DNA Denaturation Mapping in Nanochannels for Bacterial Identification

In the current work, partial denaturation mapping of DNA in nanochannels is studied with the goal to use this technique as a tool for the identification of bacteria. This method has the potential to simplify and speed up the diagnosis of bacterial infections. A partially denatured and YOYO-1-labeled DNA molecule displays a sequence specific pattern of fluorescent and non-fluorescent regions along the molecule. This pattern can be interpreted as a barcode. By stretching the DNA molecule inside nanofluidic channels, this barcode can easily be imaged with an optical microscope and compared with a database consisting of theoretically generated barcodes. The DNA is identified by finding the theoretical barcode that matches best to the experimental barcode. In this project, first fundamental studies of the denaturation pattern formation have been performed. Pattern formation has been studied as a function of time, confinement and fluorescent dye concentration. Second, experimentally acquired barcodes were compared to their corresponding theoretical barcodes to demonstrate the agreement between experiment and theory. Finally, identification of the strain specific DNA of S. pneumoniae, was attempted. The time it takes for denaturation patterns to form upon heating the DNA has been observed to be approximately 10 minutes. The difference in melting temperature between nano- and microconfinement was found to be in the range of 5-10 C. An effect of the fluorescent dye concentration on the melting temperature of DNA molecules was observed. Further, a good agreement between experimental and theoretical barcodes could be demonstrated. A large amount of denaturation maps of DNA fragments from different strains of S. pneumoniae were measured. However, successful identification of the bacterial DNA has not been realized yet. Potential ways to improve the results are discussed.The DNA (Deoxyribonucleic acid) is a molecule found in every living organism and bears information which is unique for its carrier. Reading this information would thus allow to identify any kind of organism. The existing methods to read the content of the DNA are most of the times either slow or expensive. In this project, a novel technique to visualize the information stored in the DNA is explored. This technique has the potential to simplify and speed up the reading process, making it suitable for the identification of bacteria and thus making it a powerful diagnostic tool. In this technique a DNA molecule is inserted into tiny channels (nanochannels) that force the molecule to stretch out. The DNA is then labelled with a special dye that falls off at specific regions of the DNA upon heating. These regions depend on the information of the DNA. Under the microscope, only regions containing the dye are visible. This pattern of bright and dark regions along the DNA can be interpreted as a barcode, which is unique for every organism. By comparing this barcode with a database of theoretically generated barcodes, the DNA and its carrier can be identified. In this project, fundamental studies of the technique have been performed and an attempt at identifying bacteria was made. It could be shown that it takes 10 minutes for the barcode to appear on the DNA when heated. It was also observed that the temperature at which the barcode appears depends on the amount of dye and the level of confinement of the DNA. Further, a good agreement between experimental barcodes and theoretical barcodes could be demonstrated. Even though a large amount of bacterial DNA was analysed, an identification of the bacteria could not be realized yet. Improvements in the experimental method as well as in the data analysis are believed to improve the results

