Gain of Additional BIRC3 Protein Functions through 3'-UTR-Mediated Protein Complex Formation.

Alternative 3' untranslated regions (3' UTRs) are widespread, but their functional roles are largely unknown. We investigated the function of the long BIRC3 3' UTR, which is upregulated in leukemia. The 3' UTR does not regulate BIRC3 protein localization or abundance but is required for CXCR4-mediated B cell migration. We established an experimental pipeline to study the mechanism of regulation and used mass spectrometry to identify BIRC3 protein interactors. In addition to 3'-UTR-independent interactors involved in known BIRC3 functions, we detected interactors that bind only to BIRC3 protein encoded from the mRNA with the long 3' UTR. They regulate several functions, including CXCR4 trafficking. We further identified RNA-binding proteins differentially bound to the alternative 3' UTRs and found that cooperative binding of Staufen and HuR mediates 3'-UTR-dependent complex formation. We show that the long 3' UTR is required for the formation of specific protein complexes that enable additional functions of BIRC3 protein beyond its 3'-UTR-independent functions.

Widespread intronic polyadenylation inactivates tumour suppressor genes in leukaemia.

DNA mutations are known cancer drivers. Here we investigated whether mRNA events that are upregulated in cancer can functionally mimic the outcome of genetic alterations. RNA sequencing or 3'-end sequencing techniques were applied to normal and malignant B cells from 59 patients with chronic lymphocytic leukaemia (CLL)1-3. We discovered widespread upregulation of truncated mRNAs and proteins in primary CLL cells that were not generated by genetic alterations but instead occurred by intronic polyadenylation. Truncated mRNAs caused by intronic polyadenylation were recurrent (n = 330) and predominantly affected genes with tumour-suppressive functions. The truncated proteins generated by intronic polyadenylation often lack the tumour-suppressive functions of the corresponding full-length proteins (such as DICER and FOXN3), and several even acted in an oncogenic manner (such as CARD11, MGA and CHST11). In CLL, the inactivation of tumour-suppressor genes by aberrant mRNA processing is substantially more prevalent than the functional loss of such genes through genetic events. We further identified new candidate tumour-suppressor genes that are inactivated by intronic polyadenylation in leukaemia and by truncating DNA mutations in solid tumours4,5. These genes are understudied in cancer, as their overall mutation rates are lower than those of well-known tumour-suppressor genes. Our findings show the need to go beyond genomic analyses in cancer diagnostics, as mRNA events that are silent at the DNA level are widespread contributors to cancer pathogenesis through the inactivation of tumour-suppressor genes.

Pseudoaneurysm following hamstring tendon harvest in arthroscopic anterior cruciate ligament reconstruction: a case report.

BACKGROUND: Vascular injury is a very rare complication following arthroscopic knee surgery. This is the first report of pseudoaneurysm at the saphenous branch of the descending genicular artery complicating semitendinosus tendon harvest in arthroscopic anterior cruciate ligament reconstruction. CASE PRESENTATION: A 19-year-old male had developed large ecchymosis, focal swelling and tenderness around his posteromedial knee after an arthroscopic anterior cruciate ligament reconstruction. Compartment syndrome of the lower leg and deep vein thrombosis were ruled out. A pseudoaneurysm formation was confirmed through an angiography and coil embolization was performed. At one year follow up, the patient reported improved functional outcome with good stability of the knee. However, mild paresthesia over the posteromedial calf was noted due to the compression injury of the saphenous nerve by the hematoma. CONCLUSIONS: The pseudoaneurysm was presumed to result from accidental vascular injury while dissecting the accessory bands of the semitendinosus and was successfully treated by coil embolization. Care must be taken to section the expansions of the hamstring tendon, especially when the patient presents with underlying coagulopathy or vascular disease.

The lncRNA HOTAIR impacts on mesenchymal stem cells via triple helix formation.

