Dorsomedial striatal AMPA receptor antagonism increases alcohol binge drinking in selectively bred crossed high alcohol preferring mice.

Crossed high alcohol preferring (cHAP) mice have been selectively bred to consume considerable amounts of alcohol resulting in binge drinking. The dorsomedial striatum (DMS) is a brain region involved in goal-directed action selection, and dorsolateral striatum (DLS) is a brain region involved in habitual action selection. Alcohol use disorder (AUD) may involve a disruption in the balance between the DMS and DLS. While the DLS is involved in binge drinking, the reliance on the DMS and DLS in binge drinking has not been investigated in cHAP mice. We have previously demonstrated that glutamatergic activity in the DLS is necessary for binge-like alcohol drinking in C57BL/6J mice, another high drinking mouse. Because of this, we hypothesised that DLS glutamatergic activity would gate binge-like alcohol drinking in cHAP mice. cHAP mice underwent bilateral cannulation into the DMS or DLS and were allowed free-access to 20% alcohol for 2 h each day for 11 days. Mice were microinjected with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) antagonist, NBQX, into the DMS or DLS immediately prior to alcohol access. AMPAR protein expression was also assessed in a separate group of animals in the DMS and DLS following an 11-day drinking history. We found that intra-DMS (but not intra-DLS) NBQX alters binge alcohol drinking, with intra-DMS NBQX increasing alcohol consumption. We also found that the ratio of GluA1 to GluA2 differs across dorsal striatal subregions. Together, these findings suggest that glutamatergic activity in the DMS may serve to limit binge drinking in cHAP mice.

Functional profiling of CHAP domain-containing peptidoglycan hydrolases of Staphylococcus aureus USA300 uncovers potential targets for anti-staphylococcal therapies.

The bacterial pathogen Staphylococcus aureus employs a thick cell wall for protection against physical and chemical insults. This wall requires continuous maintenance to ensure strength and barrier integrity, but also to permit bacterial growth and division. The main cell wall component is peptidoglycan. Accordingly, the bacteria produce so-called peptidoglycan hydrolases (PGHs) that cleave glycan strands to facilitate growth, cell wall remodelling, separation of divided cells and release of exported proteins into the extracellular milieu. A special class of PGHs contains so-called 'cysteine, histidine-dependent amidohydrolase/peptidase' (CHAP) domains. In the present study, we profiled the roles of 11 CHAP PGHs encoded by the core genome of S. aureus USA300 LAC. Mutant strains lacking individual CHAP PGHs were analysed for growth, cell morphology, autolysis, and invasion and replication inside human lung epithelial cells. The results show that several investigated CHAP PGHs contribute to different extents to extracellular and intracellular growth and replication of S. aureus, septation of dividing cells, daughter cell separation once the division process is completed, autolysis and biofilm formation. In particular, the CHAP PGHs Sle1 and SAUSA300_2253 control intracellular staphylococcal replication and the resistance to ß-lactam antibiotics like oxacillin. This makes the S. aureus PGHs in general, and the Sle1 and SAUSA300_2253 proteins in particular, attractive targets for future prophylactic or therapeutic anti-staphylococcal interventions. Alternatively, these cell surface-exposed enzymes, or particular domains of these enzymes, could be applied in innovative anti-staphylococcal therapies.

Society for Maternal-Fetal Medicine Statement: Antihypertensive therapy for mild chronic hypertension in pregnancy-The Chronic Hypertension and Pregnancy trial.

The recently published Chronic Hypertension and Pregnancy study provides important data to inform the management of mild chronic hypertension in pregnancy. The purpose of this statement is to review the results of this trial and provide guidance for the implementation of the study findings. Based on the available evidence, SMFM recommends treatment with antihypertensive therapy for mild chronic hypertension in pregnancy to a goal blood pressure of <140/90 mm Hg. Patients with treated chronic hypertension should continue established antihypertensive therapy during pregnancy or change to a regimen compatible with pregnancy to achieve this treatment goal.

The Drosophila CG1674 gene encodes a synaptopodin 2-like related protein that localizes to the Z-disc and is required for normal flight muscle development and function.

