Effects of Zilpaterol Hydrochloride with a Combination of Vitamin D3 on Feedlot Lambs: Growth Performance, Dietary Energetics, Carcass Traits, and Meat Quality.

This study evaluated the impact of supplementing ZH in combination with D3 on the growth performance, energy efficiency, carcass traits, and meat quality of feedlot lambs. Thirty-two Dorper × Katahdin cross lambs (37.3 ± 5.72 kg) were utilized in a 29 d experiment in a completely randomized block design with a 2 × 2 factorial structure consisting of two levels of ZH for 26 d (0 and 0.20 mg/kg PV-1) and two levels of D3 for 7 d (0 and 1.5 × 106 IU/d-1). ZH improved (p ≤ 0.05) the average daily gain (ADG) and feed efficiency by 9.9% and 17.8%, respectively, as well as hot carcass weight (HCW) and dressing carcass by 4.3% and 2.6%, respectively. (p ≤ 0.03). However, ZH increased (p < 0.01) muscle pH and Warner-Bratzler shear force (WBSF) (2.5 and 23.0%, respectively). D3 supplementation negatively affected (p ≤ 0.02) dry matter intake (DMI) (last 7 d) and ADG by 15.7% and 18.1%. On the other hand, D3 improved the pH of the longissimus thoracis muscle by 1.7% (p = 0.03) without affecting WBSF. When D3 was supplemented in combination with ZH, it was observed that meat quality was improved by reducing muscle pH compared to lambs treated only with ZH. However, D3 did not improve the meat tenderness negatively affected by ZH supplementation.

Adeno-Associated Virus Type 9-Mediated Gene Therapy of Choline Acetyltransferase-Deficient Mice.

The enzyme choline acetyltransferase (ChAT) synthesizes acetylcholine from acetyl-CoA and choline at the neuromuscular junction and at the nerve terminals of cholinergic neurons. Mutations in the ChAT gene (CHAT) result in a presynaptic congenital myasthenic syndrome (CMS) that often associates with life-threatening episodes of apnea. Knockout mice for Chat (Chat-/-) die at birth. To circumvent the lethality of this model, we crossed mutant mice possessing loxP sites flanking Chat exons 4 and 5 with mice that expressed Cre-ERT2. Injection of tamoxifen (Tx) at postnatal (P) day 11 in these mice induced downregulation of Chat, autonomic failure, weakness, and death. However, a proportion of Chatflox/flox-Cre-ERT2 mice receiving at birth an intracerebroventricular injection of 2 × 1013 vg/kg adeno-associated virus type 9 (AAV9) carrying human CHAT (AAV9-CHAT) survived a subsequent Tx injection and lived to adulthood without showing signs of weakness. Likewise, injection of AA9-CHAT by intracisternal injection at P28 after the onset of weakness also resulted in survival to adulthood. The expression of Chat in spinal motor neurons of Chatflox/flox-Cre-ERT2 mice injected with Tx was markedly reduced, but AAV-injected mice showed a robust recovery of ChAT expression, which was mainly translated by the human CHAT RNA. The biodistribution of the viral genome was widespread but maximal in the spinal cord and brain of AAV-injected mice. No significant histopathological changes were observed in the brain, liver, and heart of AAV-injected mice after 1 year follow-up. Thus, AAV9-mediated gene therapy may provide an effective and safe treatment for patients severely affected with CHAT-CMS.

Biologically aware lighting for newborn intensive care.

OBJECTIVE: We designed and implemented a novel neonatal intensive care (NICU) lighting system to support the current understanding of daylight-coupled physiology. METHODS: We created a system that generates wavelengths corresponding to the known blue and violet activation spectra of non-visual opsins. These are known to mediate energy management and related physiologic activity. RESULTS: Light produced by the system spans the visible spectrum, including violet wavelengths that are blocked by modern glazing and not emitted by standard LED fixtures. System features include automated light and dark phases that mimic dawn/dusk. The system also matches length of day seasonality. Spectral composition can be varied to support translational research protocols. Implementation required a comprehensive strategy to inform bedside providers about the value and use of the lighting system. CONCLUSION: Full-spectrum lighting for the NICU is feasible and will inform the optimization of the NICU environment of care to support optimal neonatal growth and development.