There is a growing perception that long non-coding RNAs (lncRNAs) modulate cellular function. In this study, we analyzed the role of the lncRNA HOTAIR in mesenchymal stem cells (MSCs) with particular focus on senescence-associated changes in gene expression and DNA-methylation (DNAm). HOTAIR binding sites were enriched at genomic regions that become hypermethylated with increasing cell culture passage. Overexpression and knockdown of HOTAIR inhibited or stimulated adipogenic differentiation of MSCs, respectively. Modification of HOTAIR expression evoked only very moderate effects on gene expression, particularly of polycomb group target genes. Furthermore, overexpression and knockdown of HOTAIR resulted in DNAm changes at HOTAIR binding sites. Five potential triple helix forming domains were predicted within the HOTAIR sequence based on reverse Hoogsteen hydrogen bonds. Notably, the predicted triple helix target sites for these HOTAIR domains were also enriched in differentially expressed genes and close to DNAm changes upon modulation of HOTAIR Electrophoretic mobility shift assays provided further evidence that HOTAIR domains form RNA-DNA-DNA triplexes with predicted target sites. Our results demonstrate that HOTAIR impacts on differentiation of MSCs and that it is associated with senescence-associated DNAm. Targeting of epigenetic modifiers to relevant loci in the genome may involve triple helix formation with HOTAIR.

The dynamics and prognostic potential of DNA methylation changes at stem cell gene loci in women's cancer.

Aberrant DNA methylation is an important cancer hallmark, yet the dynamics of DNA methylation changes in human carcinogenesis remain largely unexplored. Moreover, the role of DNA methylation for prediction of clinical outcome is still uncertain and confined to specific cancers. Here we perform the most comprehensive study of DNA methylation changes throughout human carcinogenesis, analysing 27,578 CpGs in each of 1,475 samples, ranging from normal cells in advance of non-invasive neoplastic transformation to non-invasive and invasive cancers and metastatic tissue. We demonstrate that hypermethylation at stem cell PolyComb Group Target genes (PCGTs) occurs in cytologically normal cells three years in advance of the first morphological neoplastic changes, while hypomethylation occurs preferentially at CpGs which are heavily Methylated in Embryonic Stem Cells (MESCs) and increases significantly with cancer invasion in both the epithelial and stromal tumour compartments. In contrast to PCGT hypermethylation, MESC hypomethylation progresses significantly from primary to metastatic cancer and defines a poor prognostic signature in four different gynaecological cancers. Finally, we associate expression of TET enzymes, which are involved in active DNA demethylation, to MESC hypomethylation in cancer. These findings have major implications for cancer and embryonic stem cell biology and establish the importance of systemic DNA hypomethylation for predicting prognosis in a wide range of different cancers.

Role of DNA methylation and epigenetic silencing of HAND2 in endometrial cancer development.

BACKGROUND: Endometrial cancer incidence is continuing to rise in the wake of the current ageing and obesity epidemics. Much of the risk for endometrial cancer development is influenced by the environment and lifestyle. Accumulating evidence suggests that the epigenome serves as the interface between the genome and the environment and that hypermethylation of stem cell polycomb group target genes is an epigenetic hallmark of cancer. The objective of this study was to determine the functional role of epigenetic factors in endometrial cancer development. METHODS AND FINDINGS: Epigenome-wide methylation analysis of >27,000 CpG sites in endometrial cancer tissue samples (n = 64) and control samples (n = 23) revealed that HAND2 (a gene encoding a transcription factor expressed in the endometrial stroma) is one of the most commonly hypermethylated and silenced genes in endometrial cancer. A novel integrative epigenome-transcriptome-interactome analysis further revealed that HAND2 is the hub of the most highly ranked differential methylation hotspot in endometrial cancer. These findings were validated using candidate gene methylation analysis in multiple clinical sample sets of tissue samples from a total of 272 additional women. Increased HAND2 methylation was a feature of premalignant endometrial lesions and was seen to parallel a decrease in RNA and protein levels. Furthermore, women with high endometrial HAND2 methylation in their premalignant lesions were less likely to respond to progesterone treatment. HAND2 methylation analysis of endometrial secretions collected using high vaginal swabs taken from women with postmenopausal bleeding specifically identified those patients with early stage endometrial cancer with both high sensitivity and high specificity (receiver operating characteristics area under the curve = 0.91 for stage 1A and 0.97 for higher than stage 1A). Finally, mice harbouring a Hand2 knock-out specifically in their endometrium were shown to develop precancerous endometrial lesions with increasing age, and these lesions also demonstrated a lack of PTEN expression. CONCLUSIONS: HAND2 methylation is a common and crucial molecular alteration in endometrial cancer that could potentially be employed as a biomarker for early detection of endometrial cancer and as a predictor of treatment response. The true clinical utility of HAND2 DNA methylation, however, requires further validation in prospective studies. Please see later in the article for the Editors' Summary.

Association between 3D Printing-Assisted Pelvic or Acetabular Fracture Surgery and the Length of Hospital Stay in Nongeriatric Male Adults.

