Head and neck paragangliomas: Recent advances in translational and clinical research and guidelines for patient care.

Head and neck paragangliomas (HNPGLs), rare neuroendocrine tumors that mainly arise from parasympathetic ganglia along the cranial nerves, are challenging due to anatomic origin, tendency to aggressive neurovascular and skull base infiltration, unpredictable metastatic potential, radio-chemoresistance, and risk of multiplicity. Symptoms range from mild to life threatening depending on location/size, but rarely relate to catecholamine excess. Risk factors include female sex and pathogenic germline variants in genes affecting hypoxia signaling (foremost succinate dehydrogenase genes). Diagnostic work-up relies on imaging, measurements of plasma free metanephrines/methoxytyramine, genetic testing, and pathology/immunohistochemistry. Management is tailored to patient/tumor characteristics and encompasses wait-scan, upfront surgery, debulking surgery, and radiotherapy. Presurgical embolization is recommended, except for small tympanic and tympanomasoid tumors. Presurgical stenting is required for internal carotid artery involvement, and two-stage surgery for intradural extension. Current treatments for metastatic/inoperable HNPGL are non-curative, and long-term follow-up should be recommended for all patients to monitor local recurrence and new tumors.

Structure and evolution of alanine/serine decarboxylases and the engineering of theanine production.

Ethylamine (EA), the precursor of theanine biosynthesis, is synthesized from alanine decarboxylation by alanine decarboxylase (AlaDC) in tea plants. AlaDC evolves from serine decarboxylase (SerDC) through neofunctionalization and has lower catalytic activity. However, lacking structure information hinders the understanding of the evolution of substrate specificity and catalytic activity. In this study, we solved the X-ray crystal structures of AlaDC from Camellia sinensis (CsAlaDC) and SerDC from Arabidopsis thaliana (AtSerDC). Tyr341 of AtSerDC or the corresponding Tyr336 of CsAlaDC is essential for their enzymatic activity. Tyr111 of AtSerDC and the corresponding Phe106 of CsAlaDC determine their substrate specificity. Both CsAlaDC and AtSerDC have a distinctive zinc finger and have not been identified in any other Group II PLP-dependent amino acid decarboxylases. Based on the structural comparisons, we conducted a mutation screen of CsAlaDC. The results indicated that the mutation of L110F or P114A in the CsAlaDC dimerization interface significantly improved the catalytic activity by 110% and 59%, respectively. Combining a double mutant of CsAlaDCL110F/P114A with theanine synthetase increased theanine production 672% in an in vitro system. This study provides the structural basis for the substrate selectivity and catalytic activity of CsAlaDC and AtSerDC and provides a route to more efficient biosynthesis of theanine.

Assessment of abnormal liver function tests and associated factors among COVID-19-infected patients in Addis Ababa, Ethiopia, 2022: a facility-based comparative cross-sectional study.

OBJECTIVE: Liver function test (LFT) abnormalities are higher in patients with severe COVID-19. Most of the studies on this theme were conducted in foreign nations, and the association with LFT abnormalities was not sufficiently addressed in the study areas. Therefore, the current study aimed to investigate the effects of COVID-19 infection on liver function of patients. SETTING: A facility-based comparative cross-sectional study was carried out from 10 April to 15 June 2022, among COVID-19 infected individuals admitted in Eka Kotebe General Hospital and Saint Petrous Specialized Hospitals, Addis Ababa, 2022. PARTICIPANTS: A total of 284 confirmed COVID-19-positive and COVID-19-negative controls matched by gender and age were included in the present study. RESULTS: Among SARS-COV-2 positive groups, 63 (44.4%) had one or more LFT abnormalities. The most common elevated level of the LFTs among patients with COVID-19 were gamma-glutamyl transferase (GGT) 50 (35.2%), while the most common lowered level was albumin 58 (40.8%). The mean values of aspartate aminotransferase (AST) (35.4±26.9 vs 22.9±12.6, p<0.001) were significantly different between patients with COVID-19 and the COVID-19-free groups. Being COVID-19-positive was significantly associated with an elevated level of AST (AOR=3.0, 95% CI 1.2 to 7.4) and GGT (AOR=4.55, 95% CI 2.02 to 10.3). Being male was significantly associated with an elevated level of total bilirubin (BILT, AOR=2.41, 95% CI 1.2 to 4.9) and direct bilirubin (BILD, AOR=3.7, 95% CI 1.72 to 8.2), and also severe stage of COVID-19 was associated with hypoalbuminaemia (AOR=3.3, 95% CI 1.4 to 7.9). SARS-COV-2 infection was independently associated with LFT abnormality. CONCLUSION: Patients with COVID-19 had decreased albumin levels, and elevated AST, GGT, BILT and BILD levels.

