Brachydactyly refers to shortening of digits due to hypoplasia or aplasia of bones forming the hands and/or feet. Isolated brachydactyly type E (BDE), which is characterized by shortened metacarpals and/or metatarsals, results in a small proportion of patients from HOXD13 or PTHLH mutations, although in the majority of cases molecular lesion remains unknown. BDE, like other brachydactylies, shows clinical heterogeneity with highly variable intrafamilial and interindividual expressivity. In this study, we investigated two Polish cases (one familial and one sporadic) presenting with BDE and additional symptoms due to novel PTHLH mutations. Apart from BDE, the affected family showed short stature, mild craniofacial dysmorphism and delayed bone age. Sanger sequencing of PTHLH revealed a novel heterozygous frameshift mutation c.258delC(p.N87Tfs*18) in two affected individuals and one relative manifesting mild brachydactyly. The sporadic patient, in addition to BDE, presented with craniofacial dysmorphism, normal stature and bone age, and was demonstrated to carry a de novo heterozygous c.166C>T(p.R56*) mutation. Our paper reports on the two novel truncating PTHLH variants, resulting in variable combination of BDE and other symptoms. Data shown here expand the knowledge on the phenotypic presentation of PTHLH mutations, highlighting significant clinical variability and incomplete penetrance of the PTHLH-related symptoms.

Short metacarpals and/or -tarsals are typically observed in pseudohypoparathyroidism (PHP) type Ia (PHP1A) or pseudo-PHP (PPHP), i.e. disorders caused by inactivating GNAS mutations involving exons encoding the alpha-subunit of the stimulatory G protein (Gsα). Skeletal abnormalities similar to those in PHP1A/PPHP were present in several members of an extended Belgian family without evidence for abnormal calcium and phosphate regulation. Direct nucleotide sequencing of genomic DNA from an affected individual (190/III-1) excluded GNAS mutations. Instead, whole exome analysis revealed a novel heterozygous A>> G change at nucleotide -3 up-stream of PTHLH exon 3 that encodes the last two amino acids of the pro-sequence and the mature PTHrP. The same nucleotide change was also found in her affected mother and maternal aunt (190/II-2, 190/II-1), and her affected twin sons (190/IV-1, 190/IV-2), but not in her unaffected daughter (190/IV-3) and sister (190/III-2). Complementary DNA derived from immortalized lymphoblastoid cells from 190/IV-2 (affected) and 190/IV-3 (unaffected) was PCR-amplified using forward primers located either in PTHLH exon 1 (non-coding) or exon 2 (pre-sequence and most of the pro-sequence), and reverse primers located in the 3'-non-coding regions of exons 3 or 4. Nucleotide sequence analysis of these amplicons revealed for 190/IV-2, but not for 190/IV-3, a heterozygous insertion of genomic nucleotides -2 and -1 causing a frame-shift after residue 34 of the prepro-sequence and thus 29 novel residues without homology to PTHrP or any other protein. Our findings extend previous reports indicating that PTHrP haploinsufficiency causes skeletal abnormalities similar to those observed with heterozygous GNAS mutations. This article is protected by copyright. All rights reserved.

Pancreatic cancer metastasis is a leading cause of cancer-related deaths, yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Here, we report that the parathyroid hormone-related protein (PTHrP encoded by PTHLH) is frequently amplified as part of the KRAS amplicon in pancreatic cancer patients. PTHrP upregulation drives the growth of both primary and metastatic tumors in mice and is highly enriched in PDAC metastases. Loss of PTHrP - either genetically or pharmacologically - dramatically reduces tumor burden, eliminates metastasis, and enhances overall survival. These effects are mediated in part through a reduction in epithelial-to-mesenchymal transition, which reduces the tumor cells' ability to initiate the metastatic cascade. Spp1, which encodes Osteopontin, is revealed to be a downstream effector of PTHrP. Our results establish a new paradigm in pancreatic cancer whereby PTHrP is a driver of disease progression and emerges as a novel therapeutic vulnerability.

