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Magnesium (Mg) alloys possess unique properties that make them ideal for use as biodegradable implants in clinical applications. However, reports on the in vivo assessment of these alloys are insufficient. Thus, monitoring the degradation of Mg and its alloys in vivo is challenging due to the dynamic process of implant degradation and tissue regeneration. Most current works focus on structural remodeling, but functional assessment is crucial in providing information about physiological changes in tissues, which can be used as an early indicator of healing. Here, we report continuous wave near-infrared spectroscopy (CW NIRS), a non-invasive technique that is potentially helpful in assessing the implant–tissue dynamic interface in a rodent model. The purpose of this study was to investigate the effects on hemoglobin changes and tissue oxygen saturation (StO2) after the implantation of Mg-alloy (WE43) and titanium (Ti) implants in rats’ femurs using a multiwavelength optical probe. Additionally, the effect of changes in the skin on these parameters was evaluated. Lastly, combining NIRS with photoacoustic (PA) imaging provides a more reliable assessment of tissue parameters, which is further correlated with principal component analysis.

Recently, the development of learning-based algorithms has shown a crucial role to extract features of vital importance from multi-spectral photoacoustic imaging. In particular, advances in spectral photoacoustic unmixing algorithms can identify tissue biomarkers without a priori information. This has the potential to enhance the diagnosis and treatment of a large number of diseases. Here, we investigated the latest progress within spectral photoacoustic unmixing approaches. We evaluated the sensitivity of different unsupervised Blind Source Separation (BSS) techniques such as Principal Component Analysis (PCA), Independent Component Analysis (ICA), and Non-negative Matrix Factorization (NNMF) to distinguish absorbers from spectral photoacoustic imaging. Besides, the performance of a recently developed superpixel photoacoustic unmixing (SPAX) framework has been also examined in detail. Near-infrared spectroscopy (NIRS) has been used to validate the performance of the different unmixing algorithms. Although the NNMF has shown superior unmixing performance than PCA and ICA in terms of correlation and processing time, this is still prone to unmixing misinterpretation due to spectral coloring artifact. Thus, the SPAX framework, which also compensates for the spectral coloring effect, has shown improved sensitivity and specificity of the unmixed components. In addition, the SPAX also reveals the most and less prominent tissue components from sPAI at a volumetric scale in a data-driven way. Phantom experimental measurements and in vivo studies have been conducted to benchmark the performance of the BSS algorithms and the SPAX framework.

Diagnoses of Autism Spectrum Disorder (ASD) have rapidly increased globally. However, the lack of comprehensive epidemiological surveys and surveillance systems, able to provide official data at a national or European level is one of the main issues in the monitoring of this condition. The present study aimed to estimate the prevalence of ASD in children and adolescents aged 3–18 years old living in the province of Lecce (Southern Italy) through official data provided by the Local Health Authority of Lecce (ASL/LE) up to 31 October 2020, and compare it with school-based data concerning the number of students needing support for ASD. Based on data provided by the ASL/LE, in 2020 there were 509 cases of ASD among children and adolescents aged 3–18 years old, corresponding to a prevalence of 0.46%. A total of 408 (80.2%) were boys and 101 (19.8%) were girls. In relation to their age, 155 ASD cases (0.90%) were diagnosed in the 3–5 age group, while 222 (0.55%) in the 6–11 age group and 132 (0.25%) in the 12–18 age group. Prevalence of ASD assessed by school-based dataset was underestimated in the 3–5 age group, while the 6–11 and 12–18 age groups were consistent with the official data provided by the ASL/LE.

The assessment of tissue chromophores at a volumetric scale is vital for an improved diagnosis and treatment of a large number of diseases. Spectral photoacoustic imaging (sPAI) co-registered with high-resolution ultrasound (US) is an innovative technology that has a great potential for clinical translation as it can assess the volumetric distribution of the tissue components. Conventionally, to detect and separate the chromophores from sPAI, an input of the expected tissue absorption spectra is required. However, in pathological conditions, the prediction of the absorption spectra is difficult as it can change with respect to the physiological state. Besides, this conventional approach can also be hampered due to spectral coloring, which is a prominent distortion effect that induces spectral changes at depth. Here, we are proposing a novel data-driven framework that can overcome all these limitations and provide an improved assessment of the tissue chromophores. We have developed a superpixel spectral unmixing (SPAX) approach that can detect the most and less prominent absorber spectra and their volumetric distribution without any user interactions. Within the SPAX framework, we have also implemented an advanced spectral coloring compensation approach by utilizing US image segmentation and Monte Carlo simulations, based on a predefined library of optical properties. The framework has been tested on tissue-mimicking phantoms and also on healthy animals. The obtained results show enhanced specificity and sensitivity for the detection of tissue chromophores. To our knowledge, this is a unique framework that accounts for the spectral coloring and provides automated detection of tissue spectral signatures at a volumetric scale, which can open many possibilities for translational research.

