Quality Control of Procollagen in Cells.

Collagen is the most abundant protein in mammals. A unique feature of collagen is its triple-helical structure formed by the Gly-Xaa-Yaa repeats. Three single chains of procollagen make a trimer, and the triple-helical structure is then folded in the endoplasmic reticulum (ER). This unique structure is essential for collagen's functions in vivo, including imparting bone strength, allowing signal transduction, and forming basement membranes. The triple-helical structure of procollagen is stabilized by posttranslational modifications and intermolecular interactions, but collagen is labile even at normal body temperature. Heat shock protein 47 (Hsp47) is a collagen-specific molecular chaperone residing in the ER that plays a pivotal role in collagen biosynthesis and quality control of procollagen in the ER. Mutations that affect the triple-helical structure or result in loss of Hsp47 activity cause the destabilization of procollagen, which is then degraded by autophagy. In this review, we present the current state of the field regarding quality control of procollagen.

A model for collagen secretion by intercompartmental continuities.

Newly synthesized secretory proteins are exported from the endoplasmic reticulum (ER) at specialized subcompartments called exit sites (ERES). Cargoes like procollagen are too large for export by the standard COPII-coated vesicle of 60 nm average diameter. We have previously suggested that procollagen is transported from the ER to the next secretory organelle, the ER-Golgi intermediate compartment (ERGIC), in TANGO1-dependent interorganelle tunnels. In the theoretical model presented here, we suggest that intrinsically disordered domains of TANGO1 in the ER lumen induce an entropic contraction, which exerts a force that draws procollagen toward the ERES. Within this framework, molecular gradients of pH and/or HSP47 between the ER and ERGIC create a force in the order of tens of femto-Newtons. This force is substantial enough to propel procollagen from the ER at a speed of approximately 1 nm · s-1. This calculated speed and the quantities of collagen secreted are similar to its observed physiological secretion rate in fibroblasts, consistent with the proposal that ER export is the rate-limiting step for procollagen secretion. Hence, the mechanism we propose is theoretically adequate to explain how cells can utilize molecular gradients and export procollagens at a rate commensurate with physiological needs.

N-glycan processing selects ERAD-resistant misfolded proteins for ER-to-lysosome-associated degradation.

Efficient degradation of by-products of protein biogenesis maintains cellular fitness. Strikingly, the major biosynthetic compartment in eukaryotic cells, the endoplasmic reticulum (ER), lacks degradative machineries. Misfolded proteins in the ER are translocated to the cytosol for proteasomal degradation via ER-associated degradation (ERAD). Alternatively, they are segregated in ER subdomains that are shed from the biosynthetic compartment and are delivered to endolysosomes under control of ER-phagy receptors for ER-to-lysosome-associated degradation (ERLAD). Demannosylation of N-linked oligosaccharides targets terminally misfolded proteins for ERAD. How misfolded proteins are eventually marked for ERLAD is not known. Here, we show for ATZ and mutant Pro-collagen that cycles of de-/re-glucosylation of selected N-glycans and persistent association with Calnexin (CNX) are required and sufficient to mark ERAD-resistant misfolded proteins for FAM134B-driven lysosomal delivery. In summary, we show that mannose and glucose processing of N-glycans are triggering events that target misfolded proteins in the ER to proteasomal (ERAD) and lysosomal (ERLAD) clearance, respectively, regulating protein quality control in eukaryotic cells.

Collagen has a unique SEC24 preference for efficient export from the endoplasmic reticulum.

SEC24 is mainly involved in cargo sorting during COPII vesicle assembly. There are four SEC24 paralogs (A-D) in vertebrates, which are classified into two subgroups (SEC24A/B and SEC24C/D). Pathological mutations in SEC24D cause osteogenesis imperfecta with craniofacial dysplasia in humans. sec24d mutant fish also recapitulate the phenotypes. Consistent with the skeletal phenotypes, the secretion of collagen was severely defective in mutant fish, emphasizing the importance of SEC24D in collagen secretion. However, SEC24D patient-derived fibroblasts show only a mild secretion phenotype, suggesting tissue-specificity in the secretion process. Using Sec24d KO mice and cultured cells, we show that SEC24A and SEC24B also contribute to endoplasmic reticulum (ER) export of procollagen. In contrast, fibronectin 1 requires either SEC24C or SEC24D for ER export. On the basis of our results, we propose that procollagen interacts with multiple SEC24 paralogs for efficient export from the ER, and that this is the basis for tissue-specific phenotypes resulting from SEC24 paralog deficiency.