Background: Osteoarthritis (OA) is the most common chronic joint disease worldwide. It is characterized by pain and limited mobility in the affected joints and may even cause disability. Effective clinical options for its prevention and treatment are still unavailable. This study aimed to identify differences in gene signatures between tissue samples from OA and normal knee joints and to explore potential gene targets for OA.

Methods: Five gene datasets, namely GSE55457, GSE55235, GSE12021, GSE10575, and GSE1919, were selected from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified using the R programming software. The functions of these DEGs were analyzed, and a protein-protein interaction (PPI) network was constructed. Subsequently, the most relevant biomarker genes were screened using a receiver operating characteristic (ROC) curve analysis. Finally, the expression of the protein encoded by the core gene PTHLH was evaluated in clinical samples.

Results: Eleven upregulated and 9 downregulated DEGs were shared between the five gene expression datasets. Based on the PPI network and the ROC curves of upregulated genes, PTHLH was identified as the most relevant gene for OA and was selected for further validation. Immunohistochemistry confirmed significantly higher PTHLH expression in OA tissues than in normal tissues. Moreover, similar PTHLH levels were detected in the plasma and knee synovial fluid of OA patients.

Conclusion: The bioinformatics analysis and preliminary experimental verification performed in this study identified PTHLH as a potential target for the treatment of OA.

Keywords: Differentially expressed genes; GEO database; Osteoarthritis; PTHLH; Protein–protein interaction network.

Parathyroid hormone-like hormone (PTHLH), also known as parathyroid hormone-related protein (PTHrP), was first identified as a parathyroid hormone (PTH)-like factor responsible for humoral hypercalcaemia in malignancies. Subsequent studies demonstrated that PTHLH has multiple physiological functions in many fetal and adult tissues, including the regulation of morphogenesis, cell proliferation and differentiation, and transplacental calcium transport. This review focuses on the biological characteristics of PTHLH and its function during embryonic development. The signaling pathway and potential mechanism involved are further discussed.

Little is known about interactions between environmental and genetic risk factors for osteoarthritis (OA). Genetic factors include variation or mutation in genes involved in parathyroid hormone signalling. Exposure to the endocrine disrupting chemicals perfluoro-octanoic acid (PFOA) or perfluorooctane sulfonate (PFOS) have been suggested as potential environmental contributors, although evidence to support this association is conflicting. Here we test the hypothesis that PFOA and PFOS may alter the mRNA expression of genes in the parathyroid signalling cascade to provide evidence on possible pathways between these chemicals and OA. We measured the relationship between PFOA or PFOS serum levels and the in vivo expression of the Parathyroid hormone 1 and 2 genes (PTH, PTH2), Parathyroid hormone 1 and 2 receptor genes (PTH1R, PTH2R) and the parathyroid hormone-like (PTHLH) gene in peripheral blood from a cross-sectional population study designed to assess the potential health effects of these chemicals. We used multivariate linear regression models and found that PFOA or PFOS was inversely correlated with parathyroid hormone 2 receptor (PTH2R) expression (coefficients=-0.43 and -0.32, p=p=0.017 and 0.006 for PFOA and PFOS respectively) in 189 female subjects. The levels of PTH2 transcripts encoding the ligand of PTH2r, were also found to be lower in women with OA (median 2.08) compared with controls (median 3.41, p=0.046). As the parathyroid signalling cascade is a known candidate for osteoarthritis risk and our findings raise the possibility that exposure to these chemicals may contribute to the pathogenesis of OA in some individuals.

Objective: Hip shape is a well-recognized risk factor for hip osteoarthritis (OA) and hip fracture. We aimed to investigate whether the genetic variants known to be associated with adult hip shape were also associated with adolescent hip shape.

