N-terminal cysteine bioconjugation with (2-cyanamidophenyl)boronic acids enables the direct formation of benzodiazaborines on peptides

Benzodiazaborines (BDABs) have emerged as a valuable tool to produce stable and functional bioconjugates via a click-type transformation. However, the current available methods to install them on peptides lack bioorthogonality, limiting their applications. Here, we report a strategy to install BDABs directly on peptide chains using (2-cyanamidophenyl)boronic acids (2CyPBAs). The resulting BDAB is stabilized through the formation of a key intramolecular B–N bond. This technology was applied in the selective modification of N-terminal cysteine-containing functional peptides

The mitogenome portrait of Umbria in Central Italy as depicted by contemporary inhabitants and pre-Roman remains

Umbria is located in Central Italy and took the name from its ancient inhabitants, the Umbri, whose origins are still debated. Here, we investigated the mitochondrial DNA (mtDNA) variation of 545 present-day Umbrians (with 198 entire mitogenomes) and 28 pre-Roman individuals (obtaining 19 ancient mtDNAs) excavated from the necropolis of Plestia. We found a rather homogeneous distribution of western Eurasian lineages across the region, with few notable exceptions. Contemporary inhabitants of the eastern part, delimited by the Tiber River and the Apennine Mountains, manifest a peculiar mitochondrial proximity to central-eastern Europeans, mainly due to haplogroups U4 and U5a, and an overrepresentation of J (30%) similar to the pre-Roman remains, also excavated in East Umbria. Local genetic continuities are further attested to by six terminal branches (H1e1, J1c3, J2b1, U2e2a, U8b1b1 and K1a4a) shared between ancient and modern mitogenomes. Eventually, we identified multiple inputs from various population sources that likely shaped the mitochondrial gene pool of ancient Umbri over time, since early Neolithic, including gene flows with central-eastern Europe. This diachronic mtDNA portrait of Umbria fits well with the genome-wide population structure identified on the entire peninsula and with historical sources that list the Umbri among the most ancient Italic populations.We are grateful to Soprintendenza Archeologia, Belle Arti e Paesaggio dell’Umbria, to Istituto Comprensivo Statale Foligno 5 (Perugia) and to all the volunteers who generously participated in this survey and made this research possible. We thank our colleagues Prof. Fausto Panara and Dr. Livia Lucentini with whom we have been discussing the feasibility and the first steps of this project, and Prof. Cristina Cereda, Dr. Gaetano Grieco, Dr. Marialuisa Valente, Dr. Nicole Huber and Jannika Oeke for technical support. We would like to thank the two anonymous reviewers for their suggestions and thoughtful comments. This research received support from: the Italian Ministry of Education, University and Research projects FIR2012 RBFR126B8I (to AO and AA), PRIN2017 20174BTC4R (to AA); Dipartimenti di Eccellenza Program (2018–2022)—Department of Biology and Biotechnology “L. Spallanzani,” University of Pavia (to AA, AO, OS and AT) and Department of Biology, University of Florence (to DC); the Fondazione Cariplo (project no. 2018–2045 to AA, AO and AT); the Fon-dazione Carifol (2008 to AA) and the Tiroler Wissenschaftsfonds (TWF) (UNI-404/1998) (to MB)

Dynamics of a Dual SARS-CoV-2 Lineage Co-Infection on a Prolonged Viral Shedding COVID-19 Case: Insights into Clinical Severity and Disease Duration

A few molecularly proven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases of symptomatic reinfection are currently known worldwide, with a resolved first infection followed by a second infection after a 48 to 142-day intervening period. We report a multiple-component study of a clinically severe and prolonged viral shedding coronavirus disease 2019 (COVID-19) case in a 17-year-old Portuguese female. She had two hospitalizations, a total of 19 RT-PCR tests, mostly positive, and criteria for releasing from home isolation at the end of 97 days. The viral genome was sequenced in seven serial samples and in the diagnostic sample from her infected mother. A human genome-wide array (>900 K) was screened on the seven samples, and in vitro culture was conducted on isolates from three late samples. The patient had co-infection by two SARS-CoV-2 lineages, which were affiliated in distinct clades and diverging by six variants. The 20A lineage was absolute at the diagnosis (shared with the patient's mother), but nine days later, the 20B lineage had 3% frequency, and two months later, the 20B lineage had 100% frequency. The 900 K profiles confirmed the identity of the patient in the serial samples, and they allowed us to infer that she had polygenic risk scores for hospitalization and severe respiratory disease within the normal distributions for a Portuguese population cohort. The early-on dynamic co-infection may have contributed to the severity of COVID-19 in this otherwise healthy young patient, and to her prolonged SARS-CoV-2 shedding profile.The authors acknowledge the support of the i3S Scientific Platforms BioSciences Screening and Genomics, members of the national infrastructure PPBI-Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122), PT-OPENSCREEN, GenomePT project (POCI-01-0145-FEDER-022184)