Medical implants made of biodegradable materials are advantageous for short-term applications as fracture fixation and mechanical support during bone healing. After completing the healing process, the implant biodegrades without any long-term side effects nor any need for surgical removal. In particular, Magnesium (Mg) implants, while degrading, can cause physiological changes in the tissues surrounding the implant. The evaluation of structural remodeling is relevant, however, the functional assessment is crucial to provide information about physiological changes in tissues, which can be applied as an early marker during the healing process. Hence, non-invasive monitoring of structural and functional changes in the surrounding tissue during the healing process is essential, and the need for new assessing methods is emerging. This paper provides an assessment of Mg based implants, and an extensive review of the literature is presented with the focus on the imaging techniques for investigation of the Mg implants' biodegradation. The potential of a hybrid analysis, including Near-Infrared Spectroscopy (NIRS) and photoacoustic imaging (PAI) technology, is further discussed. A hybrid solution may play a significant role in monitoring implants and have several advantages for monitoring tissue oxygenation in addition to tissue's acidity, which is directly connected to the Mg implants degradation process. Such a hybrid assessment system can be a simple, ambulant, and less costly technology with the potential for clinically monitoring of Mg implants at site.

Objective Transient neonatal diabetes mellitus (TNDM) is caused by activating mutations in ABCC8 and KCNJ11 genes (KATP/TNDM) or by chromosome 6q24 abnormalities (6q24/TNDM). We wanted to assess whether these different genetic aetiologies result in distinct clinical features. Design Retrospective analysis of the Italian data set of patients with TNDM. Methods Clinical features and treatment of 22 KATP/TNDM patients and 12 6q24/TNDM patients were compared. Results Fourteen KATP/TNDM probands had a carrier parent with abnormal glucose values, four patients with 6q24 showed macroglossia and/or umbilical hernia. Median age at diabetes onset and birth weight were lower in patients with 6q24 (1 week; −2.27 SD) than those with KATP mutations (4.0 weeks; −1.04 SD) (P = 0.009 and P = 0.007, respectively). Median time to remission was longer in KATP/TNDM than 6q24/TNDM (21.5 weeks vs 12 weeks) (P = 0.002). Two KATP/TNDM patients entered diabetes remission without pharmacological therapy. A proband with the ABCC8/L225P variant previously associated with permanent neonatal diabetes entered 7-year long remission after 1 year of sulfonylurea therapy. Seven diabetic individuals with KATP mutations were successfully treated with sulfonylurea monotherapy; four cases with relapsing 6q24/TNDM were treated with insulin, metformin or combination therapy. Conclusions If TNDM is suspected, KATP genes should be analyzed first with the exception of patients with macroglossia and/or umbilical hernia. Remission of diabetes without pharmacological therapy should not preclude genetic analysis. Early treatment with sulfonylurea may induce long-lasting remission of diabetes in patients with KATP mutations associated with PNDM. Adult patients carrying KATP/TNDM mutations respond favourably to sulfonylurea monotherapy.

Multispectral photoacoustic imaging has been widely explored as an emerging tool to visualize and quantify tissue chromophores noninvasively. This modality can capture the spectral absorption signature of prominent tissue chromophores, such as oxygenated, deoxygenated hemoglobin, and other biomarkers in the tissue by using spectral unmixing methods. Currently, most of the reported image processing algorithms use standard unmixing procedures, which include user interaction in the form of providing the expected spectral signatures. For translational research with patients, these types of supervised spectral unmixing can be challenging, as the spectral signature of the tissues can differ with respect to the disease condition. Imaging exogenous contrast agents and accessing their biodistribution can also be problematic, as some of the contrast agents are susceptible to change in spectral properties after the tissue interaction. In this work, we investigated the feasibility of an unsupervised spectral unmixing algorithm to detect and extract the tissue chromophores without any a-priori knowledge and user interaction. The algorithm has been optimized for multispectral photoacoustic imaging in the spectral range of 680–900 nm. The performance of the algorithm has been tested on simulated data, tissue-mimicking phantom, and also on the detection of exogenous contrast agents after the intravenous injection in mice. Our finding shows that the proposed automatic, unsupervised spectral unmixing method has great potential to extract and quantify the tissue chromophores, and this can be used in any wavelength range of the multispectral photoacoustic images.

