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Phosphopantetheinyl hydrolase, PptH (Rv2795c), is a recently discovered enzyme from Mycobacterium tuberculosis that removes 4′-phosphopantetheine (Ppt) from holo-carrier proteins (CPs) and thereby opposes the action of phosphopantetheinyl transferases (PPTases). PptH is the first structurally characterized enzyme of the phosphopantetheinyl hydrolase family. However, conditions for optimal activity of PptH have not been defined, and only one substrate has been identified. Here, we provide biochemical characterization of PptH and demonstrate that the enzyme hydrolyzes Ppt in vitro from more than one M. tuberculosis holo-CP as well as holo-CPs from other organisms. PptH provided the only detectable activity in mycobacterial lysates that dephosphopantetheinylated acyl carrier protein M (AcpM), suggesting that PptH is the main Ppt hydrolase in M. tuberculosis. We could not detect a role for PptH in coenzyme A (CoA) salvage, and PptH was not required for virulence of M. tuberculosis during infection of mice. It remains to be determined why mycobacteria conserve a broadly acting phosphohydrolase that removes the Ppt prosthetic group from essential CPs. We speculate that the enzyme is critical for aspects of the life cycle of M. tuberculosis that are not routinely modeled. IMPORTANCE Tuberculosis (TB), caused by Mycobacterium tuberculosis, was the leading cause of death from an infectious disease before COVID, yet the in vivo essentiality and function of many of the protein-encoding genes expressed by M. tuberculosis are not known. We biochemically characterize M. tuberculosis’s phosphopantetheinyl hydrolase, PptH, a protein unique to mycobacteria that removes an essential posttranslational modification on proteins involved in synthesis of lipids important for the bacterium’s cell wall and virulence. We demonstrate that the enzyme has broad substrate specificity, but it does not appear to have a role in coenzyme A (CoA) salvage or virulence in a mouse model of TB.

The linking of polysaccharide in glycoconjugate vaccine with carrier protein is an imperative step to develop a strong memory response. The excessive use of similar carrier protein known to result in bystander immunity warrants an urgent need for new carrier protein. The preparation of the glycoconjugate vaccine using cyanylation chemistry is to link the active cyanate ester site of polysaccharide with the carrier protein. In the present study, transferrin binding protein-B (Tbp-B) has been explored as a new carrier protein to develop in silico pneumococcal polysaccharide serotype-5 (PnPs-5) conjugate vaccine. The homology model of Tbp-B was constructed using the Prime module and stereochemically validated using ProSA, PDBsum and ProQ. The selected model revealed a Z-score of -5.6 within the X-ray region in ProSA analysis, LGscore: 9.776, and MaxSub: 0.8 in protein quality predictor suggesting its preferred use. Loop modeling and active site analysis followed by in silico PnPs-5 activation with cyanalyting agent CDAP was docked with Tbp-B using Glide module. The complex stability of cyanate esters with Tbp-B, analyzed by molecular dynamics (MD) simulation, revealed an average RMSD of 2.49 angstrom for its binding to the receptor. The RMSF values of cyanate ester-1, -2, and -3 were observed to be 1.06, 1.39 and 0.79 angstrom, respectively. The higher RMSF of 1.39 angstrom of cyanate ester-2 was further found unstable which corroborates its non-binding to the protein and also incurring conformational changes to a carrier protein. Molecular simulations revealed that cyanate ester-1 and cyanate ester-3 formed stable conjugates with carrier protein Tbp-B. Communicated by Ramaswamy H. Sarma

Background PrePubertal children with type 1 diabetes mellitus are shorter than their non- diabetic peers. we aimed to evaluate the role of HbA1c and IGFBP-3 in this phenomenon. Aim of the work: The aim of the study was to evaluate the effect of glycemic control on the growth and IGFBP-3, represented by height, weight, height and weight percentiles for age in a sample of prepubertal Egyptian children with T1DM. Patients and Methods This study was a cross sectional study conducted on 80 pre-pubertal Egyptian children, divided into 25 Males and 25 Females with T1DM and 30 age matched controls (15 Males and 15 Females), the participants were recruited from the Outpatient Clinic of the Pediatric Department of Ain Shams University Hospitals and the National Institute of Diabetes and Endocrinology in Cairo, Egypt during the period from July 2018 to August 2019. Anthropometric measures including height and weight were obtained and used to calculate the height and weight percentiles using the CDC calculators. HbA1c as well as IGFBP-3 levels were tested. Results The mean age (years) of the participants was (9.671±2.24) for male patients, (9.22 ± 2.19) for female patients and (8.39 ± 2.034) for controls. The height found to be lower in the children with T1DM when compared to the disease-free controls with median values of (129.287 ± 13.410) in male patients, (127.727 ±10.155) in female patients and (136.760 ± 13.431) in the controls. the height and weight percentiles (%) were found to be lower in the children with T1DM when compared to the disease-free controls, the height percentile with median values of 14.54 (IQR 27.95) in male patients, 17.93 (IQR 29.20) in female patients and 87.07 (IQR 20.48) in the controls. the weight percentile with median values of 40.68 (IQR 40.83) in male patients, 30.64(IQR 35.59) in female patients and 85.18 (IQR 20.17) in the controls. A negative correlation between HbA1c (%) and serum IGFBP-3 (ng/ml), height and height percentile were found with (0.014, 0.049 and 0.012) as well as a positive correlation between serum IGFBP-3 and height, height and weight percentiles (%), were found with (,0.004, 0.009 and 0.005 respectively). Serum IGFBP-3 levels were also found to be significantly lower in patients (P < 0.001) with a mean value of (198.6 ± 45.335) in male patients and (168.4 ± 44.317) in female patients and (285.333 ± 61.936) in the disease-free controls. Conclusion Serum IGFBP-3 levels (ng/ml) as well as growth are negatively affected in prepubertal children with T1DM in relation to the glycemic control.

