Integrative omics reveals subtle molecular perturbations following ischemic conditioning in a porcine kidney transplant model.

BACKGROUND: Remote Ischemic Conditioning (RIC) has been proposed as a therapeutic intervention to circumvent the ischemia/reperfusion injury (IRI) that is inherent to organ transplantation. Using a porcine kidney transplant model, we aimed to decipher the subclinical molecular effects of a RIC regime, compared to non-RIC controls. METHODS: Kidney pairs (n = 8 + 8) were extracted from brain dead donor pigs and transplanted in juvenile recipient pigs following a period of cold ischemia. One of the two kidney recipients in each pair was subjected to RIC prior to kidney graft reperfusion, while the other served as non-RIC control. We designed an integrative Omics strategy combining transcriptomics, proteomics, and phosphoproteomics to deduce molecular signatures in kidney tissue that could be attributed to RIC. RESULTS: In kidney grafts taken out 10 h after transplantation we detected minimal molecular perturbations following RIC compared to non-RIC at the transcriptome level, which was mirrored at the proteome level. In particular, we noted that RIC resulted in suppression of tissue inflammatory profiles. Furthermore, an accumulation of muscle extracellular matrix assembly proteins in kidney tissues was detected at the protein level, which may be in response to muscle tissue damage and/or fibrosis. However, the majority of these protein changes did not reach significance (p < 0.05). CONCLUSIONS: Our data identifies subtle molecular phenotypes in porcine kidneys following RIC, and this knowledge could potentially aid optimization of remote ischemic conditioning protocols in renal transplantation.

Caesarean scar pregnancy: descriptive paper of three different types of management on a series of clinical cases.

INTRODUCTION: A caesarean scar pregnancy is a complex iatrogenic pathology, which represents a consequence of a previous caesarean section. It increased in recent years due to parallel increase of cesarean sections. MATERIAL AND METHODS: We present a retrospective study on patients with caesarean scar pregnancy diagnosed in our department from June 2016 to June 2019. Stable women with an embryo (with or without cardiac activity) who accepted our experimental protocol were treated with single dose of methotrexate (50 mg administered locally intracavitary + 50 mg administered intramuscularly) and folinic acid (15 mg/day orally for 30 days). Clinically stable women with embryo (without cardiac activity) who decided to wait, were monitored by serial assays of b-hCG and clinical and ultrasonographic follow up. Women who were clinically unstable with embryo (without cardiac activity), were referred for urgent surgical treatment with dilation and curettage. RESULTS: Caesarean scar pregnancy was diagnosed in sixteen women. Among these women, seven were treated according to our experimental protocol with methotrexate and folinic acid and only one had profuse bleeding, which required a laparotomic hysterectomy. Four women were treated urgently with dilatation and curettage. Five women chose to wait: they were monitored and all spontaneously had a miscarriage. CONCLUSIONS: In our preliminary study, we highlighted how our experimental protocol gave encouraging results in the first 10 weeks of caesarean scar pregnancy. However, caution is needed in patients with advanced gestational age, a gestational sac with large diameter, higher CRL and presence of embryonic cardiac activity.

The T cell IFT20 interactome reveals new players in immune synapse assembly.

Sustained signalling at the immune synapse (IS) requires the synaptic delivery of recycling endosome-associated T cell antigen receptors (TCRs). IFT20, a component of the intraflagellar transport system, controls TCR recycling to the IS as a complex with IFT57 and IFT88. Here, we used quantitative mass spectrometry to identify additional interaction partners of IFT20 in Jurkat T cells. In addition to IFT57 and IFT88, the analysis revealed new binding partners, including IFT54 (also known as TRAF3IP1), GMAP-210 (also known as TRIP11), Arp2/3 complex subunit-3 (ARPC3), COP9 signalosome subunit-1 (CSN1, also known as GPS1) and ERGIC-53 (also known as LMAN1). A direct interaction between IFT20 and both IFT54 and GMAP-210 was confirmed in pulldown assays. Confocal imaging of antigen-specific conjugates using T cells depleted of these proteins by RNA interference showed that TCR accumulation and phosphotyrosine signalling at the IS were impaired in the absence of IFT54, ARPC3 or ERGIC-53. Similar to in IFT20-deficient T cells, this defect resulted from a reduced ability of endosomal TCRs to polarize to the IS despite a correct translocation of the centrosome towards the antigen-presenting cell contact. Our data underscore the traffic-related role of an IFT20 complex that includes components of the intracellular trafficking machinery in IS assembly.

