The 15-year-old’s mother thought her daughter was dead after the paraglider collided with her

Situated on the northwest coast of the picturesque island of Crete, Chania stands as a testament to a vibrant mix of cultures that have left their mark over the millennia Breathtaking, famous for rich historical fabrics and the warm hospitality of his people is not appropriate to the destination; It’s an experience waiting to be discovered. This blog post will be your gateway to why Chania, Greece is the paradise you have been searching for. What sets Chania apart? A mixture of histories The streets of Chania speak of the past, whispering stories of Venice, the Ottomans and the Egyptian conquerors, each of whom left behind them a piece of its culture, architecture and spirit and through this blend of influences no Chania becomes a living canvas with so much history and beauty. The Old Port of Venice, with its magnificent lighthouse, stands as a symbol of Chania’s enduring heritage, inviting visitors to walk and marvel at the texts carved into its ancient stones. Unsurpassed natural beauty From tranquil beaches with crystal-clear waters to majestic white mountains in the background, Chania offers natural landscapes as diverse as stunning with stunning white and pink sands like Balos The Lagoon and Elafonissi Beach are not heavenly. For adventure enthusiasts, the Samaria Gorge, one of the longest canyons in Europe, is both a challenge and a reward of unparalleled panoramic views Elegant Gastronomic Delights Chania’s cuisine is typical of Crete’s many dishes. With a culinary tradition that emphasizes innovation and local ingredients, Chania offers a quality and divine culinary experience. Olive oil, wild herbs, fresh herbs and catch of the day feature prominently, delivering authenticity. The heart of Chania: its people Perhaps, though, what really sets Chania apart is the kindness and hospitality of its people. Guests are welcomed with open arms and treated as part of the family, making every experience in Chania feel personal and authentic. Whether it’s through shared meals, guided tours or casual conversation, Chanias make sure you take a piece of their heart with you when you leave Why choose Chania for your next vacation Chania, a picturesque town on the northwest coast of Crete, Greece, captures the heart of every traveler with its breathtaking beauty, rich history and crystal clear beaches If you want to relax, you relax or go deeper into history, Chania has something special in store for you. Pristine Beaches Balos Lagoon and Elafonissi Beach Balos Lagoon: Nestled between the Gramvousa Peninsula and the coast of Crete, Balos Lagoon is famed for its wild natural beauty, exotic turquoise waters, and fine white sand. This beach is accessible by boat or a short hike, offering stunning views that are worth the journey. Elafonissi Beach: Known for its unique pink sand, Elafonissi Beach is located on the southwestern tip of Crete. The beach is a protected nature reserve, providing shallow crystal-clear waters perfect for families. The island opposite the beach is accessible by foot through shallow water, revealing rare plant species and an environment unspoiled by human activity. These beaches are not just places to sunbathe and swim; they are destinations where nature’s artistry is on full display, featuring: Crystal-clear blue waters Exotic landscapes Unique natural environments Rich History and Culture Chania is a city soaked in history, with its roots stretching back to ancient civilizations. Walking through Chania is akin to traversing through time, with each step uncovering a new layer of its past. Overview of Chania’s History From the Minoan civilization to Byzantine times, followed by Venetian, Ottoman, and Egyptian influence, Chania is a living museum showcasing the rich tapestry of human history. This diverse historical influence has shaped the city’s architecture, culture, and traditions, making it a fascinating place to explore. Historical Sites The Old Venetian Harbor: The heart of Chania, this harbor is lined with historic buildings, bustling cafes, and quaint shops. The striking lighthouse, built by the Venetians and reconstructed by the Egyptians, is one of the most iconic images of Chania. The Ancient Ruins of Aptera: Located a few kilometers outside of Chania, the ancient city of Aptera presents a captivating site with ruins dating back to the Minoan era. Visitors can explore ancient temples, Roman baths, and impressive fortifications that offer a glimpse into the past. Exquisite Cuisine Step into Chania, and