•PICP and BMP1 are associated with spinal cord compression and neurological symptoms in spinal cord injury.•Suppression of BMP1 eliminates fibrotic scars and inhibits activation and enrichment of ...astrocytes.•TGF-β, TNF-α and IL-1β promote the expression of BMP1.•Delayed inhibition of BMP1 improves axonal regeneration and myelin repair after spinal cord injury.
Fibrotic scars appear after spinal cord injury (SCI) and are mainly composed of fibroblasts and excess extracellular matrix (ECM), including different types of collagen. The temporal and spatial distribution and role of excess collagens and ECM after SCI are not yet fully understood. Here, we identified that the procollagen type I C-terminal propeptide (PICP), a marker of collagen type I deposition, and bone morphogenetic protein 1 (BMP1), a secreted procollagen c-proteinase (PCP) for type I collagen maturation, were significantly elevatedin cerebrospinal fluid of patients with SCI compared with healthy controls, and were associated with spinal cord compression and neurological symptoms. We revealed the deposition of type I collagen in the area damaged by SCI in mice and confirmed that BMP1 was the only expressed PCP and induced collagen deposition. Furthermore, transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) can activate the expression of BMP1. However, inhibition of BMP1 at the acute phase eliminated fibrotic scars in the damaged area and inhibited activation and enrichment of astrocytes, which made the damage difficult to repair and increased hematoma. Unexpectedly, knockdown of Bmp1 by adeno-associated virus or the inhibition of BMP1 biological function by specific inhibitors and monoclonal antibodies at different time points after injury led to distinct therapeutic effects. Only delayed inhibition of BMP1 improved axonal regeneration and myelin repair at the subacute stage post-injury, and led to the recovery of motor function, suggesting that scarring had a dual effect. Early inhibition of the scarring was not conducive to limiting inflammation, while excessive scar formation inhibited the growth of axons. After SCI, the collagen deposition indicators increased in both human cerebrospinal fluid and mouse spinal cord. Therefore, suppression of BMP1 during the subacute phase improves nerve function after SCI and is a potential target for scar reduction.
Background
Type 3 von Willebrand disease (VWD) is a severe bleeding disorder caused by the virtually complete absence of von Willebrand factor (VWF). Pathophysiological mechanisms of VWD like ...defective synthesis, secretion, and clearance of VWF have previously been evaluated using ratios of VWF propeptide (VWFpp) over VWF antigen (VWF:Ag) and factor (F)VIII coagulant activity (FVIII:C) over VWF:Ag.
Objective
To investigate whether the VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios may also be applied to understand the pathophysiological mechanism underlying type 3 VWD and whether VWFpp is associated with bleeding severity.
Methods
European and Iranian type 3 patients were enrolled in the 3WINTERS‐IPS study. Plasma samples and buffy coats were collected and a bleeding assessment tool was administered at enrolment. VWF:Ag, VWFpp, FVIII:C, and genetic analyses were performed centrally, to confirm patients’ diagnoses. VWFpp/VWF:Ag and FVIII:C/VWF:Ag ratios were compared among different variant classes using the Mann‐Whitney test. Median differences with 95% confidence intervals (CI) were estimated using the Hodges‐Lehmann method. VWFpp association with bleeding symptoms was assessed using Spearman’s rank correlation.
Results
Homozygosity/compound heterozygosity for missense variants showed higher VWFpp level and VWFpp/VWF:Ag ratio than homozygosity/compound heterozygosity for null variants (VWFpp median difference, 1.4 IU/dl; 95% CI, 0.2–2.7; P = .016; VWFpp/VWF:Ag median difference, 1.4; 95% CI, 0–4.2; P = .054). FVIII:C/VWF:Ag ratio was similarly increased in both. VWFpp level did not correlate with the bleeding symptoms (r = .024; P = .778).
Conclusions
An increased VWFpp/VWF:Ag ratio is indicative of missense variants, whereas FVIII:C/VWF:Ag ratio does not discriminate missense from null alleles. The VWFpp level was not associated with the severity of bleeding phenotype.
The effect of the
Escherichia coli
(
E. coli
) Rosetta (DE3) system on the expression of recombinant papain-like cysteine protease inhibitors (SnuCalCpIs) was evaluated, and the inhibition mode of ...the expressed inhibitor was determined. SnuCalCpI08 and SnuCalCpI17, which previously had not been expressed in the
E. coli
BL21 (DE3) system due to rare codons of more than 10%, were successfully expressed in
E. coli
Rosetta (DE3) since the strain provides tRNAs for six rare codons. Initially, both inhibitors were expressed as inclusion bodies; however, the water solubility of SnuCalCpI17 could be improved by lowering the incubation temperature, reducing the IPTG concentration, and increasing the induction time. In contrast, the other inhibitor could not be solubilized in water. To validate whether the inhibitor was expressed with correct protein folding, a papain inhibition assay was performed with SnuCalCpI17. SnuCalCpI17 showed a half-maximal inhibitory concentration (IC
50
) of 105.671 ± 9.857 µg/mL and a slow-binding inhibition mode against papain at pH 7.0 with a
K
i
app
of 75.80 μg/mL. The slow-binding inhibitor has a slow dissociation from the inhibitor-target complex, resulting in a long residence time in vivo, and thus can effectively inhibit the target at doses far below the IC
50
of the inhibitor.
