Presence of SARS-CoV-2 RNA in COVID-19 survivors with post-COVID symptoms: a systematic review of the literature.

INTRODUCTION: Viral persistence is one of the main hypotheses explaining the presence of post-COVID symptoms. This systematic review investigated the presence of SARS-CoV-2 RNA in plasma, stool, urine, and nasal/oral swab samples in individuals with post-COVID symptomatology. CONTENT: MEDLINE, CINAHL, PubMed, EMBASE, Web of Science databases, as well as medRxiv/bioRxiv preprint servers were searched up to November 25th, 2023. Articles investigating the persistence of SARS-CoV-2 RNA in plasma, stool, urine or nasal/oral swab samples in patients with post-COVID symptoms were included. Methodological quality was assessed using the Newcastle-Ottawa Scale or Cochrane's Risk of Bias (Rob) tool. SUMMARY: From 322 studies identified, six studies met all inclusion criteria. The sample included 678 COVID-19 survivors (52 % female, aged from 29 to 66 years). The methodological quality was moderate in 88 % of the studies (n=5/6). Three papers investigated the presence of SARS-CoV-2 RNA in plasma, three studies in nasal/oral swabs, two studies in stool samples, one in urine and one in saliva. The follow-up was shorter than two months (<60 days after) in 66 % of the studies (n=4/6). The prevalence of SARS-CoV-2 RNA ranged from 5 to 59 % in patients with post-COVID symptoms the first two months after infection, depending on the sample tested, however, SARS-CoV-2 RNA was also identified in COVID-19 survivors without post-COVID symptoms (one study). OUTLOOK: Available evidence can suggest the presence of persistent SARS-CoV-2 RNA in post-COVID patients in the short term, although the biases within the studies do not permit us to make firm assumptions. The association between post-COVID symptoms and SARS-CoV-2 RNA in the samples tested is also conflicting. The lack of comparative group without post-COVID symptoms limits the generalizability of viral persistence in post-COVID-19 condition.

A data-driven computational model on the effects of immigration policies.

Many scholars suggest that visa restrictions push individuals who would have otherwise migrated legally toward illegal channels. This expectation is difficult to test empirically for three reasons. First, unauthorized migration is clandestine and often unobservable. Second, interpersonal ties between migrants and would-be migrants form a self-perpetuating system, which adapts in ways that are difficult to observe or predict. Third, empirical evaluations of immigration policy are vulnerable to endogeneity and other issues of causal inference. In this paper, we pair tailor-made empirical designs with an agent-based computational model (ABM) to capture the dynamics of a migration system that often elude empirical analysis, while grounding agent rules and characteristics with primary data collected in Jamaica, an origin country. We find that some government-imposed restrictions on migrants can deter total migration, but others are ineffective. Relative to a system of free movement, the minimal eligibility conditions required to classify migrants into visa categories alone make migration inaccessible for many. Restrictive policies imposed on student and high-skilled visa categories have little added effect because eligible individuals are likely able to migrate through alternative legal categories. Meanwhile, restrictions on family-based visas result in significant reductions in total migration. However, they also produce the largest reorientation toward unauthorized channels-an unintended consequence that even the highest rates of apprehension do not effectively eliminate.

Glycation of type I collagen selectively targets the same helical domain lysine sites as lysyl oxidase-mediated cross-linking.

Nonenzymatic glycation of collagen has long been associated with the progressive secondary complications of diabetes. How exactly such random glycations result in impaired tissues is still poorly understood. Because of the slow turnover rate of most fibrillar collagens, they are more susceptible to accumulate time-dependent glycations and subsequent advanced glycation end-products. The latter are believed to include cross-links that stiffen host tissues. However, diabetic animal models have also displayed weakened tendons with reduced stiffness. Strikingly, not a single experimentally identified specific molecular site of glycation in a collagen has been reported. Here, using targeted MS, we have identified partial fructosyl-hydroxylysine glycations at each of the helical domain cross-linking sites of type I collagen that are elevated in tissues from a diabetic mouse model. Glycation was not found at any other collagen lysine residues. Type I collagen in mouse tendons is cross-linked intermolecularly by acid-labile aldimine bonds formed by the addition of telopeptide lysine aldehydes to hydroxylysine residues at positions α1(I)Lys87, α1(I)Lys930, α2(I)Lys87, and α2(I)Lys933 of the triple helix. Our data reveal that site-specific glycations of these specific lysines may significantly impair normal lysyl oxidase-controlled cross-linking in diabetic tendons. We propose that such N-linked glycations can hinder the normal cross-linking process, thus altering the content and/or placement of mature cross-links with the potential to modify tissue material properties.