Early Kinetics of the HLA Class I-Associated Peptidome of MVA.HIVconsv-Infected Cells.

UNLABELLED: Cytotoxic T cells substantially contribute to the control of intracellular pathogens such as human immunodeficiency virus type 1 (HIV-1). Here, we evaluated the immunopeptidome of Jurkat cells infected with the vaccine candidate MVA.HIVconsv, which delivers HIV-1 conserved antigenic regions by using modified vaccinia virus Ankara (MVA). We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify 6,358 unique peptides associated with the class I human leukocyte antigen (HLA), of which 98 peptides were derived from the MVA vector and 7 were derived from the HIVconsv immunogen. Human vaccine recipients responded to the peptide sequences identified by LC-MS/MS. Peptides derived from the conserved HIV-1 regions were readily detected as early as 1.5 h after MVA.HIVconsv infection. Four of the seven conserved peptides were monitored between 0 and 3.5 h of infection by using quantitative mass spectrometry (Q-MS), and their abundance in HLA class I associations reflected levels of the whole HIVconsv protein in the cell. While immunopeptides delivered by the incoming MVA vector proteins could be detected, all early HIVconsv-derived immunopeptides were likely synthesized de novo. MVA.HIVconsv infection generally altered the composition of HLA class I-associated human (self) peptides, but these changes corresponded only partially to changes in the whole cell host protein abundance. IMPORTANCE: The vast changes in cellular antigen presentation after infection of cells with a vectored vaccine, as shown here for MVA.HIVconsv, highlight the complexity of factors that need to be considered for efficient antigen delivery and presentation. Identification and quantitation of HLA class I-associated peptides by Q-MS will not only find broad application in T-cell epitope discovery but also inform vaccine design and allow evaluation of efficient epitope presentation using different delivery strategies.

Finite mixture clustering of human tissues with different levels of IGF-1 splice variants mRNA transcripts.

BACKGROUND: This study addresses a recurrent biological problem, that is to define a formal clustering structure for a set of tissues on the basis of the relative abundance of multiple alternatively spliced isoforms mRNAs generated by the same gene. To this aim, we have used a model-based clustering approach, based on a finite mixture of multivariate Gaussian densities. However, given we had more technical replicates from the same tissue for each quantitative measurement, we also employed a finite mixture of linear mixed models, with tissue-specific random effects. RESULTS: A panel of human tissues was analysed through quantitative real-time PCR methods, to quantify the relative amount of mRNA encoding different IGF-1 alternative splicing variants. After an appropriate, preliminary, equalization of the quantitative data, we provided an estimate of the distribution of the observed concentrations for the different IGF-1 mRNA splice variants in the cohort of tissues by employing suitable kernel density estimators. We observed that the analysed IGF-1 mRNA splice variants were characterized by multimodal distributions, which could be interpreted as describing the presence of several sub-population, i.e. potential tissue clusters. In this context, a formal clustering approach based on a finite mixture model (FMM) with Gaussian components is proposed. Due to the presence of potential dependence between the technical replicates (originated by repeated quantitative measurements of the same mRNA splice isoform in the same tissue) we have also employed the finite mixture of linear mixed models (FMLMM), which allowed to take into account this kind of within-tissue dependence. CONCLUSIONS: The FMM and the FMLMM provided a convenient yet formal setting for a model-based clustering of the human tissues in sub-populations, characterized by homogeneous values of concentrations of the mRNAs for one or multiple IGF-1 alternative splicing isoforms. The proposed approaches can be applied to any cohort of tissues expressing several alternatively spliced mRNAs generated by the same gene, and can overcome the limitations of clustering methods based on simple comparisons between splice isoform expression levels.

