Endoreduplication of the mouse genome in the absence of ORC1.

The largest subunit of the origin recognition complex (ORC1) is essential for assembly of the prereplicative complex, firing of DNA replication origins, and faithful duplication of the genome. Here, we generated knock-in mice with LoxP sites flanking exons encoding the critical ATPase domain of ORC1. Global or tissue-specific ablation of ORC1 function in mouse embryo fibroblasts and fetal and adult diploid tissues blocked DNA replication, cell lineage expansion, and organ development. Remarkably, ORC1 ablation in extraembryonic trophoblasts and hepatocytes, two polyploid cell types in mice, failed to impede genome endoreduplication and organ development and function. Thus, ORC1 in mice is essential for mitotic cell divisions but dispensable for endoreduplication. We propose that DNA replication of mammalian polyploid genomes uses a distinct ORC1-independent mechanism.

Mouse models of Slc35a2 brain mosaicism reveal mechanisms of mild malformations of cortical development with oligodendroglial hyperplasia in epilepsy.

OBJECTIVE: Brain somatic variants in SLC35A2 were recently identified as a genetic marker for mild malformations of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE). The role of SLC35A2 in cortical development and the contributions of abnormal neurons and oligodendrocytes to seizure activity in MOGHE remain largely unexplored. METHODS: Here, we generated a novel Slc35a2 floxed allele, which we used to develop two Slc35a2 conditional knockout mouse lines targeting (1) the Emx1 dorsal telencephalic lineage (excitatory neurons and glia) and (2) the Olig2 lineage (oligodendrocytes). We examined brain structure, behavior, and seizure activity. RESULTS: Knockout of Slc35a2 from the Emx1 lineage, which targets both cortical neurons and oligodendrocytes, resulted in early lethality and caused abnormal cortical development, increased oligodendroglial cell density, early onset seizures, and developmental delays akin to what is observed in patients with MOGHE. By tracing neuronal development with 5-Ethynyl-2'-deoxyuridine (EdU) birthdating experiments, we found that Slc35a2 deficiency disrupts corticogenesis by delaying radial migration of neurons from the subventricular zone. To discern the contributions of oligodendrocytes to these phenotypes, we knocked out Slc35a2 from the Olig2 lineage. This recapitulated the increased oligodendroglial cell density and resulted in abnormal electroencephalographic activity, but without a clear seizure phenotype, suggesting Slc35a2 deficiency in neurons is required for epileptogenesis. SIGNIFICANCE: This study presents two novel Slc35a2 conditional knockout mouse models and characterizes the effects on brain development, behavior, and epileptogenesis. Together, these results demonstrate a direct causal role for SLC35A2 in MOGHE-like phenotypes, including a critical role in neuronal migration during brain development, and identify neurons as key contributors to SLC35A2-related epileptogenesis.

Video-assisted anal fistula treatment (VAAFT) combined with ozonide oil dressing: standardization of technique in pediatric patients.

BACKGROUND: Anal fistula and perianal abscess are commonly acquired anorectal pathologies in children. Surgical treatment options commonly adopted are fistulotomy, fistulectomy, cutting seton placement, and more recently video-assisted anal fistula treatment (VAAFT). Optimal postoperative wound dressing remains debated. This study aimed to report our series of pediatric patients, who received VAAFT and postoperative wound dressing using ozonide oil. METHODS: All patients who underwent VAAFT between August 2018 and May 2023 were included in the study. Demographics, clinical features, pre-operative imaging, surgical details, outcome, and mid-term outcome data were retrospectively reviewed for each patient. All VAAFT procedures were performed under general anesthesia and using a 10-Ch fistuloscope. RESULTS: Thirty-three VAAFT procedures were performed in 30 patients over the study period. The median patient age was 5.7 years (range 1.75-14). Anal fistula was idiopathic in 26/30 (86.6%), iatrogenic in 2/30 (6.7%), and secondary to Crohn's disease in 2/30 (6.7%). The median duration of surgery was 23 min (range 18-40). All patients received ozonide oil dressing twice a day for 5 weeks postoperatively. The median hospital stay was 24 h (range 9-36). The median healing time was 28 days (range 17-39). With a median follow-up of 2 years (range 0.5-5), disease recurrence occurred in 3/30 (10%) patients with idiopathic fistula, who were re-operated using the same technique, with no further recurrence. No fecal incontinence or soiling was observed. CONCLUSION: Our series confirmed that VAAFT is a safe and effective technique to treat children with perianal fistula. The technique is versatile, allowing to treat fistulae of different etiologies. Postoperative course was painless and fast. Future comparative prospective studies are needed to better establish these conclusions.