Sc65-Null Mice Provide Evidence for a Novel Endoplasmic Reticulum Complex Regulating Collagen Lysyl Hydroxylation.

Collagen is a major component of the extracellular matrix and its integrity is essential for connective tissue and organ function. The importance of proteins involved in intracellular collagen post-translational modification, folding and transport was recently highlighted from studies on recessive forms of osteogenesis imperfecta (OI). Here we describe the critical role of SC65 (Synaptonemal Complex 65, P3H4), a leprecan-family member, as part of an endoplasmic reticulum (ER) complex with prolyl 3-hydroxylase 3. This complex affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Loss of Sc65 in the mouse results in instability of this complex, altered collagen lysine hydroxylation and cross-linking leading to connective tissue defects that include low bone mass and skin fragility. This is the first indication of a prolyl-hydroxylase complex in the ER controlling lysyl-hydroxylase activity during collagen synthesis.

P3h3-null and Sc65-null Mice Phenocopy the Collagen Lysine Under-hydroxylation and Cross-linking Abnormality of Ehlers-Danlos Syndrome Type VIA.

Tandem mass spectrometry was applied to tissues from targeted mutant mouse models to explore the collagen substrate specificities of individual members of the prolyl 3-hydroxylase (P3H) gene family. Previous studies revealed that P3h1 preferentially 3-hydroxylates proline at a single site in collagen type I chains, whereas P3h2 is responsible for 3-hydroxylating multiple proline sites in collagen types I, II, IV, and V. In screening for collagen substrate sites for the remaining members of the vertebrate P3H family, P3h3 and Sc65 knock-out mice revealed a common lysine under-hydroxylation effect at helical domain cross-linking sites in skin, bone, tendon, aorta, and cornea. No effect on prolyl 3-hydroxylation was evident on screening the spectrum of known 3-hydroxyproline sites from all major tissue collagen types. However, collagen type I extracted from both Sc65-/- and P3h3-/- skin revealed the same abnormal chain pattern on SDS-PAGE with an overabundance of a γ112 cross-linked trimer. The latter proved to be from native molecules that had intramolecular aldol cross-links at each end. The lysine under-hydroxylation was shown to alter the divalent aldimine cross-link chemistry of mutant skin collagen. Furthermore, the ratio of mature HP/LP cross-links in bone of both P3h3-/- and Sc65-/- mice was reversed compared with wild type, consistent with the level of lysine under-hydroxylation seen in individual chains at cross-linking sites. The effect on cross-linking lysines was quantitatively very similar to that previously observed in EDS VIA human and Plod1-/- mouse tissues, suggesting that P3H3 and/or SC65 mutations may cause as yet undefined EDS variants.

Post-translationally abnormal collagens of prolyl 3-hydroxylase-2 null mice offer a pathobiological mechanism for the high myopia linked to human LEPREL1 mutations.

Myopia, the leading cause of visual impairment worldwide, results from an increase in the axial length of the eyeball. Mutations in LEPREL1, the gene encoding prolyl 3-hydroxylase-2 (P3H2), have recently been identified in individuals with recessively inherited nonsyndromic severe myopia. P3H2 is a member of a family of genes that includes three isoenzymes of prolyl 3-hydroxylase (P3H), P3H1, P3H2, and P3H3. Fundamentally, it is understood that P3H1 is responsible for converting proline to 3-hydroxyproline. This limited additional knowledge also suggests that each isoenzyme has evolved different collagen sequence-preferred substrate specificities. In this study, differences in prolyl 3-hydroxylation were screened in eye tissues from P3h2-null (P3h2(n/n)) and wild-type mice to seek tissue-specific effects due the lack of P3H2 activity on post-translational collagen chemistry that could explain myopia. The mice were viable and had no gross musculoskeletal phenotypes. Tissues from sclera and cornea (type I collagen) and lens capsule (type IV collagen) were dissected from mouse eyes, and multiple sites of prolyl 3-hydroxylation were identified by mass spectrometry. The level of prolyl 3-hydroxylation at multiple substrate sites from type I collagen chains was high in sclera, similar to tendon. Almost every known site of prolyl 3-hydroxylation in types I and IV collagen from P3h2(n/n) mouse eye tissues was significantly under-hydroxylated compared with their wild-type littermates. We conclude that altered collagen prolyl 3-hydroxylation is caused by loss of P3H2. We hypothesize that this leads to structural abnormalities in multiple eye tissues, but particularly sclera, causing progressive myopia.