you’ll be welcomed with a feast not just for the eyes, but for the palate too. Cretan cuisine is a journey of taste, freshness, and tradition. Local Specialties: Dakos: A rustic appetizer featuring barley rusk topped with crushed tomato, mizithra cheese, and a drizzle of olive oil. Moussaka: A rich casserole layered with potatoes, eggplant, minced meat, and a creamy béchamel sauce. Kalitsounia: Sweet or savory cheese pies unique to the region. Health Benefits of the Mediterranean Diet: Rich in olive oil, legumes, and fresh produce, the Cretan diet is renowned for its health-promoting qualities. Dishes like Greek Salad and Grilled Seafood provide nutrients and antioxidants linked to longevity. Where to Find Local Cuisine: Tavernas in the Old Harbor serve fresh, local dishes with scenic views. Street vendors and local bakeries offer quick, delicious bites. Breathtaking Landscapes Chania’s dramatic landscapes invite both awe and adventure. Mountains and Gorges: The White Mountains: A towering range that gives you hiking trails and caves to explore. Samaria Gorge: A 16 km trek through a World Biosphere Reserve. Outdoor Activities: Guided treks are available for all levels of hikers. Boat trips allow you to appreciate the coastline from the water. Local Hospitality and Authentic Experiences The warmth of Chania’s people makes every moment spent here feel genuinely welcoming. Warm Hospitality Offered by Locals: Visitors are often greeted with a heartfelt “Kalimera” (good morning) and a smile. Many locals are eager to share stories or help guide you to the best spots. Immersive Experiences: Traditional Cretan Nights: Enjoy vibrant folk music and dancing. Local Markets: Visit the Agora (central market) to buy local honey, cheeses, and herbs. Planning Your Trip to Chania Nestled on the northwest coast of Crete, Greece, Chania is a captivating blend of scenic beauty, rich history, and vibrant culture. Planning your trip requires thoughtful consideration of when to visit, where to stay, how to navigate the city, and what experiences you shouldn’t miss. Best Time to Visit Analyzing Chania’s Weather Patterns: Spring (April to June): The weather starts warming up, making it ideal for outdoor activities without the summer crowds. Summer (July to August): Peak season with sunny days perfect for beach outings, but expect higher prices and more tourists. Autumn (September to October): Warm and less crowded. The sea remains warm enough for swimming. Winter (November to March): Cooler temperatures and occasional rain, suitable for those seeking tranquility and interested in exploring Chania’s cultural sites. Accommodations Chania offers a breadth of accommodation options to suit every taste and budget. Overview of Accommodation Options: Luxury Resorts: Beachfront properties providing exclusive amenities and breathtaking views. Boutique Hotels: Nestled in the Old Town, offering unique charm and close proximity to historical sites. Budget-Friendly Stays: Hostels and guesthouses that don’t compromise on comfort and provide excellent value. Getting Around Navigating Chania and its surrounding areas can be both fun and convenient. Tips on Navigating Chania: Public Transport: City buses are reliable for local travel. For adventures beyond the city, consider the KTEL buses that connect Chania to other parts of Crete. Car Rental: Offers the freedom to explore remote beaches and mountain villages at your pace. International driving permits are usually required for visitors. Must-Have Experiences Chania brims with activities and sights that cater to all interests. Curated List of Not-to-Miss Experiences: Old Venetian Harbor: Stroll around and enjoy the sunset views. Samaria Gorge: A challenging but rewarding hike offering stunning natural landscapes. Beaches: Balos Lagoon and Falassarna Beach are must-visits for beach lovers. Cretan Cuisine Tasting: Explore local tavernas for an authentic taste of Crete. conclusion Chania, with its perfect blend of climate, accommodation options, easy navigation and unique experiences, promises a memorable getaway. Chania is not just another holiday destination; It’s a place where every corner holds a story, every meal a celebration, every moment like heaven. Whether you’re wandering its cobblestone streets, sunbathing on its pristine beaches, or exploring its rich cultural heritage, Chania invites you to immerse yourself in its beauty, history and warmth is not just a place to visit; There is a world to experience. Make Chania your next holiday destination and discover a paradise that captures your heart.