Methods: Hip DXA scans, obtained in offspring from the Avon Longitudinal Study of Parents and Children (ALSPAC) at two time points (mean ages 13.8 and 17.8 years), were used to quantify hip morphology using a 53-point Statistical Shape Model (SSM). Principal component analysis was used to generate hip shape modes (HSMs). Genetic variants which had previously shown genome-wide significant association with specific HSMs in adults were tested for association with the same HSMs in adolescents (at each timepoint separately) using SNPTEST v2.

Results: Complete genotypic and phenotypic data were available for 3,550 and 3,175 individuals at 14 and 18 years, respectively. The strongest evidence for association with adolescent hip shape was for a variant located near SOX9 (rs2158915) with consistent effects across both time points for HSM1 (age 14: beta -0.05, p=9.9 x10^-8; age 18: -0.05, p=3.3 x10^-6) and HSM5 (age 14: beta -0.07, p=1.6 x10^-4; age 18: -0.1, p=2.7 x10^-6). There was also strong evidence of association between rs10743612 (near PTHLH) and HSM1 (age 14: 0.05, p=1.1 x10^-5; age 18: 0.04, p=0.003) and between rs6537291 (near HHIP) and HSM2 (age 14: -0.06, p=0.001; age 18: -0.07, p=0.001) across both time points. The genes with the strongest associations with hip shape in adolescents, (SOX9, PTHLH and HHIP) are known to be involved in endochondral bone formation. HSM1 indicates narrower aspect ratio of the upper femur, whereas both HSM2 and HSM5 reflect variation in the femoral head size and femoral neck width, features previously found to be related to the risk of OA in later life. The SOX9 locus has previously been found to associate with increased risk of hip fracture.

Conclusion: In conclusion, variants implicated in endochondral bone formation appear to consistently influence hip shape between adolescence and adulthood, including those aspects related to risk of hip OA and/or fracture in later life.

Keywords: ALSPAC; DXA; Genetic association; Hip fracture risk; Hip shape; Osteoarthritis.

Pigs are one of the earliest domesticated animals and multiple breeds have been developed to meet the various demands of consumers. EigenGWAS is a novel strategy to identify candidate genes that underlying population genetic differences and to infer candidate regions under selection as well. In this study, EigenGWAS and F_st analyses were performed using the public re-sequencing data of three typical commercial pig breeds, Duroc, Landrace and Yorkshire. The intersection of genome-wide significant SNPs detected by EigenGWAS and top-ranked 1% SNPs of F_st results were treated as signals under selection. Using the data of all three breeds, 3062 signals under selection were detected and the nearby genomic regions within 300 kb upstream and downstream covered 6.54% of whole genome. Pairs of breeds were analysed along with the pathway analysis. The gene function enrichment results indicated that many candidate genes located in the genomic regions of the signals under selection were associated with biological processes related to growth, metabolism, reproduction, sensory perception, etc. Among the candidate genes, the FSHB, AHR, PTHLH, KDR and FST genes were reported to be associated with reproductive performance; the KIT, KITLG, MITF, MC1R and EDNRB genes were previously identified to affect coat colour; the RETREG1, TXNIP, BMP5, PPARD and RBP4 genes were reported to be associated with lipid metabolism and growth traits. The identified genetic differences across the three commercial breeds will advance understanding of the artificial selection history of pigs and the signals under selection will suggest potential uses in pig genomic breeding programmes.

Three genes--parathyroid hormone-like hormone (PTHLH), insulin-like growth factor 1 (IGF 1), and retinoic acid receptor gamma (RARG)--have been mapped to sheep (Ovis aries) chromosome 3 (OAR 3). The order and genetic distances between loci on OAR 3 are similar to those on cattle (Bos taurus) chromosome 5, as expected from their close evolutionary relationship. The OAR 3 linkage map shows conserved synteny with human chromosome 12, but there are at least two rearrangements in gene order between the species.