Graphical abstract
Key points
• Propeptide inhibitor (SnuCalCpI17) containing rare codons was expressed in E. coli Rosetta (DE3).
• The slow-binding inhibition was shown by plotting the apparent first-order rate constant (k
obs
).
• Protein–protein interaction between SnuCalCpIs and papain was verified by docking simulation.
Higher levels of neurofilament light chain (NfL) and proinflammatory cytokines (i.e., tumor necrosis factor TNF-α) were observed in patients with bipolar disorder (BD) and major depressive disorder ...(MDD). Procollagen type 1 N-terminal propeptide (P1NP), a bone turnover biomarker, is related to MDD. The association among the brain–bone axis, systemic inflammation, and cognitive function remains unclear in severe affective disorders.
Overall, 25 patients with BD, 24 with MDD, and 29 matched controls were enrolled in the current study and underwent the measurements of the NfL, P1NP, and proinflammatory cytokine levels and 1-back and 2-back working memory tasks. Generalized linear models (GLMs) were used to examine the aforementioned biomarkers between the groups and clarify the association with each other.
GLMs showed increased levels of NfL (p = 0.001, p = 0.020) and P1NP (p = 0.050, p = 0.032) in the patients with BD and MDD than in the controls and suggested significant correlations between the NfL level and the mean time of the 2-back working memory task (p = 0.038) and between P1NL and TNF-α levels (p < 0.001).
Our study revealed the dysregulated brain–bone axis, indicated by elevated NfL and P1NP levels, and related cognitive impairment and systemic inflammation in the patients with BD and MDD. Additional studies are necessary to elucidate definite pathomechanisms underlying those conditions.
Purpose
Degradation of collagen has been suggested involved in the pathogenesis of inguinal hernia. In this study, we aim to evaluate circulating biomarkers of procollagen type I N-terminal ...propeptide (PINP), procollagen type III N-terminal propeptide (PIIINP), matrix metalloproteinases (MMP)-2, MMP-9, copper and zinc in primary and recurrent inguinal hernia patients.
Methods
This study included 110 inguinal hernia patients: 45 patients had primary indirect inguinal hernia, 40 patients had primary direct inguinal hernia, 15 patients had recurrent indirect inguinal hernia and 10 patients had recurrent direct inguinal hernia. Additional 45 patients operated for reasons other than hernia were included as a control group. All blood samples were obtained preoperatively. Circulating PINP, PIIINP, MMP-2 and MMP-9 were investigated using enzyme-linked immunoabsorbent assay (ELISA) methods, and copper and zinc were measured using an air acetylene flame atomic absorption spectrometer.
Results
Serum MMP-2 levels in patients with direct and recurrent inguinal hernias were significantly higher than controls. The ratios of PINP/PIIINP decreased more apparent in recurrent indirect or direct inguinal hernia group than primary indirect or direct inguinal hernia group. Based on receiver operating characteristic curve analysis, PINP/PIIINP can effectively diagnose recurrent inguinal hernia from primary inguinal hernia with area under the curve (AUC) of 0.919 for recurrent indirect inguinal hernia and 0.808 for recurrent direct inguinal hernia, respectively.
Conclusion
The ratio of serum PINP/PIIINP was lower in patients with recurrent inguinal hernia, demonstrating more serious damage of collagen metabolism in these patients. Serologic ratio of PINP/PIIINP may be used to identify the presence of recurrent inguinal hernia in patients.
Background
Spinal muscular atrophy (SMA) is caused by genetic defects in the survival motor neuron 1 (SMN1) gene that lead to SMN deficiency. Different SMN‐restoring therapies substantially prolong ...survival and function in transgenic mice of SMA. However, these therapies do not entirely prevent muscle atrophy and restore function completely. To further improve the outcome, we explored the potential of a combinatorial therapy by modulating SMN production and muscle‐enhancing approach as a novel therapeutic strategy for SMA.
Methods
The experiments were performed in a mouse model of severe SMA. A previously reported 25‐mer morpholino antisense oligomer PMO25 was used to restore SMN expression. The adeno‐associated virus‐mediated expression of myostatin propeptide was used to block the myostatin pathway. Newborn SMA mice were treated with a single subcutaneous injection of 40 μg/g (therapeutic dose) or 10 μg/g (low‐dose) PMO25 on its own or together with systemic delivery of a single dose of adeno‐associated virus‐mediated expression of myostatin propeptide. The multiple effects of myostatin inhibition on survival, skeletal muscle phenotype, motor function, neuromuscular junction maturation, and proprioceptive afferences were evaluated.