The post Why Chania, Greece Should Be Your Next Paradise Vacation Destination appeared first on Geeky Traveller.

Receba as principais notícias, análises e áudios produzidos pelas nossas equipes em São Paulo e Londres e seja o primeiro a saber dos fatos mais importantes e interessantes no Brasil e no mundo.

Pesquisas indicam que 25% das páginas web publicadas entre 2013 e 2023 não existem mais.

A morte por esfaqueamento de uma jovem húngara, assassinada logo após dar à luz, intriga a polícia há 15 anos.

A capital espanhola inaugura um espaço para enterrar bebês que não chegaram a nascer.

Em 2024 foram aplicadas quase 200 multas, a maioria por tráfego irregular de veículos, em áreas proibidas. Especialistas veem risco de erosão acelerada das dunas

A ideia de que o Estado deve se restringir a funções básicas, desregulamentar a economia e não interferir no comércio entre países ganhou força com o fim da Guerra Fria e chegou a ser promovida pelos EUA na América Latina. Essa agenda entrou em crise na última década, o que ajuda a explicar a plataforma econômica populista de Donald Trump - e seu retorno ao poder.

Discussão nas redes sociais está gerando pressões para mudança nas leis trabalhista; mas empresários são contra.

O ex-presidente Jair Bolsonaro e seus aliados políticos tentam reverter a inelegibilidade até 2030 imposta pela Justiça eleitoral com recursos e tentativas de mudar a legislação.

(Oct. 17) When Vice President Kamala Harris repeated on Oct. 15 that she thinks the idea of racial “reparations” should be studied further, she evinced a radicalism she just can’t […]

The post Harris is open to ‘reparations.’ It’s ore proof she’s a radical. appeared first on Quin Hillyer.

A friend of the New York Republican said September's accident left him paralysed from the chest down.

Paramount Plus, Peacock, and the Paramount Network all have a hand in the Yellowstone franchise, but it can be tricky to stream episodes.

The MK Party continued its campaign against accountability for corruption, with its deputy leader, John Hlophe, suggesting National Director of Public Prosecutions Shamila Batohi was "misleading the nation" about the Guptas' extradition.

American Media Periscope Published  Sep. 19, 2022 In this episode of MSOM, Sean Morgan interviews Ladislav Vrabel about the populist movement in Czech Republic. Next Cynthia Farahat explains the secret apparatus of the Muslim Brotherhood. CynthiaFarahat.com https://americanmediaperiscope.com/amp/signup Check Out This Episode’s Podcast: https://www.podbean.com/media/share/pb-chpay-12c2875

By Walter Donway, Savvy Street originally published on Dec. 4, 2022 Book Review: The Secret Apparatus: The Muslim Brotherhood’s Industry of Death by Cynthia Farahat, Post Hill Press, Bombardier Books (2022) A grim irony of twentieth-century history (but, of course, also predating it) is that the most horrendous threats to humanity have simply been too monstrously evil […]

Tamar Geiger
Mar 1, 2012; 11:M111.014050-M111.014050
Special Issue: Prospects in Space and Time

Amelia C. Peterson
Nov 1, 2012; 11:1475-1488
Technological Innovation and Resources

Ignat V. Shilov
Sep 1, 2007; 6:1638-1655
Technology

Jeffrey C. Silva
Jan 1, 2006; 5:144-156
Research

Jennifer L. Macdonald
May 1, 2005; 46:1061-1067
Methods

Robert W. Mahley
Jan 1, 1999; 40:1-16
Reviews

William R. Morrison
Oct 1, 1964; 5:600-608
Articles

Putting the Hong Kong Crisis into Historical and Comparative Perspective 14 November 2019 — 8:30AM TO 9:30AM Anonymous (not verified) 17 October 2019 Chatham House | 10 St James's Square | London | SW1Y 4LE

This roundtable will focus on current events unfolding in Hong Kong, where the territory has been convulsed with protests for several months.

The speakers will examine how class, race and poverty play into the conflict. Taking a comparative approach, they will examine the generational divide, looking at the ideological gulf between the older, more conservative and pro-Beijing population versus the younger, more pro-democracy protesters. The discussion will also draw upon the erosion of trust between police and the wider public.

While acknowledging the unique features of this wave of unrest, the speakers will draw parallels, placing the current crisis in Hong Kong beside events that have occurred in other periods and other places.

Parallels to be explored include those with Shanghai struggles of the 1910s through 1980s and upheavals and crackdowns in the former Soviet bloc during the Cold War.