Pathogenic Leptospira interrogans exoproteins are primarily involved in heterotrophic processes.

Leptospirosis is a life-threatening and emerging zoonotic disease with a worldwide annual occurrence of more than 1 million cases. Leptospirosis is caused by spirochetes belonging to the genus Leptospira. The mechanisms of disease manifestation in the host remain elusive, and the roles of leptospiral exoproteins in these processes have yet to be determined. Our aim in this study was to assess the composition and quantity of exoproteins of pathogenic Leptospira interrogans and to construe how these proteins contribute to disease pathogenesis. Label-free quantitative mass spectrometry of proteins obtained from Leptospira spirochetes cultured in vitro under conditions mimicking infection identified 325 exoproteins. The majority of these proteins are conserved in the nonpathogenic species Leptospira biflexa, and proteins involved in metabolism and energy-generating functions were overrepresented and displayed the highest relative abundance in culture supernatants. Conversely, proteins of unknown function, which represent the majority of pathogen-specific proteins (presumably involved in virulence mechanisms), were underrepresented. Characterization of various L. interrogans exoprotein mutants in the animal infection model revealed host mortality rates similar to those of hosts infected with wild-type L. interrogans. Collectively, these results indicate that pathogenic Leptospira exoproteins primarily function in heterotrophic processes (the processes by which organisms utilize organic substances as nutrient sources) to maintain the saprophytic lifestyle rather than the virulence of the bacteria. The underrepresentation of proteins homologous to known virulence factors, such as toxins and effectors in the exoproteome, also suggests that disease manifesting from Leptospira infection is likely caused by a combination of the primary and potentially moonlight functioning of exoproteins.

Body Stalk Anomaly Complicated by Ectopia Cordis: First-Trimester Diagnosis of Two Cases Using 2- and 3-Dimensional Sonography.

INTRODUCTION: Body stalk anomaly is a severe defect of the abdominal wall, characterized by the evisceration of abdominal organs and, in more severe cases, thoracic organs as well. The most serious condition in a body stalk anomaly may be complicated by ectopia cordis, an abnormal location of the heart outside the thorax. The aim of this scientific work is to describe our experience with the prenatal diagnosis of ectopia cordis as part of the first-trimester sonographic screening for aneuploidy. METHODS: We report two cases of body stalk anomalies complicated by ectopia cordis. The first case was identified during a first ultrasound examination at 9 weeks of gestation. The second was identified during an ultrasound examination at 13 weeks of gestation. Both of these cases were diagnosed using high-quality 2- and 3-dimensional ultrasonographic images obtained by the Realistic Vue and Crystal Vue techniques. The chorionic villus sampling showed that the fetal karyotype and CGH-array were both normal. RESULTS: In our clinical case reports, the patients, immediately after the diagnosis of a body stalk anomaly complicated by ectopia cordis, opted for the termination of pregnancies. CONCLUSION: Performing an early diagnosis of a body stalk anomaly that is complicated by ectopia cordis is desirable, considering their poor prognoses. Most of the reported cases in the literature suggest that an early diagnosis can be made between 10 and 14 weeks of gestation. A combination of 2- and 3-dimensional sonography could allow an early diagnosis of body stalk anomalies complicated by ectopia cordis, particularly using new ultrasonographic techniques, the Realistic Vue and the Crystal Vue.

The Effect of Maternal Dietary Patterns on Birth Weight for Gestational Age: Findings from the MAMI-MED Cohort.