Aim Hypoglycemia in childhood is very rare and can be caused by genetic mutations or insulin-secreting neoplasms. Postprandial hypoglycemia has previously been associated with insulin receptor (INSR) gene mutations. We aimed to identify the cause of postprandial hypoglycemia in a 10-year-old boy. Subjects We studied the symptomatic proband and his apparently asymptomatic mother and elder brother. All of them were lean. Methods Metabolic screening of the proband included a 5-hour oral glucose tolerance test (OGTT), angio-magnetic resonance imaging, and 18 F-dihydroxyphenylalanine positron emission tomography/computed tomography imaging of the pancreas. INSR gene sequencing and in vitro functional studies of a novel INSR mutation were also undertaken. Results Fasting hyperinsulinemia was detected during metabolic screening, and 5-hour OGTT showed hypoglycemia at 240' in the proband, his mother, and brother. Pancreatic imaging showed no evidence of neoplasia. Acanthosis nigricans with high fasting insulin levels in the proband suggested severe insulin resistance and prompted INSR gene sequencing, which revealed the novel, heterozygous p.Phe1213Leu mutation in the patient and his family members. In vitro studies showed that this mutation severely impairs insulin receptor function by abolishing tyrosine kinase activity and downstream insulin signaling. Conclusions The identification of etiological cause of hypoglycemia in childhood may be challenging. The combination of fasting hyperinsulinemia with acanthosis nigricans in a lean subject with hypoglycemia suggests severe insulin resistance and warrants INSR gene screening.

Context An etiologic diagnosis of diabetes can affect the therapeutic strategy and prognosis of chronic complications. Objective The aim of the present study was to establish the relative percentage of different diabetes subtypes in patients attending Italian pediatric diabetes centers and the influence of an etiologic diagnosis on therapy. Design, setting, and patients This was a retrospective study. The clinical records of 3781 consecutive patients (age, 0 to 18 years) referred to 15 pediatric diabetes clinics with a diagnosis of diabetes or impaired fasting glucose from January 1, 2007 to December 31, 2012 were examined. The clinical characteristics of the patients at their first referral to the centers, type 1 diabetes-related autoantibodies, molecular genetics records, and C-peptide measurements, if requested for the etiologic diagnosis, were acquired. Main outcome measures The primary outcome was to assess the percentage of each diabetes subtype in our sample. Results Type 1 diabetes represented the main cause (92.4%) of diabetes in this group of patients, followed by monogenic diabetes, which accounted for 6.3% of cases [maturity onset diabetes of the young (MODY), 5.5%; neonatal diabetes mellitus, 0.6%, genetic syndromes, 0.2%]. A genetic diagnosis prompted the transfer from insulin to sulphonylureas in 12 patients bearing mutations in the HNF1A or KCNJ11 genes. Type 2 diabetes was diagnosed in 1% of the patients. Conclusions Monogenic diabetes is highly prevalent in patients referred to Italian pediatric diabetes centers. A genetic diagnosis guided the therapeutic decisions, allowed the formulation of a prognosis regarding chronic diabetic complications for a relevant number of patients (i.e.,GCK/MODY), and helped to provide genetic counseling.

A novel class of experimental fungicides has been discovered, which consists of special N-thiazol-4-yl-salicylamides. They originated from amide reversion of lead structures from the patent literature and are highly active against important phytopathogens, such as Phytophthora infestans (potato and tomato late blight), Plasmopara viticola (grapevine downy mildew) and Pythium ultimum (damping-off disease). Structure–activity relationship studies revealed the importance of a phenolic or enolic hydroxy function in the β-position of a carboxamide. An efficient synthesis route has been worked out, which for the first time employs the carbonyldiimidazole-mediated Lossen rearrangement in the field of thiazole carboxylic acids.

