Search

This study adds insight regarding the occurrence and spread of SARS-CoV-2 Variants of Concern (VOCs) and Var-iants of Interest (VOIs) in Italy in October and November 2022, by testing urban wastewater collected through-out the country. A total of 332 wastewater samples were collected from 20 Italian Regions/Autonomous Provinces (APs) within the framework of national SARS-CoV-2 environmental surveillance. Of these, 164 were collected in the first week of October and 168 in the first week of November. A similar to 1600 bp fragment of the spike protein was sequenced by Sanger (for individual samples) and long-read nanopore sequencing (for pooled Region/AP samples).In October, mutations characteristic of Omicron BA.4/BA.5 were detected in the vast majority (91 %) of the samples amplified by Sanger sequencing. A fraction of these sequences (9 %) also displayed the R346T mutation. Despite the low prevalence documented in clinical cases at the time of sampling, amino acid substitutions characteristic of sublineages BQ.1 or BQ.1.1 were detected in 5 % of sequenced samples from four Regions/APs. A significantly higher variability of sequences and variants was documented in November 2022, when the rate of se-quences harbouring mutations of lineages BQ.1 and BQ1.1 increased to 43 %, and the number of Regions/APs positive for the new Omicron subvariant more than tripled (n = 13) compared to October. Moreover, an increase in the number of sequences with the mutation package BA.4/BA.5 + R346T (18 %), as well as the detection of variants never observed before in wastewater in Italy, such as BA.2.75 and XBB.1 (the latter in a Region where no clinical cases asso-ciated with this variant had ever been documented) was recorded.The results suggest that, as predicted by the ECDC, BQ.1/BQ.1.1 is rapidly becoming dominant in late 2022. Environ-mental surveillance proves to be a powerful tool for tracking the spread of SARS-CoV-2 variants/subvariants in the population.

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool.

Rationale: Regorafenib is an oral multikinase inhibitor and is approved as salvage therapy in the standard treatment of advanced colorectal cancer (CRC). Due to its limited efficacy, toxicity profile, and cost, it is necessary to identify those patients who may have the most benefit from regorafenib. In a previous case report, kinase insert domain receptor (KDR) mutation has been associated with exceptional clinical response (CR) in an elderly patient treated with a low dose of regorafenib; thus, it was hypothesized that it could represent a new predictive marker of drug response. Patient concerns: A heavily pretreated 67-year-old man with a wide peripancreatic recurrence of colon carcinoma and liver metastases was subjected to treatment with regorafenib. Diagnoses: After 3 months of therapy, a computed tomography scan showed an impressive reduction of disease. Interventions: Regorafenib was given at full doses (160 mg/die for 21 days, every 4 weeks). Outcomes: A lasting response without relevant toxicity. No KDR mutation relief was detected. After 13 months from the start of treatment, the patient died after the diagnosis of encephalic metastases. Lessons: Regorafenib can lead to an unexpected and durable CR with consistent progression-free survival and overall survival benefit even in patients affected by polychemotherapy refractory metastatic CRC. Further studies are needed to establish the benefit of KDR mutation as predictive marker for regorafenib sensitivity for patients with CRC. We include a detailed revision of prognostic and predictive factors of clinical outcome identified in literature to optimize the use of regorafenib in this setting.

Dystroglycan is a widely expressed adhesion complex that anchors cells to the basement membrane and is involved in embryonic development and differentiation. Dystroglycan expression is frequently reduced in human dystrophies and malignancies, and its molecular functions are not completely understood. Several posttranslational mechanisms have been identified that regulate dystroglycan expression and/or function, while little is known about how expression of the corresponding Dag1 gene is regulated. This study aimed to clone the Dag1 gene promoter and to characterize its regulatory elements. Analysis of the mouse Dag1 gene 5′-flanking region revealed a TATA and CAAT box-lacking promoter including a GC-rich region. Transfection studies with serially deleted promoter constructs allowed us to identify a minimal promoter region containing three Specificity protein 1 (Sp1) sites and an E-box. Sp1 binding was confirmed by chromatin immunoprecipitation assay, and Sp1 downregulation reduced dystroglycan expression in muscle cells. Treatment with 5-aza-2′-deoxycytidine and/or the histone deacetylase inhibitor trichostatin A increased Dag1 mRNA expression levels in myoblasts, and methylation decreased promoter activity in vitro. Furthermore, Dag1 gene promoter methylation was reduced while its expression increased during differentiation of C2C12myoblast cells in myotubes. In conclusion, for the first time we have characterized the activity of the mouse Dag1 gene promoter, confirming a complex regulation by Sp1 transcription factor, DNA methylation, and histone acetylation, which might be relevant for a better understanding of the physiopathology of the dystroglycan complex.