BACKGROUND: To identify novel myofibrillar components of the Drosophila flight muscles, we carried out a proteomic analysis of chemically demembranated flight muscle myofibrils, and characterized the knockdown phenotype of a novel gene identified in the screen, CG1674. RESULTS: The CG1674 protein has some similarity to vertebrate synaptopodin 2-like, and when expressed as a FLAG-tagged fusion protein, it was localized during development to the Z-disc and cytoplasm. Knockdown of CG1674 expression affected the function of multiple muscle types, and defective flight in adults was accompanied by large actin-rich structures in the flight muscles that resembled overgrown Z-discs. Localization of CG1674 to the Z-disc depended predominantly upon presence of the Z-disc component alpha-actinin, but also depended upon other Z-disc components, including Mask, Zasp52, and Sals. We also observed re-localization of FLAG-CG1674 to the nucleus in Alpha-actinin and sals knockdown animals. CONCLUSIONS: These studies identify and characterize a previously unreported myofibrillar component of Drosophila muscle that is necessary for proper myofibril assembly during development.

ClyJ Is a Novel Pneumococcal Chimeric Lysin with a Cysteine- and Histidine-Dependent Amidohydrolase/Peptidase Catalytic Domain.

Streptococcus pneumoniae is one of the leading pathogens that cause a variety of mucosal and invasive infections. With the increased emergence of multidrug-resistant S. pneumoniae, new antimicrobials with mechanisms of action different from conventional antibiotics are urgently needed. In this study, we identified a putative lysin (gp20) encoded by the Streptococcus phage SPSL1 using the LytA autolysin as a template. Molecular dissection of gp20 revealed a binding domain (GPB) containing choline-binding repeats (CBRs) that are high specificity for S. pneumoniae By fusing GPB to the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) catalytic domain of the PlyC lysin, we constructed a novel chimeric lysin, ClyJ, with improved activity to the pneumococcal Cpl-1 lysin. No resistance was observed in S. pneumoniae strains after exposure to incrementally doubling concentrations of ClyJ for 8 continuous days in vitro In a mouse bacteremia model using penicillin G as a control, a single intraperitoneal injection of ClyJ improved the survival rate of lethal S. pneumoniae-infected mice in a dose-dependent manner. Given its high lytic activity and safety profile, ClyJ may represent a promising alternative to combat pneumococcal infections.

The conserved actinobacterial transcriptional regulator FtsR controls expression of ftsZ and further target genes and influences growth and cell division in Corynebacterium glutamicum.

BACKGROUND: Key mechanisms of cell division and its regulation are well understood in model bacteria such as Escherichia coli and Bacillus subtilis. In contrast, current knowledge on the regulation of cell division in Actinobacteria is rather limited. FtsZ is one of the key players in this process, but nothing is known about its transcriptional regulation in Corynebacterium glutamicum, a model organism of the Corynebacteriales. RESULTS: In this study, we used DNA affinity chromatography to search for transcriptional regulators of ftsZ in C. glutamicum and identified the Cg1631 protein as candidate, which was named FtsR. Both deletion and overexpression of ftsR caused growth defects and an altered cell morphology. Plasmid-based expression of native ftsR or of homologs of the pathogenic relatives Corynebacterium diphtheriae and Mycobacterium tuberculosis in the ΔftsR mutant could at least partially reverse the mutant phenotype. Absence of ftsR caused decreased expression of ftsZ, in line with an activator function of FtsR. In vivo crosslinking followed by affinity purification of FtsR and next generation sequencing of the enriched DNA fragments confirmed the ftsZ promoter as in vivo binding site of FtsR and revealed additional potential target genes and a DNA-binding motif. Analysis of strains expressing ftsZ under control of the gluconate-inducible gntK promoter revealed that the phenotype of the ΔftsR mutant is not solely caused by reduced ftsZ expression, but involves further targets. CONCLUSIONS: In this study, we identified and characterized FtsR as the first transcriptional regulator of FtsZ described for C. glutamicum. Both the absence and the overproduction of FtsR had severe effects on growth and cell morphology, underlining the importance of this regulatory protein. FtsR and its DNA-binding site in the promoter region of ftsZ are highly conserved in Actinobacteria, which suggests that this regulatory mechanism is also relevant for the control of cell division in related Actinobacteria.

An Acid Up-Regulated Surface Protein of Lactobacillus paracasei Strain GCRL 46 is Phylogenetically Related to the Secreted Glucan- (GpbB) and Immunoglobulin-Binding (SibA) Protein of Pathogenic Streptococci.