Insulin gene mutations, either dominant or recessive, can cause permanent neonatal diabetes mellitus (INS/PNDM), which is defined as diabetes with onset within 6 months of birth [1, 2]. More rarely, INS dominant mutations give rise to diabetes that presents during infancy [3]. In most patients with heterozygous, dominant INS mutations, C-peptide is in the normal-low range at diabetes outset, but over time declines to undetectable levels as a consequence of ongoing apoptosis of the pancreatic beta cells. This process is triggered by the sustained endoplasmic reticulum (ER) stress [2] induced by misfolding of mutant insulin trapped inside the beta cell. Thus, at least in principle, strategies aimed at relieving ER stress of beta cells may help to preserve endogenous insulin production from the normal allele. During the last 8 years, we identified a total of 16 cases with dominant INS gene mutations from 13 different Italian centers [2–4]. Thirteen were diagnosed with INS/PNDM (or with diabetes with onset during infancy) (2–4 and FB unpublished observations) before year 2010 and were treated with standard insulin therapy. Currently, all of them show undetectable C-peptide values. Instead, the three patients diagnosed with a dominant INS mutation after year 2010 were started on continuous subcutaneous insulin infusion (CSII) within a week after diagnosis. The three patients carried the novel INS mutation c.149A>G, p.Tyr50Cys (patient 1) and the already described c.94G>A p.Gly32Ser (patient 2) and c.314T>C p.Leu105Pro (patient 3). Diabetes onset was at 1 month of age for patient 1, 7 months for patient 2, and 5 months and a half for patient 3. In patient 1, CSII was integrated with continuous (i.e., 24 h a day) glucose monitoring (CGM). CGM was set with alarms for hypoglycemia, with a 2-h automatic interruption of insulin delivery by the pump if glucose sensor detected a value ≤4.44 mmol/l (80 mg/dl), and for hyperglycemia, with increased insulin infusion over basal level (“correction factor”) of 0.025 U for every 5.55 mmol/l (100 mg/dl) above the glucose value of 8.33 mmol/l (150 mg/dl), in order to obtain the narrowest plasma glucose fluctuations possible. Patient 1 was discharged from the hospital after 1 month, patient 2 after 18 days, and patient 3 after 12 days. Informed consent was obtained from parents of patients. C-peptide was evaluated in patients 1 and 2 by two-site chemiluminescent immunometric assay. Assay employed for patient 1 (Liaison C-peptide, Diasorin, Saluggia, Italy) has a reported limit of detection of 0.01 ng/ml (0.003 nmol/l) and functional sensitivity of 0.03 ng/ml (0.01 nmol/l); for patient 2, the assay (Immulite 2000 C-Peptide, Siemens) has a reported limit of detection and functional sensitivity of 0.08 ng/ml (0.03 nmol/l). Blood samples for C-peptide determination were drawn after an overnight fast, without suspension of basal insulin infusion. At follow-up as outpatients, no allergic reaction, lipo-atrophy, or -hypertrophy at the site of catheter insertion were observed; in addition, no episodes of ketosis or severe hypoglycemia requiring new hospitalization were reported for any patient. HbA1c is not a reliable method to evaluate metabolic control below the age of 6 months, because of the interfering effect of fetal hemoglobin: consequently, we do not report on patient 1’s glycated hemoglobin during the first semester of CSII. HbA1c values determined in patients 1 and 2 six months after diabetes onset were nevertheless comparable: 50 mmol/mol (6.7 %) for patient 1 (age 7 months) and 48 mmol/mol (6.5 %) for patient 2 (age 13 months) (Table 1). At follow-up, HbA1c values 12, 18, and 24 months after diabetes onset were declining in patient 1, while patient 2 showed a more irregular pattern (Table 1). Of note, insulin dose used for patient 2 was much lower for the entire period of CSII treatment (Table 1), suggesting that patient 2 may be more insulin-sensitive than patient 1. At strong variance with patients 1 and 2, patient 3 showed very high HbA1c from diabetes onset and never reached optimal metabolic control (Table 1). Table 1 Insulin dose and HbA1c in the three patients during 24-month period of treatment with CSII Interestingly, C-peptide was measurable at diabetes onset (1.04 ng/ml or 0.35 nmol/l), but became undetectable in patient 2 (CSII only) after 12 and 24 months from diabetes diagnosis, consistent with other reports showing this pattern in patients with INS proteotoxic mutations [2, 3]. In contrast, after the initial decrement recorded the first month after diabetes presentation, C-peptide of patient 1 (integrated system CSII/CGM) increased with a current value at 24 months of age of 0.26 nmol/l (Fig. 1). However, it has to be noticed that because of different sensitivities between C-peptide assays utilized, a direct comparison of these two patients cannot be easily made. Nevertheless, this was a surprising finding that has no easy explanation. From the clinical standpoint, the effect of the p.Tyr50Cys mutation seemed more pronounced than p.Gly32Ser, because patient 1 had an earlier diabetes presentation combined with a higher plasma glucose at onset (509 vs 293 mg/dl of patient 2). Interestingly, five patients previously described who bear INS/G23S mutation had diabetes onset at 6 months of age or beyond [1, 3]. On the other side of the token, patient 1 had no HbA1c fluctuations (Table 1), and it is tempting to speculate that the reduction in patient’s glycemic excursions obtained with CSII/CGM integrated system may have led to a substantial reduction in beta cell apoptosis consequent to the alleviation of endoplasmic reticulum crowding [5]. Fig. 1 C-peptide plasma level of patient 1 during the 24-month period of integrated CSII–CGM system. Numbers below each point are glucose values (mg/dl) at the time of blood drawing The results presented here indicate that CSII alone as well as sensor-augmented CSII is a feasible—and safe—therapeutic strategy for neonates or infants with diabetes, not only in the hospital setting, but at home. Moreover, CSII–CGM integrated system may be superior to CSII only, though we acknowledge that more cases must be studied to draw any robust conclusion. This may be probably obtained only through an international collaborative study, taking into consideration that PNDM is a rare condition (1:200,000 live births in countries with low rate of consanguineous marriages) [4].