Ginsenoside Rc alleviates osteoporosis by the TGF-ß/Smad signaling pathway.

Osteoporosis is a common chronic bone disorder in postmenopausal women. Ginsenosides are primary active components in ginseng and the effects of various ginsenoside variants in osteoporosis treatment have been widely revealed. We planned to explore the impact of ginsenoside Rc on bone resorption in an osteoporosis rat model. We used ovariectomized rats to assess the potential impact of ginsenoside Rc on osteoporosis. µ-CT was implemented for analyzing the microstructure of the distal left femur in rats. H&E staining together with Masson staining were applied for bone histomorphometry evaluation. ELISA kits were implemented to detect serum concentrations of TRACP-5b, OCN, CTX, as well as PINP. Ginsenoside Rc treatment lessened the serum levels of TRACP-5b as well as CTX, while increasing serum levels of OCN, and PINP of OVX rats. Moreover, we found that ginsenoside Rc contributed to the synthesis of type I collagen via increasing Col1a1 and Col1a2 levels in femur tissues of ovariectomized rats. Our findings also revealed that ginsenoside Rc activated the TGF-ß/Smad pathway by increasing TGF-ß as well as phosphorylated Smad2/3 protein levels. Ginsenoside Rc alleviates osteoporosis in rats through promoting the TGF-ß/Smad pathway.

The effect of external fixator on bone metabolism in patients with open fracture of extremities.

This experiment aimed to explore the influence mechanism of external fixator on open fracture. A total of 128 patients with open tibiofibular fractures were included in this study. The patients were randomly divided into external fixator group (n=64) and control group (n=64) according to the order of admission. Double-blind controlled observation was used. The levels of osteocalcin (BGP), ß-CTX, P1 NP, BALP, including haptoglobin (Hp), ceruloplasmin (CER), serum adrenocorticotropic hormone (ACTH), cortisol (COR), C-reactive protein (CRP), white blood cell (WBC) and interleukin-6 (IL-6) were recorded in different groups. The postoperative VAS score and quality of life were recorded. Log-rank was used to analyze the difference in postoperative adverse reaction rates among different groups. External fixation stent treatment increased BGP, PINP, and BALP expression and decreased ß-CTX, Hp, CER, ACTH, COR, CRP, WBC, and IL-6 levels. Patients in the external fixation stent group had significantly lower VAS score quality of life scores and incidence of adverse events than the control group. External fixation stents protect open fracture patients by promoting bone metabolism.

Does metabolic control of the disease related with bone turnover markers in children with type 1 diabetes mellitus in Turkey?

BACKGROUND: The aim was to evaluate the effect of metabolic control on bone biomarkers in children with type I diabetes. MATERIALS AND METHODS: The children were divided into two groups according to their glycated hemoglobin (HbA1c) (%) levels: a group with HbA1c levels < 8% (n = 16) and: a group with HbA1c levels > 8% (n = 18). The serum total oxidative status (TOS) (µmol/L), total antioxidant status (TAS) (mmol/L), alkaline phosphatase (ALP) (IU/L), osteocalcin (OC) (ng/ml), procollagen type-1-N-terminal peptide (P1NP) (ng/ml), and vitamin D (IU) levels and food consumption frequencies were determined. RESULTS: When patients were classified according to HbA1c (%) levels, those with HbA1c levels < 8% were found to have lower TOS (µmol/L) values (8.7 ± 6.16, 9.5 ± 5.60) and higher serum OC (ng/mL) (24.2 ± 16.92, 22.0 ± 6.21) levels than those with HbA1c levels > 8% (p < 0.05). Regardless of the level of metabolic control, there was a statistically significant association between serum TOS (µmol/L) and P1NP (ng/ml) (p < 0.05) levels, with no group-specific relationship (HbA1c levels <%8 or HbA1c levels >%8). CONCLUSION: HbA1c and serum TOS levels had an effect on bone turnover biomarkers in individuals with type I diabetes.