Bacterial cell wall hydrolases, including amidases and peptidases, play a critical role in peptidoglycan turnover during growth, impacting daughter cell separation, and cell death, through autolysis. When exploring the regulation of protein expression across the growth cycle of an acid-resistant strain of Lactobacillus paracasei, GCRL 46, we observed temporal up-regulation of proteins in the 40⁻45 kDa molecular weight range for whole-cell extracts when culturing in fermenters at a controlled pH of 4.0 versus optimum growth pH of 6.3. Up-regulation of proteins in this size range was not detected in SDS-PAGE gels of the cytosolic fraction, but was routinely detected following growth at low pH in whole cells and cell debris obtained after bead beating and centrifugation, indicating a cell surface location. N-terminal sequencing and in silico analyses showed sequence similarity with proteins in the L. casei group (L. casei, L. paracasei and L. rhamnosus) which were variously annotated as uncharacterized proteins, surface antigens, possible TrsG proteins, CHAP (cysteine, histidine-dependent amidohydrolases/peptidases)-domain proteins or putative peptidoglycan d,l-endopeptidase due to the presence of a CwlO domain. This protein is a homologue of the p40 (Msp2) secreted protein of L. rhamnosus LGG, which is linked to probiotic functionality in this species, and is phylogenetically related to structurally-similar proteins found in Enterococcus, Streptococcus and Bifidobacterium species, including the glucan-binding (GbpB), surface antigen (SagA) proteins detected in pathogenic group A streptococci species as secreted, immunoglobulin-binding (SibA) proteins (also named PcsB). Three-dimensional (3D) modelling predicted structural similarities in the CHAP proteins from the L. casei group and streptococcal species, indicating retention of overall architecture despite sequence divergence, and an implied retention of function during evolution. A phylogenetically-related hydrolase also contained the CwlO domain with a NLPC_P60 domain, and showed similar overall but distinct architecture to the CHAP proteins. We concluded that the surface-located, CHAP protein in L. casei is up-regulated during long-term exposure to acidic conditions during growth but not during acid shock.

A Quorum Sensing-Regulated Protein Binds Cell Wall Components and Enhances Lysozyme Resistance in Streptococcus pyogenes.

The Rgg2/3 quorum sensing (QS) system is conserved among all sequenced isolates of group A Streptococcus (GAS; Streptococcus pyogenes). The molecular architecture of the system consists of a transcriptional activator (Rgg2) and a transcriptional repressor (Rgg3) under the control of autoinducing peptide pheromones (SHP2 and SHP3). Activation of the Rgg2/3 pathway leads to increases in biofilm formation and resistance to the bactericidal effects of the host factor lysozyme. In this work, we show that deletion of a small gene, spy49_0414c, abolished both phenotypes in response to pheromone signaling. The gene encodes a small, positively charged, secreted protein, referred to as StcA. Analysis of recombinant StcA showed that it can directly interact with GAS cell wall preparations containing phosphodiester-linked carbohydrate polymers but not with preparations devoid of them. Immunofluorescence microscopy detected antibody against StcA bound to the surface of paraformaldehyde-fixed wild-type cells. Expression of StcA in bacterial culture induced a shift in the electrostatic potential of the bacterial cell surface, which became more positively charged. These results suggest that StcA promotes phenotypes by way of ionic interactions with the GAS cell wall, most likely with negatively charged cell wall-associated polysaccharides.IMPORTANCE This study focuses on a small protein, StcA, that is expressed and secreted under induction of Rgg2/3 QS, ionically associating with negatively charged domains on the cell surface. These data present a novel mechanism of resistance to the host factor lysozyme by GAS and have implications in the relevance of this circuit in the interaction between the bacterium and the human host that is mediated by the bacterial cell surface.

Mechanisms of zinc binding to the solute-binding protein AztC and transfer from the metallochaperone AztD.