Biopolymer nanoparticle production for controlled release of biopharmaceuticals.

For drug applications, nanoparticles, used as drug carriers, offer the advantage of controlled release, therapeutic impact and targeted delivery. In drug delivery applications, biodegradable polymers can be extracted from natural sources or prepared synthetically by polymerization. Natural polymers typically have varying compositions and physiochemical properties. As a result, methods which utilize natural polymers to encapsulate drugs are more varied and polymer dependent. The following polymers are discussed in this review article: alginate, chitosan, gelatin, albumin, gliadin, pullulan, and dextran. Specialized encapsulation nanotechnologies will be discussed such as ionotropic gelation, complexation, the reverse microemulsion technique, cross-linking methods, emulsion-dependent methods, desolvation methods and self-assembly methods. For each biopolymer an overview of the structure is presented with the corresponding encapsulation techniques. Understanding the structure of the biopolymer is important as to not only understand the rational for current encapsulation techniques but to continue to develop new encapsulation techniques in pursuit of the ideal drug carrier for application in therapeutic treatments.

Characterization of TGFß1-induced tendon-like structure in the scaffold-free three-dimensional tendon cell culture system.

The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well-established, partly due to the lack of reliable in vitro systems that produce highly aligned collagenous tissues. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells in a differentially adherent growth channel. Transforming Growth Factor-ß (TGFß) signaling is involved in various biological processes in the tendon, regulating tendon cell fate, recruitment and maintenance of tenocytes, and matrix organization. This known function of TGFß signaling in tendon prompted us to utilize TGFß1 to induce tendon-like structures in 3D tendon constructs. TGFß1 treatment promoted a tendon-like structure in the peripheral layer of the constructs characterized by increased thickness with a gradual decrease in cell density and highly aligned collagen matrix. TGFß1 also enhanced cell proliferation, matrix production, and morphological maturation of cells in the peripheral layer compared to vehicle treatment. TGFß1 treatment also induced early tenogenic differentiation and resulted in sufficient mechanical integrity, allowing biomechanical testing. The current study suggests that this scaffold-free 3D tendon cell culture system could be an in vitro platform to investigate underlying biological mechanisms that regulate tenogenic cell differentiation and matrix organization.

Clinical, Health-Related Quality of Life, and Gait Differences Among Obesity Classes in Adults With Knee Osteoarthritis.

OBJECTIVE: The purpose of this study was to determine whether clinical, health-related quality of life (HRQL), and gait characteristics in adults with knee osteoarthritis (OA) differed by obesity category. METHODS: This cross-sectional analysis of 823 older adults (mean age 64.6 years, SD 7.8 years) with knee OA and overweight or obesity compared clinical, HRQL, and gait outcomes among obesity classifications (overweight or class I, body mass index [BMI] 27.0-34.9; class II, BMI 35.0-39.9; class III BMI ≥40.0). RESULTS: Patients with class III obesity had worse Western Ontario McMasters Universities Arthritis Index knee pain (0-20) than the overweight or class I (mean 8.6 vs 7.0; difference 1.5; 95% confidence interval [CI] 1.0-2.1; P < 0.0001) and class II (mean 8.6 vs 7.4; difference 1.1; 95% CI 0.6-1.7; P = 0.0002) obesity groups. The Short Form 36 physical HRQL measure was lower in the class III obesity group compared to the overweight or class I (mean 31.0 vs 37.3; difference -6.2; 95% CI -7.8 to -4.7; P < 0.0001) and class II (mean 31.0 vs 35.0; difference -3.9; 95% CI -5.6 to -2.2; P < 0.0001) obesity groups. The class III obesity group had a base of support (cm) during gait that was wider than that for the overweight or class I (mean 14.0 vs 11.6; difference 3.3; 95% CI 2.6-4.0; P < 0.0001) and class II (mean 14.0 vs 11.6; difference 2.4; 95% CI 1.6-3.2; P < 0.0001) obesity groups. CONCLUSION: Among adults with knee OA, those with class III obesity had significantly higher pain levels and worse physical HRQL and gait characteristics compared to adults with overweight or class I or class II obesity.

