Deciphering Breast Cancer Metastasis Cascade: A Systems Biology Approach Integrating Transcriptome and Interactome Insights for Target Discovery.

Breast cancer is the lead cause of cancer-related deaths among women globally. Breast cancer metastasis is a complex and still inadequately understood process and a key dimension of mortality attendant to breast cancer. This study reports dysregulated genes across metastatic stages and tissues, shedding light on their molecular interplay in disease pathogenesis and new possibilities for drug discovery. Comprehensive analyses of gene expression data from primary breast tumor, circulating tumor cells, and distant metastatic sites in the brain, lung, liver, and bone were conducted. Genes dysregulated across multiple stages and tissues were identified as metastatic cascade genes, and are further classified based on functional associations with metastasis-related mechanisms. Their interactions with HUB genes in interactome networks were scrutinized, followed by pathway enrichment analysis. Validation for their potential as targets included assessments for survival, druggability, prognostic marker status, secretome annotation, protein expression, and cell type marker association. Results displayed critical genes in the metastatic cascade and those specific to metastatic sites, revealing the involvement of the collagen degradation and assembly of collagen fibrils and other multimeric structure pathways in driving metastasis. Notably, pivotal cascade genes FABP4, CXCL12, APOD, and IGF1 emerged with high metastatic potential, linked to significant druggability and survival scores, establishing them as potential molecular targets. The significance of this research lies in its potential to uncover novel biomarkers for early detection, therapeutic targets, and a deeper understanding of the molecular mechanisms underpinning the metastatic cascade in breast cancer, and with an eye to precision/personalized medicine.

COLQ-Congenital myasthenic syndrome in an Iranian cohort: the clinical and genetics spectrum.

BACKGROUND: Congenital myasthenic syndrome (CMS) is a group of neuromuscular disorders caused by abnormal signal transmission at the motor endplate. Mutations in the collagen-like tail subunit gene (COLQ) of acetylcholinesterase are responsible for recessive forms of synaptic congenital myasthenic syndromes with end plate acetylcholinesterase deficiency. Clinical presentation includes ptosis, ophthalmoparesis, and progressive weakness with onset at birth or early infancy. METHODS: We followed 26 patients with COLQ-CMS over a mean period of 9 years (ranging from 3 to 213 months) and reported their clinical features, electrophysiologic findings, genetic characteristics, and therapeutic management. RESULTS: In our population, the onset of symptoms ranged from birth to 15 years. Delayed developmental motor milestones were detected in 13 patients (∼ 52%), and the most common presenting signs were ptosis, ophthalmoparesis, and limb weakness. Sluggish pupils were seen in 8 (∼ 30%) patients. All patients who underwent electrophysiologic study showed a significant decremental response (> 10%) following low-frequency repetitive nerve stimulation. Moreover, double compound muscle action potential was evident in 18 patients (∼ 75%). We detected 14 variants (eight novel variants), including six missense, three frameshift, three nonsense, one synonymous and one copy number variation (CNV), in the COLQ gene. There was no benefit from esterase inhibitor treatment, while treatment with ephedrine and salbutamol was objectively efficient in all cases. CONCLUSION: Despite the rarity of the disease, our findings provide valuable information for understanding the clinical and electrophysiological features as well as the genetic characterization and response to the treatment of COLQ-CMS.

Fibroblasts transfection by electroporation in 3D reconstructed human dermal tissue.

The understanding of the mechanisms involved in DNA electrotransfer in human skin remains modest and limits the clinical development of various biomedical applications, such as DNA vaccination. To elucidate some mechanisms of DNA transfer in the skin following electroporation, we created a model of the dermis using a tissue engineering approach. This model allowed us to study the electrotransfection of fibroblasts in a three-dimensional environment that included multiple layers of fibroblasts as well as the self-secreted collagen matrix. With the aim of improving transfection yield, we applied electrical pulses with electric field lines perpendicular to the reconstructed model tissue. Our results indicate that the fibroblasts of the reconstructed skin tissue can be efficiently permeabilized by applied millisecond electrical pulses. However, despite efficient permeabilization, the transfected cells remain localized only on the surface of the microtissue, to which the plasmid was deposited. Second harmonic generation microscopy revealed the extensive extracellular collagen matrix around the fibroblasts, which might have affected the mobility of the plasmid into deeper layers of the skin tissue model. Our results show that the used skin tissue model reproduces the structural barriers that might be responsible for the limited gene electrotransfer in the skin.