The concept of monogenic diabetes emerged 25 years ago with a paper reporting the glucokinase locus linkage to maturity-onset diabetes of the young, an autosomal dominant disorder of glucose metabolism. Since then a huge leap forward has been made with the discovery of other clinical forms of monogenic diabetes, such as neonatal diabetes mellitus (NDM), and the identification of literally tens of genes that cause diabetes, either in isolation or syndromic. Of note, NDM genetics not only shed light on several aspects of pancreatic β-cell biology, but revealed new and unexpected therapeutic options for patients carrying mutations in specific genes, such as oral hypoglycemic agents (mutations of KATP genes) or stem cell transplantation (IPEX). In this chapter, we describe the genetic defects leading to neonatal diabetes that recognize dominant, recessive, and X-linked modes of inheritance or the association with incorrect parental origins of chromosomes and disturbances of imprinting.

Heterozygous, activating mutations of KCNJ11 encoding the pore-forming subunit (Kir 6.2) of the ATP-sensitive potassium channel (KATP) are the most prevalent cause of diabetes with onset in the first 6 months of life [1]. Rarely, KCNJ11 mutations are associated with diabetes that presents later in childhood or adulthood [2]. Kir6.2 is expressed not only in the pancreatic beta cell, but also in skeletal muscle, peripheral nerves, and especially brain [3], likely the explanation for a more severe condition termed developmental delay, epilepsy, and neonatal diabetes (DEND syndrome) that can result from rare KCNJ11 missense mutations [3, 4]. Patients with DEND syndrome have motor and speech delay and are often unable to talk and walk without aid. In addition, they suffer from epilepsy that generally occurs before 1 yr of age [3, 4]. In contrast, an intermediate form of the syndrome (iDEND), i.e.. with no epilepsy. is more frequent and less severe [1, 3, 5-8]. Identification of KCNJ11 mutations has important therapeutic consequences, as most patients respond to oral treatment with the sulfonylurea (SU) class of anti-diabetic drugs and can be totally weaned from insulin injections, reaching an excellent, long-lasting metabolic control [9-11]. In addition, motor function and other neurological consequences may be improved by SU treatment [3, 7, 12-14], although some patients with DEND may fail to favorably respond even to very high doses of SU [4, 9].

The fragment of the cytochrome b (cyt b) gene responsible for the binding site of QoI fungicides was sequenced for different Puccinia species by using DNA and RNA as template for PCR and RT-PCR, respectively. Degenerated primers for the cyt b gene amplified in P. recondita f.sp. tritici a 450 bp fragment, which was cloned and sequenced. At cDNA level, several Thermal Asymmetric InterLaced (TAIL)-PCR cycles were needed to produce a 996 bp long fragment, which corresponded to almost the whole cyt b gene (about 1160–1180 bp, without introns). This fragment was sequenced and specific primers were designed. Amplification with cyt b specific primers using genomic DNA as template revealed the presence of an intron of about 1500 bp length after the codon for glycine at amino acid position 143. By using the same primer pair, the cyt b gene fragment was amplified and sequenced both at cDNA and genomic DNA level also for other rust species, including P. graminis f.sp. tritici (length: 506 bp), P. striiformis f.sp. tritici (755 bp), P. coronata f.sp. avenae (644 bp), P. hordei (660 bp), P. recondita f.sp. secalis (687 bp), P. sorghi (709 bp), and P. horiana (478 bp). At the same position as for P. recondita f.sp. tritici, an intron of about 1500–1600 bp length was detected also in all other Puccinia species. High homologies were observed among all Puccinia species for both the exonic and intronic fragments of the cyt b gene. Specific primers for the cyt b gene of all eight Puccinia species were developed, which easily amplified the fragment of the gene including all possible mutations known to confer resistance to QoIs in several plant pathogens. However, in all tested isolates of the Puccinia species included in this study, the sequence of cyt b gene fragment did not contain any point mutations.