Correlation study between bone metabolic markers, bone mineral density, and sarcopenia.

OBJECTIVE: To investigate the correlation between bone metabolism markers, bone mineral density (BMD), and sarcopenia. METHODS: A total of 331 consecutive patients aged ≥ 60 years who were hospitalized between November 2020 and December 2021 were enrolled. Participants were divided into sarcopenia and non-sarcopenia groups according to the Asian Working Group on Sarcopenia criteria (AWGS, 2019). The clinical data, bone metabolism markers (ß-CTX, N-MID, and TP1NP), and BMD were compared between the two groups. RESULTS: Age, ß-CTX, and N-MID of the sarcopenia group were higher than those of the non-sarcopenia group (P < 0.05), but the BMD T values were lower than those of the non-sarcopenia group (P < 0.05). Binary logistic regression analysis showed that increased femoral neck BMD (FNBMD) was a protective factor for sarcopenia, while increased ß-CTX was a risk factor. Pearson/Spearman correlation analysis showed that the diagnostic indices of sarcopenia were positively correlated with FNBMD and negatively correlated with ß-CTX and N-MID. Multiple linear regression analysis revealed that BMI and FNBMD significantly positively affected muscle strength and appendicular skeletal muscle mass (ASM). The FNBMD significantly positively affected physical performance, while ß-CTX significantly negatively affected muscle strength, ASM, and physical performance. CONCLUSION: Increased FNBMD may be a protective factor against sarcopenia, and increased ß-CTX may be a risk factor. The FNBMD significantly positively affected the diagnostic indices of sarcopenia, while ß-CTX significantly negatively affected them. BMD and bone metabolism marker levels may be considered in early screening for sarcopenia.

Collagen's enigmatic, highly conserved N-glycan has an essential proteostatic function.

Intracellular procollagen folding begins at the protein's C-terminal propeptide (C-Pro) domain, which initiates triple-helix assembly and defines the composition and chain register of fibrillar collagen trimers. The C-Pro domain is later proteolytically cleaved and excreted from the body, while the mature triple helix is incorporated into the extracellular matrix. The procollagen C-Pro domain possesses a single N-glycosylation site that is widely conserved in all the fibrillar procollagens across humans and diverse other species. Given that the C-Pro domain is removed once procollagen folding is complete, the N-glycan might be presumed to be important for folding. Surprisingly, however, there is no difference in the folding and secretion of N-glycosylated versus non-N-glycosylated collagen type-I, leaving the function of the N-glycan unclear. We hypothesized that the collagen N-glycan might have a context-dependent function, specifically, that it could be required to promote procollagen folding only when proteostasis is challenged. We show that removal of the N-glycan from misfolding-prone C-Pro domain variants does indeed cause serious procollagen and ER proteostasis defects. The N-glycan promotes folding and secretion of destabilized C-Pro variants by providing access to the ER's lectin-based chaperone machinery. Finally, we show that the C-Pro N-glycan is actually critical for the folding and secretion of even wild-type procollagen under ER stress conditions. Such stress is commonly incurred during development, wound healing, and other processes in which collagen production plays a key role. Collectively, these results establish an essential, context-dependent function for procollagen's previously enigmatic N-glycan, wherein the carbohydrate moiety buffers procollagen folding against proteostatic challenge.

Assessment of Osteogenic Exercise Efficacy via Bone Turnover Markers in Premenopausal Women: A Randomized Controlled Trial.