T. De Ryck and S. Mishra
Math. Comp. 93 (), 2643-2677.
Abstract, references and article information

Shoham Letzter
Trans. Amer. Math. Soc. 377 (), 5583-5615.
Abstract, references and article information

Olha Prykhodko and Kostiantyn Ralchenko
Theor. Probability and Math. Statist. 111 (), 123-135.
Abstract, references and article information

RNase J enzymes are metallohydrolases that are involved in RNA maturation and RNA recycling, govern gene expression in bacteria, and catalyze both exonuclease and endonuclease activity. The catalytic activity of RNase J is regulated by multiple mechanisms which include oligomerization, conformational changes to aid substrate recognition, and the metal cofactor at the active site. However, little is known of how RNase J paralogs differ in expression and activity. Here we describe structural and biochemical features of two Staphylococcus epidermidis RNase J paralogs, RNase J1 and RNase J2. RNase J1 is a homodimer with exonuclease activity aided by two metal cofactors at the active site. RNase J2, on the other hand, has endonuclease activity and one metal ion at the active site and is predominantly a monomer. We note that the expression levels of these enzymes vary across Staphylococcal strains. Together, these observations suggest that multiple interacting RNase J paralogs could provide a strategy for functional improvisation utilizing differences in intracellular concentration, quaternary structure, and distinct active site architecture despite overall structural similarity.

The dimeric ectonucleotidase CD73 catalyzes the hydrolysis of AMP at the cell surface to form adenosine, a potent suppressor of the immune response. Blocking CD73 activity in the tumor microenvironment can have a beneficial effect on tumor eradication and is a promising approach for cancer therapy. Biparatopic antibodies binding different regions of CD73 may be a means to antagonize its enzymatic activity. A panel of biparatopic antibodies representing the pairwise combination of 11 parental monoclonal antibodies against CD73 was generated by Fab-arm exchange. Nine variants vastly exceeded the potency of their parental antibodies with ≥90% inhibition of activity and subnanomolar EC50 values. Pairing the Fabs of parents with nonoverlapping epitopes was both sufficient and necessary whereas monovalent antibodies were poor inhibitors. Some parental antibodies yielded potent biparatopics with multiple partners, one of which (TB19) producing the most potent. The structure of the TB19 Fab with CD73 reveals that it blocks alignment of the N- and C-terminal CD73 domains necessary for catalysis. A separate structure of CD73 with a Fab (TB38) which complements TB19 in a particularly potent biparatopic shows its binding to a nonoverlapping site on the CD73 N-terminal domain. Structural modeling demonstrates a TB19/TB38 biparatopic antibody would be unable to bind the CD73 dimer in a bivalent manner, implicating crosslinking of separate CD73 dimers in its mechanism of action. This ability of a biparatopic antibody to both crosslink CD73 dimers and fix them in an inactive conformation thus represents a highly effective mechanism for the inhibition of CD73 activity.

The life cycle of malaria parasites in both their mammalian host and mosquito vector consists of multiple developmental stages that ensure proper replication and progeny survival. The transition between these stages is fueled by nutrients scavenged from the host and fed into specialized metabolic pathways of the parasite. One such pathway is used by Plasmodium falciparum, which causes the most severe form of human malaria, to synthesize its major phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Much is known about the enzymes involved in the synthesis of these phospholipids, and recent advances in genetic engineering, single-cell RNA-Seq analyses, and drug screening have provided new perspectives on the importance of some of these enzymes in parasite development and sexual differentiation and have identified targets for the development of new antimalarial drugs. This Minireview focuses on two phospholipid biosynthesis enzymes of P. falciparum that catalyze phosphoethanolamine transmethylation (PfPMT) and phosphatidylserine decarboxylation (PfPSD) during the blood stages of the parasite. We also discuss our current understanding of the biochemical, structural, and biological functions of these enzymes and highlight efforts to use them as antimalarial drug targets.