Maturity-onset diabetes of the young (MODY; MIM# 606391) is a genetically and clinically heterogeneous form of diabetes, accounting for 1–2% of all diabetes cases (1). MODY is characterized by mild hyperglycemia or overt diabetes usually detected in three consecutive generations, with onset before the age of 25 years and absence of type 1 diabetes autoantibodies. Among the thirteen MODY genes identified, two subtypes, GCK -MODY and HNF1A -MODY, account for most of cases (1). The prevalence of GCK -MODY has been reported higher in Southern Europe (2), while HNF1A -MODY is the most common MODY subtype in Northern Europe (3). This difference might be attributable to the clinical setting in which genetic screening is performed, especially when pediatric and adult diabetes clinics are distinct entities. We addressed this issue by investigating MODY patients identified in the pediatric or in the adult diabetes clinics of the same research-based …

A novel class of experimental fungicides has been discovered, which consists of special quinolin-6-yloxyacetamides. They are highly active against important phytopathogens, such as Phytophthora infestans (potato and tomato late blight), Mycosphaerella graminicola (wheat leaf blotch) and Uncinula necator (grape powdery mildew). Their fungicidal activity is due to their ability to inhibit fungal tubulin polymerization, leading to microtubule destabilization. An efficient synthesis route has been worked out, which allows the diverse substitution of four identified key positions across the molecular scaffold.

Transglutaminase 2 (TG2) is a multifunctional protein with Ca2 +-dependent transamidating and G protein activity. Previously we reported that the role of TG2 in insulin secretion may involve cytoplasmic actin remodeling and a regulative action on other proteins during granule movement. The aim of this study was to gain a better insight into the role of TG2 transamidating activity in mitochondria and in the nucleus of INS-1E rat insulinoma cell line (INS-1E) during insulin secretion. To this end we labeled INS-1E with an artificial donor (biotinylated peptide), in basal condition and after stimulus with glucose for 2, 5, and 8 min. Biotinylated proteins of the nuclear/mitochondrial-enriched fraction were analyzed using two-dimensional electrophoresis and mass spectrometry. Many mitochondrial proteins involved in Ca2 + homeostasis (e.g. voltage-dependent anion-selective channel protein, prohibitin and different ATP synthase subunits) and many nuclear proteins involved in gene regulation (e.g. histone H3, barrier to autointegration factor and various heterogeneous nuclear ribonucleoprotein) were identified among a number of transamidating substrates of TG2 in INS-1E. The combined results provide evidence that a temporal link exists between glucose-stimulation, first phase insulin secretion and the action of TG on histone H3 both in INS-1E and human pancreatic islets. Biological significance Research into the role of transglutaminase 2 during insulin secretion in INS-1E rat insulinoma cellular model is depicting a complex role for this enzyme. Transglutaminase 2 acts in the different INS-1E compartments in the same way: catalyzing a post-translational modification event of its substrates. In this work we identify some mitochondrial and nuclear substrates of INS-1E during first phase insulin secretion. The finding that TG2 interacts with nuclear proteins that include BAF and histone H3 immediately after (2–5 min) glucose stimulus of INS-1E suggests that TG2 may be involved not only in insulin secretion, as suggested by our previous studies in cytoplasmic INS-1E fraction, but also in the regulation of glucose-induced gene transcription., Graphical abstract, Highlights • Transglutaminase 2 localizes in the nucleus and in the mitochondrion of INS-1E. • TG2 acts as a modifying enzyme in both compartments during FPIS. • TG2 may contribute to Ca2 + sensing in mitochondrion through its substrates. • TG2 may contribute to chromatin condensation in nucleus through its substrates.

