Urinary proteomics studies have primarily focused on identifying markers of chronic kidney disease (CKD) progression. Here, we aimed to determine urinary markers of CKD renal parenchymal injury through proteomics analysis in animal kidney tissues and cells and in the urine of patients with CKD. Label-free quantitative proteomics analysis based on liquid chromatography-tandem mass spectrometry was performed on urine samples obtained from 6 normal controls and 9, 11, and 10 patients with CKD stages 1, 3, and 5, respectively, and on kidney tissue samples from a rat CKD model by 5/6 nephrectomy. Tandem mass tag-based quantitative proteomics analysis was performed for primary cultured glomerular endothelial cells (GECs) and proximal tubular epithelial cells (PTECs) before and after inducing 24-h hypoxia injury. Upon hierarchical clustering, out of 858 differentially expressed proteins (DEPs) in the urine of CKD patients, the levels of 416 decreased and 403 increased sequentially according to the disease stage, respectively. Among 2965 DEPs across 5/6 nephrectomized and sham-operated rat kidney tissues, 86 DEPs showed same expression patterns in the urine and kidney tissue. After cross-validation with two external animal proteome datasets, 38 DEPs were organized; only 10 DEPs, including serotransferrin, gelsolin, poly ADP-ribose polymerase 1, neuroblast differentiation-associated protein AHNAK, microtubule-associated protein 4, galectin-1, protein S, thymosin beta-4, myristoylated alanine-rich C-kinase substrate, and vimentin were finalized by screening human GECs and PTECs data. Among these ten potential candidates for universal CKD marker, validation analyses for protein S and galectin-1 were conducted. Galectin-1 was observed to have a significant inverse correlation with renal function as well as higher expression in glomerulus with chronic injury than protein S. This constitutes the first multi-sample proteomics study for identifying key renal-expressed proteins associated with CKD progression. The discovered proteins represent potential markers of chronic renal cell and tissue damage and candidate contributors to CKD pathophysiology.

In nucleotide excision repair, bulky DNA lesions such as UV-induced cyclobutane pyrimidine dimers are removed from the genome by concerted dual incisions bracketing the lesion, followed by gap filling and ligation. So far, two dual-incision patterns have been discovered: the prokaryotic type, which removes the damage in 11–13-nucleotide-long oligomers, and the eukaryotic type, which removes the damage in 24–32-nucleotide-long oligomers. However, a recent study reported that the UvrC protein of Mycobacterium tuberculosis removes damage in a manner analogous to yeast and humans in a 25-mer oligonucleotide arising from incisions at 15 nt from the 3´ end and 9 nt from the 5´ end flanking the damage. To test this model, we used the in vivo excision assay and the excision repair sequencing genome-wide repair mapping method developed in our laboratory to determine the repair pattern and genome-wide repair map of Mycobacterium smegmatis. We find that M. smegmatis, which possesses homologs of the Escherichia coli uvrA, uvrB, and uvrC genes, removes cyclobutane pyrimidine dimers from the genome in a manner identical to the prokaryotic pattern by incising 7 nt 5´ and 3 or 4 nt 3´ to the photoproduct, and performs transcription-coupled repair in a manner similar to E. coli.

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.

DNA polymerase from bacteriophage T7 undergoes large, substrate-induced conformational changes that are thought to account for high replication fidelity, but prior studies were adversely affected by mutations required to construct a Cys-lite variant needed for site-specific fluorescence labeling. Here we have optimized the direct incorporation of a fluorescent un-natural amino acid, (7-hydroxy-4-coumarin-yl)-ethylglycine, using orthogonal amber suppression machinery in Escherichia coli. MS methods verify that the unnatural amino acid is only incorporated at one position with minimal background. We show that the single fluorophore provides a signal to detect nucleotide-induced conformational changes through equilibrium and stopped-flow kinetic measurements of correct nucleotide binding and incorporation. Pre-steady-state chemical quench methods show that the kinetics and fidelity of DNA replication catalyzed by the labeled enzyme are largely unaffected by the unnatural amino acid. These advances enable rigorous analysis to establish the kinetic and mechanistic basis for high-fidelity DNA replication.

