- 1.6 mm vs. white: 14.1 +/- 1.1), thickness (black: 3.3 +/- 0.3 mm vs. white: 2.1 +/- 1.9), and the cross-sectional area (black: 58.2 +/- 6.7 mm(2) vs. white: 26.5 +/- 2.7 mm(2), p < 0.001) of the SAFL. Conventional light microscopy revealed no obvious differences. However, transmission electron microscopy revealed notably larger collagen fibril diameter in black than white subjects. In conclusion, the interbody distances were greater in the black group, indicating a greater intervertebral disc thickness, compared to that of the white. Furthermore, the SAFL was significantly longer and thicker in the black than in the white group. Albeit unsubstantiated, these race-specific macroscopic findings may have implications for understanding the etiology of various low back stress problems.

Using histological and ultrastructural techniques the aims of this study were to investigate whether the mineralization pattern and surface microanatomy of the caries–susceptible fissure enamel were different from those on the caries–inactive lingual surface. The material consisted of 31 unerupted third mandibular molars. The specimens were initially grouped into four categories: (1) without, (2) with initial, (3) with almost completed and (4) with completed root formation. One ground section with fissure–like morphology was selected from each tooth. Using water as a medium the observed birefringence was negative along the lingual and fissure transverses in specimens with almost completed and with completed root formation, while the observed birefringence was positive at different distances in the enamel in sections representing less maturation stages. Qualitative imbibition studies revealed hypomineralized enamel in the lower part of the fissures in specimens representing almost and completed root formation. Imbibed in quinoline, parts of the hypomineralized enamel behaved like a molecular sieve due to the presence of micropores, indicating that the structural arrangement is different from that in the enamel adjacent to this areas. After division of the sections into a lingual and a buccal part, SEM features were described from lower and upper parts of the buccal fissure wall and on lingual enamel in the area corresponding to the bottom part of the fissure. The surface microanatomy varied greatly. Negative developmental irregularities such as fissures and holes were associated with the immature enamel, while matured enamel – particularly fissures – housed many positive developmental irregularities such as enamel caps and protrusions. The crystal size in the mature specimens appeared smaller and more uniform than the crystals from the immature specimens. Apart from the occurrence of hypomineralized enamel in fissures and numerous positive developmental irregularities on the fissure surface, no major differences between fissure and lingual enamel were noticed, neither with respect to mineralization pattern during final stages of tooth development nor to the degree of surface porosity prior to tooth emergence.

A recent investigation has suggested that the chief cells of the endolymphatic sac produce an endogenous inhibitor of sodium resorption in the kidneys, tentatively named saccin. In the current study, the ultrastructure of the endolymphatic sac and in particular the chief cells are described to demonstrate that this organ fulfils the morphological criteria of a potential endocrine gland. Accordingly, the chief cells are shown to exhibit all the organelles and characteristics of cells that simultaneously synthesize, secrete, absorb and digest proteins.

A previously unknown ligament, the superficial anulus fibrosus ligament (SAFL), situated on the ventral part of the L5 intervertebral disc (ID) was observed and described from autopsy material. Twenty-eight cadaveric specimens from 12 black and 16 white persons aged 17–30 years were studied during routine forensic autopsies. The anterior longitudinal ligament was separated from the ID and the ventral part of the SAFL was visualized. The SAFL samples were removed, measured and studied with both conventional histology and examination by transmission electron microscopy. The SAFL was a completely separate ligament at the level of the L5-S1 ID situated between the anterior longitudinal ligament and the anulus fibrosus of the ID. The fibers of the ligament were vertically oriented. A difference in distance between the L5-S1 vertebral bodies ventrally was noted in the two groups studied (18.7 ± 1.2 mm in the black vs. 15.2 ± 1.0 mm in the white, p < 0.001), indicating a difference in the ventral thickness of the intervertebral disc. Also, there was a difference in the length (black: 17.7 ± 1.6 mm vs. white: 14.1 ± 1.1), thickness (black: 3.3 ± 0.3 mm vs. white: 2.1 ± 1.9), and the cross-sectional area (black: 58.2 ± 6.7 mm 2 vs. white: 26.5 ± 2.7 mm 2 , p < 0.001) of the SAFL. Conventional light microscopy revealed no obvious differences. However, transmission electron microscopy revealed notably larger collagen fibril diameter in black than white subjects. In conclusion, the interbody distances were greater in the black group, indicating a greater intervertebral disc thickness, compared to that of the white. Furthermore, the SAFL was significantly longer and thicker in the black than in the white group. Albeit unsubstantiated, these race-specific macroscopic findings may have implications for understanding the etiology of various low back stress problems.