REST is a hypoxia-responsive transcriptional repressor

Cellular exposure to hypoxia results in altered gene expression in a range of physiologic and pathophysiologic states. Discrete cohorts of genes can be either up- or down-regulated in response to hypoxia. While the Hypoxia-Inducible Factor (HIF) is the primary driver of hypoxia-induced adaptive gene expression, less is known about the signalling mechanisms regulating hypoxia-dependent gene repression. Using RNA-seq, we demonstrate that equivalent numbers of genes are induced and repressed in human embryonic kidney (HEK293) cells. We demonstrate that nuclear localization of the Repressor Element 1-Silencing Transcription factor (REST) is induced in hypoxia and that REST is responsible for regulating approximately 20% of the hypoxia-repressed genes. Using chromatin immunoprecipitation assays we demonstrate that REST-dependent gene repression is at least in part mediated by direct binding to the promoters of target genes. Based on these data, we propose that REST is a key mediator of gene repression in hypoxia

Species differential regulation of COX2 can be described by an NFκB-dependent logic AND gate

Cyclooxygenase 2 (COX2), a key regulatory enzyme of the prostaglandin/eicosanoid pathway, is an important target for anti-inflammatory therapy. It is highly induced by pro-inflammatory cytokines in a Nuclear factor kappa B (NFκB)-dependent manner. However, the mechanisms determining the amplitude and dynamics of this important pro-inflammatory event are poorly understood. Furthermore, there is significant difference between human and mouse COX2 expression in response to the inflammatory stimulus tumor necrosis factor alpha (TNFα). Here, we report the presence of a molecular logic AND gate composed of two NFκB response elements (NREs) which controls the expression of human COX2 in a switch-like manner. Combining quantitative kinetic modeling and thermostatistical analysis followed by experimental validation in iterative cycles, we show that the human COX2 expression machinery regulated by NFκB displays features of a logic AND gate. We propose that this provides a digital, noise-filtering mechanism for a tighter control of expression in response to TNFα, such that a threshold level of NFκB activation is required before the promoter becomes active and initiates transcription. This NFκB-regulated AND gate is absent in the mouse COX2 promoter, most likely contributing to its differential graded response in promoter activity and protein expression to TNFα. Our data suggest that the NFκB-regulated AND gate acts as a novel mechanism for controlling the expression of human COX2 to TNFα, and its absence in the mouse COX2 provides the foundation for further studies on understanding species-specific differential gene regulation

The Gracilis Myocutaneous Free Flap: A Quantitative Analysis of the Fasciocutaneous Blood Supply and Implications for Autologous Breast Reconstruction

BACKGROUND: Mastectomies are one of the most common surgical procedures in women of the developed world. The gracilis myocutaneous flap is favoured by many reconstructive surgeons due to the donor site profile and speed of dissection. The distal component of the longitudinal skin paddle of the gracilis myocutaneous flap is unreliable. This study quantifies the fasciocutaneous vascular territories of the gracilis flap and offers the potential to reconstruct breasts of all sizes. METHODS: Twenty-seven human cadaver dissections were performed and injected using lead oxide into the gracilis vascular pedicles, followed by radiographic studies to identify the muscular and fasciocutaneous perforator patterns. The vascular territories and choke zones were characterized quantitatively using the 'Lymphatic Vessel Analysis Protocol' (LVAP) plug-in for Image J® software. RESULTS: We found a step-wise decrease in the average vessel density from the upper to middle and lower thirds of both the gracilis muscle and the overlying skin paddle with a significantly higher average vessel density in the skin compared to the muscle. The average vessel width was greater in the muscle. Distal to the main pedicle, there were either one (7/27 cases), two (14/27 cases) or three (6/27 cases) minor pedicles. The gracilis angiosome was T-shaped and the maximum cutaneous vascular territory for the main and first minor pedicle was 35 × 19 cm and 34 × 10 cm, respectively. CONCLUSION: Our findings support the concept that small volume breast reconstructions can be performed on suitable patients, based on septocutaneous perforators from the minor pedicle without the need to harvest any muscle, further reducing donor site morbidity. For large reconstructions, if a 'T' or tri-lobed flap with an extended vertical component is needed, it is important to establish if three territories are present. Flap reliability and size may be optimized following computed tomographic angiography and surgical delay

Understanding complexity in the HIF signaling pathway using systems biology and mathematical modeling

Hypoxia is a common micro-environmental stress which is experienced by cells during a range of physiologic and pathophysiologic processes. The identification of the hypoxia-inducible factor (HIF) as the master regulator of the transcriptional response to hypoxia transformed our understanding of the mechanism underpinning the hypoxic response at the molecular level and identified HIF as a potentially important new therapeutic target. It has recently become clear that multiple levels of regulatory control exert influence on the HIF pathway giving the response a complex and dynamic activity profile. These include positive and negative feedback loops within the HIF pathway as well as multiple levels of crosstalk with other signaling pathways. The emerging model reflects a multi-level regulatory network that affects multiple aspects of the physiologic response to hypoxia including proliferation, apoptosis, and differentiation. Understanding the interplay between the molecular mechanisms involved in the dynamic regulation of the HIF pathway at a systems level is critically important in defining new appropriate therapeutic targets for human diseases including ischemia, cancer, and chronic inflammation. Here, we review our current knowledge of the regulatory circuits which exert influence over the HIF response and give examples of in silico model-based predictions of the dynamic behaviour of this system

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field