Results
We show that myostatin inhibition acts synergistically with SMN‐restoring antisense therapy in SMA mice treated with the higher therapeutic dose PMO25 (40 μg/g), by increasing not only body weight (21% increase in male mice at Day 40), muscle mass (38% increase), and fibre size (35% increase in tibialis anterior muscle in 3 month female SMA mice), but also motor function and physical performance as measured in hanging wire test (two‐fold increase in time score) and treadmill exercise test (two‐fold increase in running distance). In SMA mice treated with low‐dose PMO25 (10 μg/g), the early application of myostatin inhibition prolongs survival (40% increase), improves neuromuscular junction maturation (50% increase) and innervation (30% increase), and increases both the size of sensory neurons in dorsal root ganglia (60% increase) and the preservation of proprioceptive synapses in the spinal cord (30% increase).
Conclusions
These data suggest that myostatin inhibition, in addition to the well‐known effect on muscle mass, can also positively influence the sensory neural circuits that may enhance motor neurons function. While the availability of the antisense drug Spinraza for SMA and other SMN‐enhancing therapies has provided unprecedented improvement in SMA patients, there are still unmet needs in these patients. Our study provides further rationale for considering myostatin inhibitors as a therapeutic intervention in SMA patients, in combination with SMN‐restoring drugs.
Myostatin (MSTN) was identified as a negative regulator of skeletal muscle growth. MSTN inhibition by myostatin propeptide (MSPP) increased skeletal muscle mass, myofiber growth and muscle force. ...Thus, this study was designed to produce wild-type porcine MSPP (WT-MSPP) and its mutated form (D75A-MSPP) in yeast Pichia pastoris and to investigate its potential enhancement of myoblast growth and differentiation. In an in vitro study, C2C12 myoblasts were treated with the purified WT-MSPP or D75A-MSPP (10 μg/mL) in either a regular culture medium or in a differentiation medium for 72 h. In an animal trial, post-weaning C57BL/6 mice fed with a high-fat diet (HFD) were administered WT-MSPP or D75A-MSPP for 6 weeks. The results showed that C2C12 myoblasts treated with the purified WT-MSPP or D75A-MSPP could dramatically promote cell proliferation. Both myoD and myogenin were significantly increased (p < .05) after WT-MSPP or D75A-MSPP treatment. D75A-MSPP was particularly more effective than WT-MSPP in promoting myotube formation (p < .05). The post-weaning mice treated with D75A-MSPP significantly increased both body and muscle weights compared with the mock and WT-MSPP groups (p < .05). Furthermore, the mice treatment with D75A-MSPP could prevent increased glucose injection from inducing glucose elevation. Our data indicated that a mutant-type MSPP (D75A-MSPP) was superior to WT-MSPP in effectively enhancing myofiber growth due to the highly resistant to proteolytic cleavage by the bone morphogenetic protein-1/tolloid (BMP-1/TLD) and thus has potential applications for clinical muscle wasting diseases or for increasing muscle mass in meat-producing animals.
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•The originality of this study is production of high-level secreted porcine MSPP in Pichia pastoris yeast culture.•High-level MSPP expression in yeast culture exhibits 25-fold performance compared to that in the E. coli system.•D75A-MSPP performs a highly resistant to proteolytic cleavage by BMP-1/tolloid (BMP-1/TLD) than WT-MSPP.•In C2C12 myoblast cells test, D75A-MSPP exhibits more effective than WT-MSPP in promoting myotube formation.•In an animal trial, post-weaning mice treated with D75A-MSPP significantly increased in both body and muscle weights.
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•Introducing hydrophobic mutation and replacing propeptide improved the expression of trypsin.•An efficient signal peptide was designed in Pichia pastoris.•The key autolysis residue ...K101 was identified from SGT trypsin.•The production of trypsin was 227.65±6.51U·mL−1 and a yield of 185.71±5.68mg·L−1.
Streptomyces griseus trypsin (SGT) possesses enzymatic properties similar to mammalian trypsins and has great potential applications in the leather processing, bioethanol, detergent and pharmaceutical industry. Here, a new strategy was reported for improving its stable, active secretory production through engineering of its propeptide and self-degradation sites. By rationally introducing hydrophobic mutations into the N-terminus of SGT Exmt (R145I), replacing the propeptide with FPVDDDDK and engineering the α-factor signal peptide, trypsin production (amidase activity) was improved to 177.85±2.83U·mL−1 in a 3-L fermenter (a 3.75-fold increase). Subsequently, all of the residues involved in autolysis that were identified by mass spectrometry were mutated and the resulting proteins were evaluated. In particular, the variant tbcf (K101A) demonstrated high stability and production (227.65±6.51U·mL−1 and 185.71±5.68mg·L−1, respectively). The recombinant strain constructed here has great potential for large-scale production of active trypsin.
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