Assessing bone's response to physical activity interventions is challenging. This randomized controlled trial investigates if changes in bone turnover markers can offer an early evaluation of a physical activity intervention's effectiveness in improving bone mineral density (BMD) in premenopausal women. Participants in the intervention group (n = 27, with 24 completing the trial) were instructed to walk at least 10,000 steps every day on a brisk walk and to execute 60 jumps daily, each surpassing 4g of acceleration, using an accelerometer-based wearable device. Meanwhile, the control group (n = 26, with 18 completing the trial) continued with their usual lifestyle. Bone turnover markers, comprising of C-terminal telopeptide of Type I collagen, procollagen Type 1 N-terminal propeptide, and total osteocalcin (carboxylated and undercarboxylated) were measured at baseline and midway through the intervention (3 months). Dual-energy X-ray absorptiometry scans of the hip and lumbar spine were conducted at baseline and the end of the intervention (6 months) to estimate BMD. Analysis of covariance exhibited significant differences between groups in procollagen Type 1 N-terminal propeptide (-6.74 µg/L, p = .023) and C-terminal telopeptide of Type I collagen (-83 ng/L, p = .043) after 3 months, and in femoral neck BMD (+0.024 g/cm2, p = .016), total hip BMD (+0.036 g/cm2, p = .004), and lumbar spine BMD (+0.026 g/cm2, p = .020) after 6 months. A significant correlation (r = -.73; p < .001) was detected between reductions in C-terminal telopeptide of Type I collagen and increases in femoral neck BMD. In conclusion, this intervention improved BMD in premenopausal women, with bone turnover markers potentially useful for early intervention assessment, though further research is needed.

Effectiveness of teriparatide in in improving healing rates and bone-turnover markers of osteoporotic hip fracture: a meta-analysis.

Objective: To evaluate the effect of subcutaneous teriparatide therapy on fracture healing rate and change in bone mass density in osteoporotic hip fractures. METHODS: The meta-analysis was done from September to December 2022, and comprised literature search on Wanfang, CNKI, VIP, PubMed, Embase, Cochrane Library, and Web of Science databases from the establishment of the respective database till December 2022. The relevant journals of the library of Macao University of Science and Technology, China, were manually searched for randomised controlled trials of teriparatide in the treatment of osteoporotic hip fractures. The shortlisted studies were subjectd to Cochrane Risk of Bias tool and the Jadad Rating Scale. Meta-analysis was done using the RevMan 5.4 software provided by the Cochrane Collaboration Network. Fracture healing rate and bone mineral density were the primary outcome measures, while mortality, adverse events, malformations, complications, subsequent fractures, timed-up-and-go test, visual analogue scale score, and procollagen type I N-terminal propeptide were the secondary outcome measures. RESULTS: Of the 1,094 articles retrieved, 8(0.7%) randomised controlled trials were analysed. There were 744 patients; 372(50%) in the teriparatide group and 372(50%) in the control group. Fracture healing rate was not significantly different (p=0.82), while bone mineral density was significantly different between the groups (p<0.001). Mortality, adverse events, deformity, and complications were not significantly different (p>0.05), while subsequent fractures, timed-up-and-go score, visual analogue scale score and procollagen type I N-terminal propeptide were significantly different between the groups (p<0.05). Conclusion: The literature did not support teriparatide's ability to improve the healing rate of osteoporotic hip fractures, or to reduce mortality, adverse events, malformations, and complications. In addition, teriparatide could increase bone mineral density of osteoporotic hip fractures and the procollagen type I N-terminal propeptide value, alleviate hip pain, and reduce subsequent fracture rates. This trial is registered with PROSPERO with registration number CRD42022379832.

miR-449a ameliorates acute rejection after liver transplantation via targeting procollagen-lysine1,2-oxoglutarate5-dioxygenase 1 in macrophages.

Acute rejection (AR) is an important factor that leads to poor prognosis after liver transplantation (LT). Macrophage M1-polarization is an important mechanism in AR development. MicroRNAs play vital roles in disease regulation; however, their effects on macrophages and AR remain unclear. In this study, rat models of AR were established following LT, and macrophages and peripheral blood mononuclear cells were isolated from rats and humans, respectively. We found miR-449a expression to be significantly reduced in macrophages and peripheral blood mononuclear cells. Overexpression of miR-449a not only inhibited the M1-polarization of macrophages in vitro but also improved the AR of transplant in vivo. The mechanism involved inhibiting the noncanonical nuclear factor-kappaB (NF-κB) pathway. We identified procollagen-lysine1,2-oxoglutarate5-dioxygenase 1 (PLOD1) as a target gene of miR-449a, which could reverse miR-449a's inhibition of macrophage M1-polarization, amelioration of AR, and inhibition of the NF-κB pathway. Overall, miR-449a inhibited the NF-κB pathway in macrophages through PLOD1 and also inhibited the M1-polarization of macrophages, thus attenuating AR after LT. In conclusion, miR-449a and PLOD1 may be new targets for the prevention and mitigation of AR.