The power lies in the glycans.

Glycans play an important role in modulating the interactions between natural killer cells and antibodies to fight pathogens and harmful cells.

Characterization model of the post COVID-19 condition based on immunological, biochemical, and cytokine markers.

Post-coronavirus disease condition (PCC) continues to affect many people globally, yet there remains a lack of diagnostic biomarkers to distinguish PCC from those recovered from acute COVID-19. This study compared biomarkers between two age- and gender-matched groups: PCC individuals and those recovered within three months of acute COVID-19 in 2020 (n = 85 each). Biomarkers were assessed 12-24 months after initial diagnosis, examining biochemical profiles, blood cell counts, coagulation status, antibody serology, lymphocyte populations, and cytokine levels. PCC individuals exhibited significant alterations in 49 of 167 markers, including K+ levels, αGAD antibodies, antithrombin III, insulin-like growth factor-binding protein 3 (IGFBP3), and interleukin-10 (IL-10). A panel of αGAD, IL-10, potassium levels, and CD16brightCD56- cell presence distinguished PCC individuals from recovered patients with >88% accuracy and <92% precision.

Impact of graded doses of enrofloxacin on the safety and biological responses of Nile tilapia Oreochromis niloticus.

The cultivation of tilapias, the third most farmed fish group globally, has been rapidly growing, especially in Southeast Asia. This surge in tilapia farming intensification has led to increased use of antibiotics to control bacterial diseases. This study investigated the safety implications of administering graded doses of enrofloxacin (ENF) at 0 (control), 10, 30, 50 and 100 mg/kg biomass/day orally to Oreochromis niloticus. The 43-day study comprised 7 days of pre-dosing, 15 days of ENF-dosing, and a 21-day recovery period with a periodical assessment of the biological responses of fish. The results revealed that the overdosed groups experienced up to 21% reduction in feed consumption, 11% mortalities, and adverse impacts on hematology, including a decrease in erythrocytes, and monocytes and an increase in leukocytes, thrombocytes, lymphocytes, and neutrophils. Haematological indices like mean corpuscular volume and mean corpuscular hemoglobin decreased, while mean corpuscular hemoglobin concentration increased. The plasma biochemical parameters including glucose and liver and kidney enzymes unveiled a significant dose- and time-dependent increase, while calcium and chloride levels decreased. Erythrocytes displayed several erythrocyte cellular and nuclear abnormalities. The frequency of micronucleus increased with dose and time, suggesting potential genotoxicity of ENF. Additionally, a dose-dependent increase in residues in the tissues with the highest accumulation in muscle was documented. Nevertheless, the recovery of the measured parameters upon dose termination indicated that the ENF-induced alterations are reversible. The study affirmed the safety of ENF at the recommended dose (10 mg) in O. niloticus and their adoptive responses to higher doses.

Protocol for CRISPR-Cas12a genome editing of protein tyrosine phosphatases in human pluripotent stem cells and functional ß-like cell generation.

Gene editing of human pluripotent stem cells is a promising approach for developing targeted gene therapies for metabolic diseases. Here, we present a protocol for generating a CRISPR-Cas12a gene knockout of protein tyrosine phosphatases in human embryonic stem cells. We describe steps for differentiating the edited clones into pancreatic islet-like spheroids rich in ß-like cells. We then detail procedures for implanting these spheroids under the murine kidney capsule for in vivo maturation.

Protocol for preparing SUV420H1 in complex with the nucleosome containing H2A.Z and H4K20Ecx for structure determination.