In hens, follicle selection is an important process affecting egg laying traits. This study investigated the role of parathyroid hormone-like hormone (PTHLH) in chicken follicle selection, its transcriptional regulation and genetic effects on egg laying traits. PTHLH and its receptor PTH1R were mainly expressed in follicles of 6-8 mm in diameter, exhibits differential expression pattern in the theca and granulosa cells of pre- and hierarchal follicles. PTHLH stimulates the proliferation of follicular granulosa and theca cells, the expression of StAR and CYP11A1 mRNA and the production of progesterone (P4) in pre-hierarchal follicles. Treatment with FSH increased PTHLH mRNA expression in pre-hierarchal follicular theca cells and hierarchal follicular granulosa cells. Two critical regions regulating chicken PTHLH transcription were revealed, each of which harbored a SNP: C>T (chr1: 72530014) for AP-1 and a SNP: A>G (chr1: 72531676). Hens with diplotype AC/GT were younger at first laying and laid more eggs at 32 weeks. The haplotype (G^-1827T^-165) with double mutations had the greatest promoter activity of chicken PTHLH transcription. Collectively, PTHLH plays an important role in chicken follicle selection by stimulating cell proliferation and steroidogenesis. Polymorphisms in chicken PTHLH promoter region are associated with egg laying traits by affecting the binding of transcription factor AP-1.

Susceptibly to the induction of rat tongue cancer (TC) by oral 4-nitroquinoline 1-oxide (4NQO) exposure is a polygenic trait. Among several quantitative trait loci identified by crosses between TC-susceptible Dark Agouti (DA) rats and TC-resistant Wistar-Furth (WF) rats, we focused on tongue cancer susceptibility locus (Tcas3) of chromosome 4. We examined tongue carcinogenesis in the reciprocal congenic strains DA.WF-Tcas3 and WF.DA-Tcas3 and in their parental strains. The Tcas3DA allele, and not the Tcas3WF allele, significantly favored tumor latency, incidence and TC number/size. In genomic DNA of TCs induced in (DA x WF) F1 rats, the resistant Tcas3WF allele was frequently and selectively lost, particularly in larger tumors. Thus, we searched the possible candidate genes in the Tcas3 region using microarray analysis of TCs in F1 rats and revealed significant upregulation of 2 cancer-related genes, parathyroid hormone-like hormone (Pthlh) and Kras2. The relevance of the WF allele of Pthlh as a cancer modifier was indicated by 3 single nucleotide polymorphisms specific to this strain. In contrast, no consistent strain-specific variations were found in Kras2. Moreover, the plasma Ca2+ level was consistently higher in DA rats when compared to the level in WF rats bearing TCs; moreover, the Pthlh-mRNA expression level was >30-fold higher in TCs when compared to this level in the normal tongue mucosa. Immunostaining experiments showed strong PTHrP protein expression in TCs of DA rats, and the signal was intensified in larger TCs. Kras2 was also upregulated in TCs, but to a lesser degree than PTHrP. Thus, Pthlh is a promising candidate modifier gene in the development and progression of rat TCs.

OBJECTIVE

Investigate the effect of hydrostatic pressure (HP) on 3', 5'-cyclic adenosine monophosphate (cAMP) levels and downstream signaling in cultures of normal optic nerve head (ONH) astrocytes from Caucasian American (CA) and African American (AA) donors.

METHODS

Intracellular cAMP levels were assayed after exposing ONH astrocytes to HP for varying times. Quantitative RT-PCR was used to determine the expression levels of selected cAMP pathway genes in human ONH astrocytes after HP treatment. Western blots were used to measure changes in the phosphorylation state of cAMP response element binding protein (CREB) in astrocytes subjected to HP, ATP, and phosphodiesterase or kinase inhibitors.