The HIF-1α/PLOD2 axis integrates extracellular matrix organization and cell metabolism leading to aberrant musculoskeletal repair.

While hypoxic signaling has been shown to play a role in many cellular processes, its role in metabolism-linked extracellular matrix (ECM) organization and downstream processes of cell fate after musculoskeletal injury remains to be determined. Heterotopic ossification (HO) is a debilitating condition where abnormal bone formation occurs within extra-skeletal tissues. Hypoxia and hypoxia-inducible factor 1α (HIF-1α) activation have been shown to promote HO. However, the underlying molecular mechanisms by which the HIF-1α pathway in mesenchymal progenitor cells (MPCs) contributes to pathologic bone formation remain to be elucidated. Here, we used a proven mouse injury-induced HO model to investigate the role of HIF-1α on aberrant cell fate. Using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics analyses of the HO site, we found that collagen ECM organization is the most highly up-regulated biological process in MPCs. Zeugopod mesenchymal cell-specific deletion of Hif1α (Hoxa11-CreERT2; Hif1afl/fl) significantly mitigated HO in vivo. ScRNA-seq analysis of these Hoxa11-CreERT2; Hif1afl/fl mice identified the PLOD2/LOX pathway for collagen cross-linking as downstream of the HIF-1α regulation of HO. Importantly, our scRNA-seq data and mechanistic studies further uncovered that glucose metabolism in MPCs is most highly impacted by HIF-1α deletion. From a translational aspect, a pan-LOX inhibitor significantly decreased HO. A newly screened compound revealed that the inhibition of PLOD2 activity in MPCs significantly decreased osteogenic differentiation and glycolytic metabolism. This suggests that the HIF-1α/PLOD2/LOX axis linked to metabolism regulates HO-forming MPC fate. These results suggest that the HIF-1α/PLOD2/LOX pathway represents a promising strategy to mitigate HO formation.

Collagen prolyl 3-hydroxylation: a major role for a minor post-translational modification?

Prolyl 3-hydroxylation is a rare but conserved post-translational modification in many collagen types and, when defective, may be linked to a number of human diseases with musculoskeletal and potentially ocular and renal pathologies. Prolyl 3-hydroxylase-1 (P3H1), the enzyme responsible for converting proline to 3-hydroxyproline (3Hyp) in type I collagen, requires the coenzyme CRTAP for activity. Mass spectrometric analysis showed that the Crtap-/- mouse was missing 3-hydroxyproline in type I collagen α-chains. This finding led to the discovery of mutations in genes encoding the P3H1 complex as a cause of recessively inherited osteogenesis imperfecta (brittle bone disease). Since then, many additional 3Hyp sites have been identified in various collagen types and classified based on observed substrate and tissue specificity. P3H1 is part of a family of gene products that also includes isoenzymes P3H2 and P3H3 as well as CRTAP and Sc65. It is believed these isoenzymes and coenzyme proteins have evolved different collagen substrate site and tissue specificities in their activities. The post-translational fingerprinting of collagens will be essential in understanding the basic role and extent of regulated variations of prolyl 3-hydroxylation in collagen. We believe that prolyl 3-hydroxylation is a functionally significant collagen post-translational modification and can be a cause of disease when absent.

An image dataset of diverse safflower (Carthamus tinctorius L.) genotypes for salt response phenotyping.

This article describes a dataset of high-resolution visible-spectrum images of safflower (Carthamus tinctorius L.) plants obtained from a LemnaTec Scanalyser automated phenomics platform along with the associated image analysis output and manually acquired biomass data. This series contains 1832 images of 200 diverse safflower genotypes, acquired at the Plant Phenomics Victoria, Horsham, Victoria, Australia. Two Prosilica GT RGB (red-green-blue) cameras were used to generate 6576 × 4384 pixel portable network graphic (PNG) images. Safflower genotypes were either subjected to a salt treatment (250 mM NaCl) or grown as a control (0 mM NaCl) and imaged daily from 15 to 36 days after sowing. Each snapshot consists of four images collected at a point in time; one of which is taken from above (top-view) and the remainder from the side at either 0°, 120° or 240°. The dataset also includes analysis output quantifying traits and describing phenotypes, as well as manually collected biomass and leaf ion content data. The usage of the dataset is already demonstrated in Thoday-Kennedy et al. (2021) [1]. This dataset describes the early growth differences of diverse safflower genotypes and identified genotypes tolerant or susceptible to salinity stress. This dataset provides detailed image analysis parameters for phenotyping a large population of safflower that can be used for the training of image-based trait identification pipelines for a wide range of crop species.