Targeting OCT3 attenuates doxorubicin-induced cardiac injury.

Doxorubicin is a commonly used anticancer agent that can cause debilitating and irreversible cardiac injury. The initiating mechanisms contributing to this side effect remain unknown, and current preventative strategies offer only modest protection. Using stem-cell-derived cardiomyocytes from patients receiving doxorubicin, we probed the transcriptomic landscape of solute carriers and identified organic cation transporter 3 (OCT3) (SLC22A3) as a critical transporter regulating the cardiac accumulation of doxorubicin. Functional validation studies in heterologous overexpression models confirmed that doxorubicin is transported into cardiomyocytes by OCT3 and that deficiency of OCT3 protected mice from acute and chronic doxorubicin-related changes in cardiovascular function and genetic pathways associated with cardiac damage. To provide proof-of-principle and demonstrate translational relevance of this transport mechanism, we identified several pharmacological inhibitors of OCT3, including nilotinib, and found that pharmacological targeting of OCT3 can also preserve cardiovascular function following treatment with doxorubicin without affecting its plasma levels or antitumor effects in multiple models of leukemia and breast cancer. Finally, we identified a previously unrecognized, OCT3-dependent pathway of doxorubicin-induced cardiotoxicity that results in a downstream signaling cascade involving the calcium-binding proteins S100A8 and S100A9. These collective findings not only shed light on the etiology of doxorubicin-induced cardiotoxicity, but also are of potential translational relevance and provide a rationale for the implementation of a targeted intervention strategy to prevent this debilitating side effect.

Quality of life of pediatric patients operated for pilonidal sinus disease.

Quality of life (QOL) outcome is an ideal method for determining the efficacy of a surgical treatment. In children operated for pilonidal sinus disease (PSD), open procedures imply prolonged wound care, significant morbidity, and high recurrence rates. Endoscopic treatment (PEPSIT) overcomes these limitations. We report our experience in the management of PSD to evaluate the QOL of patients undergoing open and endoscopic treatment. The records of 177 patients undergoing surgery for PSD from 2008 to 2021 were retrospectively reviewed. Twenty patients were operated with open surgery (G1) and 157 with PEPSIT (G2). We analyzed QOL through the following criteria: hospital stay (HS), healing time (HT), return to sport (RTSp), return to school (RTSc), resumption of social life (RSL), and recurrence rate and reoperation (RRR). Moreover, we used Pediatric Quality of Life Enjoyment and Satisfaction Questionnaire (PQ-LES-Q) for a more subjective evaluation of life satisfaction. We found significant differences in all the analyzed criteria: HS varied from 3 to 7 days in G1 and from 1 to 2 days in G2; HT from 40 to 75 days in G1 while from 20 to 41 days in G2; RTSp from 50 to 80 days in G1 while from 7 to 21 days in G2; RTSc from 9 to 15 days in G1 while from 2 to 4 days in G2; RSL from 13 to 20 days in G1 while from 2 to 5 days in G2; RRR was 25% in G1 and 4.4% in G2. CONCLUSION: Endoscopic treatment (PEPSIT) significantly improves the quality of life of patients operated for PSD. Compared to open surgery, PEPSIT presents shorter hospital stay, faster healing time, return to sport activities, return to school and resumption of a normal social life, and lower rates of recurrence and reoperation. In addition, PQ-LES-Q demonstrated a good overall quality of life and life satisfaction. Further prospective studies should be obtained to consider PEPSIT as the gold standard for the treatment of PSD in pediatric patients. WHAT IS KNOWN: • Many techniques have been proposed in the last 20 years for the surgical treatment of PSD. • PEPSIT is showing promising results in terms of safety and long-term efficacy. WHAT IS NEW: • The main impact in QOL of patients operated with PEPSIT is on their daily activity, including a shorter hospital stay, faster healing time, return to sport activities, return to school and resumption of a normal social life, lower rates of recurrence and reoperation. • After PEPSIT, children maintain a satisfactory quality of life according to the analysis of PQ-LES-Q.