Colorectal Cancer (CRC) in developed countries is the major causes of cancer death. It may have sporadic or hereditary origin. Familial Adenomatous Polyposis (FAP) is a very frequent hereditary syndromes predisposing to Colorectal Cancer and is characterized by the development of numerous precancerous polyps (from hundreds to thousands) in the second decade of life. Classical FAP is caused by germline mutations in the APC gene (Adenomatous Polyposis Coli) a tumor suppressor gene. Often the results of the genetic mutation are a truncating protein that lacks functional domains, such losing its function and promoting tumorigenesis. A subject who presents a monoallelic germline variation in APC gene inherits in a dominant manner the predisposition to polyps, which will turn in CRC, through the occurrence of a somatic alteration in the wild-type copy of gene. Molecular analyses of APC gene confirms diagnosis in FAP patients and allow to detect the specific pathological mutation in order to screen the unaffected members of the same family, identify pre-symptomatic highrisk subjects and insert them in endoscopic surveillance protocols. We analyzed the APC mutational status in a Ukrainian woman with a history of CRC and proctocolectomy. By direct sequencing of the encoding exons of APC gene we found a c.3778 C>T heterozygous substitution which causes the substitution of a Glutamine with a stop codon, resulting in a premature truncation of APC protein (Q1260X). This mutation had never been described before and it may be considered as a “novel mutation”. We extended the analyses to 1st grade related, proving that one of the patient’s son was heterozygous for the same mutation and that to the endoscopic evaluation he showed an early develop of hundreds of polyps. This finding confirmed our speculation about the pathologic effect of Q1260X.

Totipotent cDNA libraries representative of all the potentially expressed sequences in a genome would be of great benefit to gene expression studies. Here, we report on an innovative method for creating such a library for durum wheat (Triticum turgidum L. var. durum) and its application for gene discovery. The use of suitable quantities of 5-azacytidine during the germination phase induced the demethylation of total DNA, and the resulting seedlings potentially express all of the genes present in the genome. A new wheat microarray consisting of 4925 unigenes was developed from the totipotent cDNA library and used to screen for genes that may contribute to differences in the disease resistance of two near-isogenic lines, the durum wheat cultivar Latino and the line 5BIL-42, which are respectively susceptible and resistant to powdery mildew. Fluorescently labeled cDNA was prepared from the RNA of seedlings of the two near-isogenic wheat lines after infection with a single powdery mildew isolate under controlled conditions in the greenhouse. Hybridization to the microarray identified six genes that were differently expressed in the two lines. Four of the sequences could be assigned putative functions based on their similarity to known genes in public databases. Physical mapping of the six genes localized them to two regions of the genome: the centromeric region of chromosome 5B, where the Pm36 resistance gene was previously localized, and chromosome 6B.

Escherichia coli has been recognized as the principal indicator of fecal contamination of water. Indeed, E. coli is the only species in the coliform group found in relationship with gastrointestinal tract of human and warm-blooded animals and subsequently excreted in large numbers in the human feces. To obtain a complete picture of water quality and therefore, a better protection of public health, different techniques for water analysis have been proposed. In this article, we describe an alternative method that uses SYBR green real time-polymerase chain reaction (RT-PCR) technology to identify and quantify all E. coli biotypes in a group of wastewater samples collected from a wastewater depurator located in South of Italy. This new RT-PCR protocol is accurate in measuring the concentration of chromosomal E. coli DNA using the amplification of three new specific fragments of the following bacteria genes: CadC, HNS, and Allan whose sequence is specific for E. coli family and conserved in all E. coli subtypes. This method allowed us to detect the presence of all E. coli biotypes directly in wastewater samples and estimated the correspondence between colony forming units and bacterial DNA concentrations. The availability of a rapid and sensitive method may be useful to monitor the persistence of E. coli in water, to evaluate the efficiency of wastewater purification treatments and the possible recycle for agricultural use. Furthermore, the development of a simple and routine method to monitor water quality with RT-PCR analysis can encourage the testing of a higher number of samples. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1106–1113, 2012