Defining a species-specific K constant in American Bullfrogs (Lithobates catesbeianus) through use of computed tomography.

OBJECTIVE: To use CT measurements to define the body surface area (BSA) formula in American bullfrogs (Lithobates catesbeianus) and calculate the species-specific shape constant (K) to suggest chemotherapeutic doses. ANIMALS: 12 American bullfrogs owned by the North Carolina State College of Veterinary Medicine Department of Laboratory Animal Resources underwent CT scans without anesthesia or sedation in November 2022. METHODS: As part of this prospective study, each American bullfrog underwent a complete physical exam and CT scan. 3-D surface models were created using CT data, and the resulting measurements were used for BSA calculations. Animals were grouped by sex. Nonlinear regression analysis of BSA versus body weight was performed, and a species-specific formula was derived for calculating BSA in American bullfrogs. RESULTS: The mean body weight of the bullfrogs was 354 grams. The mean CT-derived BSA was 414.92 cm2. The calculated K constant was 8.28 for the 12 American bullfrogs, and the CT-derived BSA formula was BSA in cm2 = 8.28 X (body weight in g)2/3. The K constant was 8.07 for females and 8.44 for males and was not significantly different between sexes (P = .5). CLINICAL RELEVANCE: Results indicated that the species-specific K constant for American bullfrogs is 8.28. This is the first calculated K constant that exists for amphibians to our knowledge.

phox2ba: The Potential Genetic Link behind the Overlap in the Symptomatology between CHARGE and Central Congenital Hypoventilation Syndromes.

CHARGE syndrome typically results from mutations in the gene encoding chromodomain helicase DNA-binding protein 7 (CHD7). CHD7 is involved in regulating neural crest development, which gives rise to tissues of the skull/face and the autonomic nervous system (ANS). Individuals with CHARGE syndrome are frequently born with anomalies requiring multiple surgeries and often experience adverse events post-anesthesia, including oxygen desaturations, decreased respiratory rates, and heart rate abnormalities. Central congenital hypoventilation syndrome (CCHS) affects ANS components that regulate breathing. Its hallmark feature is hypoventilation during sleep, clinically resembling observations in anesthetized CHARGE patients. Loss of PHOX2B (paired-like homeobox 2b) underlies CCHS. Employing a chd7-null zebrafish model, we investigated physiologic responses to anesthesia and compared these to loss of phox2b. Heart rates were lower in chd7 mutants compared to the wild-type. Exposure to tricaine, a zebrafish anesthetic/muscle relaxant, revealed that chd7 mutants took longer to become anesthetized, with higher respiratory rates during recovery. chd7 mutant larvae demonstrated unique phox2ba expression patterns. The knockdown of phox2ba reduced larval heart rates similar to chd7 mutants. chd7 mutant fish are a valuable preclinical model to investigate anesthesia in CHARGE syndrome and reveal a novel functional link between CHARGE syndrome and CCHS.

Back to the future: evolutionary biology reveals a key regulatory switch in neuroblastoma pathogenesis.

While MYCN expression is an important contributing factor to heterogeneity in the natural history of neuroblastoma (NBL), a mechanistic understanding of this often mutationally quiet tumor has remained elusive. In this issue of the JCI, Weichert-Leahey and authors focused on the adrenergic and mesenchymal core regulatory circuitries (CRC) as NBL transcriptional programs. The authors previously showed that overexpression of LIM-domain-only 1 (LMO1), a transcriptional coregulator, synergizes with MYCN to accelerate tumor formation and metastasis in an NBL-zebrafish model. They now demonstrate experimentally, using genome-edited zebrafish, that a polymorphism in the human rs2168101 locus of the LMO1 gene determines which CRC is active in a tumor. In some cases, LMO3 compensated for LMO1 loss and drove the adrenergic CRC in MYCN-positive NBL. This study exemplifies the value of evolutionary relationships and zebrafish models in the investigation of human disease and reveals pathways of NBL development that may affect prevention or intervention strategies.