Noncatalytic PTEN missense mutation predisposes to organ-selective cancer development in vivo.

Inactivation of phosphatase and tensin homology deleted on chromosome 10 (PTEN) is linked to increased PI3K-AKT signaling, enhanced organismal growth, and cancer development. Here we generated and analyzed Pten knock-in mice harboring a C2 domain missense mutation at phenylalanine 341 (Pten(FV)), found in human cancer. Despite having reduced levels of PTEN protein, homozygous Pten(FV/FV) embryos have intact AKT signaling, develop normally, and are carried to term. Heterozygous Pten(FV/+) mice develop carcinoma in the thymus, stomach, adrenal medulla, and mammary gland but not in other organs typically sensitive to Pten deficiency, including the thyroid, prostate, and uterus. Progression to carcinoma in sensitive organs ensues in the absence of overt AKT activation. Carcinoma in the uterus, a cancer-resistant organ, requires a second clonal event associated with the spontaneous activation of AKT and downstream signaling. In summary, this PTEN noncatalytic missense mutation exposes a core tumor suppressor function distinct from inhibition of canonical AKT signaling that predisposes to organ-selective cancer development in vivo.

IFITM3 protects the heart during influenza virus infection.

Influenza virus can disseminate from the lungs to the heart in severe infections and can induce cardiac pathology, but this has been difficult to study due to a lack of small animal models. In humans, polymorphisms in the gene encoding the antiviral restriction factor IFN-induced transmembrane protein 3 (IFITM3) are associated with susceptibility to severe influenza, but whether IFITM3 deficiencies contribute to cardiac dysfunction during infection is unclear. We show that IFITM3 deficiency in a new knockout (KO) mouse model increases weight loss and mortality following influenza virus infections. We investigated this enhanced pathogenesis with the A/PR/8/34 (H1N1) (PR8) influenza virus strain, which is lethal in KO mice even at low doses, and observed increased replication of virus in the lungs, spleens, and hearts of KO mice compared with wild-type (WT) mice. Infected IFITM3 KO mice developed aberrant cardiac electrical activity, including decreased heart rate and irregular, arrhythmic RR (interbeat) intervals, whereas WT mice exhibited a mild decrease in heart rate without irregular RR intervals. Cardiac electrical dysfunction in PR8-infected KO mice was accompanied by increased activation of fibrotic pathways and fibrotic lesions in the heart. Infection with a sublethal dose of a less virulent influenza virus strain (A/WSN/33 [H1N1]) resulted in a milder cardiac electrical dysfunction in KO mice that subsided as the mice recovered. Our findings reveal an essential role for IFITM3 in limiting influenza virus replication and pathogenesis in heart tissue and establish IFITM3 KO mice as a powerful model for studying mild and severe influenza virus-induced cardiac dysfunction.

Formation and contraction of multicellular actomyosin cables facilitate lens placode invagination.