The histone lysine methyltransferase SUV420H1 preferentially targets the H2A.Z-containing nucleosome core particle (H2A.Z-NCP) and catalyzes the H4K20me2 modification at replication origins. Here, we present a protocol for preparing SUV420H1 in complex with the nucleosome containing H2A.Z and H4K20Ecx for structure determination. We describe steps for the installation of S-ethyl-cysteine (Ecx), nucleosome and complex preparation, and performing the cryoelectron microscopy (cryo-EM) sample check. This protocol substitutes lysine 20 in histone H4 with S-ethyl-cysteine (H4K20Ecx), which enhances the stability of the interaction between SUV420H1 and nucleosomes. For complete details on the use and execution of this protocol, please refer to Huang et al.1.

A 2-hydroxybutyrate-mediated feedback loop regulates muscular fatigue.

Several metabolites have been shown to have independent and at times unexpected biological effects outside of their metabolic pathways. These include succinate, lactate, fumarate, and 2-hydroxyglutarate. 2-Hydroxybutyrate (2HB) is a byproduct of endogenous cysteine synthesis, produced during periods of cellular stress. 2HB rises acutely after exercise; it also rises during infection and is also chronically increased in a number of metabolic disorders. We show here that 2HB inhibits branched-chain aminotransferase enzymes, which in turn triggers a SIRT4-dependent shift in the compartmental abundance of protein ADP-ribosylation. The 2HB-induced decrease in nuclear protein ADP-ribosylation leads to a C/EBPß-mediated transcriptional response in the branched-chain amino acid degradation pathway. This response to 2HB exposure leads to an improved oxidative capacity in vitro. We found that repeated injection with 2HB can replicate the improvement to oxidative capacity that occurs following exercise training. Together, we show that 2-HB regulates fundamental aspects of skeletal muscle metabolism.

The deubiquitinase Ubp3/Usp10 constrains glucose-mediated mitochondrial repression via phosphate budgeting.

Many cells in high glucose repress mitochondrial respiration, as observed in the Crabtree and Warburg effects. Our understanding of biochemical constraints for mitochondrial activation is limited. Using a Saccharomyces cerevisiae screen, we identified the conserved deubiquitinase Ubp3 (Usp10), as necessary for mitochondrial repression. Ubp3 mutants have increased mitochondrial activity despite abundant glucose, along with decreased glycolytic enzymes, and a rewired glucose metabolic network with increased trehalose production. Utilizing ∆ubp3 cells, along with orthogonal approaches, we establish that the high glycolytic flux in glucose continuously consumes free Pi. This restricts mitochondrial access to inorganic phosphate (Pi), and prevents mitochondrial activation. Contrastingly, rewired glucose metabolism with enhanced trehalose production and reduced GAPDH (as in ∆ubp3 cells) restores Pi. This collectively results in increased mitochondrial Pi and derepression, while restricting mitochondrial Pi transport prevents activation. We therefore suggest that glycolytic flux-dependent intracellular Pi budgeting is a key constraint for mitochondrial repression.

Combined transcriptomics and metabolomics to reveal the effects of copper exposure on the liver of rainbow trout(Oncorhynchus mykiss).

Copper (Cu) is recognized as an essential trace elements for the body; However, excessive levels of Cu can lead to toxic effects. We investigated the effects of Cu2+(75 µg/L, 150 µg/L, and 300 µg/L) on the rainbow trout liver. Combination of transcriptome and metabolome analyses, the regulatory mechanisms of the liver under Cu stress were elucidated. The results showed that Cu affected the antioxidant levels, leading to disruptions in the normal tissue structure of the liver. Combined transcriptome and metabolome analyses revealed significant enrichment of the insulin signaling pathway and the adipocytokine signaling pathway. Additionally, Cu2+ stress altered the amino acid metabolism in rainbow trout by reducing serine and arginine levels while increasing proline content. Apoptosis is inhibited and autophagy and lipid metabolism are suppressed; In summary, Cu2+ stress affects energy and lipid metabolism, and the reduction of serine and arginine represents a decrease in the antioxidant capacity, whereas the increase in proline and the promotion of apoptosis potentially serving as crucial strategies for Cu2+ resistance in rainbow trout. These findings provided insights into the regulatory mechanisms of rainbow trout under Cu2+ stress and informed the prevention of heavy metal pollution and the selection of biomarkers under Cu pollution.