In the present study we have evaluated whether (i) 5-azacytidine (AZA), a well-known demethylating agent, could be able to modify the phenotype of human preadipocytes and (ii) the modified cells could possess multilineage differentiation potential. Human preadipocytes at the 3rd passage were treated for 48 or 96 h with 10 μM AZA and then expanded up to passage 5. Stem cell markers, such as OCT-4, Nanog, and Sox2, were upregulated after 96 h of treatment with the demethylating treatment. Further, decreases in the expression of genes, such as adipose differentiation-related protein, characterizing the preadipocytes were noted. Our data showed that AZA-treated preadipocytes differentiated into cell lineages derived from mesoderm. Indeed, after incubation with inductive media for 3 weeks, osteblast-, chondrocyte-, and myoblast-like cells were detected in the cultures. Interestingly, both upregulation of stem cell markers and differentiation potential were maintained by the treated cultures expanded until the 5th passage. Taken together, our results suggest that AZA, without the use of transduction methods, convert preadipocytes to a less differentiated state that can be induced, under suitable stimuli, to the formation of mesoderm-derived cell lineages.

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by recurrent chromosomal aberrations of prognostic significance. We aimed to evaluate the potential of the multiplex ligation-dependent probe amplification (MLPA) assay to detect genomic alterations in CLL. Highly purified (>90%) peripheral mononuclear CD19+ cell populations from 100 untreated CLL patients (pts) in early stage disease (Binet stage A) were included in this study. All samples were investigated by fluorescence in situ hybridization (FISH) for the presence of trisomy 12 and 17p13.1, 11q22.3, and 13q14.3 deletions. For MPLA analysis, DNA was amplified by means of two commercially available probes sets allowing the simultaneous screening of 56 genomic sequences. Overall, a high degree of concordance (95%) between MPLA and FISH results was found, if the abnormal clone was present in more than 30% of the leukemic cell population. The use of multiple MPLA probes allowed the fine-mapping of the 13q14 deletion and the identification of intragenic or small alterations undetected by FISH. Moreover, additional alterations in 2p24 (MYCN) (3 pts), 8q24 (MYC) (1 pt), 9p21 (CDKN2A2B) (1 pt), 1q21 (LMNA) (1 pt), and 6q25-26 (1 pt) regions not covered by a standard FISH assay were detected and all confirmed by FISH. Our data extend previously limited evidence that MLPA may represent a useful technique for the characterization of well-known lesions as well as the investigation of additional genomic changes in CLL.

Genus Nocardia belongs to aerobic Actinomycetes, a wide group of polymorphous Gram+, fixed and acapsusalated, branching, partially acid-fast bacilli. Nocardia organism are ubiquitous, soil-borne actinomycetes that usually infect humans as a result of the inhalation of airborne bacilli or traumatic inoculation. Nocardiosis is a rare opportunistic disease that affects mainly patients with impaired cellmediated immunity, such as those with acquired immunodeficiency syndrome (AIDS) or transplant recipients, patients with pulmonary disease, haematological malignancies etc. Pulmonary disease is the most common presentation in immounosuppressed patients. Nocardial organism have a tendency to disseminate hematogenously from the primary site of infection.The central nervous system (CNS) is one of the most frequent sites of dissemination. Herein we describe a rare and fatal case of diffuse meningo-encephalytis due to Nocardia farcinica in a young patient kidney transplant recipient. Nocardia has been isolated from Cerebrospinal fluid in a micobacteriology laboratory with identification of the strain using sequencing of hsp65 gene. The diagnosis can be challenging, as signs and symptoms are not specific and a high degree of clinical suspicion is required. Identification of Nocardia farcinica is important because of its aggressiveness, its tendency to disseminate, and its resistance to antibiotics. Susceptibilities to 10 antimicrobial agents were determined by E-test.The isolate was resistant to Gentamicin, Clarithromycin, Doxycicline, Cefotaxime and susceptible to Amikacin,Amoxicillin clavulanate, Imipenem, Ciprofloxacin, Linezolid and Trimethoprim–Sulfamethoxazole. The susceptibility profile was favourable since, in North Italy, the strains are generally resistant to Trimethoprim-Sulfamethoxazole.