A selective ER-phagy exerts procollagen quality control via a Calnexin-FAM134B complex.

Autophagy is a cytosolic quality control process that recognizes substrates through receptor-mediated mechanisms. Procollagens, the most abundant gene products in Metazoa, are synthesized in the endoplasmic reticulum (ER), and a fraction that fails to attain the native structure is cleared by autophagy. However, how autophagy selectively recognizes misfolded procollagens in the ER lumen is still unknown. We performed siRNA interference, CRISPR-Cas9 or knockout-mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells. We found that the ER-resident lectin chaperone Calnexin (CANX) and the ER-phagy receptor FAM134B are required for autophagy-mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co-receptor that recognizes ER luminal misfolded procollagens and interacts with the ER-phagy receptor FAM134B. In turn, FAM134B binds the autophagosome membrane-associated protein LC3 and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation. Thus, a crosstalk between the ER quality control machinery and the autophagy pathway selectively disposes of proteasome-resistant misfolded clients from the ER.

Baseline serum PINP level is associated with the increase in hip bone mineral density seen with Romosozumab treatment in previously untreated women with osteoporosis.

Baseline serum PINP value was significantly and independently associated with the increased bone mineral density (≥ 3%) in both total hip and femoral necks by 12 months of romosozumab treatment in patients with treatment-naive postmenopausal osteoporosis. PURPOSE: Some patients fail to obtain a sufficiently increased hip bone mineral density (BMD) by romosozumab (ROMO) treatment. This study aimed to investigate the prognostic factor for increased hip BMD with ROMO in patients with treatment-naive postmenopausal osteoporosis. METHODS: This prospective, observational, and multicenter study included patients (n = 63: mean age, 72.6 years; T-scores of the lumbar spine [LS], - 3.3; total hip [TH], - 2.6; femoral neck [FN], - 3.3; serum type I procollagen N-terminal propeptide [PINP], 68.5 µg/L) treated by ROMO for 12 months. BMD and serum bone turnover markers were evaluated at each time point. A responder analysis was performed to assess the patient percentage, and both univariate and multivariate analyses were performed to investigate the factors associated with clinically significant increased BMD (≥ 3%) in both TH and FN. RESULTS: Percentage changes of BMD from baseline in the LS, TH, and FN areas were 17.5%, 4.9%, and 4.3%, respectively. In LS, 96.8% of patients achieved ≥ 6% increased LS-BMD, although 57.1% could not achieve ≥ 3% increased BMD in either TH or FN. Multiple regression analysis revealed that only the baseline PINP value was significantly and independently associated with ≥ 3% increased BMD in both TH and FN (p = 0.019, 95% confidence interval = 1.006-1.054). The optimal cut-off PINP value was 53.7 µg/L with 54.3% sensitivity and 92.3% specificity (area under the curve = 0.752). CONCLUSIONS: In a real-world setting, baseline PINP value was associated with the increased BMD of TH and FN by ROMO treatment in treatment-naive patients.

Bone Turnover Markers in Children: From Laboratory Challenges to Clinical Interpretation.

Bone turnover markers (BTMs) have been developed many years ago to study, in combination with imaging techniques, bone remodeling in adults. In children and adolescents, bone metabolism differs from adults since it implies both growth and bone remodeling, suggesting an age- and gender-dependent BTM concentration. Therefore, specific studies have evaluated BTMs in not only physiological but also pathological conditions. However, in pediatrics, the use of BTMs in clinical practice is still limited due to these many children-related specificities. This review will discuss about physiological levels of BTMs as well as their modifications under pathological conditions in children and adolescents. A focus is also given on analytical and clinical challenges that restrain BTM usefulness in pediatrics.

Impact of essential amino acid intake, resistance exercise, and aging on the concentration of Achilles peritendinous amino acids and procollagen Iα1 in humans.