Bacteria can acquire the essential metal zinc from extremely zinc-limited environments by using ATP-binding cassette (ABC) transporters. These transporters are critical virulence factors, relying on specific and high-affinity binding of zinc by a periplasmic solute-binding protein (SBP). As such, the mechanisms of zinc binding and release among bacterial SBPs are of considerable interest as antibacterial drug targets. Zinc SBPs are characterized by a flexible loop near the high-affinity zinc-binding site. The function of this structure is not always clear, and its flexibility has thus far prevented structural characterization by X-ray crystallography. Here, we present intact structures for the zinc-specific SBP AztC from the bacterium Paracoccus denitrificans in the zinc-bound and apo-states. A comparison of these structures revealed that zinc loss prompts significant structural rearrangements, mediated by the formation of a sodium-binding site in the apo-structure. We further show that the AztC flexible loop has no impact on zinc-binding affinity, stoichiometry, or protein structure, yet is essential for zinc transfer from the metallochaperone AztD. We also found that 3 His residues in the loop appear to temporarily coordinate zinc and then convey it to the high-affinity binding site. Thus, mutation of any of these residues to Ala abrogated zinc transfer from AztD. Our structural and mechanistic findings conclusively identify a role for the AztC flexible loop in zinc acquisition from the metallochaperone AztD, yielding critical insights into metal binding by AztC from both solution and AztD. These proteins are highly conserved in human pathogens, making this work potentially useful for the development of novel antibiotics.

Activator protein-1 (AP-1) and response to pathogen infection in the Hong Kong oyster (Crassostrea hongkongensis).

Growing evidence suggests that the transcription factor activator protein-1 (AP-1), a downstream target of mitogen-activated protein kinase (MAPK) signaling, plays a major role in stimulating the synthesis of immune effector molecules during innate immune responses. We have characterized ChAP-1, an AP-1-like protein in Crassostrea hongkongensis that is a member of the AP-1 family of proteins. ChAP-1 is composed of 290 amino acid residues with a Jun and bZIP domain at the N- and C-termini, respectively, a structure similar to that of known Ap-1 proteins. ChAP-1 mRNA is expressed in several tissues analyzed, with highest expression in the mantle. Expression of ChAP-1 increases in response to Vibrio alginolyticus, Salmo haemolyticus or Salmo cerevisiae infection and, despite the location of GFP-tagged full-length ChAP-1 protein in the cytoplasm, ChAP-1 activates the transcription of an L8G5-luc reporter gene, and its over-expression can also activate the AP-1-Luc reporter gene in HEK293T cells.

SP-CHAP, an endolysin with enhanced activity against biofilm pneumococci and nasopharyngeal colonization.

Streptococcus pneumoniae (Spn), a Gram-positive bacterium, is responsible for causing a wide variety of invasive infections. The emergence of multi-drug antibiotic resistance has prompted the search for antimicrobial alternatives. Phage-derived peptidoglycan hydrolases, known as endolysins, are an attractive alternative. In this study, an endolysin active against Spn, designated SP-CHAP, was cloned, produced, purified, biochemically characterized, and evaluated for its antimicrobial properties. Cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domains are widely represented in bacteriophage endolysins but have never previously been reported for pneumococcal endolysins. Here, we characterize the first pneumococcal endolysin with a CHAP catalytic domain. SP-CHAP was antimicrobial against all Spn serovars tested, including capsular and capsule-free pneumococci, and it was found to be more active than the most widely studied pneumococcal endolysin, Cpl-1, while not affecting various oral or nasal commensal organisms tested. SP-CHAP was also effective in eradicating Spn biofilms at concentrations as low as 1.56 µg/mL. In addition, a Spn mouse nasopharyngeal colonization model was employed, which showed that SP-CHAP caused a significant reduction in Spn colony-forming units, even more than Cpl-1. These results indicate that SP-CHAP may represent a promising alternative to combating Spn infections. IMPORTANCE: Considering the high rates of pneumococcal resistance reported for several antibiotics, alternatives are urgently needed. In the present study, we report a Streptococcus pneumoniae-targeting endolysin with even greater activity than Cpl-1, the most characterized pneumococcal endolysin to date. We have employed a combination of biochemical and microbiological assays to assess the stability and lytic potential of SP-CHAP and demonstrate its efficacy on pneumococcal biofilms in vitro and in an in vivo mouse model of colonization. Our findings highlight the therapeutic potential of SP-CHAP as an antibiotic alternative to treat Streptococcus pneumoniae infections.

Home cage voluntary alcohol consumption increases binge drinking without affecting abstinence-related depressive-like behaviors or operant responding in crossed high alcohol-preferring mice (cHAPs).