Ribosome-mediated biosynthesis of pyridazinone oligomers in vitro.

The ribosome is a macromolecular machine that catalyzes the sequence-defined polymerization of L-α-amino acids into polypeptides. The catalysis of peptide bond formation between amino acid substrates is based on entropy trapping, wherein the adjacency of transfer RNA (tRNA)-coupled acyl bonds in the P-site and the α-amino groups in the A-site aligns the substrates for coupling. The plasticity of this catalytic mechanism has been observed in both remnants of the evolution of the genetic code and modern efforts to reprogram the genetic code (e.g., ribosomal incorporation of non-canonical amino acids, ribosomal ester formation). However, the limits of ribosome-mediated polymerization are underexplored. Here, rather than peptide bonds, we demonstrate ribosome-mediated polymerization of pyridazinone bonds via a cyclocondensation reaction between activated γ-keto and α-hydrazino ester monomers. In addition, we demonstrate the ribosome-catalyzed synthesis of peptide-hybrid oligomers composed of multiple sequence-defined alternating pyridazinone linkages. Our results highlight the plasticity of the ribosome's ancient bond-formation mechanism, expand the range of non-canonical polymeric backbones that can be synthesized by the ribosome, and open the door to new applications in synthetic biology.

Ca2+ channels couple spiking to mitochondrial metabolism in substantia nigra dopaminergic neurons.

How do neurons match generation of adenosine triphosphate by mitochondria to the bioenergetic demands of regenerative activity? Although the subject of speculation, this coupling is still poorly understood, particularly in neurons that are tonically active. To help fill this gap, pacemaking substantia nigra dopaminergic neurons were studied using a combination of optical, electrophysiological, and molecular approaches. In these neurons, spike-activated calcium (Ca2+) entry through Cav1 channels triggered Ca2+ release from the endoplasmic reticulum, which stimulated mitochondrial oxidative phosphorylation through two complementary Ca2+-dependent mechanisms: one mediated by the mitochondrial uniporter and another by the malate-aspartate shuttle. Disrupting either mechanism impaired the ability of dopaminergic neurons to sustain spike activity. While this feedforward control helps dopaminergic neurons meet the bioenergetic demands associated with sustained spiking, it is also responsible for their elevated oxidant stress and possibly to their decline with aging and disease.

Organo-mercury species in a polluted agricultural flood plain: Combining speciation methods and polymerase chain reaction to investigate pathways of contamination.

The analysis of organic mercury (Hg) species in polluted soils is a necessary tool to assess the environmental risk(s) of mercury in contaminated legacy sites. The artificial formation of monomethylmercury (MeHg) during soil extraction and/or analysis is a well-known limitation and is especially relevant in highly polluted areas where MeHg/Hg ratios are notoriously low. Although this has been known for almost 30 years, the thorough characterisation of artificial formation rates is rarely a part of the method development in scientific literature. Here we present the application of two separate procedures (inorganic Hg (iHg) spiking and double-spike isotope dilution analyses (DSIDA)) to determine and correct for artificial Hg methylation in MeHg-selective acid-leaching/organic solvent extraction procedure. Subsequently, we combined corrected MeHg and ethylmercury (EtHg) measurements with PCR amplification of hgcA genes to distinguish between naturally formed MeHg from primary deposited MeHg in soils from a legacy site in a Swiss mountain valley. We found the DSIDA procedure incompatible with the organomercury selective extraction method due to the quantitative removal of iHg. Methylation factors from iHg spiking were in the range of (0.0075 ± 0.0001%) and were consistent across soils and sediment matrices. Further, we suggest that MeHg was deposited and not formed in-situ in two out of three studied locations. Our line of evidence consists of 1) the concomitant detection of EtHg, 2) the elevated MeHg concentrations (up to 4.84 µg kg-1), and 3) the absence of hgcA genes at these locations. The combination of Hg speciation and methylation gene (hgcA) abundance analyses are tools suited to assess Hg pollution pathways at Hg legacy sites.