RESULTS

The basal intracellular cAMP level is similar among AA and CA astrocytes. After exposure to HP for 15 min and 30 min in the presence of a phosphodiesterase inhibitor a further increase of intracellular cAMP was observed in AA astrocytes, but not in CA astrocytes. Consistent with activation of the cAMP-dependent protein kinase pathway, CREB phosphorylation (Ser-133) was increased to a greater extent in AA than in CA astrocytes after 3 h of HP. Exposure to elevated HP for 3-6 h differentially altered the expression levels of selected cAMP pathway genes (ADCY3, ADCY9, PTHLH, PDE7B) in AA compared to CA astrocytes. Treatment with ATP increased more CREB phosphorylation in CA than in AA astrocytes, suggesting differential Ca(2+) signaling in these populations.

CONCLUSIONS

Activation of the cAMP-dependent signaling pathway by pressure may be an important contributor to increased susceptibility to elevated intraocular pressure and glaucoma in AA, a population at higher risk for the disease.

Background: Cutaneous squamous cell carcinoma (cSCC) often follows actinic keratosis (AK) and is the second most common skin cancer worldwide. To reduce metastasis risk, it is important to diagnose and treat cSCC early. This study aimed to identify hub genes associated with cSCC and AK.

Methods: This study used three datasets GSE45216, GSE98774, and GSE108008. We combined samples from the GSE45216 and GSE98774 datasets into the new dataset GSE45216-98774. We applied a weighted gene co-expression network analysis (WGCNA) to investigate key modules and hub genes associated with cSCC and AK. We considered the hub genes found in both the GSE45216-98774 and GSE108008 datasets as validated hub genes. We tested whether the expression of hub genes could predict patient survival outcomes in other cancers using TCGA pan-cancer data.

Results: We identified modules most relevant to cSCC and AK. Additionally, we identified and validated seven hub genes of cSCC: GATM, ARHGEF26, PTHLH, MMP1, POU2F3, MMP10 and GATA3. We did not find validated hub genes for AK. Each hub gene was significantly associated with the survival of various cancer types. Only GATA3 was significantly associated with melanoma survival.

Conclusions: We applied WGCNA to find seven hub genes that play important roles in cSCC tumorigenesis. These results provide new insights that help explain the pathogenesis of cSCC. These hub genes may become biomarkers or therapeutic targets for accurate diagnosis and treatment of cSCC in the future.

Keywords: Cutaneous squamous cell carcinoma; Hub gene; Survival; Weighted gene co-expression network analysis.

Metastatic breast cancer is one of the leading causes of cancer related death in women. Limited studies have been done on the genomic evolution between primary and metastatic breast cancer. We reconstructed the genomic evolution through the sixteen year history of an ER+ HER2- breast cancer patient to investigate molecular mechanisms of disease relapse and treatment resistance after long term exposure to hormonal therapy. Genomic and transcriptome profiling was performed on primary breast tumor (2002), initial recurrence (2012) and liver metastasis (2015) samples. Cell free DNA analysis was performed at eleven timepoints (2015-2017). Mutational analysis revealed a low mutational burden in the primary tumor which doubled at the time of progression, with driver mutations in PI3K-Akt and RAS-RAF signaling pathways. Phylogenetic analysis showed an early branching off between primary tumor and metastasis. Liquid biopsies, while initially negative, started to detect an ESR1 E380Q mutation in 2016 with increasing allele frequency till end of 2017. Transcriptome analysis revealed 721 (193 up, 528 down) genes to be differentially expressed between primary tumor and first relapse. Most significantly down-regulated genes were TFF1 and PGR, indicating resistance to AI therapy. Most up-regulated genes included PTHLH, S100P, and SOX2 promoting tumor growth and metastasis. This phylogenetic reconstruction of the life history of a single patient's cancer as well as monitoring tumor progression through liquid biopsies allowed for uncovering the molecular mechanisms leading to initial relapse, metastatic spread and treatment resistance.

Keywords: Multifocal breast carcinoma.