Embryonic morphogenesis relies on the intrinsic ability of cells, often through remodeling the cytoskeleton, to shape epithelial tissues during development. Epithelial invagination is an example of morphogenesis that depends on this remodeling but the cellular mechanisms driving arrangement of cytoskeletal elements needed for tissue deformation remain incompletely characterized. To elucidate these mechanisms, live fluorescent microscopy and immunohistochemistry on fixed specimens were performed on chick and mouse lens placodes. This analysis revealed the formation of peripherally localized, circumferentially orientated and aligned junctions enriched in F-actin and MyoIIB. Once formed, the aligned junctions contract in a Rho-kinase and non-muscle myosin dependent manner. Further molecular characterization of these junctions revealed a Rho-kinase dependent accumulation of Arhgef11, a RhoA-specific guanine exchange factor known to regulate the formation of actomyosin cables and junctional contraction. In contrast, the localization of the Par-complex protein Par3, was reduced in these circumferentially orientated junctions. In an effort to determine if Par3 plays a negative role in MyoIIB accumulation, Par3-deficient mouse embryos were analyzed which not only revealed an increase in bicellular junctional accumulation of MyoIIB, but also a reduction of Arhgef11. Together, these results highlight the importance of the formation of the multicellular actomyosin cables that appear essential to the initiation of epithelial invagination and implicate the potential role of Arhgef11 and Par3 in their contraction and formation.

Technical standardization of ICG near-infrared fluorescence (NIRF) laparoscopic partial nephrectomy for duplex kidney in pediatric patients.

PURPOSE: This study aimed to standardize the operative technique of indocyanine green (ICG) near-infrared fluorescence (NIRF) laparoscopic partial nephrectomy (LPN) and compare it with the standard technique. METHODS: In the last 4 years, we performed 22 LPN (14 right-sided, 8 left-sided) in children with non-functioning moiety of duplex kidney. Patients included 12 girls and 10 boys with a median age of 3.9 years (range 1-10). Patients were grouped according to the use of ICG-NIRF: G1 included 12 patients operated using ICG-NIRF and G2 included 10 patients receiving the standard technique. We standardized the technique of injection of ICG in three different steps. RESULTS: The median operative time was significantly lower in G1 [87 min (range 68-110)] compared with G2 [140 min (range 70-220)] (p = 0.001). One intra-operative complication occurred in G2. At post-operative ultrasound (US), the residual moiety was normal in all patients. An asymptomatic renal cyst related to the site of surgery was visualized at US in 8/22 (36%), with a significantly higher incidence in G2 (6/10, 60%) compared with G1 (2/12, 16.6%) (p = 0.001). Renogram demonstrated no loss of function of residual moiety. No allergic reactions to ICG occurred. CONCLUSION: ICG-NIRF LPN is technically easier, quicker, and safer compared with the standard technique. The main advantages of using ICG-NIRF during LPN are the clear identification of normal ureter, vasculature of non-functioning pole, and demarcation line between the avascular and the perfused pole. The main limitation of ICG technology remains the need for specific laparoscopic equipment that is not always available.

Evaluation of efficacy of oxygen-enriched oil-based gel dressing in patients who underwent surgical repair of distal hypospadias: a prospective randomised clinical trial.

PURPOSE: This study aimed to evaluate the efficacy of oxygen-enriched oil-based gel dressing on wound healing and postoperative outcome in children who underwent distal hypospadias repair. METHODS: We included all patients with distal hypospadias, who underwent Snodgrass urethroplasty and preputioplasty over an 18-months period. The patients were randomized in two groups according to the type of medication: oxygen-enriched oil-based gel (G1) and hyaluronic acid cream (G2). After discharge, parents changed the dressing twice a day for 2-3 weeks postoperatively. The patients were evaluated at 7, 14, 21, 30, 60 and 180 postoperative days and thereafter annually. RESULTS: One-hundred and fourteen patients (median age 18 months) were included in the study and randomized in two groups, each of 57 patients. The wound healing was significantly faster in G1 compared with G2 (p = 0.001). G1 reported significantly higher SWAS and modified HOPE scores compared with G2 (p = 0.001) at all steps of follow-up. No adverse skin reactions occurred. Foreskin dehiscence and re-operations rates were significantly lower in G1 compared with G2 (p = 0.001). Postoperative foreskin retractability was better in G1, with a significantly higher incidence of secondary phimosis in G2 (p = 0.001). The median treatment costs were significantly lower in G1 compared with G2 (p = 0.001). CONCLUSION: Postoperative dressing using oxygen-enriched oil-based gel was highly effective, promoting a faster wound healing in patients who underwent distal hypospadias repair. It reported a lower incidence of foreskin dehiscence and better foreskin retractability compared with the control group. It was cost-effective and clinically safe without allergy or intolerance to the product.