7 August 2020

Visual Abstract

Simone Kurz
Dec 29, 2020; 0:RA120.002266v1-mcp.RA120.002266
Research

Johannes Griss
Dec 1, 2020; 19:2115-2124
Technological Innovation and Resources

Jianhua Wang
Nov 4, 2020; 0:RA120.002375v1-mcp.RA120.002375
Research

Oxylipins are potent lipid mediators involved in a variety of physiological processes. Their profiling has the potential to provide a wealth of information regarding human health and disease and is a promising technology for translation into clinical applications. However, results generated by independent groups are rarely comparable, which increases the need for the implementation of internationally agreed upon protocols. We performed an interlaboratory comparison for the MS-based quantitative analysis of total oxylipins. Five independent laboratories assessed the technical variability and comparability of 133 oxylipins using a harmonized and standardized protocol, common biological materials (i.e., seven quality control plasmas), standard calibration series, and analytical methods. The quantitative analysis was based on a standard calibration series with isotopically labeled internal standards. Using the standardized protocol, the technical variance was within ±15% for 73% of oxylipins; however, most epoxy fatty acids were identified as critical analytes due to high variabilities in concentrations. The comparability of concentrations determined by the laboratories was examined using consensus value estimates and unsupervised/supervised multivariate analysis (i.e., principal component analysis and partial least squares discriminant analysis). Interlaboratory variability was limited and did not interfere with our ability to distinguish the different plasmas. Moreover, all laboratories were able to identify similar differences between plasmas. In summary, we show that by using a standardized protocol for sample preparation, low technical variability can be achieved. Harmonization of all oxylipin extraction and analysis steps led to reliable, reproducible, and comparable oxylipin concentrations in independent laboratories, allowing the generation of biologically meaningful oxylipin patterns.

Despite the continued analysis of HDAC inhibitors in clinical trials, the heterogeneous nature of the protein complexes they target limits our understanding of the beneficial and off-target effects associated with their application. Among the many HDAC protein complexes found within the cell, Sin3 complexes are conserved from yeast to humans and likely play important roles as regulators of transcriptional activity. The presence of two Sin3 paralogs in humans, SIN3A and SIN3B, may result in a heterogeneous population of Sin3 complexes and contributes to our poor understanding of the functional attributes of these complexes. Here, we profile the interaction networks of SIN3A and SIN3B to gain insight into complex composition and organization. In accordance with existing data, we show that Sin3 paralog identity influences complex composition. Additionally, chemical cross-linking MS identifies domains that mediate interactions between Sin3 proteins and binding partners. The characterization of rare SIN3B proteoforms provides additional evidence for the existence of conserved and divergent elements within human Sin3 proteins. Together, these findings shed light on both the shared and divergent properties of human Sin3 proteins and highlight the heterogeneous nature of the complexes they organize.

After ejaculation, mammalian spermatozoa must undergo a process known as capacitation in order to successfully fertilize the oocyte. Several post-translational modifications occur during capacitation, including sialylation, which despite being limited to a few proteins, seems to be essential for proper sperm-oocyte interaction. Regardless of its importance, to date, no single study has ever identified nor quantified which glycoproteins bearing terminal sialic acid (Sia) are altered during capacitation. Here we characterize sialylation during mouse sperm capacitation. Using tandem MS coupled with liquid chromatography (LC–MS/MS), we found 142 nonreductant peptides, with 9 of them showing potential modifications on their sialylated oligosaccharides during capacitation. As such, N-linked sialoglycopeptides from C4b-binding protein, endothelial lipase (EL), serine proteases 39 and 52, testis-expressed protein 101 and zonadhesin were reduced following capacitation. In contrast, mitochondrial aconitate hydratase (aconitase; ACO2), a TCA cycle enzyme, was the only protein to show an increase in Sia content during capacitation. Interestingly, although the loss of Sia within EL (N62) was accompanied by a reduction in its phospholipase A₁ activity, a decrease in the activity of ACO2 (i.e. stereospecific isomerization of citrate to isocitrate) occurred when sialylation increased (N612). The latter was confirmed by N612D recombinant protein tagged with both His and GFP. The replacement of Sia for the negatively charged Aspartic acid in the N612D mutant caused complete loss of aconitase activity compared with the WT. Computer modeling show that N612 sits atop the catalytic site of ACO2. The introduction of Sia causes a large conformational change in the alpha helix, essentially, distorting the active site, leading to complete loss of function. These findings suggest that the switch from oxidative phosphorylation, over to glycolysis that occurs during capacitation may come about through sialylation of ACO2.