Pelvic and acetabular fractures are challenging for orthopedic surgeons, but 3D printing has many benefits in treating these fractures and has been applied worldwide. This study aimed to determine whether 3D printing can shorten the length of hospital stay (LHS) in nongeriatric male adult patients with these fractures. This is a single-center retrospective study of 167 nongeriatric male adult participants from August 2009 to December 2021. Participants were divided into two groups based on whether they received 3D printing assistance. Subgroup analyses were performed. Pearson's correlation and multivariable linear regression models were used to analyze the LHS and the parameters. Results showed that 3D printing-assisted surgery did not affect LHS in the analyzed patients. The LHS was positively correlated with the Injury Severity Score (ISS). Initial hemoglobin levels were negatively associated with LHS in patients aged 18−40 and non-major trauma (ISS < 16) patients. In 40−60-year-old and non-major trauma patients, the duration from fracture to admission was significantly associated with LHS. This study indicates that 3D-assisted technology for pelvic or acetabular fracture surgery for nongeriatric male adults does not influence the LHS. More importantly, the initial evaluation of patients in the hospital was the main predictor of the LHS.

Does 3D Printing-Assisted Acetabular or Pelvic Fracture Surgery Shorten Hospitalization Durations among Older Adults?

Acetabular or anterior pelvic ring fractures are rare but extremely complicated and challenging injuries for orthopedic trauma surgeons. Three-dimensional (3D) printing technology is widely used in the management of these two fracture types for surgical benefits. Our study aimed to explore whether 3D printing-assisted acetabular or pelvic surgery is beneficial in terms of shortening the length of hospital stay (LHS) and intensive care unit (ICU) stay (ICU LS) for older patients. This retrospective study included two groups of 76 participants over 60 years old who underwent operations with (n = 41) or without (n = 35) guidance by 3D printing. The Mann-Whitney U test was used to analyze continuous variables. Chi-square analysis was applied for categorical variables. Univariable and multivariable linear regression models were used to analyze the factors associated with LHS. The median LHS in the group without 3D printing assistance was 16 (12-21) days, and the median ICU LS was 0 (0-2) days. The median LHS in the group with 3D printing assistance was 17 (12.5-22.5) days, and the median ICU LS was 0 (0-3) days. There was no significant difference in LHS associated with 3D printing assistance vs. that without 3D printing among patients who underwent open reduction and internal fixation for pelvic or acetabular fractures. The LHS positively correlated with the ICU LS whether the operation was 3D printing assisted or not. For fracture surgery in older patients, in addition to the advancement of surgical treatment and techniques, medical teams require more detailed preoperative evaluations, and more personalized medical plans regarding postoperative care to achieve the goals of shortening LHS, reducing healthcare costs, and reducing complication rates.

A 3D-ACEK/SERS system for highly efficient and selectable electrokinetic bacteria concentration/detection/ antibiotic-susceptibility-test on whole blood.

This study demonstrates a novel multi-functional microfluidic system, designated three dimensional Alternative Current Electrokinetic/Surface Enhanced Raman Scattering (3D-ACEK/SERS), which can concentrate bacteria from whole blood, identify bacterial species, and determine antibiotic susceptibilities of the bacteria rapidly. The system consists of a hybrid electrokinetic mechanism, integrating AC-electroosmosis (AC-EO) and dielectrophoresis (DEP) that allows thousand-fold concentration of bacteria, including S. aureus, Escherichia coli, and Chryseobacterium indologenes, in the center of an electrode with a wide range of working distance (hundreds to thousands of µm), while exclusion of blood cells through negative DEP forces. This microchip employs SERS assay to determine the identity of the concentrated bacteria in approximately 2 min with a limit of detection of 3 CFU/ml, 5 orders of magnitude lower than that using standard centrifugation-purification process. Finally, label-free antibiotic susceptibility testing has been successfully demonstrated on the platform using both antibiotic-sensitive and multidrug-resistant bacterial strains illustrating a potential utility of the system to clinical applications.

Variable methylation of the imprinted gene, SNRPN, supports a relationship between intracranial germ cell tumours and neural stem cells.