Biallelic insulin receptor (INSR) gene mutations cause congenital syndromes of severe insulin resistance (SIR) known as Donohue syndrome (DS) and Rabson-Mendenhall syndrome (RMS). At presentation, DS and RMS are difficult to differentiate since they share many clinical features; however, while patients with DS usually die within 1 year of birth, individuals classified as RMS can reach adult age. INSR mutations can be also found in pubertal females with hyperinsulinism, hyperandrogenism, and acanthosis nigricans (type A SIR). We studied the INSR gene in five subjects with congenital SIR and in a patient with type A SIR. Nine biallelic INSR gene mutations (eight novels, including an in-frame deletion of INSR signal peptide) were identified in patients with congenital SIR; a heterozygous, spontaneous INSR mutation was detected in the patient with type A SIR. Two probands, presenting severe hirsutism at birth, died at the age of 3 months and were classified as DS, while other 2, currently 2 and 3 years old, were diagnosed with RMS (patients 3 and 4). The fifth patient with congenital SIR died when 14 months old. Nephrocalcinosis, hyperaldosteronism, hyperreninemia, and hypokalemia, in the absence of hypertension, were discovered in patients 3 and 5 when 24 and 4 months old, respectively. Patient 3, now 3 years/3 months old, still shows hyperreninemic hyperaldosteronism requiring potassium supplementation. We conclude that renal abnormalities resembling antenatal Bartter's syndrome type II, recently reported also by others, is a common observation in patients with congenital SIR.

Gain-of-function mutations of KCNJ11 can cause permanent neonatal diabetes mellitus, but only rarely after 6 months of age. Specific uncommon mutations KCNJ11give rise to a syndrome defined as developmental delay, epilepsy, and neonatal diabetes (DEND), or - more frequently - to a milder sub-type lacking epilepsy, denoted as intermediate-DEND (iDEND). Our aim was to consider a possible monogenic etiology in a 12-yr-old boy with early onset diabetes and mild neurological features. We studied a subject diagnosed with diabetes at 21 months of age, and negative to type 1 diabetes autoantibodies testing. He had learning difficulties during primary school, and a single episode of seizures at the age of 10 yr. We performed direct DNA sequencing of the KCNJ11 gene with subsequent functional study of mutated channels in COSm6 cells. The patient's clinical response to oral glyburide (Glyb) was assessed. Motor coordination was evaluated before and after 6 and 12 months of Glyb therapy. Sequencing of the KCNJ11 gene detected the novel, spontaneous mutation S225T, combined with deletion of amino acids 226-232. In vitro studies revealed that the mutation results in a K(ATP) channel with reduced sensitivity to the inhibitory action of ATP. Glyb improved diabetes control (hemoglobin A1c on insulin: 52 mmol/mol/6.9%; on Glyb: 36 mmol/mol/5.4%) and also performance on motor coordination tests that were impaired before the switch of therapy. We conclude that KCNJ11/S225T, del226-232 mutation caused a mild iDEND form in our patient. KCNJ11 should be considered as the etiology of diabetes even beyond the neonatal period if present in combination with negative autoantibody testing and even mild neurological symptoms.

The cytochrome b (cyt b) gene structure was characterized for different agronomically important plant pathogens, such as Puccinia recondita f sp tritici (Erikss) CO Johnston, P graminis f sp tritici Erikss and Hennings, P striiformis f sp tritici Erikss, P coronata f sp avenae P Syd & Syd, P hordei GH Otth, P recondita f sp secalis Roberge, P sorghi Schwein, P horiana Henn, Uromyces appendiculatus (Pers) Unger, Phakopsora pachyrhizi Syd & P Syd, Hemileia vastatrix Berk & Broome, Alternaria solani Sorauer, A alternata (Fr) Keissl and Plasmopara viticola (Berk & Curt) Berlese & de Toni. The sequenced fragment included the two hot spot regions in which mutations conferring resistance to QoI fungicides may occur. The cyt b gene structure of these pathogens was compared with that of other species from public databases, including the strobilurin-producing fungus Mycena galopoda (Pers) P Kumm, Saccharomyces cerevisiae Meyer ex Hansen, Venturia inaequalis (Cooke) Winter and Mycosphaerella fijiensis Morelet. In all rust species, as well as in A solani, resistance to QoI fungicides caused by the mutation G143A has never been reported. A type I intron was observed directly after the codon for glycine at position 143 in these species. This intron was absent in pathogens such as A alternata, Blumeria graminis (DC) Speer, Pyricularia grisea Sacc, Mycosphaerella graminicola (Fuckel) J Schrot, M fijiensis, V inaequalis and P viticola, in which resistance to QoI fungicides has occurred and the glycine is replaced by alanine at position 143 in the resistant genotype. The present authors predict that a nucleotide substitution in codon 143 would prevent splicing of the intron, leading to a deficient cytochrome b, which is lethal. As a consequence, the evolution of resistance to QoI fungicides based on G143A is not likely to evolve in pathogens carrying an intron directly after this codon.