TAS-102 (trifluridine–tipiracil) has shown a significant overall survival benefit compared with placebo in patients with chemorefractory metastatic colorectal cancer. Inspired by the encouraging results of a small phase 1–2 study, C-TASK FORCE, which evaluated the combination of TAS-102 plus bevacizumab in patients with chemorefractory metastatic colorectal cancer, we aimed to compare the efficacy of TAS-102 plus bevacizumab versus TAS-102 monotherapy in patients receiving refractory therapy for metastatic colorectal cancer . This investigator-initiated, open-label, randomised, phase 2 study enrolled patients (aged ≥18 years) with metastatic colorectal from four cancer centres in Denmark. The main inclusion criteria were histopathologically confirmed metastatic colorectal cancer refractory or intolerant to a fluoropyrimidine, irinotecan, oxaliplatin, and cetuximab or panitumumab (only for RAS wild-type), and WHO performance status of 0 or 1. Previous therapy with bevacizumab, aflibercept, ramucirumab, or regorafenib was allowed but not mandatory. Participants were enrolled and randomly assigned (1:1) in block sizes of two, four, or six by a web-based tool to receive oral TAS-102 (35 mg/m2 twice daily on days 1–5 and 8–12 every 28 days) alone or combined with intravenous bevacizumab (5 mg/kg on days 1 and 15) until progression, unacceptable toxicity, or patient decision to withdraw. Treatment assignment was not masked, and randomisation was stratified by institution and RAS mutation status. The primary endpoint was investigator-evaluated progression-free survival. All analyses were based on intention to treat. This trial is registered with EudraCT, 2016–005241–23. From Aug 24, 2017, to Oct 31, 2018, 93 patients were enrolled and randomly assigned to TAS-102 (n=47) or TAS-102 plus bevacizumab (n=46). The clinical cut-off date was Feb 15, 2019, after a median follow-up of 10·0 months (IQR 6·8–14·0). Median progression-free survival was 2·6 months (95% CI 1·6–3·5) in the TAS-102 group versus 4·6 months (3·5–6·5) in the TAS-102 plus bevacizumab group (hazard ratio 0·45 [95% CI 0·29–0·72]; p=0·0015). The most frequent grade 3 or worse adverse event was neutropenia (18 [38%] of 47 in the TAS-102 monotherapy group vs 31 [67%] of 46 in the TAS-102 plus bevacizumab group). Serious adverse events were observed in 21 (45%) patients in the TAS-102 group and 19 (41%) in the TAS-102 plus bevacizumab group. No deaths were deemed treatment related. In patients with chemorefractory metastatic colorectal cancer, TAS-102 plus bevacizumab, as compared with TAS-102 monotherapy, was associated with a significant and clinically relevant improvement in progression-free survival with tolerable toxicity. The combination of TAS-102 plus bevacizumab could be a new treatment option for patients with refractory metastatic colorectal cancer and could be a practice-changing development. Servier.

Secretory vesicles in endocrine cells store hormones such as growth hormone (GH) and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN) and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs) domain protein PICK1 (protein interacting with C kinase 1) as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient Drosophila and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the Drosophila brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a Drosophila model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of peptidergic endocrine cells and support an important role of PICK1/ICA69 in maintenance of metabolic homeostasis.