Recent studies have shown that consuming amino acid-rich compounds improves tendon collagen content and biomechanical properties. Yet, it is unclear if the consumption of amino acids alters local (peritendinous) amino acid concentrations. If aging or exercise influence local amino acid concentrations in conjunction with an amino acid bolus is also not known. We conducted two studies. In Study 1, young women (n = 7, 25 ± 2 years) completed two identical resistance training sessions with either essential amino acid (EAA) or placebo consumption. In Study 2, an EAA bolus identical to Study 1 was given to younger (n = 7; 27 ± 1 year) and older adults (n = 6; 68 ± 2 years). Microdialysis was used to determine Achilles peritendinous amino acid and pro-collagen Iα1 (a marker of collagen synthesis) concentrations. In Study 1, amino acid consumption increased peritendinous concentrations of all EAA except histidine (p < 0.05). In Study 2, the peritendinous concentration of EAAs except for methionine, histidine, and lysine (p > 0.05) increased with time (p < 0.05). Further, the concentrations of most measured amino acids were greater in older adults (p < 0.05). Pro-collagen Iα1 concentration (p > 0.05) was unaffected by exercise, EAA, or aging (p > 0.05). Our findings demonstrate the following: (1) when not combined with exercise, an oral EAA bolus leads to only modest increases in Achilles peritendinous amino acid concentrations; (2) when combined with resistance exercise, EAA consumption resulted in greater peritendinous amino acid concentrations compared to no exercise; (3) the basal concentrations of most amino acids were greater in older adults, and (4) neither the EAA bolus nor exercise altered peritendinous pro-collagen concentrations.

Hypoxic regulation of ADAMTS-2 and -3 (a disintegrin and matrix metalloproteinase with thrombospondin motifs 2 and 3) procollagen N proteinases by HIF-1α in endothelial cells.

ADAMTS-2 and ADAMTS-3, known as procollagen amino proteases (PNP), are primarily responsible for processing the amino ends of the fibrillar collagen precursors. ADAMTS-2 is a highly expressed gene in type I collagen-rich tissues, such as skin, bones, tendons, and aorta. ADAMTS-3 is mainly expressed in cartilage, where it colocalizes with type II procollagen and in the nervous system. Studies about ADAMTS-2 and ADAMTS-3 enzymes primarily focused on their collagen processing activity. Knowledge about the transcriptional regulations of these genes is rather limited. Here we analyzed the transcriptional regulations of ADAMTS-2 and ADAMTS-3 genes under chemically induced hypoxic conditions in endothelial cell model, HUVECs. We elucidated that hypoxia is the potent positive regulator of ADAMTS-2 and ADAMTS-3 genes. qRT-PCR and western blotting studies revealed that ADAMTS-2 and ADAMTS-3 expressions were increased at mRNA and protein levels under chemically induced hypoxic conditions in HUVECs. In addition, Transient transfection experiments of ADAMTS-2 and ADAMTS-3 promoter-reporter constructs indicated that low oxygen conditions increased ADAMTS-2 and ADAMTS-3 promoter activities. Furthermore, the DNA-protein interaction assay provided evidence of the functional binding of HIF-1α on bioinformatically determined HRE regions on the ADAMTS-2 and ADAMTS-3 promoters.

Negative impact on bone homeostasis in postmenopausal women with non-metastatic breast cancer during cytotoxic chemotherapy.

INTRODUCTION: The burden and mechanisms of endocrine therapy-related bone loss are well known, while there are limited data on chemotherapy-induced bone resorption. The study aimed to evaluate the effect of cytotoxic chemotherapy on bone homeostasis among postmenopausal women with non-metastatic breast cancer. MATERIALS AND METHODS: Early and locally advanced postmenopausal non-metastatic breast cancer patients aged 45 to 65 planned for three cycles of anthracycline and four cycles of taxane chemotherapy administered along with dexamethasone (cumulative dose-256 mg) as an antiemetic from June 2018 to December 2021 were included. Bone mineral density (BMD), bone turnover markers, calciotropic hormones, pro-inflammatory cytokines, oxidative stress, and total antioxidant levels (TAS) were measured. RESULTS: We recruited 109 patients, with early 34 (31.2%) and locally advanced breast cancer 75 (68.8%) with median age 53 (45-65) years. There was a significant decrease in the % BMD at the lumbar spine, neck of the femur, and total hip post-chemotherapy. There was a significant increase in serum C-terminal telopeptide of type I collagen (CTX) and procollagen type I N-terminal propeptide (PINP) levels post-chemotherapy. PINP/CTX ratio significantly decreased post-chemotherapy. Serum 25-OH vitamin D was significantly reduced with a compensatory increase in plasma iPTH levels. The change in CTX, PINP/CTX ratio, 25-OH vitamin D, iPTH, and oxidative stress index was more pronounced during anthracycline as taxane chemotherapy. There were no significant changes in pro-inflammatory cytokine levels. CONCLUSION: Chemotherapy and dexamethasone as antiemetic resulted in significant bone loss, as evidenced by bone turnover markers. Further studies are required to understand the mechanism of chemotherapy-induced bone loss and the need for bone-strengthening agents during chemotherapy.