ADARs regulate cuticle collagen expression and promote survival to pathogen infection.

BACKGROUND: In all organisms, the innate immune system defends against pathogens through basal expression of molecules that provide critical barriers to invasion and inducible expression of effectors that combat infection. The adenosine deaminase that act on RNA (ADAR) family of RNA-binding proteins has been reported to influence innate immunity in metazoans. However, studies on the susceptibility of ADAR mutant animals to infection are largely lacking. RESULTS: Here, by analyzing adr-1 and adr-2 null mutants in well-established slow-killing assays, we find that both Caenorhabditis elegans ADARs are important for organismal survival to gram-negative and gram-positive bacteria, all of which are pathogenic to humans. Furthermore, our high-throughput sequencing and genetic analysis reveal that ADR-1 and ADR-2 function in the same pathway to regulate collagen expression. Consistent with this finding, our scanning electron microscopy studies indicate adr-1;adr-2 mutant animals also have altered cuticle morphology prior to pathogen exposure. CONCLUSIONS: Our data uncover a critical role of the C. elegans ADAR family of RNA-binding proteins in promoting cuticular collagen expression, which represents a new post-transcriptional regulatory node that influences the extracellular matrix. In addition, we provide the first evidence that ADAR mutant animals have altered susceptibility to infection with several opportunistic human pathogens, suggesting a broader role of ADARs in altering physical barriers to infection to influence innate immunity.

Downregulation of salusins alleviates hypertrophic cardiomyopathy via attenuating oxidative stress and autophagy.

INTRODUCTION: Salusins, which are translated from the alternatively spliced mRNA of torsin family 2 member A (TOR2A), play a vital role in regulation of various cardiovascular diseases. However, it remains unclear precisely regarding their roles in hypertrophic cardiomyopathy (HCM). Therefore, this study was conducted to explore therapeutic effect and the underlying mechanisms of salusins on HCM. MATERIAL AND METHODS: In vivo experiments, Sprague-Dawley rats were used to induce HCM model by angiotensin (Ang) II infusion for 4 weeks. The rats were randomly divided into four groups, namely, Saline + Control shRNA (n = 7), Ang II + Control shRNA (n = 8), Saline + TOR2A shRNA (n = 7), and Ang II + TOR2A shRNA groups (n = 8). After HCM induction, doppler echocardiography is recommended to evaluate heart function. In vitro experiments, primary neonatal rat cardiomyocytes (NRCMs) and cardiac fibroblasts (NRCFs) were obtained from newborn rats, and were treated with Ang II (10-6 M) for 24 h. RESULTS: After treatment with Ang II, levels of salusin-α and salusin-ß were elevated in serum and cardiac tissues of rats and in the neonatal rat cardiomyocytes and cardiac fibroblasts. Downregulation of salusins alleviated the Ang II-induced cardiac hypertrophy by suppressing the increased atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and beta-myosin heavy chain (ß-MHC) and cardiac fibrosis by blocking collagen I, collagen III and transforming growth factor-beta (TGF-ß), and it also attenuated oxidative stress by suppressing the increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels and reversing the decreased superoxide dismutase (SOD) activity and autophagy by inhibiting the increased microtubule-associated protein light chain 3B (LC3B), Beclin1, autophagy related gene (Atg) 3 and Atg5 in the cardiac tissues of Ang II-infused rats and in the Ang II-treated NRCMs. CONCLUSIONS: All these findings suggest that the levels of salusins were elevated in the HCM, and targeting of salusins contributes to alleviation of cardiac hypertrophy and fibrosis probably via attenuating oxidative stress and autophagy. Accordingly, targeting of salusins may be a strategy for HCM therapy.

Novel KMT2B gene mutation in MUC4 positive low-grade fibromyxoid sarcoma.