Collagen breaks at weak sacrificial bonds taming its mechanoradicals.

Collagen is a force-bearing, hierarchical structural protein important to all connective tissue. In tendon collagen, high load even below macroscopic failure level creates mechanoradicals by homolytic bond scission, similar to polymers. The location and type of initial rupture sites critically decide on both the mechanical and chemical impact of these micro-ruptures on the tissue, but are yet to be explored. We here use scale-bridging simulations supported by gel electrophoresis and mass spectrometry to determine breakage points in collagen. We find collagen crosslinks, as opposed to the backbone, to harbor the weakest bonds, with one particular bond in trivalent crosslinks as the most dominant rupture site. We identify this bond as sacrificial, rupturing prior to other bonds while maintaining the material's integrity. Also, collagen's weak bonds funnel ruptures such that the potentially harmful mechanoradicals are readily stabilized. Our results suggest this unique failure mode of collagen to be tailored towards combatting an early onset of macroscopic failure and material ageing.

Peptidyl 3-hydroxyproline binding properties of type I collagen suggest a function in fibril supramolecular assembly.

Proline residues in collagens are extensively hydroxylated post-translationally. A rare form of this modification, (3S,2S)-l-hydroxyproline (3Hyp), remains without a clear function. Disruption of the enzyme complex responsible for prolyl 3-hydroxylation results in severe forms of recessive osteogenesis imperfecta (OI). These OI types exhibit a loss of or reduction in the level of 3-hydroxylation at two proline residues, α1(I) Pro986 and α2(I) Pro707. Whether the resulting brittle bone phenotype is caused by the lack of the 3-hydroxyl addition or by another function of the enzyme complex is unknown. We have speculated that the most efficient mechanism for explaining the chemistry of collagen intermolecular cross-linking is for pairs of collagen molecules in register to be the subunit that assembles into fibrils. In this concept, the exposed hydroxyls from 3Hyp are positioned within mutually interactive binding motifs on adjacent collagen molecules that contribute through hydrogen bonding to the process of fibril supramolecular assembly. Here we report observations on the physical binding properties of 3Hyp in collagen chains from experiments designed to explore the potential for interaction using synthetic collagen-like peptides containing 3Hyp. Evidence of self-association was observed between a synthetic peptide containing 3Hyp and the CB6 domain of the α1(I) chain, which contains the single fully 3-hydroxylated proline. Using collagen from a case of severe recessive OI with a CRTAP defect, in which Pro986 was minimally 3-hydroxylated, such binding was not observed. Further study of the role of 3Hyp in supramolecular assembly is warranted for understanding the evolution of tissue-specific variations in collagen fibril organization.

A novel 3-hydroxyproline (3Hyp)-rich motif marks the triple-helical C terminus of tendon type I collagen.

Because of its unique physical and chemical properties, rat tail tendon collagen has long been favored for crystallographic and biochemical studies of fibril structure. In studies of the distribution of 3-hydroxyproline in type I collagen of rat bone, skin, and tail tendon by mass spectrometry, the repeating sequences of Gly-Pro-Pro (GPP) triplets at the C terminus of α1(I) and α2(I) chains were shown to be heavily 3-hydroxylated in tendon but not in skin and bone. By isolating the tryptic peptides and subjecting them to Edman sequence analysis, the presence of repeating 3-hydroxyprolines in consecutive GPP triplets adjacent to 4-hydroxyproline was confirmed as a unique feature of the tendon collagen. A 1960s study by Piez et al. (Piez, K. A., Eigner, E. A., and Lewis, M. S. (1963) Biochemistry 2, 58-66) in which they compared the amino acid compositions of rat skin and tail tendon type I collagen chains indeed showed 3-4 residues of 3Hyp in tendon α1(I) and α2(I) chains but only one 3Hyp residue in skin α1(I) and none in α2(I). The present work therefore confirms this difference and localizes the additional 3Hyp to the GPP repeat at the C terminus of the triple-helix. We speculate on the significance in terms of a potential function in contributing to the unique assembly mechanism and molecular packing in tendon collagen fibrils and on mechanisms that could regulate 3-hydroxylation at this novel substrate site in a tissue-specific manner.