Antimony release and volatilization from rice paddy soils: Field and microcosm study.

The fate of antimony (Sb) in submerged soils and the impact of common agricultural practices (e.g., manuring) on Sb release and volatilization is understudied. We investigated porewater Sb release and volatilization in the field and laboratory for three rice paddy soils. In the field study, the porewater Sb concentration (up to 107.1 µg L-1) was associated with iron (Fe) at two sites, and with pH, Fe, manganese (Mn), and sulfate (SO42-) at one site. The surface water Sb concentrations (up to 495.3 ± 113.7 µg L-1) were up to 99 times higher than the regulatory values indicating a potential risk to aquaculture and rice agriculture. For the first time, volatile Sb was detected in rice paddy fields using a validated quantitative method (18.1 ± 5.2 to 217.9 ± 160.7 mg ha-1 y-1). We also investigated the influence of two common rice agriculture practices (flooding and manuring) on Sb release and volatilization in a 56-day microcosm experiment using the same soils from the field campaign. Flooding induced an immediate, but temporary, Sb release into the porewater that declined with SO42-, indicating that SO42- reduction may reduce porewater Sb concentrations. A secondary Sb release, corresponding to Fe reduction in the porewater, was observed in some of the microcosms. Our results suggest flooding-induced Sb release into rice paddy porewaters is temporary but relevant. Manuring the soils did not impact the porewater Sb concentration but did enhance Sb volatilization. Volatile Sb (159.6 ± 108.4 to 2237.5 ± 679.7 ng kg-1 y-1) was detected in most of the treatments and was correlated with the surface water Sb concentration. Our study indicates that Sb volatilization could be occurring at the soil-water interface or directly in the surface water and highlights that future works should investigate this potentially relevant mechanism.

Chemical insights into flexizyme-mediated tRNA acylation.

A critical step in repurposing the cellular translation machinery for the synthesis of polymeric products is the acylation of transfer RNA (tRNA) with unnatural monomers. Toward this goal, flexizymes, ribozymes capable of aminoacylation, have emerged as a uniquely adept tool for charging tRNA with ever increasingly diverse substrates. In this review, we present a library of monomer substrates that have been tested for tRNA acylation with the flexizyme system. From this mile-high view, we provide insights for understanding the chemical factors that influence flexizyme-mediated tRNA acylation. We conclude that flexizymes are primitive esterification catalysts that display a modest binding affinity to the monomer's aromatic recognition element. Together, these robust, yet flexible, flexizyme systems provide researchers with unprecedented access for preparing unnatural acyl-tRNA and the opportunity to repurpose the translation machinery for the synthesis of novel biologically derived structures beyond native proteins and peptides.

Does the Configuration at the Metal Matter in Noyori-Ikariya Type Asymmetric Transfer Hydrogenation Catalysts?

Noyori-Ikariya type [(arene)RuCl(TsDPEN)] (TsDPEN, sulfonated diphenyl ethylenediamine) complexes are widely used C=O and C=N reduction catalysts that produce chiral alcohols and amines via a key ruthenium-hydride intermediate that determines the stereochemistry of the product. Whereas many details about the interactions of the pro-chiral substrate with the hydride complex and the nature of the hydrogen transfer from the latter to the former have been investigated over the past 25 years, the role of the stereochemical configuration at the stereogenic ruthenium center in the catalysis has not been elucidated so far. Using operando FlowNMR spectroscopy and nuclear Overhauser effect spectroscopy, we show the existence of two diastereomeric hydride complexes under reaction conditions, assign their absolute configurations in solution, and monitor their interconversion during transfer hydrogenation catalysis. Configurational analysis and multifunctional density functional theory (DFT) calculations show the λ-(R,R)S Ru configured [(mesitylene)RuH(TsDPEN)] complex to be both thermodynamically and kinetically favored over its λ-(R,R)R Ru isomer with the opposite configuration at the metal. Computational analysis of both diastereomeric catalytic manifolds show the major λ-(R,R)S Ru configured [(mesitylene)RuH(TsDPEN)] complex to dominate asymmetric ketone reduction catalysis with the minor λ-(R,R)R Ru [(mesitylene)RuH(TsDPEN)] stereoisomer being both less active and less enantioselective. These findings also hold true for a tethered catalyst derivative with a propyl linker between the arene and TsDPEN ligands and thus show enantioselective transfer hydrogenation catalysis with Noyori-Ikariya complexes to proceed via a lock-and-key mechanism.