BACKGROUND: Low-grade Fibromyxoid Sarcoma(LGFM)is a rare fibrosarcoma, which mainly occurs in young people and is mostly seen in the trunk and limbs. The tumor is usually FUS-CREB3L2 fusion caused by t(7;16)(q32-34;p11)chromosome translocation, and rarely FUS-CREB3L1 and EWSR1-CREB3L1 fusion. MUC4 diffuse strong positive can be used as a specific index of LGFM. LGFM is similar to Sclerosing Epithelioid Fibrosarcoma(SEF) and may have the same origin. CASE PRESENTATION: We report a case of LGFM in the chest wall. A female who is 59 years old. In 2016, CT showed dense nodule shadow and focal thickening of the left pleura, the patient underwent surgery, Pathological report that low to moderate malignant fibrosarcoma(fibromyxoid type). The CT re-examination in 2021 showed that the tumors on the left chest wall were significantly larger than before. Pathological examination showed the disease is composed of alternating collagen like and mucinous areas. Under high-power microscope, the tumor cells are consistent in shape, spindle or short spindle, and the tumor cells are arranged in bundles. In local areas, the density of tumor cells is significantly increased, mixed with collagen fibers, and small focal SEF appear. The result of immunohistochemistry showed that SMA, Desmin, CD34, STAT6, S100, SOX10, HMB45 and Melan A were negative, EMA was weakly positive, MUC4 was diffuse and strongly positive, and Ki67 index was low (3%). CONCLUSION: Sequencing results showed that MET, EGFR, KMT2B and RET gene were mutated in LGFM, and KMT2B gene had cancer promoting effect, but there was no literature report in LGFM, which may be of certain significance for the diagnosis and treatment of LGFM.

Association of genetic variation in COL11A1 with adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a common and progressive spinal deformity in children that exhibits striking sexual dimorphism, with girls at more than fivefold greater risk of severe disease compared to boys. Despite its medical impact, the molecular mechanisms that drive AIS are largely unknown. We previously defined a female-specific AIS genetic risk locus in an enhancer near the PAX1 gene. Here, we sought to define the roles of PAX1 and newly identified AIS-associated genes in the developmental mechanism of AIS. In a genetic study of 10,519 individuals with AIS and 93,238 unaffected controls, significant association was identified with a variant in COL11A1 encoding collagen (α1) XI (rs3753841; NM_080629.2_c.4004C>T; p.(Pro1335Leu); p=7.07E-11, OR = 1.118). Using CRISPR mutagenesis we generated Pax1 knockout mice (Pax1-/-). In postnatal spines we found that PAX1 and collagen (α1) XI protein both localize within the intervertebral disc-vertebral junction region encompassing the growth plate, with less collagen (α1) XI detected in Pax1-/- spines compared to wild-type. By genetic targeting we found that wild-type Col11a1 expression in costal chondrocytes suppresses expression of Pax1 and of Mmp3, encoding the matrix metalloproteinase 3 enzyme implicated in matrix remodeling. However, the latter suppression was abrogated in the presence of the AIS-associated COL11A1P1335L mutant. Further, we found that either knockdown of the estrogen receptor gene Esr2 or tamoxifen treatment significantly altered Col11a1 and Mmp3 expression in chondrocytes. We propose a new molecular model of AIS pathogenesis wherein genetic variation and estrogen signaling increase disease susceptibility by altering a PAX1-COL11a1-MMP3 signaling axis in spinal chondrocytes.

Adolescent idiopathic scoliosis (AIS) is a twisting deformity of the spine that occurs during periods of rapid growth in children worldwide. Children with severe cases of AIS require surgery to stop it from getting worse, presenting a significant financial burden to health systems and families. Although AIS is known to cluster in families, its genetic causes and its inheritance pattern have remained elusive. Additionally, AIS is known to be more prevalent in females, a bias that has not been explained. Advances in techniques to study the genetics underlying diseases have revealed that certain variations that increase the risk of AIS affect cartilage and connective tissue. In humans, one such variation is near a gene called Pax1, and it is female-specific. The extracellular matrix is a network of proteins and other molecules in the space between cells that help connect tissues together, and it is particularly important in cartilage and other connective tissues. One of the main components of the extracellular matrix is collagen. Yu, Kanshour, Ushiki et al. hypothesized that changes in the extracellular matrix could affect the cartilage and connective tissues of the spine, leading to AIS. To show this, the scientists screened over 100,000 individuals and found that AIS is associated with variants in two genes coding for extracellular matrix proteins. One of these variants was found in a gene called Col11a1, which codes for one of the proteins that makes up collagen. To understand the relationship between Pax1 and Col11a1, Yu, Kanshour, Ushiki et al. genetically modified mice so that they would lack the Pax1 gene. In these mice, the activation of Col11a1 was reduced in the mouse spine. They also found that the form of Col11a1 associated with AIS could not suppress the activation of a gene called Mmp3 in mouse cartilage cells as effectively as unmutated Col11a1. Going one step further, the researchers found that lowering the levels of an estrogen receptor altered the activation patterns of Pax1, Col11a1, and Mmp3 in mouse cartilage cells. These findings suggest a possible mechanism for AIS, particularly in females. The findings of Yu, Kanshour, Ushiki et al. highlight that cartilage cells in the spine are particularly relevant in AIS. The results also point to specific molecules within the extracellular matrix as important for maintaining proper alignment in the spine when children are growing rapidly. This information may guide future therapies aimed at maintaining healthy spinal cells in adolescent children, particularly girls.