Chronic alcohol consumption can lead to tolerance and escalation of drinking in humans and animals, but mechanisms underlying these changes are not fully characterized. Preclinical models can delineate which mechanisms are involved. The chronic intermittent ethanol exposure (CIE) procedure uses forced exposure to vaporized alcohol that elicits withdrawal and increased responding for alcohol in operant tasks in C57BL/6J inbred mice. Chronic two-bottle choice (2BC) drinking in the same strain elicits abstinent-related depression-like behavior, suggestive of allostatic changes. Selected lines such as crossed High Alcohol Preferring (cHAP) mice voluntarily drink to blood alcohol concentrations comparable to those attained in CIE and could be used to assess how alcohol affects these same endpoints without the confounds of involuntary vapor inhalation. In three experiments, we assess how 2BC drinking in cHAP mice affects abstinence-related depressive- and anxiety-like behavior, operant responding for alcohol, and binge consumption using drinking-in-the-dark (DID). We hypothesized that cHAPs with home-cage drinking experience would exhibit more depressive behavior after abstinence, increased responding for alcohol in the operant box, and increased DID intake. Of these, a drinking history increased DID intake in female cHAPs only and increased sucrose preference and intake following abstinence, but had no effects on operant responding or NSFT latency and FST immobility following forced abstinence. These results are consistent with recent findings using slice electrophysiology showing tolerance to alcohol's actions on the dorsolateral striatum following 2BC drinking in female, but not male cHAP mice. Overall, these data suggest that cHAPs may require procedures allowing rapid intoxication, such as DID, to demonstrate changes in alcohol's rewarding effects.

Crystallization of the CHAP domain of the endolysin from Staphylococcus aureus bacteriophage K.

CHAP(K) is the N-terminal cysteine, histidine-dependent amidohydrolase/peptidase domain (CHAP domain) of the Staphylococcus aureus bacteriophage K endolysin LysK. It is formed from the first 165 residues of LysK and functions by cleaving specific peptidoglycan peptide bonds, causing bacterial lysis. CHAP(K) can lyse S. aureus when applied exogenously, making it a good candidate for the treatment of multidrug-resistant Staphylococcus aureus infections. Here, the crystallization of CHAP(K) and the collection of native and derivative data to high resolution, which allowed structure solution, are reported. The structure may help to elucidate the mechanism of action and in the design of chimeric proteins or mutants with improved antibacterial activity.

Two-dimensional solid-state NMR spectroscopy investigations of surface precipitation of phosphate onto calcite.

The interaction of phosphate with typical soil minerals is important for understanding P cycling in natural and agricultural systems. We investigated the mechanisms of kinetics of phosphate uptake onto calcite using solid-state NMR spectroscopy. At a low phosphate concentration of 0.5 mM, the 31P single-pulse solid-state NMR peak revealed the formation of amorphous calcium phosphate (ACP) within the initial 30 min, which transformed to carbonated hydroxyapatite (CHAP) after 12 d. At a high phosphate concentration (5 mM), the results showed transformation from ACP to OCP, later to brushite, and eventually to CHAP. The formation of brushite is further supported by 31P{1H} heteronuclear correlation (HETCOR) spectra via a correlation of δP-31 = 1.7 ppm and the 1H peak at δH-1 = 6.4 ppm, which denotes the structure water of brushite. Furthermore, 13C NMR directly revealed both A-type and B-type CHAP. Generally, this work provides a detailed understanding of the aging effect on the phase transition scale of phosphate surface precipitation onto calcite in soil environments.

First principal observation documenting the three-dimensional uptake of cadmium and spatial distribution of cadmium hydroxyapatite mineral in bone char.

The 3-D matrix scale ion-exchange mechanism was explored for high-capacity cadmium (Cd) removal using bone chars (BC) chunks (1-2 mm) made at 500 °C (500BC) and 700 °C (700BC) in aqueous solutions. The Cd incorporation into the carbonated hydroxyapatite (CHAp) mineral of BC was examined using a set of synchrotron-based techniques. The Cd removal from solution and incorporation into mineral lattice were higher in 500BC than 700BC, and the diffusion depth was modulated by the initial Cd concentration and charring temperature. A higher carbonate level of BC, more pre-leached Ca sites, and external phosphorus input enhanced Cd removal. The 500BC showed a higher CO32-/PO43- ratio and specific surface area (SSA) than the 700BC, providing more vacant sites by dissolution of Ca2+. In situ observations revealed the refilling of sub-micron pore space in the mineral matrix because of Cd incorporation.The X-ray nanodiffraction (XND) analyses revealed that Cd was mainly removed from water by incorporation into the mineral lattice of 500BC via ion exchange, rather than surface sorption and precipitation, and the mineral phase was transformed from hydroxyapatite (HAp) to cadmium hydroxyapatite (Cd-HAp). The Rietveld's refinement of X-ray diffraction (XRD) data resolved up to 91% of the crystal displacement of Ca2+ by Cd2+. The specific phase and stoichiometry of the new Cd-HAp mineral was dependent on the level of ion exchange. This mechanistic study confirmed that 3-D ion exchange was the most important path for heavy metal removal from aqueous solution and immobilization in BC mineral matrix, and put forward a novel and sustainable remediation strategy for Cd removal in wastewater and soil clean-up.