Limited evidence exists on the effects of maternal dietary patterns on birth weight, and most studies conducted so far did not adjust their findings for gestational age and sex, leading to potentially biased conclusions. In the present study, we applied a novel method, namely the clustering on principal components, to derive dietary patterns among 667 pregnant women from Catania (Italy) and to evaluate the associations with birth weight for gestational age. We identified two clusters reflecting distinct dietary patterns: the first one was mainly characterized by plant-based foods (e.g., potatoes, cooked and raw vegetables, legumes, soup, fruits, nuts, rice, wholemeal bread), fish and white meat, eggs, butter and margarine, coffee and tea; the second one consisted mainly of junk foods (sweets, dips, salty snacks, and fries), pasta, white bread, milk, vegetable and olive oils. Regarding small gestational age births, the main predictors were employment status and primiparity, but not the adherence to dietary patterns. By contrast, women belonging to cluster 2 had higher odds of large for gestational age (LGA) births than those belonging to cluster 1 (OR = 2.213; 95%CI = 1.047-4.679; p = 0.038). Moreover, the odds of LGA increased by nearly 11% for each one-unit increase in pregestational BMI (OR = 1.107; 95%CI = 1.053-1.163; p < 0.001). To our knowledge, the present study is the first to highlight a relationship between adherence to an unhealthy dietary pattern and the likelihood of giving birth to a LGA newborn. This evidence adds to the current knowledge about the effects of diet on birth weight, which, however, remains limited and controversial.

Uterine Rupture in Pregnancy following Two Abdominal Myomectomies and IVF.

Objective: Uterine rupture (UR) during pregnancy is an obstetric emergency that could determine poor maternal and neonatal outcomes. There are many factors that could increase the risk of UR, such as a previous myomectomy. The aim of this study is to evaluate the role of a previous myomectomy in a spontaneous UR in pregnancy. Methods: A 33-year-old primigravida comes to our obstetric emergency room for pelvic pain at 29 weeks of gestation. In her medical history, there were two previous surgical operations of abdominal myomectomy, one in 2015 and one in January 2021 (6 months before conception). After 34 minutes, a pubo-subumbilical longitudinal laparotomy was performed for pathological decelerations in the cardiotocography. In the peritoneal cavity, there was 500 mL of blood serum liquid. The right arm and shoulder of the fetus were extending out of the uterus across a breach of 5 cm near the right tubal corner. A corporal incision was performed, and a healthy baby was born and moved to neonatal intensive unit care. Results: A UR can occur at any stage of pregnancy, mostly during the third trimester of pregnancy. Risk factors that increase the incidence of a uterine rupture after myomectomy include a short period (i.e., <12 months) between the myomectomy and conception, the opening of the endometrial cavity, and large myomas (with a maximum diameter above 4 cm). Uterine rupture during pregnancy after abdominal myomectomy seems to be less frequent than after a laparoscopic one. Conclusion: Uterine rupture is an obstetric emergency; it is mandatory to consider this eventuality in pregnancy, particularly in the third trimester, if there was a previous laparoscopic myomectomy in the anamnesis of the patient.

The Impact of the COVID-19 Pandemic on Dietary Patterns of Pregnant Women: A Comparison between Two Mother-Child Cohorts in Sicily, Italy.

A maternal diet, before and during pregnancy, plays a key role in ensuring maternal and newborn health. The COVID-19 pandemic, however, may have compromised dietary habits in the general population and in specific subgroups of individuals. Here, we evaluated the impact of COVID-19 on the diet of pregnant women, using data from two mother-child cohorts in Sicily (Italy). Dietary data were collected using a food frequency questionnaire and analyzed through the Mediterranean diet (MD) score and principal component analysis (PCA). The comparison of maternal dietary consumption before and during the COVID-19 pandemic showed differences in terms of vegetables (p < 0.001), fruit (p < 0.001), dairy products (p < 0.001), fish (p < 0.001), and legumes (p = 0.001). Accordingly, after adjusting for covariates, mothers enrolled during the pandemic were more likely to report low adherence to MD than those enrolled before (OR = 1.65; 95%CI = 1.12−2.42; p = 0.011). A similar result was obtained by analyzing the adherence to a prudent dietary pattern, derived through PCA and characterized by high intake of cooked and row vegetables, legumes, fruit, fish, and soup. Overall, these findings suggested that the COVID-19 pandemic may have influenced maternal diet during pregnancy. However, further efforts are needed to investigate the main causes and consequences of this change.