Collagen and actin network mediate antiviral immunity against Orsay virus in C. elegans intestinal cells.

C. elegans is a free-living nematode that is widely used as a small animal model for studying fundamental biological processes and disease mechanisms. Since the discovery of the Orsay virus in 2011, C. elegans also holds the promise of dissecting virus-host interaction networks and innate antiviral immunity pathways in an intact animal. Orsay virus primarily targets the worm intestine, causing enlarged intestinal lumen as well as visible changes to infected cells such as liquefaction of cytoplasm and convoluted apical border. Previous studies of Orsay virus identified that C. elegans is able to mount antiviral responses by DRH-1/RIG-I mediated RNA interference and Intracellular Pathogen Response, a uridylyltransferase that destabilizes viral RNAs by 3' end uridylation, and ubiquitin protein modifications and turnover. To comprehensively search for novel antiviral pathways in C. elegans, we performed genome-wide RNAi screens by bacterial feeding using existing bacterial RNAi libraries covering 94% of the entire genome. Out of the 106 potential antiviral gene hits identified, we investigated those in three new pathways: collagens, actin remodelers, and epigenetic regulators. By characterizing Orsay virus infection in RNAi and mutant worms, our results indicate that collagens likely form a physical barrier in intestine cells to inhibit viral infection by preventing Orsay virus entry. Furthermore, evidence suggests that actin remodeling proteins (unc-34, wve-1 and wsp-1) and chromatin remodelers (nurf-1 and isw-1) exert their antiviral activities by regulating the intestinal actin (act-5), a critical component of the terminal web which likely function as another physical barrier to prevent Orsay infection.

Collagen prolyl 4-hydroxylase isoenzymes I and II have sequence specificity towards different X-Pro-Gly triplets.

Collagen biosynthesis requires several co- and post-translational modifications of lysine and proline residues to form structurally and functionally competent collagen molecules. Formation of 4-hydroxyproline (4Hyp) in Y-position prolines of the repetitive -X-Y-Gly- sequences provides thermal stability for the triple-helical collagen molecules. 4Hyp formation is catalyzed by a collagen prolyl 4-hydroxylase (C-P4H) family consisting of three isoenzymes. Here we identify specific roles for the two main C-P4H isoenzymes in collagen hydroxylation by a detailed 4Hyp analysis of type I and IV collagens derived from cell and tissue samples. Loss of C-P4H-I results in underhydroxylation of collagen where the affected prolines are not uniformly distributed, but mainly present in sites where the adjacent X-position amino acid has a positively charged or a polar uncharged side chain. In contrast, loss of C-P4H-II results in underhydroxylation of triplets where the X-position is occupied by a negatively charged amino acid glutamate or aspartate. Hydroxylation of these triplets was found to be important as loss of C-P4H-II alone resulted in reduced collagen melting temperature and altered assembly of collagen fibrils and basement membrane. The observed C-P4H isoenzyme differences in substrate specificity were explained by selective binding of the substrate to the active site resulting in distinct differences in Km and Vmax values. Furthermore, our results clearly show that the substrate proline selection is not dependent on the collagen type, but the main determinant is the X-position amino acid of the -X-Pro-Gly- triplet. Although our data clearly shows the necessity of both C-P4H-I and II for normal prolyl 4-hydroxylation and function of collagens, the mRNA expression of the isoenzymes with various procollagens was, surprisingly, not tightly coordinated, suggesting additional levels of control. In conclusion, this study provides a molecular level explanation for the need of multiple C-P4H isoenzymes to generate collagen molecules capable to assemble into intact extracellular matrix structures.