Architecture of the ESCPE-1 membrane coat

Recycling of membrane proteins enables the reuse of receptors, ion channels and transporters. A key component of the recycling machinery is the endosomal sorting complex for promoting exit 1 (ESCPE-1), which rescues transmembrane proteins from the endolysosomal pathway for transport to the trans-Golgi network and the plasma membrane. This rescue entails the formation of recycling tubules through ESCPE-1 recruitment, cargo capture, coat assembly and membrane sculpting by mechanisms that remain largely unknown. Herein, we show that ESCPE-1 has a single-layer coat organization and suggest how synergistic interactions between ESCPE-1 protomers, phosphoinositides and cargo molecules result in a global arrangement of amphipathic helices to drive tubule formation. Our results thus define a key process of tubule-based endosomal sorting.This work was funded by MCIN/AEI/10.13039/501100011033 (PID2020- 119132GB-I00, CEX2021-001136-S) (to A.H.), the Intramural Program of NICHD, NIH (ZIA HD001607 to J.S.B.), the Swiss National Science Foundation grant 205321 179041 (to D.C.-D.), the Human Frontiers Science Program grant RGP0017/2020 (to D.C.-D.) and the PID2021- 127309NB-I00 funded by AEI/10.13039/501100011033/ FEDER, UE (to D.C.-D.). This study made use of the Diamond Light Source proposal MX20113, ALBA synchrotron beamline BL13-XALOC, the cryo-EM facilities at the UK Electron Bio-Imaging Centre, proposals EM17171- 6 and EM17171, and the Midlands Regional Cryo-EM Facility at the Leicester Institute of Structural and Chemical Biology (LISCB). We thank C. Savva (LISCB, University of Leicester) for his help in cryo-EM data collection. With funding from the Spanish government through the Severo Ochoa Centre of Excellence’ accreditation (CEX2021-001136-S

The Silent Epidemic of Diabetic Ketoacidosis at Diagnosis of Type 1 Diabetes in Children and Adolescents in Italy During the COVID-19 Pandemic in 2020

To compare the frequency of diabetic ketoacidosis (DKA) at diagnosis of type 1 diabetes in Italy during the COVID-19 pandemic in 2020 with the frequency of DKA during 2017-2019

Has COVID-19 Delayed the Diagnosis and Worsened the Presentation of Type 1 Diabetes in Children?

Objective: To evaluate whether the diagnosis of pediatric type 1 diabetes or its acute complications changed during the early phase of the coronavirus disease 2019 (COVID-19) pandemic in Italy. Research design and methods: This was a cross-sectional, Web-based survey of all Italian pediatric diabetes centers to collect diabetes, diabetic ketoacidosis (DKA), and COVID-19 data in patients presenting with new-onset or established type 1 diabetes between 20 February and 14 April in 2019 and 2020. Results: Fifty-three of 68 centers (77.9%) responded. There was a 23% reduction in new diabetes cases in 2020 compared with 2019. Among those newly diagnosed patient who presented in a state of DKA, the proportion with severe DKA was 44.3% in 2020 vs. 36.1% in 2019 (P = 0.03). There were no differences in acute complications. Eight patients with asymptomatic or mild COVID-19 had laboratory-confirmed severe acute respiratory syndrome coronavirus 2. Conclusions: The COVID-19 pandemic might have altered diabetes presentation and DKA severity. Preparing for any "second wave" requires strategies to educate and reassure parents about timely emergency department attendance for non-COVID-19 symptoms

A Multicenter Retrospective Survey regarding Diabetic Ketoacidosis Management in Italian Children with Type 1 Diabetes

We conducted a retrospective survey in pediatric centers belonging to the Italian Society for Pediatric Diabetology and Endocrinology. The following data were collected for all new-onset diabetes patients aged 0-18 years: DKA (pH < 7.30), severe DKA (pH < 7.1), DKA in preschool children, DKA treatment according to ISPAD protocol, type of rehydrating solution used, bicarbonates use, and amount of insulin infused. Records (n = 2453) of children with newly diagnosed diabetes were collected from 68/77 centers (87%), 39 of which are tertiary referral centers, the majority of whom (n = 1536, 89.4%) were diagnosed in the tertiary referral centers. DKA was observed in 38.5% and severe DKA in 10.3%. Considering preschool children, DKA was observed in 72%, and severe DKA in 16.7%. Cerebral edema following DKA treatment was observed in 5 (0.5%). DKA treatment according to ISPAD guidelines was adopted in 68% of the centers. In the first 2 hours, rehydration was started with normal saline in all centers, but with different amount. Bicarbonate was quite never been used. Insulin was infused starting from third hour at the rate of 0.05-0.1 U/kg/h in 72% of centers. Despite prevention campaign, DKA is still observed in Italian children at onset, with significant variability in DKA treatment, underlying the need to share guidelines among centers