STSs, which have been used to build and format clone contigs, have been used here to assemble a transcriptional map across a cytogenetic band. Of fifty one STSs in Xq28, 20 were positive by RT-PCR. Thus, an additional 20 possible ESTs were detected among the STSs, and seven of these also identified cDNAs in at least one library. The transcripts confirm the high expression level of this region, correlated with its GC compositional map and CpG island content.

Abstract 1863 Background: Clonal chromosome abnormalities are present in the marrow cells in about 50% of patients with myelodysplastic syndromes (MDS) at the time of presentation. Cytogenetic analysis of the bone marrow is not only indicated in MDS for diagnostic purposes, but also to assess the individual prognosis according to IPSS scoring guidelines and plan tailored therapy. Conventional cytogenetics (CC) analysis is performed in clinical practice to detect chromosomal abnormalities. It has been reported that fluorescence in situ hybridization (FISH) is a more sensitive approach, but this analysis is limited to detection of the more frequent abnormalities on chromosomes 5, 7, 8, 11, and 20, and reports from the literature provide contradictory data. A new method has recently been described for the measurement of gene/chromosome copy number using genomic DNA: Multiplex Ligation-dependent Probe Amplification (MLPA). Aims: The purpose of this study was to perform the MLPA assay in a series of 29 MDS patients (M: 21, F: 8, median age 71 years, range 44–84), and to compare the results obtained with CC data. According to the WHO classification, 7 cases were classified as RA, 7 as refractory cytopenia with multilineage dysplasia, 2 as RAEB-1, 8 as RAEB-2, 1 as MDS_U, and 4 as CMML. According to the IPSS score, 8 were considered low risk, 11 intermediate-1 risk, 5 intermediate-2 risk, and 5 high risk. Methods: The MLPA assay was performed for all samples in two independent reactions, one for each probe mix (SALSA Probe-Mix P144 and P145) according to the manufacturer's recommendations (MRC-Holland). This mix contains 61 target sequences specific for different chromosome regions commonly involved in MDS: 5q (9 probes) + 5p (1 probe), 7q (8 probes) + 7p (2 probes), 8q (8 probes) + 8p (2 probes), 11q (8 probes), 12p (6 probes), 17q (2 probes) + 17p (4 probes), 20q (5 probes) + 20p (1 probe) and 21q (5 probes). The Probe mixes also include 21 reference probes selected from chromosomal regions that appear to be “quiet” in MDS. Data were analyzed with Coffalyser Software (MRC-Holland) using DNA from ten healthy donors as controls. The CC study was performed following standard protocols and at least 20 metaphases were analyzed. Results and Conclusions: Our study showed a good correlation between the MLPA and CC results, as shown in Table I, since most of the alterations were detected by both techniques. Discrepancies were found in 5 (17%) samples. MLPA analysis did not detect: in sample n°5 the presence of a chromosomal (chr.) translocation; in sample n°12 a chr. deletion and a chr. translocation; in sample n°17 a chr. deletion; in sample n°22 several chr. translocations and deletions; in sample n°28 a chr. gain. In fact, MLPA is not able to detect chr. translocations because it can reveal only chr. loss or gain; it can only analyse the chr. regions commonly involved in MDS (5, 7, 8, 11, 12, 17, 20 and 21); it can reveal chr. abnormalities only if the percentage of cells carrying the alterations is about 30–35% and do not show mosaicism. On the other hand, with CC we observed a karyotype failure (no metaphases) in 3 samples. MLPA proved to be rapid, cost effective, relatively easy to perform, had high throughput and enabled simultaneous analysis of many samples by automated data processing. MLPA and CC result complementary techniques, and MLPA is particularly useful in MDS cases with Karyotype failure. Disclosures: No relevant conflicts of interest to declare.