Transcriptome analysis reveals dysfunction of the endoplasmic reticulum protein processing in the sonic muscle of small yellow croaker (Larimichthys polyactis) following noise exposure.

Noise pollution is increasingly prevalent in aquatic ecosystems, causing detrimental effects on growth and behavior of marine fishes. The physiological responses of fish to underwater noise are poorly understood. In this study, we used RNA-sequencing (RNA-seq) to study the transcriptome of the sonic muscle in small yellow croaker (Larimichthys polyactis) after exposure to a 120 dB noise for 30 min. The behavioral experiment revealed that noise exposure resulted in accelerated tail swimming behavior at the beginning of the exposure period, followed by loss of balance at the end of experiment. Transcriptomic analysis found that most highly expressed genes in the sonic muscle, including parvalbumin, slc25a4, and troponin C were related with energy metabolism and locomotor function. Further, a total of 1261 differentially expressed genes (DEGs) were identified, including 284 up-regulated and 977 down-regulated genes in the noise exposure group compared with the control group. Gene ontology (GO) analysis indicated that the most enriched categories of DEGs included protein folding and response to unfolding protein. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis found over-represented pathways including protein processing in the endoplasmic reticulum, chaperones and folding catalysts, as well as arginine and proline metabolism. Specifically, many genes related to fatty acid and collagen metabolism were up-regulated in the noise exposure group. Taken together, our results indicate that exposure to noise stressors alters the swimming behavior of croaker, inducing endoplasmic reticulum stress, disrupting lipid metabolism, and causing collagen degradation in the sonic muscle of L. polyactis.

An incipient late-onset retinal degeneration with a C1QTNF5 mutation: a case report with an 11-year follow-up.

PURPOSE: The purpose of this study was to describe and diagnose the difficulty in a long-term follow-up (eleven years) patient with a very early presentation of late-onset retinal degeneration (L-ORD) and the significance of electrophysiological examinations and follow-up in assessing undiagnosed inherited retinal diseases. METHODS: This is an observational case report of a 56-year-old woman, with scattered multiple yellow-white retinal dots firstly diagnosed as fundus albipunctatus. Ten years after presentation, a deterioration in rod and cone responses in ff-ERG was detected, which allowed us to discard the first diagnostic hypothesis and proceed with a genetic testing. RESULTS: Ten years after presentation, she presented a clear progression of the abnormal photoreceptor response with a cone and rod involvement in ff-ERG, which was not compatible with the previous suspicion of fundus albipunctatus. Six months later, genetic testing results together with the typical progression of atrophic patchy lesions in multimodal imaging allowed a certain diagnosis of L-ORD, caused by an already reported pathogenic variant in the C1QTNF5 gene (c.563C > T; p. Pro188 Leu). CONCLUSIONS: We demonstrate the importance of the ff-ERG examination and the follow-up (or ERG and imaging repetition) in the differential diagnosis of an incipient L-ORD, which can be easily misdiagnosed in the early stages, before the appearance of the characteristic chorioretinal atrophy seen with the progression of this rare disease.

Identification of key genes and molecular pathways associated with claw regeneration in mud crab (Scylla paramamosain).

The mud crab (Scylla paramamosain) possesses extensive regenerative abilities, enabling it to replace missing body parts, including claws, legs, and even eyes. Studying the genetic and molecular mechanisms underlying regenerative ability in diverse animal phyla has the potential to provide new insights into regenerative medicine in humans. In the present study, we performed mRNA sequencing to reveal the genetic mechanisms underlying the claw regeneration in mud crab. Several differentially expressed genes (DEGs) were expressed in biological pathways associated with cuticle synthase, collagen synthase, tissue regeneration, blastema formation, wound healing, cell cycle, cell division, and cell migration. The top GO enrichment terms were microtubule-based process, collagen trimer, cell cycle process, and extracellular matrix structural constituent. The most enriched KEGG pathways were ECM-receptor interaction and focal adhesion. The genes encoding key functional proteins, such as collagen alpha, cuticle protein, early cuticle protein, arthrodial cuticle protein, dentin sialophosphoprotein (DSPP), epidermal growth factor receptor (EGFR), kinesin family member C1 (KIFC1), and DNA replication licensing factor mcm2-like (MCM2) were the most significant and important DEGs suspected to participate in claw regeneration. The findings of this research offer a comprehensive and insightful understanding of the genetic and molecular mechanisms underlying claw regeneration in S. paramamosain. By elucidating the specific genes and molecular pathways implicated in this process, our study contributes significantly to the broader field of regenerative biology and offers potential avenues for further exploration in crustacean limb regeneration.