Disruption of mitochondrial complex I induces progressive parkinsonism.

Loss of functional mitochondrial complex I (MCI) in the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson's disease1. Yet, whether this change contributes to Parkinson's disease pathogenesis is unclear2. Here we used intersectional genetics to disrupt the function of MCI in mouse dopaminergic neurons. Disruption of MCI induced a Warburg-like shift in metabolism that enabled neuronal survival, but triggered a progressive loss of the dopaminergic phenotype that was first evident in nigrostriatal axons. This axonal deficit was accompanied by motor learning and fine motor deficits, but not by clear levodopa-responsive parkinsonism-which emerged only after the later loss of dopamine release in the substantia nigra. Thus, MCI dysfunction alone is sufficient to cause progressive, human-like parkinsonism in which the loss of nigral dopamine release makes a critical contribution to motor dysfunction, contrary to the current Parkinson's disease paradigm3,4.

Efficient molecular encoding in multifunctional self-immolative urethanes.

Molecular encoding in sequence-defined polymers shows promise as a new paradigm for data storage. Here, we report what is, to our knowledge, the first use of self-immolative oligourethanes for storing and reading encoded information. As a proof of principle, we describe how a text passage from Jane Austen's Mansfield Park was encoded in sequence-defined oligourethanes and reconstructed via self-immolative sequencing. We develop Mol.E-coder, a software tool that uses a Huffman encoding scheme to convert the character table to hexadecimal. The oligourethanes are then generated by a high-throughput parallel synthesis. Sequencing of the oligourethanes by self-immolation is done concurrently in a parallel fashion, and the liquid chromatography-mass spectrometry (LC-MS) information decoded by our Mol.E-decoder software. The passage is capable of being reproduced wholly intact by a third-party, without any purifications or the use of tandem MS (MS/MS), despite multiple rounds of compression, encoding, and synthesis.

The impact of the host intestinal microbiome on carcinogenesis and the response to chemotherapy.

The microbiome consists of all microbes present on and within the human body. An unbalanced, or 'dysbiotic' intestinal microbiome is associated with inflammatory bowel disease, diabetes and some cancer types. Drug treatment can alter the intestinal microbiome composition. Additionally, some chemotherapeutics interact with microbiome components, leading to changes in drug safety and/or efficacy. The intestinal microbiome is a modifiable target, using strategies such as antibiotic treatment, fecal microbial transplantation or probiotic administration. Understanding the impact of the microbiome on the safety and efficacy of cancer treatment may result in improved treatment outcome. The present review seeks to summarize relevant research and look to the future of cancer treatment, where the intestinal microbiome is recognized as an actionable treatment target.

Lay abstract The microbiome describes all of the microorganisms (including bacteria, viruses and fungi) that are normally present on and inside the human body. Some diseases, including cancer, can be caused or worsened by an 'unbalanced' or 'unhealthy' gut microbiome. Some drugs that are given to people who have cancer can change the microbiome. Importantly, components of the gut microbiome can also change how a cancer drug will work in someone. We can change the microbiome in certain ways, like by giving someone antibiotics. Understanding how the microbiome influences the way anticancer drugs work is important because it could help us understand how to make cancer treatment safer and more effective. This review article summarizes available research on the impact of the microbiome on cancer treatment.

Validation and deployment of a quantitative trapping method to measure volatile antimony emissions.