A novel phage-encoded endolysin EN534-C active against clinical strain Streptococcus agalactiae GBS.

Streptococcus agalactiae (Group B Streptococcus, GBS) is primarily known as a major neonatal pathogen. In adults, these bacteria often colonize the gastrointestinal and urogenital tracts. Treatment of infections using antibiotics is often complicated by recurrences caused by multi-resistant streptococci. Endolysin EN534 from prophage A2 of human isolate Streptococcus agalactiae KMB-534 has a modular structure consisting of two terminal catalytic domains, amidase_3 and CHAP, and one central binding domain, LysM. The EN534 gene was cloned into an expression vector, and the corresponding recombinant protein EN534-C was expressed in Escherichia coli in a soluble form and isolated by affinity chromatography. The lytic activity of this endolysin was tested on cell wall substrates from different GBS serotypes, B. subtilis, L. jensenii, and E. coli. The enzyme lysed streptococci, but not beneficial vaginal lactobacilli. The isolated protein is stable in a temperature range of 20-37 °C. Calcium ions enhanced the activity of the enzyme in the pH range from 5.0 to 8.0. The exolytic activity of EN534-C was observed by time-lapse fluorescence microscopy on a S. agalactiae CCM 6187 substrate. Recombinant endolysin EN534-C may have the potential to become an antimicrobial agent for the treatment of S. agalactiae infections.

3D printing of PEEK and its composite to increase biointerfaces as a biomedical material- A review.

Poly ether-ether-ketone (PEEK) is a polymer with better lignin biocompatibility than other polymers. It is good for biomedical engineering applications. This research summarises the outcomes of an evaluation conducted on PEEK material composites, such as cellular calcium hydroxyapatite (CHAp) for medical applications. Prospects of PEEK for medical implant are highlighted. Critical analysis and review on 3D printing of PEEK, CHAp and their biological macromolecular behaviours are presented. An electronic search was carried out on Scupos database, Google search and peer-reviewed papers published in the last ten years. Because of the extraordinary strength and biological behaviours of PEEK and its composite of CHAp, 3D-printed PEEK has several biomedical applications, and its biological macromolecular behaviour leads to health sustainability. This work highlights its biological macromolecular behaviours as a bone implant material and the optimum 3D printing process for PEEK and CHAp for medical applications. The current problems with printing PEEK and CHAp are investigated along with their possible uses. Possible solutions to improve the 3D printability of PEEK and CHAp are explained based on scientific mechanisms. This detailed report stands to benefit both scientific community and medical industry to enhance 3D printing concepts for PEEK and CHAp.

Effect of ketamine on binge drinking patterns in crossed high alcohol-preferring (cHAP) mice.