Markers of adipose tissue fibrogenesis associate with clinically significant liver fibrosis and are unchanged by synbiotic treatment in patients with NAFLD.

BACKGROUND AND AIMS: Subcutaneous adipose tissue (SAT) dysfunction contributes to NAFLD pathogenesis and may be influenced by the gut microbiota. Whether transcript profiles of SAT are associated with liver fibrosis and are influenced by synbiotic treatment (that changes the gut microbiome) is unknown. We investigated: (a) whether the presence of clinically significant, ≥F2 liver fibrosis associated with adipose tissue (AT) dysfunction, differential gene expression in SAT, and/or a marker of tissue fibrosis (Composite collagen gene expression (CCGE)); and (b) whether synbiotic treatment modified markers of AT dysfunction and the SAT transcriptome. METHODS: Sixty-two patients with NAFLD (60 % men) were studied before and after 12 months of treatment with synbiotic or placebo and provided SAT samples. Vibration-controlled transient elastography (VCTE)-validated thresholds were used to assess liver fibrosis. RNA-sequencing and histological analysis of SAT were performed to determine differential gene expression, CCGE and the presence of collagen fibres. Regression modelling and receiver operator characteristic curve analysis were used to test associations with, and risk prediction for, ≥F2 liver fibrosis. RESULTS: Patients with ≥F2 liver fibrosis (n = 24) had altered markers of AT dysfunction and a SAT gene expression signature characterised by enrichment of inflammatory and extracellular matrix-associated genes, compared to those with

Collagen gene cluster expression and liver fibrogenesis in patients with biliary atresia: a preliminary study.

OBJECTIVE: Biliary atresia (BA) is a progressive fibro-obliterative disease of the biliary tract, which results in end-stage liver disease. However, liver fibrosis progression may continue even after Kasai surgery. Recent evidence showed that collagen plays a pivotal role in the progression of liver fibrosis in BA. However, most studies were conducted in developed countries. We investigated the expressions of the collagen gene cluster (COL6A1, COL6A2, COL6A3, and COL1A1) in BA patients in Indonesia. RESULTS: There was a significant down-regulated expression of COL6A1 (ΔCT 9.06 ± 2.64 vs. 5.42 ± 2.41; p = 0.0009), COL6A2 (ΔCT 8.25 ± 2.07 vs. 5.77 ± 3.51; p = 0.02), COL6A3 (ΔCT 11.2 ± 6.08 vs. 6.78 ± 3.51; p = 0.024), and COL1A1 (ΔCT 3.26 ± 1.71 vs. 0.19 ± 2.76; p = 0.0015) in BA patients compared to controls. Interestingly, the collagen gene cluster expressions were significantly associated with the presence of cirrhosis (p = 0.0085, 0.04, and 0.0283 for COL6A1, COL6A2, and COL6A3, respectively). In conclusion, our study shows the changes in the collagen gene cluster, particularly collagen type I and VI, expressions in patients with BA in a particular developing country. Our findings suggest the role of these collagen gene clusters in the liver fibrogenesis of BA.

Defect of TIMP4 Is Associated with High Myopia and Participates in Rat Ocular Development in a Dose-Dependent Manner.