Parathyroid hormone-like hormone (PTHLH) plays an important role in bone formation. Several skeletal dysplasias have been described that are associated with disruption of PTHLH functioning. Here we report on a new patient with a 898 Kb duplication on chromosome 12p11.22 including the PTHLH gene. The boy has multiple skeletal abnormalities including chondrodysplasia, lesions radiographically resembling enchondromas and posterior rib deformities leading to a severe chest deformity. Severe pulmonary symptoms were thought to be caused by limited mobility and secondary sputum evacuation problems due to the chest deformity. Imaging studies during follow-up revealed progression of the number of skeletal lesions over time. This case extends the phenotypic spectrum associated with copy number variation of PTHLH.

Keywords: AP, anteroposterior; CT, computed tomography; Chondrodysplasia; DEXA, dual-energy X-ray absorptiometry; Enchondroma; IHH, Indian hedgehog; PA, posteroanterior; PTHLH; PTHLH, parathyroid hormone-like hormone; PTHrP, parathyroid hormone related peptide; Parathyroid hormone-like hormone; SDS, standard deviation score.

Skeletal disorders, including both isolated and syndromic brachydactyly type E, derive from genetic defects affecting the fine tuning of the network of pathways involved in skeletogenesis and growth-plate development. Alterations of different genes of this network may result in overlapping phenotypes, as exemplified by disorders due to the impairment of the parathyroid hormone/parathyroid hormone-related protein pathway, and obtaining a correct diagnosis is sometimes challenging without a genetic confirmation. Five patients with "AHO-like" (Albright's Hereditary Osteodystrophy) skeletal malformations without a clear clinical diagnosis were analyzed by whole exome sequencing and novel potentially pathogenic variants in PTHLH (BDE with short stature, BDE2) and TRPS1 (tricho-rhino-phalangeal syndrome, TRPS) were discovered. The pathogenic impact of these variants was confirmed by in vitro functional studies. This study expands the spectrum of genetic defects associated with BDE2 and TRPS and demonstrates the pathogenicity of TRPS1 missense variants located outside both the nuclear localization signal and the GATA and Ikaros-like binding domains. Unfortunately, we could not find distinctive phenotypic features that might have led to an earlier clinical diagnosis, further highlighting the high degree of overlap among skeletal syndromes associated with brachydactyly and AHO-like features, and the need for a close interdisciplinary workout in these rare patients. This article is protected by copyright. All rights reserved.

Secreted signals in patterning systems often induce repressive signals that shape their distributions in space and time. In developing growth plates (GPs) of endochondral long bones, Parathyroid hormone-like hormone (Pthlh) inhibits Indian hedgehog (Ihh) to form a negative feedback loop that controls GP progression and bone size. Whether similar systems operate in other bones and how they arise during embryogenesis remain unclear. We show that Pthlha expression in the zebrafish craniofacial skeleton precedes chondrocyte differentiation and restricts where cells undergo hypertrophy, thereby initiating a future GP. Loss of Pthlha leads to an expansion of cells expressing a novel early marker of the hypertrophic zone (HZ), entpd5a, and later HZ markers such as ihha, while local Pthlha misexpression induces ectopic entpd5a expression. Formation of this early pre-HZ correlates with onset of muscle contraction and requires mechanical force; paralysis leads to loss of entpd5a and ihha expression in the pre-HZ, mislocalized pthlha expression, and no subsequent ossification. These results suggest that local Pthlh sources combined with force determine HZ locations, establishing the negative feedback loop that later maintains GPs.

Keywords: Cartilage hypertrophy; Danio rerio; Growth plate; Growth zone.