Amino acid hydroxylation is a common post-translational modification, which generally regulates protein interactions or adds a functional group that can be further modified. Such hydroxylation is currently considered irreversible, necessitating the degradation and re-synthesis of the entire protein to reset the modification. Here we present evidence that the cellular machinery can reverse FIH-mediated asparagine hydroxylation on intact proteins. These data suggest that asparagine hydroxylation is a flexible and dynamic post-translational modification akin to modifications involved in regulating signaling networks, such as phosphorylation, methylation and ubiquitylation.

Pathway analyses are key methods to analyze 'omics experiments. Nevertheless, integrating data from different 'omics technologies and different species still requires considerable bioinformatics knowledge.

Here we present the novel ReactomeGSA resource for comparative pathway analyses of multi-omics datasets. ReactomeGSA can be used through Reactome's existing web interface and the novel ReactomeGSA R Bioconductor package with explicit support for scRNA-seq data. Data from different species is automatically mapped to a common pathway space. Public data from ExpressionAtlas and Single Cell ExpressionAtlas can be directly integrated in the analysis. ReactomeGSA greatly reduces the technical barrier for multi-omics, cross-species, comparative pathway analyses.

We used ReactomeGSA to characterize the role of B cells in anti-tumor immunity. We compared B cell rich and poor human cancer samples from five of the Cancer Genome Atlas (TCGA) transcriptomics and two of the Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteomics studies. B cell-rich lung adenocarcinoma samples lacked the otherwise present activation through NFkappaB. This may be linked to the presence of a specific subset of tumor associated IgG+ plasma cells that lack NFkappaB activation in scRNA-seq data from human melanoma. This showcases how ReactomeGSA can derive novel biomedical insights by integrating large multi-omics datasets.

Protein tyrosine kinases (PTKs) play key roles in cellular signal transduction, cell cycle regulation, cell division, and cell differentiation. Dysregulation of PTK-activated pathways, often by receptor overexpression, gene amplification, or genetic mutation, is a causal factor underlying numerous cancers. In this study, we have developed a parallel reaction monitoring (PRM)-based assay for quantitative profiling of 83 PTKs. The assay detects 308 proteotypic peptides from 54 receptor tyrosine kinases and 29 nonreceptor tyrosine kinases in a single run. Quantitative comparisons were based on the labeled reference peptide method. We implemented the assay in four cell models: 1) a comparison of proliferating versus epidermal growth factor (EGF)-stimulated A431 cells, 2) a comparison of SW480Null (mutant APC) and SW480APC (APC restored) colon tumor cell lines, and 3) a comparison of 10 colorectal cancer cell lines with different genomic abnormalities, and 4) lung cancer cell lines with either susceptibility (11-18) or acquired resistance (11-18R) to the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. We observed distinct PTK expression changes that were induced by stimuli, genomic features or drug resistance, which were consistent with previous reports. However, most of the measured expression differences were novel observations. For example, acquired resistance to erlotinib in the 11-18 cell model was associated not only with previously reported upregulation of MET, but also with upregulation of FLK2 and downregulation of LYN and PTK7. Immunoblot analyses and shotgun proteomics data were highly consistent with PRM data. Multiplexed PRM assays provide a targeted, systems-level profiling approach to evaluate cancer-related proteotypes and adaptations. Data are available through Proteome eXchange Accession PXD002706.

This article has been withdrawn by the authors. This article did not comply with the editorial guidelines of MCP. Specifically, single peptide based protein identifications of 9-19% were included in the analysis and discussed in the results and conclusions. We wish to withdraw this article and resubmit a clarified, corrected manuscript for review.

We developed a simple and rapid method to enrich protein N-terminal peptides, in which the protease TrypN is first employed to generate protein N-terminal peptides without Lys or Arg and internal peptides with two positive charges at their N-termini, and then the N-terminal peptides with or without N-acetylation are separated from the internal peptides by strong cation exchange chromatography according to a retention model based on the charge/orientation of peptides. This approach was applied to 20 μg of human HEK293T cell lysate proteins to profile the N-terminal proteome. On average, 1,550 acetylated and 200 unmodified protein N-terminal peptides were successfully identified in a single LC/MS/MS run with less than 3% contamination with internal peptides, even when we accepted only canonical protein N-termini registered in the Swiss-Prot database. Since this method involves only two steps, protein digestion and chromatographic separation, without the need for tedious chemical reactions, it should be useful for comprehensive profiling of protein N-termini, including proteoforms with neo-N-termini.