Knockout of the non-essential gene SUGCT creates diet-linked, age-related microbiome disbalance with a diabetes-like metabolic syndrome phenotype.

SUGCT (C7orf10) is a mitochondrial enzyme that synthesizes glutaryl-CoA from glutarate in tryptophan and lysine catabolism, but it has not been studied in vivo. Although mutations in Sugct lead to Glutaric Aciduria Type 3 disease in humans, patients remain largely asymptomatic despite high levels of glutarate in the urine. To study the disease mechanism, we generated SugctKO mice and uncovered imbalanced lipid and acylcarnitine metabolism in kidney in addition to changes in the gut microbiome. After SugctKO mice were treated with antibiotics, metabolites were comparable to WT, indicating that the microbiome affects metabolism in SugctKO mice. SUGCT loss of function contributes to gut microbiota dysbiosis, leading to age-dependent pathological changes in kidney, liver, and adipose tissue. This is associated with an obesity-related phenotype that is accompanied by lipid accumulation in kidney and liver, as well as "crown-like" structures in adipocytes. Furthermore, we show that the SugctKO kidney pathology is accelerated and exacerbated by a high-lysine diet. Our study highlights the importance of non-essential genes with no readily detectable early phenotype, but with substantial contributions to the development of age-related pathologies, which result from an interplay between genetic background, microbiome, and diet in the health of mammals.

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays.

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development.

Standardization of Pre- and Postoperative Management Using Laser Epilation and Oxygen-Enriched Oil-Based Gel Dressing in Pediatric Patients Undergoing Pediatric Endoscopic Pilonidal Sinus Treatment (PEPSiT).

BACKGROUND AND OBJECTIVE: Pediatric endoscopic pilonidal sinus treatment (PEPSiT) has become the new standard of care for pilonidal sinus disease (PSD) in pediatric patients. This study aimed to compare our current wound treatment protocol (laser epilation (LE) and oxygen-enriched oil-based gel dressing) with our previous protocol (silver sulfadiazine spray) and demonstrate its efficacy as means to prevent PSD recurrence in children undergoing PEPSiT. STUDY DESIGN/MATERIALS AND METHODS: We retrospectively reviewed the data of 87 pediatric patients, 52 boys and 35 girls, with an average age of 17.1 years (range, 12-18) affected by chronic PSD, who underwent PEPSiT over a 24-month period (December 2017-December 2019). The patients were divided into two groups: G1 (n = 47) treated with pre- and postoperative LE and oxygen-enriched oil-based gel dressing; and G2 (n = 40) treated with only postoperative dressing using silver sulfadiazine spray. The two groups were compared regarding the operative outcome, wound-healing time, disease recurrence, wound infections, and other complications. Furthermore, efficacy, safety, and tolerability of LE were assessed in G1. RESULTS: No significant difference emerged between the two groups regarding the median operating time, postoperative pain score, hospital stay length, and time to full daily activities (P = 0.33). The median healing time significantly decreased in G1 (21 days) compared with G2 (28.1 days) (P = 0.001]. The disease recurrence rate was significantly lower in G1 (n = 1, 2.1%) compared with G2 (n = 6, 15%) (P = 0.001), and the wound infection rate was significantly lower in G1 (n = 1, 2.1%) compared with G2 (n = 4, 10%) (P = 0.001). All patients with wound infection were treated with oral antibiotics and, after the resolution of the acute episode, received LE with no further infections (Clavien II). Granuloma of the wound occurred in two G2 patients (5%), who were treated with topical silver nitrate (Clavien II). LE was well-tolerated and without complications in all G1 patients; a median number of 7 LE sessions (range, 4-10) at 4-6 weeks interval was required to achieve definitive hair removal. CONCLUSION: The results of this study confirmed that our standardized pre- and postoperative wound management, including LE and oxygen-enriched oil-based gel dressing, was extremely safe and effective in reducing PSD recurrence and wound infection rate in pediatric patients undergoing PEPSiT. LE should be routinely offered as adjunctive treatment to all patients who receive PEPSiT and is strongly advocated to be started before surgery and continued after wound healing. More importantly, LE showed to have a role as a preventive modality in patients with recurrent folliculitis or infections at the intergluteal crease. It was also associated with significant improvement and acceleration of wound-healing time. LE and oxygen-enriched oil-based gel dressings were clinically safe and well-tolerated in all patients, with no adverse skin reactions or injuries to both therapies. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Selective targeting of point-mutated KRAS through artificial microRNAs.