The periodontium is essential for supporting the functionality of the tooth, composed of diversity of mineralized and non-mineralized tissues such as the cementum, the periodontal ligament (PDL) and the alveolar bone. The periodontium is developmentally derived from the dental follicle (DF), a fibrous tissue surrounding the developing tooth bud. We previously showed through in vivo lineage-tracing experiments that DF contains mesenchymal progenitor cells expressing parathyroid hormone-related protein (PTHrP), which give rise to cells forming the periodontal attachment apparatus in a manner regulated by autocrine signaling through the PTH/PTHrP receptor. However, the developmental relationships between PTHrP⁺ DF cells and diverse cell populations constituting the periodontium remain undefined. Here, we performed single-cell RNA-sequencing (scRNA-seq) analyses of cells in the periodontium by integrating the two datasets, i.e. PTHrP-mCherry⁺ DF cells at P6 and 2.3kb Col1a1 promoter-driven GFP⁺ periodontal cells at P25 that include descendants of PTHrP⁺ DF cells, cementoblasts, osteoblasts and periodontal ligament cells. This integrative scRNA-seq analysis revealed heterogeneity of cells of the periodontium and their cell type-specific markers, as well as their relationships with DF cells. Most importantly, our analysis identified a cementoblast-specific metagene that discriminate cementoblasts from alveolar bone osteoblasts, including Pthlh (encoding PTHrP) and Tubb3. RNA velocity analysis indicated that cementoblasts were directly derived from PTHrP⁺ DF cells in the early developmental stage and did not interconvert with other cell types. Further, CellPhoneDB cell-cell communication analysis indicated that PTHrP derived from cementoblasts acts on diversity of cells in the periodontium in an autocrine and paracrine manner. Collectively, our findings provide insights into the lineage hierarchy and intercellular interactions of cells in the periodontium at a single-cell level, aiding to understand cellular and molecular basis of periodontal tissue formation.

Keywords: dental follicle; mesenchymal progenitor cells; mouse genetic models; parathyroid hormone-related protein; periodontium; single cell analysis.

Alu are high copy number interspersed repeats that have accumulated near genes during primate and human evolution. They are a pervasive source of structural variation in modern humans. Impacts that Alu insertions may have on gene expression are not well understood, although some have been associated with expression quantitative trait loci (eQTLs). Here, we directly test regulatory effects of polymorphic Alu insertions in isolation of other variants on the same haplotype. To screen insertion variants for those with such effects, we used ectopic luciferase reporter assays and evaluated 110 Alu insertion variants, including more than 40 with a potential role in disease risk. We observed a continuum of effects with significant outliers that up- or down-regulate luciferase activity. Using a series of reporter constructs, which included genomic context surrounding the Alu, we can distinguish between instances in which the Alu disrupts another regulator and those in which the Alu introduces new regulatory sequence. We next focused on three polymorphic Alu loci associated with breast cancer that display significant effects in the reporter assay. We used CRISPR to modify the endogenous sequences, establishing cell lines varying in the Alu genotype. Our findings indicate that Alu genotype can alter expression of genes implicated in cancer risk, including PTHLH, RANBP9, and MYC These data show that commonly occurring polymorphic Alu elements can alter transcript levels and potentially contribute to disease risk.

Despite recent advances in the treatment of non-small cell lung cancer (NSCLC), less than 10% of patients survive the first five years when the disease has already spread at primary diagnosis.

Methods: Blood samples were taken from 118 NSCLC patients at primary diagnosis or at progression of the disease before the start of a new treatment line and enriched for circulating tumor cells (CTCs) by microfluidic Parsortix™ (Angle plc, Guildford GU2 7AF, UK) technology. The gene expression of epithelial cancer stem cell (CSC), epithelial to mesenchymal (EMT), and lung-related markers was assessed by qPCR, and the association of each marker with overall survival (OS) was evaluated using log-rank tests.

Results: EpCAM was the most prevalent transcript, with 53.7% positive samples at primary diagnosis and 25.6% at recurrence. EpCAM and CK19, as well as NANOG, PROM1, TERT, CDH5, FAM83A, and PTHLH transcripts, were associated with worse OS. However, only the CSC-specific NANOG and PROM1 were related to the outcome both at primary diagnosis (NANOG: HR 3.21, 95%CI 1.02-10.14, p = 0.016; PROM1: HR 4.23, 95% CI 0.65-27.56, p = 0.007) and disease progression (NANOG: HR 4.17, 95%CI 0.72-24.14, p = 0.025; PROM1: HR 4.77, 95% CI 0.29-78.94, p = 0.032).