Isonitrile biosynthesis by non-heme iron(II)-dependent oxidases/decarboxylases.

The isonitrile group is a compact, electron-rich moiety coveted for its commonplace as a building block and bioorthogonal functionality in synthetic chemistry and chemical biology. Hundreds of natural products containing an isonitrile group with intriguing bioactive properties have been isolated from diverse organisms. Our recent discovery of a conserved biosynthetic gene cluster in some Actinobacteria species highlighted a novel enzymatic pathway to isonitrile formation involving a non-heme iron(II) and α-ketoglutarate-dependent dioxygenase. Here, we focus this chapter on recent advances in understanding and probing the biosynthetic machinery for isonitrile synthesis by non-heme iron(II) and α-ketoglutarate-dependent dioxygenases. We will begin by describing how to harness isonitrile enzymatic machinery through heterologous expression, purification, synthetic strategies, and in vitro biochemical/kinetic characterization. We will then describe a generalizable strategy to probe the mechanism for isonitrile formation by combining various spectroscopic methods. The chapter will also cover strategies to study other enzyme homologs by implementing coupled assays using biosynthetic pathway enzymes. We will conclude this chapter by addressing current challenges and future directions in understanding and engineering isonitrile synthesis.

Analysis of the genus B othrops snake venom: An inter and intraspecific comparative study.

The genus Bothrops are considered Category 1 of medical importance by the World Health Organization, responsible for approximately 85 % of snakebites occurring throughout Brazil. Main factors determining snake venom variations can be genetics, diet, gender, geographic distribution, age, or even seasonality. In this study, we compared the composition of protein profile, biochemical activities, and immunorecognition of toxins present in the venom of eight adults of Bothrops species (B. alternatus, B. atrox, B. jararaca, B. jararacussu, B. leucurus, B. moojeni, B. neuwiedi and B. pauloensis). The following methods were used to analyze the venoms: protein dosage; electrophoresis in polyacrylamide gel containing SDS; High Performance Liquid Chromatography - Reverse Phase; enzymatic activities, western blotting and Enzyme Linked Immuno Sorbent Assay. The results show inter and intraspecific differences in the electrophoretic profile. LAAO and PLA2 activities, in general, were higher in males than females and proteolytic activity was higher in females than males. The bothropic antivenom produced by Instituto Butantan recognized most of the protein bands in all Bothrops species analyzed, with only the regions between 37 and 25 kDa presenting lower intensity. A notable variability in the chromatograms was observed. Bothrops venom demonstrated inter-intraspecific disparities in protein composition and biochemical activity.

Identification of blood at simulated crime scenes using silver nanoparticles with SERS.

The analysis of substances and samples obtained from a crime scene is very important in solving forensic cases. To determine the variables involved in a crime and to expedite the investigation process, the rapid analysis of body fluids in small quantities and within environments containing diverse components is particularly necessary. For this reason, it is of great importance to analyze biological fluids with rapid, noncontaminating, nondestructive, low-cost, and accurate techniques. In recent years, with advancements in laser technology, spectroscopic methods have been introduced as analytical techniques in forensic medicine and chemical studies. This study focuses on surface-enhanced Raman spectroscopy (SERS) to demonstrate the detection of blood samples in simulated crime scenes. To minimize the background signal from fluorescent biomolecules in blood, dilution was performed with two different components and Raman analysis was performed for four different concentrations of blood. In general, a decrease in noise in the spectra was observed as the blood was diluted. Crime scenes consisting of pure blood, blood diluted with ethanol and distilled water (1:2, 1:4, and 1:8), a blood-mineral water mixture, a blood-cherry juice mixture, and silver nanoparticle-added mixtures were simulated, and their spectra were examined. Chemometric analyses of the data were performed. Despite high noise and low peak intensities, blood-identifying signals were detected when examining different blood concentrations. It was observed that silver nanoparticles provided high enhancement of blood peaks thanks to their strong plasmonic properties.