Prenatal diagnosis of 45,X/46,XY mosaicism with cleft lip and epispadias.

INTRODUCTION: 45,X/46,XY mosaicism is an uncommon chromosomal anomaly with a range of phenotypes from normal males to cases of multiple congenital anomalies. MATERIALS AND METHODS: We report a case with associated cleft lip and epispadias prenatally diagnosed with autopsy evidences. CONCLUSION: Our case, with an uncommon association of congenital anomalies, stresses the difficulty of prenatal counselling regarding 45,X/46,XY mosaicism and discuss the possible role of sex chromosome genes that may be involved in the pathogenesis of both types of midline defect.

Unilateral ovarian and fallopian tube agenesis.

INTRODUCTION: The unilateral agenesis of the ovary and fallopian tube is an extremely uncommon event with only few cases described in literature. CASE REPORT: Here, we report a case of a 30-year-old woman admitted to our Unit for increasing pelvic pain. In the suspect of a corpus luteum hemorrhage, we performed a laparoscopy identifying the rare anomaly. DISCUSSION: Together with the predisposing genetic and/or environmental factor not yet discovered, two are the hypotheses explaining the absence of one or both the adnexes, the mechanical hypothesis, i.e., the asymptomatic torsion of both Fallopian tube and ovary with consequent organs' ischemia and atresia and the embryological hypothesis, i.e., the congenital absence of the adnexes.

Metabolically Active Three-Dimensional Brown Adipose Tissue Engineered from White Adipose-Derived Stem Cells.

Brown adipose tissue (BAT) has a unique capacity to expend calories by decoupling energy expenditure from ATP production, therefore BAT could realize therapeutic potential to treat metabolic diseases such as obesity and type 2 diabetes. Recent studies have investigated markers and function of native BAT, however, successful therapies will rely on methods that supplement the small existing pool of brown adipocytes in adult humans. In this study, we engineered BAT from both human and rat adipose precursors and determined whether these ex vivo constructs could mimic in vivo tissue form and metabolic function. Adipose-derived stem cells (ASCs) were isolated from several sources, human white adipose tissue (WAT), rat WAT, and rat BAT, then differentiated toward both white and brown adipogenic lineages in two-dimensional and three-dimensional (3D) culture conditions. ASCs derived from WAT were successfully differentiated in 3D poly(ethylene glycol) hydrogels into mature adipocytes with BAT phenotype and function, including high uncoupling protein 1 (UCP1) mRNA and protein expression and increased metabolic activity (basal oxygen consumption, proton leak, and maximum respiration). By utilizing this "browning" process, the abundant and accessible WAT stem cell population can be engineered into 3D tissue constructs with the metabolic capacity of native BAT, ultimately for therapeutic intervention in vivo and as a tool for studying BAT and its metabolic properties.

Space pruning monotonic search for the non-unique probe selection problem.

Identification of targets, generally viruses or bacteria, in a biological sample is a relevant problem in medicine. Biologists can use hybridisation experiments to determine whether a specific DNA fragment, that represents the virus, is presented in a DNA solution. A probe is a segment of DNA or RNA, labelled with a radioactive isotope, dye or enzyme, used to find a specific target sequence on a DNA molecule by hybridisation. Selecting unique probes through hybridisation experiments is a difficult task, especially when targets have a high degree of similarity, for instance in a case of closely related viruses. After preliminary experiments, performed by a canonical Monte Carlo method with Heuristic Reduction (MCHR), a new combinatorial optimisation approach, the Space Pruning Monotonic Search (SPMS) method, is introduced. The experiments show that SPMS provides high quality solutions and outperforms the current state-of-the-art algorithms.