In eukaryotic DNA replication, DNA polymerase ε (Polε) is responsible for leading strand synthesis, whereas DNA polymerases α and δ synthesize the lagging strand. The human Polε (hPolε) holoenzyme is comprised of the catalytic p261 subunit and the noncatalytic p59, p17, and p12 small subunits. So far, the contribution of the noncatalytic subunits to hPolε function is not well understood. Using pre-steady-state kinetic methods, we established a minimal kinetic mechanism for DNA polymerization and editing catalyzed by the hPolε holoenzyme. Compared with the 140-kDa N-terminal catalytic fragment of p261 (p261N), which we kinetically characterized in our earlier studies, the presence of the p261 C-terminal domain (p261C) and the three small subunits increased the DNA binding affinity and the base substitution fidelity. Although the small subunits enhanced correct nucleotide incorporation efficiency, there was a wide range of rate constants when incorporating a correct nucleotide over a single-base mismatch. Surprisingly, the 3'→5' exonuclease activity of the hPolε holoenzyme was significantly slower than that of p261N when editing both matched and mismatched DNA substrates. This suggests that the presence of p261C and the three small subunits regulates the 3'→5' exonuclease activity of the hPolε holoenzyme. Together, the 3'→5' exonuclease activity and the variable mismatch extension activity modulate the overall fidelity of the hPolε holoenzyme by up to 3 orders of magnitude. Thus, the presence of p261C and the three noncatalytic subunits optimizes the dual enzymatic activities of the catalytic p261 subunit and makes the hPolε holoenzyme an efficient and faithful replicative DNA polymerase.

The origin recognition complex (ORC), composed of six subunits, ORC1–6, binds to origins of replication as a ring-shaped heterohexameric ATPase that is believed to be essential to recruit and load MCM2–7, the minichromosome maintenance protein complex, around DNA and initiate DNA replication. We previously reported the creation of viable cancer cell lines that lacked detectable ORC1 or ORC2 protein without a reduction in the number of origins firing. Here, using CRISPR-Cas9–mediated mutations, we report that human HCT116 colon cancer cells also survive when ORC5 protein expression is abolished via a mutation in the initiator ATG of the ORC5 gene. Even if an internal methionine is used to produce an undetectable, N terminally deleted ORC5, the protein would lack 80% of the AAA+ ATPase domain, including the Walker A motif. The ORC5-depleted cells show normal chromatin binding of MCM2–7 and initiate replication from a similar number of origins as WT cells. In addition, we introduced a second mutation in ORC2 in the ORC5 mutant cells, rendering both ORC5 and ORC2 proteins undetectable in the same cells and destabilizing the ORC1, ORC3, and ORC4 proteins. Yet the double mutant cells grow, recruit MCM2–7 normally to chromatin, and initiate DNA replication with normal number of origins. Thus, in these selected cancer cells, either a crippled ORC lacking ORC2 and ORC5 and present at minimal levels on the chromatin can recruit and load enough MCM2–7 to initiate DNA replication, or human cell lines can sometimes recruit MCM2–7 to origins independent of ORC.

Timely repair of DNA double-strand breaks (DSBs) is essential to maintaining genomic integrity and preventing illnesses induced by genetic abnormalities. We previously demonstrated that the E3 ubiquitin ligase SMURF2 plays a critical tumor suppressing role via its interaction with RNF20 (ring finger protein 20) in shaping chromatin landscape and preserving genomic stability. However, the mechanism that mobilizes SMURF2 in response to DNA damage remains unclear. Using biochemical approaches and MS analysis, we show that upon the onset of the DNA-damage response, SMURF2 becomes phosphorylated at Ser384 by ataxia telangiectasia mutated (ATM) serine/threonine kinase, and this phosphorylation is required for its interaction with RNF20. We demonstrate that a SMURF2 mutant with an S384A substitution has reduced capacity to ubiquitinate RNF20 while promoting Smad3 ubiquitination unabatedly. More importantly, mouse embryonic fibroblasts expressing the SMURF2 S384A mutant show a weakened ability to sustain the DSB response compared with those expressing WT SMURF2 following etoposide treatment. These data indicate that SMURF2-mediated RNF20 ubiquitination and degradation controlled by ataxia telangiectasia mutated–induced phosphorylation at Ser384 constitutes a negative feedback loop that regulates DSB repair.

A Qantas airlines flight made an emergency landing at Sydney Airport on Friday afternoon after suffering engine failure shortly after takeoff, the company said.

Mysterious black balls that washed up on Sydney, Australia, beaches were initially suspected to be tar balls but turned out to be miniature "fatbergs" containing human feces.

Nov. 11, 2024 — University of Sydney quantum researchers Dominic Williamson and Nouédyn Baspin have revealed a new architecture for managing errors that emerge in the operation of quantum computers. Their […]

The post University of Sydney Scientists Unveil Quantum Code to Enhance Error Correction with Fewer Qubits appeared first on HPCwire.