In two separate trials during 2000–2002, the efficacy of slow sand filtration and UV treatment in eliminating Phytophthora cryptogea propagules, artificially added in the recirculating nutrient solution, was evaluated with gerbera plants grown in closed soilless systems. A slow sand filtration technique was tested both alone and in combination with different antagonistic strains belonging to Fusarium spp. and Trichoderma spp., isolated from gerbera rhizosphere and applied into the soilless system. The dynamics of these fungi in the recirculating nutrient solution and in the sand filter was also investigated by plate counts on selective media. Slow sand filtration and UV treatment were both effective in reducing P. cryptogea root rot. However, slow sand filtration may be a more feasible disinfection method than UV because of lower costs of installation and maintenance and for its adaptability to a wide range of production systems. Moreover, this disinfection technique can be successfully combine with the application of antagonistic microorganisms.

ATP-sensitive potassium (K(ATP)) channels are widely distributed in various tissues and cell types where they couple cell metabolism to cell excitability. Gain of channel function (GOF) mutations in the genes encoding Kir6.2 (KCNJ11) or the associated regulatory ssulfonylurea receptor 1 subunit (ABCC8), cause developmental delay, epilepsy and neonatal diabetes (DEND) due to suppressed cell excitability in pancreatic β-cells and neurons. The objective of this study was to determine the molecular basis of infancy-onset diabetes and a mild form of intermediate DEND, resulting from a novel KCNJ11 in frame mutation plus deletion. The naturally occurring Kir6.2 mutation plus deletion (Ser225Thr, Pro226_Pro232del) as well as the isolated S225T mutation or isolated del226-232 deletion were coexpressed with SUR1 in COS cells in homozygous or heterozygous states. The protein expression and gating effects of the resulting channels were assessed biochemically and electrophysiologically. For both the deletion and point mutations, simulated heterozygous expression resulted in overall increased conductance in intact cells in basal conditions and rightward shifted ATP dose-response curves in excised patches, due to increased intrinsic open probability. Interestingly, homomeric channels for the combined deletion/mutation, or for the deletion alone, showed dramatically reduced channel expression at the cell membrane, which would underlie a reduced function in vivo. These results demonstrate that both the mis-sense mutation and the deleted region in the Kir6.2 subunit are important for control of the intrinsic channel gating and suggest that the clinical presentation could be affected by the competition between loss-of-function by reduced trafficking and enhanced channel gating.

Until early 2000, permanent and transient neonatal diabetes mellitus (NDM), defined as diabetes with onset within 6 weeks from birth that requires insulin therapy for at least 2 weeks, were considered exceedingly rare conditions, with a global incidence of 1:500,000–1:400,000 live births. The new definition of NDM recently adopted, that includes patients with diabetes onset within 6 months of age, has prompted studies that have set the incidence of the permanent form alone between 1:210,000 and 1:260,000 live births. Aim of the present work was to ascertain the incidence of NDM (i.e. permanent + transient form) in Italy for years 2005–2010. Patients referred to the Italian reference laboratory for NDM between years 2005 and 2010 and screened for mutations in common NDM genes (KCNJ11, ABCC8, and INS) and for uniparental isodisomy of chromosome 6 (UDP6) were reviewed. A questionnaire aimed at identifying NDM cases investigated in other laboratories was sent to 54 Italian reference centers for pediatric diabetes. Twenty-seven patients with NDM born between 2005 and 2010 were referred to the reference laboratory. In this group, a mutation of either KCNJ11, ABCC8 or INS was found in 18 patients, and a case with UDP6 was identified. Questionnaires revealed 4 additional cases with transient neonatal diabetes due to UDP6. Incidence of NDM was calculated at 1:90,000 (CI: 1:63,000–1:132,000) live births. Thus, with the definition currently in use, about 6 new cases with NDM are expected to be born in Italy each year.

Spectral Photoacoustic (sPAI) is an innovative imaging technology with great potential to detect and quantify the tissue chromophores. Since tissue chromophores have distinct spectral absorption signatures, sPAI is intrinsically sensitive to molecular components distribution in tissues. Linear unmixing is commonly used to differentiate the underlying components from sPAI. Although this fitting-based approach yields acceptable results, it requires user interaction to provide the source spectral curves as an input. For translational research with patients, this approach can be challenging, as the spectral signatures could differ concerning to the disease conditions. Imaging exogenous contrast can also be challenging as some of the agents are susceptible to spectral changes after the interaction with living tissues. Besides, light fluence attenuation along the imaging depth might induce spectral coloring that compromise the accurate quantification. Here we propose a novel unsupervised sPAI framework that enables fully automatic spectral unmixing and accurate quantification of molecular components. The algorithm has shown improved sensitivity and specificity in tissue-mimicking phantom and in vivo experiments.