Characterization and mechanism investigation of salt-activated methionine sulfoxide reductase A from halophiles.

Halophiles, thriving in harsh saline environments, capture scientific interest due to their remarkable ability to prosper under extreme salinity. This study unveils the distinct salt-induced activation of methionine sulfoxide reductases (MsrA) from Halobacterium hubeiense, showcasing a significant enhancement in enzymatic activity across various salt concentrations ranging from 0.5 to 3.5 M. This contrasts sharply with the activity profiles of non-halophilic counterparts. Through comprehensive molecular dynamics simulations, we demonstrate that salt ions stabilize and compact the enzyme's structure, notably enhancing its substrate affinity. Mutagenesis analysis further confirms the essential role of salt bridges formed by the basic Arg168 residue in salt-induced activation. Mutating Arg168 to an acidic or neutral residue disrupts salt-induced activation, substantially reducing the enzyme activity under salt conditions. Our research provides evidence of salt-activated MsrA activity in halophiles, elucidating the molecular basis of halophilic enzyme activity in response to salts.

gp78-regulated KAP1 phosphorylation induces radioresistance in breast cancer by facilitating PPP1CC/PPP2CA ubiquitination.

Adjuvant radiation therapy is a common treatment for breast cancer, yet its effectiveness is often limited by radioresistance in patients. Identifying novel targets to combat this radioresistance is imperative. Recent investigations show that gp78 is upregulated in drug-resistant breast cancer cells. Our study reveals that gp78 markedly increased the phosphorylation of KAP1 and promoted DNA damage repair caused by ionizing radiation. Mechanistically, gp78 degrades phosphatases (PPP1CC/PPP2CA) in a ubiquitination-dependent manner. PPP1CC and PPP2CA are crucial regulators of KAP1 phosphorylation in response to DNA damage. Therefore, gp78 leads to a notable elevation in the phosphorylation of KAP1 by degrading phosphatases, thereby promoting the DNA damage repair process and increasing the radioresistance of tumor cells. The identification of gp78 as a pivotal regulator in radioresistance suggests a promising avenue for intervention. Combining blockade strategies targeting gp78 holds a signification potential for reversing radioresistance and improving the efficacy of breast cancer radiotherapy.

Molecular glue-mediated targeted protein degradation: A novel strategy in small-molecule drug development.

Small-molecule drugs are effective and thus most widely used. However, their applications are limited by their reliance on active high-affinity binding sites, restricting their target options. A breakthrough approach involves molecular glues, a novel class of small-molecule compounds capable of inducing protein-protein interactions (PPIs). This opens avenues to target conventionally undruggable proteins, overcoming limitations seen in conventional small-molecule drugs. Molecular glues play a key role in targeted protein degradation (TPD) techniques, including ubiquitin-proteasome system-based approaches such as proteolysis targeting chimeras (PROTACs) and molecular glue degraders and recently emergent lysosome system-based techniques like molecular degraders of extracellular proteins through the asialoglycoprotein receptors (MoDE-As) and macroautophagy degradation targeting chimeras (MADTACs). These techniques enable an innovative targeted degradation strategy for prolonged inhibition of pathology-associated proteins. This review provides an overview of them, emphasizing the clinical potential of molecular glues and guiding the development of molecular-glue-mediated TPD techniques.

Effects of Bacillus subtilis and Lactobacillus on growth performance, serum biochemistry, nutrient apparent digestibility, and cecum flora in heat-stressed broilers.