Thinning of the sclera happens in myopia eyes owing to extracellular matrix (ECM) remodeling, but the initiators of the ECM remodeling in myopia are mainly unknown. The matrix metalloproteinase (MMPs) and tissue inhibitors of matrix metalloproteinase (TIMPs) regulate the homeostasis of the ECM. However, genetic studies of the MMPs and TIMPs in the occurrence of myopia are poor and limited. This study systematically investigated the association between twenty-nine genes of the TIMPs and MMPs families and early-onset high myopia (eoHM) based on whole exome sequencing data. Two TIMP4 heterozygous loss-of-function (LoF) variants, c.528C>A in six patients and c.234_235insAA in one patient, were statistically enriched in 928 eoHM probands compared to that in 5469 non-high myopia control (p = 3.7 × 10-5) and that in the general population (p = 2.78 × 10-9). Consequently, the Timp4 gene editing rat was further evaluated to explore the possible role of Timp4 on ocular and myopia development. A series of ocular morphology abnormalities in a dose-dependent manner (Timp4-/- < Timp4+/- < Timp4+/+) were observed in a rat model, including the decline in the retinal thickness, the elongation in the axial length, more vulnerable to the form deprivation model, morphology changes in sclera collagen bundles, and the decrease in collagen contents of the sclera and retina. Electroretinogram revealed that the b-wave amplitudes of Timp4 defect rats were significantly reduced, consistent with the shorter length of the bipolar axons detected by HE and IF staining. Heterozygous LoF variants in the TIMP4 are associated with early onset high myopia, and the Timp4 defect disturbs ocular development by influencing the morphology and function of the ocular tissue.

Col1A-2 Mutation in Osteogenesis Imperfecta Mice Contributes to Long Bone Fragility by Modifying Cell-Matrix Organization.

Osteogenesis imperfecta (OI) is a rare congenital bone dysplasia generally caused by a mutation of one of the type I collagen genes and characterized by low bone mass, numerous fractures, and bone deformities. The collagen organization and osteocyte lacuna arrangement were investigated in the long bones of 17-week-old wildtype (WT, n = 17) and osteogenesis imperfecta mice (OIM, n = 16) that is a validated model of severe human OI in order to assess their possible role in bone fragility. Fractures were counted after in vivo scanning at weeks 5, 11, and 17. Humerus, femur, and tibia diaphyses from both groups were analyzed ex vivo with pQCT, polarized and ordinary light histology, and Nano-CT. The fractures observed in the OIM were more numerous in the humerus and femur than in the tibia, whereas the quantitative bone parameters were altered in different ways among these bones. Collagen fiber organization appeared disrupted, with a lower birefringence in OIM than WT bones, whereas the osteocyte lacunae were more numerous, more spherical, and not aligned in a lamellar pattern. These modifications, which are typical of immature and less mechanically competent bone, attest to the reciprocal alteration of collagen matrix and osteocyte lacuna organization in the OIM, thereby contributing to bone fragility.

Influence of polymorphisms in TNF-α and IL1ß on susceptibility to alcohol induced liver diseases and therapeutic potential of miR-124-3p impeding TNF-α/IL1ß mediated multi-cellular signaling in liver microenvironment.