Mutated protein-coding genes drive the molecular pathogenesis of many diseases, including cancer. Specifically, mutated KRAS is a documented driver for malignant transformation, occurring early during the pathogenesis of cancers such as lung and pancreatic adenocarcinomas. Therapeutically, the indiscriminate targeting of wild-type and point-mutated transcripts represents an important limitation. Here, we leveraged on the design of miRNA-like artificial molecules (amiRNAs) to specifically target point-mutated genes, such as KRAS, without affecting their wild-type counterparts. Compared with an siRNA-like approach, the requirement of perfect complementarity of the microRNA seed region to a given target sequence in the microRNA/target model has proven to be a more efficient strategy, accomplishing the selective targeting of point-mutated KRAS in vitro and in vivo.

Speedy A-Cdk2 binding mediates initial telomere-nuclear envelope attachment during meiotic prophase I independent of Cdk2 activation.

Telomere attachment to the nuclear envelope (NE) is a prerequisite for chromosome movement during meiotic prophase I that is required for pairing of homologous chromosomes, synapsis, and homologous recombination. Here we show that Speedy A, a noncanonical activator of cyclin-dependent kinases (Cdks), is specifically localized to telomeres in prophase I male and female germ cells in mice, and plays an essential role in the telomere-NE attachment. Deletion of Spdya in mice disrupts telomere-NE attachment, and this impairs homologous pairing and synapsis and leads to zygotene arrest in male and female germ cells. In addition, we have identified a telomere localization domain on Speedy A covering the distal N terminus and the Cdk2-binding Ringo domain, and this domain is essential for the localization of Speedy A to telomeres. Furthermore, we found that the binding of Cdk2 to Speedy A is indispensable for Cdk2's localization on telomeres, suggesting that Speedy A and Cdk2 might be the initial components that are recruited to the NE for forming the meiotic telomere complex. However, Speedy A-Cdk2-mediated telomere-NE attachment is independent of Cdk2 activation. Our results thus indicate that Speedy A and Cdk2 might mediate the initial telomere-NE attachment for the efficient assembly of the telomere complex that is essential for meiotic prophase I progression.

Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth.