Germ cell tumours (GCTs) are a diverse group of neoplasms all of which are generally believed to arise from germ cell progenitors (PGCs). Even those that form in the nervous system are likewise believed to be PGC-derived, despite being found a great distance from the normal location of germ cells. The primary evidence in favour of this model for the origins of intracranial GCTs is that they share molecular features with other GCTs. Those features include shared gene expression and a lack of methylation of imprinted genes, including SNRPN. Contrary to this model, we have proposed that endogenous neural stem cells of the brain are a more likely origin for these tumours. We show here that the lack of methylation of SNRPN that has previously been taken to indicate an origin for GCTs from PGCs is also seen in neural stem cells of mice and humans. We believe that, in the light of these and other recent observations, endogenous neural precursors of the brain are a more plausible origin for intracranial GCTs than are misplaced PGCs.

Dynamic methylation and expression of Oct4 in early neural stem cells.

Neural stem cells are a multipotent population of tissue-specific stem cells with a broad but limited differentiation potential. However, recent studies have shown that over-expression of the pluripotency gene, Oct4, alone is sufficient to initiate a process by which these can form 'induced pluripotent stem cells' (iPS cells) with the same broad potential as embryonic stem cells. This led us to examine the expression of Oct4 in endogenous neural stem cells, as data regarding its expression in neural stem cells in vivo are contradictory and incomplete. In this study we have therefore analysed the expression of Oct4 and other genes associated with pluripotency throughout development of the mouse CNS and in neural stem cells grown in vitro. We find that Oct4 is still expressed in the CNS by E8.5, but that this expression declines rapidly until it is undetectable by E15.5. This decline is coincident with the gradual methylation of the Oct4 promoter and proximal enhancer. Immunostaining suggests that the Oct4 protein is predominantly cytoplasmic in location. We also found that neural stem cells from all ages expressed the pluripotency associated genes, Sox2, c-Myc, Klf4 and Nanog. These data provide an explanation for the varying behaviour of cells from the early neuroepithelium at different stages of development. The expression of these genes also provides an indication of why Oct4 alone is sufficient to induce iPS formation in neural stem cells at later stages.

Widespread intronic polyadenylation diversifies immune cell transcriptomes.

Alternative cleavage and polyadenylation (ApA) is known to alter untranslated region (3'UTR) length but can also recognize intronic polyadenylation (IpA) signals to generate transcripts that lose part or all of the coding region. We analyzed 46 3'-seq and RNA-seq profiles from normal human tissues, primary immune cells, and multiple myeloma (MM) samples and created an atlas of 4927 high-confidence IpA events represented in these cell types. IpA isoforms are widely expressed in immune cells, differentially used during B-cell development or in different cellular environments, and can generate truncated proteins lacking C-terminal functional domains. This can mimic ectodomain shedding through loss of transmembrane domains or alter the binding specificity of proteins with DNA-binding or protein-protein interaction domains. MM cells display a striking loss of IpA isoforms expressed in plasma cells, associated with shorter progression-free survival and impacting key genes in MM biology and response to lenalidomide.

HOTAIR and its surrogate DNA methylation signature indicate carboplatin resistance in ovarian cancer.

BACKGROUND: Understanding carboplatin resistance in ovarian cancer is critical for the improvement of patients' lives. Multipotent mesenchymal stem cells or an aggravated epithelial to mesenchymal transition phenotype of a cancer are integrally involved in pathways conferring chemo-resistance. Long non-coding RNA HOTAIR (HOX transcript antisense intergenic RNA) is involved in mesenchymal stem cell fate and cancer biology. METHODS: We analyzed HOTAIR expression and associated surrogate DNA methylation (DNAme) in 134 primary ovarian cancer cases (63 received carboplatin, 55 received cisplatin and 16 no chemotherapy). We validated our findings by HOTAIR expression and DNAme analysis in a multicentre setting of five additional sets, encompassing 946 ovarian cancers. Chemo-sensitivity has been assessed in cell culture experiments. RESULTS: HOTAIR expression was significantly associated with poor survival in carboplatin-treated patients with adjusted hazard ratios for death of 3.64 (95 % confidence interval [CI] 1.78-7.42; P < 0.001) in the discovery and 1.63 (95 % CI 1.04-2.56; P = 0.032) in the validation set. This effect was not seen in patients who did not receive carboplatin (0.97 [95 % CI 0.52-1.80; P = 0.932]). HOTAIR expression or its surrogate DNAme signature predicted poor outcome in all additional sets of carboplatin-treated ovarian cancer patients while HOTAIR expressors responded preferentially to cisplatin (multivariate interaction P = 0.008). CONCLUSIONS: Non-coding RNA HOTAIR or its more stable DNAme surrogate may indicate the presence of a subset of cells which confer resistance to carboplatin and can serve as (1) a marker to personalise treatment and (2) a novel target to overcome carboplatin resistance.