Cystine-mediated oligomerization of the Atlantic salmon serum C-type lectin.

The Atlantic salmon (Salmo salar) serum lectin (SSL) is a C-type lectin that binds to bacteria including salmon pathogens. SSL has been shown to be oligomeric in salmon serum and it displays a stoichiometric band-laddering pattern when analyzed by SDS-PAGE under non-reducing conditions. In this study, a model was generated for SSL isoform 2 in silico in order to identify cysteines that are available to form intermolecular disulfide bonds facilitating oligomerization. Then, recombinant SSL was expressed in E. coli and mutants were produced at positions Cys72 and Cys149. The SSL preparations were purified by metal-affinity chromatography and shown to be functional by carbohydrate-affinity chromatography. The recombinant SSL formed oligomers, which were evident by non-reducing covalent cross-linking and non-reducing SDS-PAGE; however, the band patterns were different for the mutants, with the maximal and predominant multimer sizes distinct from the wild-type recombinant lectin. Further examination of oligomerization by size exclusion chromatography revealed a subunit number from 35 to at least 110 for the wild-type recombinant SSL and subunit numbers below 9 for each mutant SSL oligomer. Thus, both cysteines were found to contribute to oligomerization of SSL.

Functional outcomes and local control are acceptable following hand-sewn colo-anal anastomoses for rectal cancer: Results of long-term follow-up.

BACKGROUND: Rectal cancer that grows so close to the anal canal that an adequate distal margin cannot be achieved with a double-stapled anastomosis (DSA) has been managed with abdominoperineal resection. Inter-sphincteric dissection and hand-sewn colo-anal anastomoses (HSCAA) allows anastomosis in some cases where DSA is impossible. There are concerns that HSCAA may lead to complications, local recurrence and poor continence. Our aim was to assess our experience with HSCAA in terms of recurrence, complications, continence and quality of life. METHODS: Consecutive patients at two metropolitan hospitals who underwent an ultra-low anterior resection with hand-sewn colo-anal anastomoses for low rectal cancer during a 10-year period were asked to complete a questionnaire which allowed continence and quality-of-life scores to be calculated. Recurrence and complication rates were obtained from a retrospective medical record review. RESULTS: A total of 26 patients underwent HSCAA. Six patients were not sent a questionnaire (3 deaths, 1 with ileostomy, 1 with ileostomy reversal within 3 months and one who had transferred care to another hospital). Fifteen patients returned questionnaires. Local recurrence occurred in zero cases. Two developed systemic recurrence. Four patients developed anastomotic stricture and three had anastomotic leak. Median Faecal Incontinence Severity Index score was 28 and median FIQoL scores were 3.00, 2.78, 3.86 and 3.00. One patient wished that they had had a permanent stoma rather than anastomosis. CONCLUSION: HSCAA delivered good local control of rectal cancer and high avoidance of permanent stoma. Faecal continence is impaired; however, the results are acceptable to the majority of patients.

Novel regulatory roles of small leucine-rich proteoglycans in remodeling of the uterine cervix in pregnancy.

The cervix undergoes rapid and dramatic shifts in collagen and elastic fiber structure to achieve its disparate physiological roles of competence during pregnancy and compliance during birth. An understanding of the structure-function relationships of collagen and elastic fibers to maintain extracellular matrix (ECM) homeostasis requires an understanding of the mechanisms executed by non-structural ECM molecules. Small-leucine rich proteoglycans (SLRPs) play key functions in biology by affecting collagen fibrillogenesis and regulating enzyme and growth factor bioactivities. In the current study, we evaluated collagen and elastic fiber structure-function relationships in mouse cervices using mice with genetic ablation of decorin and/or biglycan genes as representative of Class I SLRPs, and lumican gene representative of Class II SLRP. We identified structural defects in collagen fibril and elastic fiber organization in nonpregnant mice lacking decorin, or biglycan or lumican with variable resolution of defects noted during pregnancy. The severity of collagen and elastic fiber defects was greater in nonpregnant mice lacking both decorin and biglycan and defects were maintained throughout pregnancy. Loss of biglycan alone reduced tissue extensibility in nonpregnant mice while loss of both decorin and biglycan manifested in decreased rupture stretch in late pregnancy. Collagen cross-link density was similar in the Class I SLRP null mice as compared to wild-type nonpregnant and pregnant controls. A broader range in collagen fibril diameter along with an increase in mean fibril spacing was observed in the mutant mice compared to wild-type controls. Collectively, these findings uncover functional redundancy and hierarchical roles of Class I and Class II SLRPs as key regulators of cervical ECM remodeling in pregnancy. These results expand our understating of the critical role SLRPs play to maintain ECM homeostasis in the cervix.