Approximately 2% of colorectal cancer is linked to pre-existing inflammation known as colitis-associated cancer, but most develops in patients without underlying inflammatory bowel disease. Colorectal cancer often follows a genetic pathway whereby loss of the adenomatous polyposis coli (APC) tumour suppressor and activation of ß-catenin are followed by mutations in K-Ras, PIK3CA and TP53, as the tumour emerges and progresses. Curiously, however, 'inflammatory signature' genes characteristic of colitis-associated cancer are also upregulated in colorectal cancer. Further, like most solid tumours, colorectal cancer exhibits immune/inflammatory infiltrates, referred to as 'tumour-elicited inflammation'. Although infiltrating CD4(+) T(H)1 cells and CD8(+) cytotoxic T cells constitute a positive prognostic sign in colorectal cancer, myeloid cells and T-helper interleukin (IL)-17-producing (T(H)17) cells promote tumorigenesis, and a 'T(H)17 expression signature' in stage I/II colorectal cancer is associated with a drastic decrease in disease-free survival. Despite its pathogenic importance, the mechanisms responsible for the appearance of tumour-elicited inflammation are poorly understood. Many epithelial cancers develop proximally to microbial communities, which are physically separated from immune cells by an epithelial barrier. We investigated mechanisms responsible for tumour-elicited inflammation in a mouse model of colorectal tumorigenesis, which, like human colorectal cancer, exhibits upregulation of IL-23 and IL-17. Here we show that IL-23 signalling promotes tumour growth and progression, and development of a tumoural IL-17 response. IL-23 is mainly produced by tumour-associated myeloid cells that are likely to be activated by microbial products, which penetrate the tumours but not adjacent tissue. Both early and late colorectal neoplasms exhibit defective expression of several barrier proteins. We propose that barrier deterioration induced by colorectal-cancer-initiating genetic lesions results in adenoma invasion by microbial products that trigger tumour-elicited inflammation, which in turn drives tumour growth.

Human anti-nucleolin recombinant immunoagent for cancer therapy.

Nucleolin (NCL) is a nucleocytoplasmic protein involved in many biological processes, such as ribosomal assembly, rRNA processing, and mRNA stabilization. NCL also regulates the biogenesis of specific microRNAs (miRNAs) involved in tumor development and aggressiveness. Interestingly, NCL is expressed on the surface of actively proliferating cancer cells, but not on their normal counterparts. Therefore, NCL is an attractive target for antineoplastic treatments. Taking advantage of phage-display technology, we engineered a fully human single-chain fragment variable, named 4LB5. This immunoagent binds NCL on the cell surface, it is translocated into the cytoplasm of target cells, and it abrogates the biogenesis of NCL-dependent miRNAs. Binding of 4LB5 to NCL on the cell surface of a variety of breast cancer and hepatocellular carcinoma cell lines, but not to normal-like MCF-10a breast cells, dramatically reduces cancer cell viability and proliferation. Finally, in orthotopic breast cancer mouse models, 4LB5 administration results in a significant reduction of the tumor volume without evident side effects. In summary, here we describe, to our knowledge, the first anti-NCL single-chain fragment variable displaying antineoplastic activity against established solid tumors, which could represent the prototype of novel immune-based NCL-targeting drugs with clinical potential as diagnostic and therapeutic tools in a wide variety of human cancers.

Scorpins in the DNA Damage Response.

The DNA Damage Response (DDR) is a complex signaling network that comes into play when cells experience genotoxic stress. Upon DNA damage, cellular signaling pathways are rewired to slow down cell cycle progression and allow recovery. However, when the damage is beyond repair, cells activate complex and still not fully understood mechanisms, leading to a complete proliferative arrest or cell death. Several conventional and novel anti-neoplastic treatments rely on causing DNA damage or on the inhibition of the DDR in cancer cells. However, the identification of molecular determinants directing cancer cells toward recovery or death upon DNA damage is still far from complete, and it is object of intense investigation. SPRY-containing RAN binding Proteins (Scorpins) RANBP9 and RANBP10 are evolutionarily conserved and ubiquitously expressed proteins whose biological functions are still debated. RANBP9 has been previously implicated in cell proliferation, survival, apoptosis and migration. Recent studies also showed that RANBP9 is involved in the Ataxia Telangiectasia Mutated (ATM) signaling upon DNA damage. Accordingly, cells lacking RANBP9 show increased sensitivity to genotoxic treatment. Although there is no published evidence, extensive protein similarities suggest that RANBP10 might have partially overlapping functions with RANBP9. Like RANBP9, RANBP10 bears sites putative target of PIK-kinases and high throughput studies found RANBP10 to be phosphorylated following genotoxic stress. Therefore, this second Scorpin might be another overlooked player of the DDR alone or in combination with RANBP9. This review focuses on the relatively unknown role played by RANBP9 and RANBP10 in responding to genotoxic stress.