Circulating levels of endotrophin and cross-linked type III collagen reflect liver fibrosis in people with HIV.

BACKGROUND AND AIMS: Liver-associated complications still frequently lead to mortality in people with HIV (PWH), even though combined antiretroviral treatment (cART) has significantly improved overall survival. The quantification of circulating collagen fragments released during collagen formation and degradation correlate with the turnover of extracellular matrix (ECM) in liver disease. Here, we analysed the levels of ECM turnover markers PC3X, PRO-C5, and PRO-C6 in PWH and correlated these with hepatic fibrosis and steatosis. METHODS: This monocentre, retrospective study included 141 PWH. Liver stiffness and liver fat content were determined using transient elastography (Fibroscan) with integrated CAP function. Serum levels of formation of cross-linked type III collagen (PC3X), formation of type V collagen (PRO-C5) and formation type VI collagen (PRO-C6), also known as the hormone endotrophin, were measured with ELISA. RESULTS: Twenty-five (17.7%) of 141 PWH had clinical significant fibrosis with liver stiffness ≥ 7.1 kPa, and 62 PWH (44.0%) had steatosis with a CAP value > 238 dB/m. Study participants with fibrosis were older (p = 0.004) and had higher levels of AST (p = 0.037) and lower number of thrombocytes compared to individuals without fibrosis (p = 0.0001). PC3X and PRO-C6 were markedly elevated in PWH with fibrosis. Multivariable cox regression analysis confirmed PC3X as independently associated with hepatic fibrosis. PRO-C5 was significantly elevated in participants with presence of hepatic steatosis. CONCLUSION: Serological levels of cross-linked type III collagen formation and endotrophin were significantly associated with liver fibrosis in PWH receiving cART and thus may be suitable as a non-invasive evaluation of liver fibrosis in HIV disease.

Development of a rapid, efficient, and reusable magnetic bead-based immunocapture system for recombinant human procollagen type II isolation from yeast fermentation broth.

Recombinant collagen production, especially using yeasts as expression systems, could represent a promising alternative over traditional extractive methods from animal sources, offering controllable, scalable, and high-quality products. Monitoring the efficiency and efficacy of procollagen/collagen expression, especially in the initial fermentation phases, can be difficult and time consuming, as biological matrices necessitate purification and commonly used analytical methods are only partially informative. We propose a straightforward, efficient, and reusable immunocapture system able to specifically isolate human procollagen type II from fermentation broths and to release it in few experimental steps. A recovered sample allows for a detailed characterization providing information on structural identity and integrity, which can strongly support the monitoring of fermentation processes. The immunocapture system relies on the use of protein A-coated magnetic beads which have been functionalized and cross-linked with a human anti-procollagen II antibody (average immobilization yield of 97.7%) to create a stable and reusable support for the specific procollagen fishing. We set up the binding and release conditions ensuring specific and reproducible binding with a synthetic procollagen antigen. The absence of non-specific interaction with the support and binding specificity was demonstrated, and the latter was also confirmed by a peptide mapping epitope study in reversed-phase liquid chromatography high-resolution mass spectrometry (RP-LC-HRMS). The bio-activated support proved to be reusable and stable over 21 days from the initial use. Finally, the system was successfully tested on a raw yeast fermentation sample to provide a proof of concept of the applicability within recombinant collagen production.