Potential impacts from simulated vessel noise and sonar on commercially important invertebrates.

Human usage of coastal water bodies continues to increase and many invertebrates face a broad suite of anthropogenic stressors (e.g., warming, pollution, acidification, fishing pressure). Underwater sound is a stressor that continues to increase in coastal areas, but the potential impact on invertebrates is not well understood. In addition to masking natural sound cues which may be important for behavioral interactions, there is a small but increasing body of scientific literature indicating sublethal physiological stress may occur in invertebrates exposed to high levels of underwater sound, particularly low frequency sounds such as vessel traffic, construction noise, and some types of sonar. Juvenile and sub-adult blue crabs (Callinectes sapidus) and American lobsters (Homarus americanus) were exposed to simulated low-frequency vessel noise (a signal was low-pass filtered below 1 kHz to ensure low-frequency content only) and mid-frequency sonar (a 1-s 1.67 kHz continuous wave pulse followed by a 2.5 to 4.0 kHz 1-s linear frequency modulated chirp) and behavioral response (the animal's activity level) was quantified during and after exposure using EthoVision XT™ from overhead video recordings. Source noise was quantified by particle acceleration and pressure. Physiological response to the insults (stress and recovery) were also quantified by measuring changes in hemolymph heat shock protein (HSP27) and glucose over 7 days post-exposure. In general, physiological indicators returned to baseline levels within approximately 48 h, and no observable difference in mortality between treatment and control animals was detected. However, there was a consistent amplified hemolymph glucose signal present 7 days after exposure for those animals exposed to mid-frequency sound and there were changes to C. sapidus competitive behavior within 24 h of exposure to sound. These results stress the importance of considering the impacts of underwater sound among the suite of stressors facing marine and estuarine invertebrates, and in the discussion of management actions such as protected areas, impact assessments, and marine spatial planning efforts.

Location of 3-hydroxyproline residues in collagen types I, II, III, and V/XI implies a role in fibril supramolecular assembly.

Collagen triple helices are stabilized by 4-hydroxyproline residues. No function is known for the much less common 3-hydroxyproline (3Hyp), although genetic defects inhibiting its formation cause recessive osteogenesis imperfecta. To help understand the pathogenesis, we used mass spectrometry to identify the sites and local sequence motifs of 3Hyp residues in fibril-forming collagens from normal human and bovine tissues. The results confirm a single, essentially fully occupied 3Hyp site (A1) at Pro(986) in A-clade chains alpha1(I), alpha1(II), and alpha2(V). Two partially modified sites (A2 and A3) were found at Pro(944) in alpha1(II) and alpha2(V) and Pro(707) in alpha2(I) and alpha2(V), which differed from A1 in sequence motif. Significantly, the distance between sites 2 and 3, 237 residues, is close to the collagen D-period (234 residues). A search for additional D-periodic 3Hyp sites revealed a fourth site (A4) at Pro(470) in alpha2(V), 237 residues N-terminal to site 3. In contrast, human and bovine type III collagen contained no 3Hyp at any site, despite a candidate proline residue and recognizable A1 sequence motif. A conserved histidine in mammalian alpha1(III) at A1 may have prevented 3-hydroxylation because this site in chicken type III was fully hydroxylated, and tyrosine replaced histidine. All three B-clade type V/XI collagen chains revealed the same three sites of 3Hyp but at different loci and sequence contexts from those in A-clade collagen chains. Two of these B-clade sites were spaced apart by 231 residues. From these and other observations we propose a fundamental role for 3Hyp residues in the ordered self-assembly of collagen supramolecular structures.