Malaria elimination is still pending on the development of novel tools that rely on a deep understanding of parasite biology. Proteins of all living cells undergo a myriad number of posttranslational modifications (PTMs) that are critical to multifarious life processes. An extensive proteome-wide dissection revealed a fine PTM map of most proteins in both Plasmodium falciparum, the causative agent of severe malaria, and the infected red blood cells. More than two-thirds of proteins of the parasite and its host cell underwent extensive and dynamic modification throughout the erythrocytic developmental stage. PTMs critically modulate the virulence factors involved in the host-parasite interaction and pathogenesis. Furthermore, P. falciparum stabilized the supporting proteins of erythrocyte origin by selective de-modification. Collectively, our multiple omic analyses, apart from having furthered a deep understanding of the systems biology of P. falciparum and malaria pathogenesis, provide a valuable resource for mining new antimalarial targets.

High performance liquid chromatography has been employed for decades to enhance detection sensitivity and quantification of complex analytes within biological mixtures. Among these analytes, glycans released from glycoproteins and glycolipids have been characterized as underivatized or fluorescently tagged derivatives by HPLC coupled to various detection methods. These approaches have proven extremely useful for profiling the structural diversity of glycoprotein and glycolipid glycosylation but require the availability of glycan standards and secondary orthogonal degradation strategies to validate structural assignments. A robust method for HPLC separation of glycans as their permethylated derivatives, coupled with in-line MSn fragmentation to assign structural features independent of standards, would significantly enhance the depth of knowledge obtainable from biological samples. Here, we report an optimized workflow for LC-MS analysis of permethylated glycans that includes sample preparation, mobile phase optimization, and MSn method development to resolve structural isomers on-the-fly. We report baseline separation and MSn fragmentation of isomeric N- and O-glycan structures, aided by supplementing mobile phases with Li+, which simplifies adduct heterogeneity and facilitates cross-ring fragmentation to obtain valuable monosaccharide linkage information. Our workflow has been adapted from standard proteomics-based workflows and, therefore, provides opportunities for laboratories with expertise in proteomics to acquire glycomic data with minimal deviation from existing buffer systems, chromatography media, and instrument configurations. Furthermore, our workflow does not require a mass spectrometer with high-resolution/accurate mass capabilities. The rapidly evolving appreciation of the biological significance of glycans for human health and disease requires the implementation of high-throughput methods to identify and quantify glycans harvested from sample sets of sufficient size to achieve appropriately powered statistical significance. The LC-MSn approach we report generates glycan isomeric separations, robust structural characterization, and is amenable to auto-sampling with associated throughput enhancements.

The faithful segregation, or “partition,” of many low-copy number bacterial plasmids is driven by plasmid-encoded ATPases that are represented by the P1 plasmid ParA protein. ParA binds to the bacterial nucleoid via an ATP-dependent nonspecific DNA (nsDNA)-binding activity, which is essential for partition. ParA also has a site-specific DNA-binding activity to the par operator (parOP), which requires either ATP or ADP, and which is essential for it to act as a transcriptional repressor but is dispensable for partition. Here we examine how DNA binding by ParA contributes to the relative distribution of its plasmid partition and repressor activities, using a ParA with an alanine substitution at Arg351, a residue previously predicted to participate in site-specific DNA binding. In vivo, the parAR351A allele is compromised for partition, but its repressor activity is dramatically improved so that it behaves as a “super-repressor.” In vitro, ParAR351A binds and hydrolyzes ATP, and undergoes a specific conformational change required for nsDNA binding, but its nsDNA-binding activity is significantly damaged. This defect in turn significantly reduces the assembly and stability of partition complexes formed by the interaction of ParA with ParB, the centromere-binding protein, and DNA. In contrast, the R351A change shows only a mild defect in site-specific DNA binding. We conclude that the partition defect is due to altered nsDNA binding kinetics and affinity for the bacterial chromosome. Furthermore, the super-repressor phenotype is explained by an increased pool of non-nucleoid bound ParA that is competent to bind parOP and repress transcription.

School districts must make amends for their racist history, writes Daarel Burnette II. What should that look like?