Twist1-induced dissemination preserves epithelial identity and requires E-cadherin.

Dissemination of epithelial cells is a critical step in metastatic spread. Molecular models of dissemination focus on loss of E-cadherin or repression of cell adhesion through an epithelial to mesenchymal transition (EMT). We sought to define the minimum molecular events necessary to induce dissemination of cells out of primary murine mammary epithelium. Deletion of E-cadherin disrupted epithelial architecture and morphogenesis but only rarely resulted in dissemination. In contrast, expression of the EMT transcription factor Twist1 induced rapid dissemination of cytokeratin-positive epithelial cells. Twist1 induced dramatic transcriptional changes in extracellular compartment and cell-matrix adhesion genes but not in cell-cell adhesion genes. Surprisingly, we observed disseminating cells with membrane-localized E-cadherin and ß-catenin, and E-cadherin knockdown strongly inhibited Twist1-induced single cell dissemination. Dissemination can therefore occur with retention of epithelial cell identity. The spread of cancer cells during metastasis could similarly involve activation of an epithelial motility program without requiring a transition from epithelial to mesenchymal character.

Prenatal diagnosis of a fetus with anencephaly and thumb agenesis.

Severe anomalies of the forebrain together with reduction limb anomalies are a rare congenital anomalies association. We report a prenatal diagnosis of acalvaria, anencephaly and thumb agenesis in a voluntary terminated fetus and discuss the role of genetic counseling.

Prenatal Diagnosis of a Fetus with de novo Supernumerary Ring Chromosome 16 Characterized by Array Comparative Genomic Hybridization.

A fetus with de novo ring chromosome 16 is presented. At 20 weeks' gestation, ultrasound examination demonstrated bilateral clubfoot, bilateral renal pyelectasis, hypoplasia of the corpus callosum, and transposition of the great vessel. Amniocentesis was performed. Chromosome analysis identified a ring chromosome 16 [47,XY,r(16)] and array comparative genomic hybridization (a-CGH) demonstrated that the ring included the euchromatic portion 16p11.2. Postmortem examination confirmed prenatal findings. This is the first case of de novo ring chromosome 16 diagnosed prenatally with a new phenotypic pattern and also reinforces the importance of offering amniocentesis with a-CGH if fetal anomalies are detected.

Uterine rupture after prostaglandin analogues to induce midtrimester abortion.

Although prostaglandins are largely used and considered safe drugs to induce midtrimester abortion, the literature reports several cases of uterine rupture consequent to their administration. We report the second ever-described case of uterine rupture after administration of gemeprost and sulprostone for midtrimester abortion in a 45 years-old women with scarred uterus.She was admitted to our Unit for termination at 20 weeks' gestation because of trisomy 21 diagnosed by chromosomal analysis of amniotic liquid at 16 weeks' gestation. Five pessaries of gemeprost (one pessary, every 3 hours) were administered into the posterior vaginal fornix. Since the cervix remained closed and uneffaced, another cycle of 5 gemeprost administration was conducted. When the cervix changed in consistency and dilatation, we decided to administrate sulprostone. At the obstetric examination any visible fetus was evidenced. The abdominal ultrasonography showed an empty uterine cavity and the gestational sac with the dead fetus in abdomen. Emergency laparotomy was therefore undertaken. Primary suture of the ruptured uterus was initially attempted but in vain. Therefore, total abdominal hysterectomy was performed to control bleeding and eventual hypovolemic shock.Given the lack of strong evidence in literature and the fact that case reports are not an optimal method for assessing frequency of an event nor the overall risks of a procedure since they frequently report rare single events, other larger studies are needed to assess whether women with multiple risk factors (e.g. advanced age and previous uterine surgery), and administered with prostaglandins' association have a higher risk of uterine rupture.