This study investigates the effect of dietary Bacillus subtilis and Lactobacillus on the growth performance, serum biochemistry, nutrient apparent digestibility, and cecum flora of broilers under heat stress (HS) and provides a theoretical basis for the application of probiotic additives to alleviate the stress of poultry under HS. A total of 200 Cobb broilers were randomly assigned to four replicates of 10 broilers in each of the five groups. The growth performance, serum biochemistry, nutrient apparent digestibility, and cecum flora of broilers were detected on the 28th, 35th, and 42nd days, respectively. Results revealed that HS can affect the growth performance and serum biochemical indexes of broilers, lowered the number of intestinal bifidobacteria and Lactobacillus, and increase the number of Escherichia coli in comparsion to the CON group. Compared with the HS group, the ADFI of HS broilers in the BS group and the combined group significantly increased (P < 0.05) at 22-28 days of age, and the serum calcium and phosphorus increased (P < 0.05) significantly at 42 days of age. Meanwhile, the number of Lactobacillus in the BS group and LAB group increased significantly at 42 days of age (P < 0.05). The number of Escherichia coli in the LAB group and combination group decreased significantly at 35 days of age (P < 0.01). The present study revealed that the addition of Bacillus subtilis or Lactobacillus to diets increased ADFI, increased probiotic counts, and lowered Escherichia coli counts in HS broilers, while probiotics alone work well.

Two-way Dispatched function in Sonic hedgehog shedding and transfer to high-density lipoproteins.

The Sonic hedgehog (Shh) signaling pathway controls embryonic development and tissue homeostasis after birth. This requires regulated solubilization of dual-lipidated, firmly plasma membrane-associated Shh precursors from producing cells. Although it is firmly established that the resistance-nodulation-division transporter Dispatched (Disp) drives this process, it is less clear how lipidated Shh solubilization from the plasma membrane is achieved. We have previously shown that Disp promotes proteolytic solubilization of Shh from its lipidated terminal peptide anchors. This process, termed shedding, converts tightly membrane-associated hydrophobic Shh precursors into delipidated soluble proteins. We show here that Disp-mediated Shh shedding is modulated by a serum factor that we identify as high-density lipoprotein (HDL). In addition to serving as a soluble sink for free membrane cholesterol, HDLs also accept the cholesterol-modified Shh peptide from Disp. The cholesteroylated Shh peptide is necessary and sufficient for Disp-mediated transfer because artificially cholesteroylated mCherry associates with HDL in a Disp-dependent manner, whereas an N-palmitoylated Shh variant lacking C-cholesterol does not. Disp-mediated Shh transfer to HDL is completed by proteolytic processing of the palmitoylated N-terminal membrane anchor. In contrast to dual-processed soluble Shh with moderate bioactivity, HDL-associated N-processed Shh is highly bioactive. We propose that the purpose of generating different soluble forms of Shh from the dual-lipidated precursor is to tune cellular responses in a tissue-type and time-specific manner.

Health assessment of nesting loggerhead sea turtles (Caretta caretta) in one of their largest rookeries (central eastern Florida coast, USA).

Reproduction is a physiologically demanding process for sea turtles. Health indicators, including morphometric indices and blood analytes, provide insight into overall health, physiology and organ function for breeding sea turtles as a way to assess population-level effects. The Archie Carr National Wildlife Refuge (ACNWR) on Florida's central eastern coast is critical nesting habitat for loggerhead sea turtles (Caretta caretta), but health variables from this location have not been documented. Objectives of the study were to (1) assess morphometrics and blood analyte data (including haematology, plasma biochemistry, protein electrophoresis, ß-hydroxybutyrate, trace nutrients, vitamins and fatty acid profiles) from loggerheads nesting on or near the beaches of the ACNWR, (2) investigate correlations of body condition index (BCI) with blood analytes and (3) analyse temporal trends in morphometric and blood analyte data throughout the nesting season. Morphometric and/or blood analyte data are reported for 57 nesting loggerheads encountered between 2016 and 2019. Plasma copper and iron positively correlated with BCI. Mass tended to decline across nesting season, whereas BCI did not. Many blood analytes significantly increased or decreased across nesting season, reflecting the catabolic state and haemodynamic variations of nesting turtles. Twenty-three of 34 fatty acids declined across nesting season, which demonstrates the physiological demands of nesting turtles for vitellogenesis and reproductive activities, thus suggesting potential utility of fatty acids for the assessment of foraging status and phases of reproduction. The findings herein are relevant for future spatiotemporal and interspecies comparisons, investigating stressor effects and understanding the physiological demands in nesting sea turtles. This information provides comparative data for individual animals in rescue or managed care settings and for assessment of conservation strategies.