Kevin Costner says he didn't know about his "Yellowstone" character John Dutton's death until after the episode aired on Sunday.

Is it possible to meet the world's seemingly infinite demand for data storage while also caring for the natural environment? Biomedical researcher Keolu Fox and professional surfer and scientist Cliff Kapono believe that Indigenous knowledge combined with the science of genetics may offer such a solution: using the DNA of plant cells (like those found in sugar cane) as mini data warehouses. Learn more about the incredible potential of this technology — and how it could help foster ecosystem resilience in a high-tech world.

Thousands of teachers will head to the state capital on Wednesday to call for a nearly $10,000 raise over four years and an increase to per-pupil spending.

Private schools in New York soon will be required to report suspected sexual abuse of students in their schools to law enforcement, bringing the independent schools under the same rules as public schools.

Cochlear hair cells (HCs) sense sound waves and allow us to hear. Loss of HCs will cause irreversible sensorineural hearing loss. It is well known that DNA damage repair plays a critical role in protecting cells in many organs. However, how HCs respond to DNA damage and how defective DNA damage repair contributes to hearing loss remain elusive. In this study, we showed that cisplatin induced DNA damage in outer hair cells (OHCs) and promoted OHC loss, leading to hearing loss in mice of either sex. Cisplatin induced the expression of Brca1, a DNA damage repair factor, in OHCs. Deficiency of Brca1 induced OHC and hearing loss, and further promoted cisplatin-induced DNA damage in OHCs, accelerating OHC loss. This study provides the first in vivo evidence demonstrating that cisplatin mainly induces DNA damage in OHCs and that BRCA1 promotes repair of DNA damage in OHCs and prevents hearing loss. Our findings not only demonstrate that DNA damage–inducing agent generates DNA damage in postmitotic HCs but also suggest that DNA repair factors, like BRCA1, protect postmitotic HCs from DNA damage–induced cell death and hearing loss.

Two of the preeminent researchers of gene therapy hope to improve their patients' sight in an experimental operation (Stephen Voss/WPN)

On March 10, 1933, a major earthquake caught the Los Angeles area by surprise. The devastation was of sufficient scale to spur scientific interest in earthquakes—and how to predict them.

The viewfinders are outfitted with special lenses that help people with red-green colorblindness distinguish between hues

Researchers recently identified James Fitzjames, a captain on the ill-fated HMS Erebus that went looking for the Northwest Passage in 1845

New research has revealed that the mysterious white substance found alongside three ancient mummies was once a soft cheese called kefir

The notorious predators, nicknamed the “Man-Eaters of Tsavo,” terrorized railway workers in Kenya for roughly nine months

The baby boy’s recovered genome suggests he’s related to a famous Ice Age population

The robbers only made away with two of the screen prints, which they swiped from a gallery in the Netherlands. They abandoned the other artworks on the street

A new study has shattered historians' long-held assumptions about some of the people who died in Mount Vesuvius' eruption in 79 C.E.

The tongues of bird-like dinosaurs and pterosaurs, however, may have been more mobile

Quebec provincial police have confirmed that the body found in a Montreal nature park on Oct. 30 was that of kidnapping victim and cryptocurrency influencer Kevin Mirshahi.

Mosquitoes take blood meals from a diverse range of host animals and their host associations vary by species. Characterizing these associations is an important element of the transmission dynamics of mosquito-vectored pathogens. To characterize mosquito host associations, various molecular techniques have been developed, which are collectively referred to as blood meal analysis. DNA barcoding has diverse biological applications and is well-suited to mosquito blood meal analysis. The standard DNA barcoding marker for animals is a 5' fragment of the cytochrome c oxidase I (COI) gene. A major advantage of this marker is its taxonomic coverage in DNA sequence reference databases, making it feasible to identify a wider range of mosquito host species than with any other gene. However, the COI gene contains high sequence variation at potential priming sites between vertebrate orders. Coupled with the need for primer sequences to be mismatched with mosquito priming sites so that annealing to mosquito DNA is inhibited, it can be difficult to design primers suitable for blood meal analysis applications. Several primers are available that perform well in mosquito blood meal analysis, annealing to priming sites for most vertebrate host taxa, but not to those of mosquitoes. Because priming site sequence variation among vertebrate taxa can cause amplification to fail, a hierarchical approach to DNA barcoding-based blood meal analysis can be applied. In such an approach, no single primer set is expected to be effective for 100% of potential host species. If amplification fails in the initial reaction, a subsequent reaction is attempted with primers that anneal to different priming sites, and so on, until amplification is successful.