Putative binding sites for mir-125 family miRNAs in the mouse Lfng 3'UTR affect transcript expression in the segmentation clock, but mir-125a-5p is dispensable for normal somitogenesis.

BACKGROUND: In vertebrate embryos, a "segmentation clock" times somitogenesis. Clock-linked genes, including Lunatic fringe (Lfng), exhibit cyclic expression in the presomitic mesoderm (PSM), with a period matching the rate of somite formation. The clock period varies widely across species, but the mechanisms that underlie this variability are not clear. The half-lives of clock components are proposed to influence the rate of clock oscillations, and are tightly regulated in the PSM. Interactions between Lfng and mir-125a-5p in the embryonic chicken PSM promote Lfng transcript instability, but the conservation of this mechanism in other vertebrates has not been tested. Here, we examine whether this interaction affects clock activity in a mammalian species. RESULTS: Mutation of mir-125 binding sites in the Lfng 3'UTR leads to persistent, nonoscillatory reporter transcript expression in the caudal-most mouse PSM, although dynamic transcript expression recovers in the central PSM. Despite this, expression of endogenous mir-125a-5p is dispensable for mouse somitogenesis. CONCLUSIONS: These results suggest that mir-125a sites in the Lfng 3' untranslated region influence transcript turnover in both mouse and chicken embryos, and support the existence of position-dependent regulatory mechanisms in the PSM. They further suggest the existence of compensatory mechanisms that can rescue the loss of mir-125a-5p in mice. Developmental Dynamics 246:740-748, 2017. © 2017 Wiley Periodicals, Inc.

miR-216 and miR-217 expression is reduced in transgenic mouse models of pancreatic adenocarcinoma, knockout of miR-216/miR-217 host gene is embryonic lethal.

Mice harboring a G12D activating Kras mutation are among the most heavily studied models in the field of pancreatic adenocarcinoma (PDAC) research. miRNAs are differentially expressed in PDAC from patients and mouse models of PDAC. To better understand the relationship that Kras activation has on miRNA expression, we profiled the expression of 629 miRNAs in RNA isolated from the pancreas of control, young, and old P48+/Cre;LSL-KRASG12D as well as PDX-1-Cre;LSL-KRASG12D mice. One hundred of the differentially expressed miRNAs had increased expression in the advanced disease (old) P48+/Cre;LSL-KRASG12D compared to wild-type mice. Interestingly, the expression of three miRNAs, miR-216a, miR-216b, and miR-217, located within a ∼30-kbp region on 11qA3.3, decreased with age (and phenotype severity) in these mice. miR-216/-217 expression was also evaluated in another acinar-specific ELa-KrasG12D mouse model and was downregulated as well. As miR-216/-217 are acinar enriched, reduced in human PDAC and target KRAS, we hypothesized that they may maintain acinar differentiation or represent tumor suppressive miRNAs. To test this hypothesis, we deleted a 27.9-kbp region of 11qA3.3 containing the miR-216/-217 host gene in the mouse's germ line. We report that germ line deletion of this cluster is embryonic lethal in the mouse. We estimate that lethality occurs shortly after E9.5. qPCR analysis of the miR-216b and miR-217 expression in the heterozygous animals showed no difference in expression, suggesting haplosufficiency by some type of compensatory mechanism. We present the differential miRNA expression in KrasG12D transgenic mice and report lethality from deletion of the miR-216/-217 host gene in the mouse's germ line.