Conclusions: The present study further underlines the relevance of the molecular characterization of CTCs. Our multi-marker analysis highlighted the prognostic value of cancer stem cell-related transcripts at primary diagnosis and disease progression.

Keywords: cancer stem cell; circulating tumor cells; gene expression analysis; microfluidics; non-small cell lung cancer.

Comprehensive studies on cancer patients with different smoking histories, including non-smokers, former smokers, and current smokers, remain elusive. Therefore, we conducted a multi-omics analysis to explore the effect of smoking history on cancer patients. Patients with smoking history were screened from The Cancer Genome Atlas database, and their multi-omics data and clinical information were downloaded. A total of 2,317 patients were included in this study, whereby current smokers presented the worst prognosis, followed by former smokers, while non-smokers showed the best prognosis. More importantly, smoking history was an independent prognosis factor. Patients with different smoking histories exhibited different immune content, and former smokers had the highest immune cells and tumor immune microenvironment. Smokers are under a higher incidence of genomic instability that can be reversed following smoking cessation in some changes. We also noted that smoking reduced the sensitivity of patients to chemotherapeutic drugs, whereas smoking cessation can reverse the situation. Competing endogenous RNA network revealed that mir-193b-3p, mir-301b, mir-205-5p, mir-132-3p, mir-212-3p, mir-1271-5p, and mir-137 may contribute significantly in tobacco-mediated tumor formation. We identified 11 methylation driver genes (including EIF5A2, GBP6, HGD, HS6ST1, ITGA5, NR2F2, PLS1, PPP1R18, PTHLH, SLC6A15, and YEATS2), and methylation modifications of some of these genes have not been reported to be associated with tumors. We constructed a 46-gene model that predicted overall survival with good predictive power. We next drew nomograms of each cancer type. Interestingly, calibration diagrams and concordance indexes are verified that the nomograms were highly accurate for the prognosis of patients. Meanwhile, we found that the 46-gene model has good applicability to the overall survival as well as to disease-specific survival and progression-free intervals. The results of this research provide new and valuable insights for the diagnosis, treatment, and follow-up of cancer patients with different smoking histories.

Keywords: TCGA; bioinformatics; cessation; current smokers; former smokers; multi-omics; tobacco.

The peripartal cow experiences a rapid change in calcium metabolism at the onset of lactation. Research has focused on understanding how mammary-derived factors, such as serotonin (5HT) and parathyroid hormone like hormone (PTHLH), aid in coordinating these calcemic adaptations to lactation. Therefore, the aim of our study was to determine how induced subclinical hypocalcemia influences physiological responses, specifically the 5HT-PTHLH-Ca axis, in lactating and nonlactating dairy cows to elucidate the potential contribution of the mammary gland. Twelve nonlactating, nonpregnant (NL) multiparous Holstein cows and 12 early-lactation (EL) multiparous Holstein cows received either (1) a continuous 24-h intravenous solution of 0.9% NaCl or (2) 5% ethylene glycol tetraacetic acid (EGTA) solution in 0.9% NaCl (n = 6 EL, n = 6 NL per treatment) with the aim of maintaining blood ionized calcium (iCa) less than 1.0 mM. Mammary gland biopsies were taken immediately after and 48 h after termination of infusion. Blood was sampled hourly during infusion and 4, 8, 12, 24, 48, and 72 h after termination of infusion. Infusion of EGTA successfully decreased blood iCa concentrations. However, EL EGTA-infused cows required increased rates of EGTA infusion to maintain iCa below 1.0 mM. Circulating and mammary serotonin concentrations were increased in EL relative to NL cows, with no difference as a result of EGTA infusion. Mammary PTHLH expression was increased in EL cows, with highest expression observed in EL EGTA-infused cows. Collectively, these data demonstrate the robust adaptations EL cows have to maintain Ca homeostasis and the supporting roles 5HT and PTHLH may play.

Keywords: EGTA; PTHLH; hypocalcemia; serotonin.