BACKGROUND: Previous research has demonstrated the utility of subanesthetic doses of ketamine in decreasing binge (Drinking-in-the-Dark, or DID) 20% alcohol intake in female inbred (C57BL/6J) mice when administered 12 hours prior to alcohol access (Crowley et al., 2019). In the current study, we assess the efficacy of a similar ketamine pretreatment using male and female selectively bred, crossed High Alcohol Preferring (cHAP) mice, which also drink to intoxication, but are not inbred. We hypothesized that ketamine would decrease binge alcohol intake without impacting locomotor activity. METHODS AND RESULTS: Subjects were 28 adult cHAP mice. Mice first received a 2-week DID drinking history using 2-h/day alcohol access. On day 12, prior to ketamine treatment, the average blood ethanol concentration (BEC) was 130 mg/dL, confirming that mice reliably reached intoxicating BECs. On day 15, mice were given 0, 3, or 10 mg/kg of ketamine 12 hours prior to the DID session. Ketamine did not decrease total (2-h) alcohol consumption or locomotion. Interestingly, the 10 mg/kg dose of ketamine did alter the drinking pattern in male mice, decreasing front-loading for a single day. We opted to then increase the doses to 32 or 100 mg/kg (i.e., an anesthetic dose) two days after the initial treatment, keeping the saline control. Mice of both sexes decreased total binge alcohol intake at the 100 mg/kg dose only, but again, the effect only lasted one day. CONCLUSIONS: The current study found that cHAP mice reached more than double the BECs observed in C57BL/6J mice during DID, but did not respond to subanesthetic ketamine. Modest efficacy was found for ketamine pretreatment at anesthetic doses. Differences in findings may be due to differential intake during DID, or genetic differences between C57Bl/6J mice and cHAP mice. Drug efficacy in multiple models is important for discovering reliable pharmacotherapies for alcoholism.

Systematic changes of bone hydroxyapatite along a charring temperature gradient: An integrative study with dissolution behavior.

The applicability of bone char as a long-term phosphorus nutrient source was assessed by integrating their mineral transformation and physicochemical properties with their dissolution behavior. We have explored synchrotron-based spectroscopic and imaging techniques (FTIR, XRD, and TXM) to investigate the physicochemical changes of bone and bone char along a charring temperature gradient (300-1200 °C) and used a lab incubation experiment to study their dissolution behaviors in solutions of different pH (4, 6, and 6.9). The thermal decomposition of inorganic carbonate (CO32-) and the loss of organic components rendered a crystallographic rearrangement (blueshift of the PO43- peak) and mineral transformation with increasing temperatures. The mineral transformation from B-type to AB- and A-type carbonate substitution occurred mainly at <700 °C, while the transformation from carbonated hydroxyapatite (CHAp) to more mineralogically and chemically stable HAp occurred at >800 °C. The loss of inorganic carbonate and the increase of structural OH- with increasing temperatures explained the change of pH buffering capacity and increase of pH and their dissolution behaviors. The higher peak area ratios of phosphate to carbonate and phosphate to amide I band with increasing temperatures corroborated the higher stability and resistivity to acidic dissolution by bone chars made at higher temperatures. Our findings suggest that bone char made at low to intermediate temperatures can be a substantial source of phosphorus for soil fertility via waste management and recycling. The bone char made at 500 °C exhibited a high pH buffering capacity in acidic and near-neutral solutions. The 700 °C bone char was proposed as a suitable liming agent for raising the soil pH and abating soil acidity. Our study has underpinned the systematic changes of bone char and interlinked the charring effect with their dissolution behavior, providing a scientific base for understanding the applicability of different bone chars as suitable P-fertilizers.

Structural and Functional Insights Into Skl and Pal Endolysins, Two Cysteine-Amidases With Anti-pneumococcal Activity. Dithiothreitol (DTT) Effect on Lytic Activity.

We have structurally and functionally characterized Skl and Pal endolysins, the latter being the first endolysin shown to kill effectively Streptococcus pneumoniae, a leading cause of deathly diseases. We have proved that Skl and Pal are cysteine-amidases whose catalytic domains, from CHAP and Amidase_5 families, respectively, share an α3ß6-fold with papain-like topology. Catalytic triads are identified (for the first time in Amidase_5 family), and residues relevant for substrate binding and catalysis inferred from in silico models, including a calcium-binding site accounting for Skl dependence on this cation for activity. Both endolysins contain a choline-binding domain (CBD) with a ß-solenoid fold (homology modeled) and six conserved choline-binding loci whose saturation induced dimerization. Remarkably, Pal and Skl dimers display a common overall architecture, preserved in choline-bound dimers of pneumococcal lysins with other catalytic domains and bond specificities, as disclosed using small angle X-ray scattering (SAXS). Additionally, Skl is proved to be an efficient anti-pneumococcal agent that kills multi-resistant strains and clinical emergent-serotype isolates. Interestingly, Skl and Pal time-courses of pneumococcal lysis were sigmoidal, which might denote a limited access of both endolysins to target bonds at first stages of lysis. Furthermore, their DTT-mediated activation, of relevance for other cysteine-peptidases, cannot be solely ascribed to reversal of catalytic-cysteine oxidation.