Microbial-mediated Sb volatilization is a poorly understood part of the Sb biogeochemical cycle. This is mostly due to a lack of laboratory and field-deployable methods that are capable of quantifying low-level emissions of Sb from diffuse sources. In this study, we validated two methods using a H2O2 -HNO3 liquid chemotrap and an activated coconut shell charcoal solid-phase trap, achieving an absolute limit of detection of 4.6 ng and below 2.0 ng Sb, respectively. The activated charcoal solid-phase trapping method, the most easily operated method, was then applied to contaminated shooting range soils. Four treatments were tested: 1) flooded, 2) manure amended + flooded, 3) 70 % water holding capacity, and 4) manure amendment +70 % water holding capacity, since agricultural practices and flooding events may contribute to Sb volatilization. Volatile Sb was only produced from flooded microcosms and manure amendment greatly influenced the onset and amount of volatile Sb produced. The highest amount of volatile Sb produced, up to 62.1 ng kg-1 d-1, was from the flooded manure amended soil. This suggests that anaerobic microorganisms may potentially be drivers of Sb volatilization. Our results show that polluted shooting range soils are a source of volatile Sb under flooded conditions, which may lead to an increase in the mobility of Sb. Some of these volatile Sb species are toxic and genotoxic, highlighting the role of Sb volatilization on environmental health, especially for individuals living in contaminated areas exposed to wetlands or flooded conditions (e.g., rice paddy agriculture surrounding mining areas). This work paves way for research on Sb volatilization in the environment.

Exploring the effect of alcohol on disease activity and outcomes in rheumatoid arthritis through systematic review and meta-analysis.

To evaluate the effects of alcohol consumption on disease activity in rheumatoid arthritis. EMBASE, Pubmed, the Cochrane Library, and Web of Science were searched until July 29, 2020. English language studies that reported disease activity outcomes in rheumatoid arthritis were included. Studies were excluded if they were reviews, case reports, had fewer than 20 patients, or reported on prevalence but not disease activity in RA. Forest plots were used to determine pooled mean difference and were generated on RevMan5.3. Linear regression was used to determine correlations between alcohol and antibody status, gender, and smoking status. The search identified 4126 citations of which 14 were included. The pooled mean difference in DAS28 (95% CI) was 0.34 (0.24, 0.44) (p < 10-5) between drinkers and non-drinkers with lower DAS28 in non-drinkers, 0.33 (0.05, 0.62) (p = 0.02) between heavy drinkers and non-drinkers with lower DAS28 in heavy drinkers, and 0.00 (- 0.30, 0.30) (p = 0.98) between low- and high-risk drinkers. The mean difference of HAQ assessments was significantly different between those who drink alcohol compared to those who do not, with drinkers reporting lower HAQ scores (0.3 (0.18, 0.41), p < 10-5). There was no significant correlation between drinking and gender, smoking status, or antibody positivity. Alcohol consumption is associated with lower disease activity and self-reported health assessment in rheumatoid arthritis. However, drinking has no correlation with smoking, gender, or antibody status.

Mutation in the Ciliary Protein C2CD3 Reveals Organ-Specific Mechanisms of Hedgehog Signal Transduction in Avian Embryos.

Primary cilia are ubiquitous microtubule-based organelles that serve as signaling hubs for numerous developmental pathways, most notably the Hedgehog (Hh) pathway. Defects in the structure or function of primary cilia result in a class of diseases called ciliopathies. It is well known that primary cilia participate in transducing a Hh signal, and as such ciliopathies frequently present with phenotypes indicative of aberrant Hh function. Interestingly, the exact mechanisms of cilia-dependent Hh signaling transduction are unclear as some ciliopathic animal models simultaneously present with gain-of-Hh phenotypes in one organ system and loss-of-Hh phenotypes in another. To better understand how Hh signaling is perturbed across different tissues in ciliopathic conditions, we examined four distinct Hh-dependent signaling centers in the naturally occurring avian ciliopathic mutant talpid2 (ta2). In addition to the well-known and previously reported limb and craniofacial malformations, we observed dorsal-ventral patterning defects in the neural tube, and a shortened gastrointestinal tract. Molecular analyses for elements of the Hh pathway revealed that the loss of cilia impact transduction of an Hh signal in a tissue-specific manner at variable levels of the pathway. These studies will provide increased knowledge into how impaired ciliogenesis differentially regulates Hh signaling across tissues and will provide potential avenues for future targeted therapeutic treatments.