Background and aims: Alcoholic liver disease (ALD) is the leading cause of the liver cirrhosis related death worldwide. Excessive alcohol consumption resulting enhanced gut permeability which trigger sensitization of inflammatory cells to bacterial endotoxins and induces secretion of cytokines, chemokines leading to activation of stellate cells, neutrophil infiltration and hepatocyte injury followed by steatohepatitis, fibrosis and cirrhosis. But all chronic alcoholics are not susceptible to ALD. This study investigated the causes of differential immune responses among ALD patients and alcoholic controls (ALC) to identify genetic risk factors and assessed the therapeutic potential of a microRNA, miR-124-3p. Materials and methods: Bio-Plex Pro™ Human Chemokine analysis/qRT-PCR array was used for identification of deregulated immune genes. Sequencing/luciferase assay/ELISA detected and confirmed the polymorphisms. THP1 co-cultured with HepG2/LX2/HUVEC and apoptosis assay/qRT-PCR/neutrophil migration assay were employed as required. Results: The combined data analysis of the GSE143318/Bio-Plex Pro™ Human Chemokine array and qRT-PCR array revealed that six genes (TNFα/IL1ß/IL8/MCP1/IL6/TGFß) were commonly overexpressed in both serum/liver tissue of ALD-patients compared to ALC. The promoter sequence analysis of these 6 genes among ALD (n=322)/ALC (n=168) samples revealed that only two SNPs, rs361525(G/A) at -238 in TNF-α/rs1143627(C/T) at -31 in IL1ß were independently associated with ALD respectively. To evaluate the functional implication of these SNPs on ALD development, the serum level of TNF-α/IL1ß was verified and observed significantly higher in ALD patients with risk genotypes TNF-α-238GA/IL1ß-31CT+TT than TNF-α-238GG/IL1ß-31CC. The TNF-α/IL1ß promoter Luciferase-reporter assays showed significantly elevated level of luciferase activities with risk genotypes -238AA/-31TT than -238GG/-31CC respectively. Furthermore, treatment of conditioned medium of TNF-α/IL1ß over-expressed THP1 cells to HepG2/LX2/HUVEC cells independently showed enhanced level of ER stress and apoptosis in HepG2/increased TGFß and collagen-I production by LX2/huge neutrophil infiltration through endothelial layer. However, restoration of miR-124-3p in THP1 attenuated such inter-cellular communications and hepatocyte damage/collagen production/neutrophil infiltration were prohibited. Target analysis/luciferase-reporter assays revealed that both TNF-α/IL1ß were inhibited by miR-124-3p along with multiple genes from TLR4 signaling/apoptosis/fibrogenesis pathways including MYD88, TRAF3/TRADD, Caspase8/PDGFRA, TGFßR2/MCP1, and ICAM1 respectively. Conclusion: Thus, rs361525(G/A) in TNF-α and rs1143627(C/T) in IL1ß gene may be used as early predictors of ALD susceptibility among East Indian population. Impeding overexpressed TNF-α/IL1ß and various genes from associated immune response pathways, miR-124-3p exhibits robust therapeutic potential for ALD patients.

Treatment Strategy With Gene Editing for Late-Onset Retinal Degeneration Caused by a Founder Variant in C1QTNF5.

Purpose: Genome editing is an emerging group of technologies with the potential to ameliorate dominant, monogenic human diseases such as late-onset retinal degeneration (L-ORD). The goal of this study was to identify disease stages and retinal locations optimal for evaluating the efficacy of a future genome editing trial. Methods: Twenty five L-ORD patients (age range, 33-77 years; median age, 59 years) harboring the founder variant S163R in C1QTNF5 were enrolled from three centers in the United Kingdom and United States. Patients were examined with widefield optical coherence tomography (OCT) and chromatic perimetry under dark-adapted and light-adapted conditions to derive phenomaps of retinal disease. Results were analyzed with a model of a shared natural history of a single delayed exponential across all subjects and all retinal locations. Results: Critical age for the initiation of photoreceptor loss ranged from 48 years at the temporal paramacular retina to 74 years at the inferior midperipheral retina. Subretinal deposits (sRET-Ds) became more prevalent as critical age was approached. Subretinal pigment epithelial deposits (sRPE-Ds) were detectable in the youngest patients showing no other structural or functional abnormalities at the retina. The sRPE-D thickness continuously increased, reaching 25 µm in the extrafoveal retina and 19 µm in the fovea at critical age. Loss of light sensitivity preceded shortening of outer segments and loss of photoreceptors by more than a decade. Conclusions: Retinal regions providing an ideal treatment window exist across all severity stages of L-ORD.

Nanoscale patterning of collagens in C. elegans apical extracellular matrix.

Apical extracellular matrices (aECMs) are complex extracellular compartments that form important interfaces between animals and their environment. In the adult C. elegans cuticle, layers are connected by regularly spaced columnar structures known as struts. Defects in struts result in swelling of the fluid-filled medial cuticle layer ('blistering', Bli). Here we show that three cuticle collagens BLI-1, BLI-2, and BLI-6, play key roles in struts. BLI-1 and BLI-2 are essential for strut formation whereas activating mutations in BLI-6 disrupt strut formation. BLI-1, BLI-2, and BLI-6 precisely colocalize to arrays of puncta in the adult cuticle, corresponding to struts, initially deposited in diffuse stripes adjacent to cuticle furrows. They eventually exhibit tube-like morphology, with the basal ends of BLI-containing struts contact regularly spaced holes in the cuticle. Genetic interaction studies indicate that BLI strut patterning involves interactions with other cuticle components. Our results reveal strut formation as a tractable example of precise aECM patterning at the nanoscale.