Mosquito species vary in their host associations. Although some species are relative generalists, most specialize, to varying extents, on particular types of host animals. Mosquito host associations are among the most important factors that influence the transmission dynamics of mosquito-vectored pathogens, and understanding these associations can provide insight on how such pathogens move within ecosystems. Characterization of the host associations of mosquito species requires applying blood meal analysis to the largest possible sample size of mosquito blood meals. Processing large samples of mosquito blood meals can be time-consuming, especially when chain-termination sequencing is used, necessitating individual processing of each specimen. Various methods and commercially available kits and products are available for extracting DNA from mosquito blood meals. The hot sodium hydroxide and Tris (HotSHOT) method is a rapid and inexpensive method of DNA extraction that is compatible with the recovery of DNA from mosquito blood meals preserved on QIAcard Flinders Technology Associates (FTA) Classic Cards (FTA cards). FTA cards allow nucleic acids found in blood meals to be preserved easily, even in field conditions. DNA prepared using this method is suitable for polymerase chain reaction (PCR)-based blood meal analysis.

Headlines for October 30, 2024; Report from Pennsylvania: Marc Lamont Hill on Harris’s Closing Speech & Dangers of a Trump Victory; Imara Jones: Transphobia Is “Key Pillar” of Trump’s Push for a “Patriarchal Fascist Regime”

Headlines for November 06, 2024; “The Confederacy Won”: Why Donald Trump’s Reelection Is a Win for White Supremacy, Xenophobia & Hate; “This Is a Collapse of the Democratic Party”: Ralph Nader on Roots of Trump’s Win Over Harris; “A Devastating Result”: John Nichols on GOP Taking White House and the Senate; Linda Sarsour: Harris’s Embrace of Pro-Israel Policies at Odds with Democratic Base; Keeanga-Yamahtta Taylor: Democrats Demobilized Their Base. A Movement Is Now Needed to Oppose Trump; Rami Khouri: U.S. Voters Are Sick of Foreign Wars. Can Trump Strike a Grand Bargain in Middle East?; 7 States Vote to Protect Abortion Rights in Busy Year for Ballot Initiatives

Figure skating pair Evelyn Walsh and Trennt Michaud didn't qualify for the 2022 Beijing Winter Olympics next month, their coach has all the faith that the pair will come back even stronger. 

Canadian swingman RJ Barrett was upgraded to day-to-day and engaged in non-contact practise on Monday, two days before Toronto hosts the Cleveland Cavaliers at Scotiabank Arena.

A couple in Montenegro, working with children in a tough neighbourhood in Bar, desire to find ways to reach them with Jesus’ love.

Virginia Tech Students Advancing the Possibilities with SolidWorks 3D CAD Software

When God directed Ellianna to Ireland instead of Austria, she discovered a new opportunity to meet refugees.

A team of OM workers and local believers share New Testaments and relief packs of food in a Muslim village in Israel.

A story of one OM worker’s struggle, triumph and hope.

OM Costa Rica team members and volunteers share in the joy of giving more than 1,000 indigenous children a dream Christmas.

Leung Wai, from Hong Kong, is burned to pray for Japan after dressing as Santa Claus and being warmly greeted by passers-by last December.

Don’t forget to vote for your school board, writes Charlie Wilson. It has direct consequences for the education children receive.

Don’t forget to vote for your school board, writes Charlie Wilson. It has direct consequences for the education children receive.

World Kindness Day is Wednesday, Nov. 13, but at Penn State Altoona, we are celebrating all week. Events include a friendship-themed Taco Tuesday, sweet treats, and displays of affirmations on Kindness Trees throughout campus.

A single woman tells what it's like to serve God in the Arabian Peninsula.

Antofagasta, Chile :: A team of Logos Hope crewmembers pray for patients and their relatives at a hospital.

In a political twist almost no one saw coming, on the morning of November 23, 2019, Devendra Fadnavis took oath as the Maharashtra Chief Minister for a second term.

A day after the political row over a routine check of Shiv Sena (BT) chief Uddhav Thackeray's luggage, the BJP today posted a video of airport security frisking Maharashtra Deputy Chief Minister Devendra Fadnavis' bags and took a dig at Mr Thackeray

It's named Bathydevius due to its "devious" nature that fooled the scientists at first, while caudactylus refers to the dactyls -- the fingerlike projections on its tail.

A day after the political row over a routine check of Shiv Sena (BT) chief Uddhav Thackeray's luggage, the BJP today posted a video of airport security frisking Maharashtra Deputy Chief Minister Devendra Fadnavis' bags and took a dig at Mr Thackeray