Introduction The public health burden caused by pathogenic Gram-negative bacteria is increasingly prominent due to antimicrobial resistance. The surface carbohydrates are potential antigens for vaccines against Gram-negative bacteria. The enhanced immunogenicity of the O-specific polysaccharide (O-SP) moiety of LPS when coupled to a carrier protein may protect against bacterial pathogens. However, because of the toxic lipid A moiety and relatively high costs of O-SP isolation, LPS has not been a popular vaccine antigen until recently. Areas covered In this review, we discuss the rationales for developing LPS-based glycoconjugate vaccines, principles of glycoconjugate-induced immunity, and highlight the recent developments and challenges faced by LPS-based glycoconjugate vaccines. Expert opinion Advances in LPS harvesting, LPS chemical synthesis, and newer carrier proteins in the past decade have propelled LPS-based glycoconjugate vaccines toward further development, through to clinical evaluation. The development of LPS-based glycoconjugates offers a new horizon for vaccine prevention of Gram-negative bacterial infection.

Outer Membrane Vesicles (OMV) constitute a promising platform for the development of efficient vaccines. OMV can be decorated with heterologous antigens (proteins or polysaccharides), becoming attractive novel carriers for the development of multicomponent vaccines. Chemical conjugation represents a tool for linking antigens, also from phylogenetically distant pathogens, to OMV. Here we develop two simple and widely applicable conjugation chemistries targeting proteins or lipopolysaccharides on the surface of Generalized Modules for Membrane Antigens (GMMA), OMV spontaneously released from Gram-negative bacteria mutated to increase vesicle yield and reduce potential reactogenicity. A Design of Experiment approach was used to identify optimal conditions for GMMA activation before conjugation, resulting in consistent processes and ensuring conjugation efficiency. Conjugates produced by both chemistries induced strong humoral response against the heterologous antigen and GMMA. Additionally, the use of the two orthogonal chemistries allowed to control the linkage of two different antigens on the same GMMA particle. This work supports the further advancement of this novel platform with great potential for the design of effective vaccines.

Nanoparticle systems are being explored for the display of carbohydrate antigens, characterized by multimeric presentation of glycan epitopes and special chemico-physical properties of nano-sized particles. Among them, outer membrane vesicles (OMVs) are receiving great attention, combining antigen presentation with the immunopotentiator effect of the Toll-like receptor agonists naturally present on these systems. In this context, we are testing Generalized Modules for Membrane Antigens (GMMA), OMVs naturally released from Gram-negative bacteria mutated to increase blebbing, as carrier for polysaccharides. Here, we investigated the impact of saccharide length, density, and attachment site on the immune response elicited by GMMA in animal models, using a variety of structurally diverse polysaccharides from different pathogens (i.e.,Neisseria meningitidisserogroup A and C,Haemophilus influenzaetype b, and streptococcus Group A Carbohydrate andSalmonellaTyphi Vi). Anti-polysaccharide immune response was not affected by the number of saccharides per GMMA particle. However, lower saccharide loading can better preserve the immunogenicity of GMMA as antigen. In contrast, saccharide length needs to be optimized for each specific antigen. Interestingly, GMMA conjugates induced strong functional immune response even when the polysaccharides were linked to sugars on GMMA. We also verified that GMMA conjugates elicit a T-dependent humoral immune response to polysaccharides that is strictly dependent on the nature of the polysaccharide. The results obtained are important to design novel glycoconjugate vaccines using GMMA as carrier and support the development of multicomponent glycoconjugate vaccines where GMMA can play the dual role of carrier and antigen. In addition, this work provides significant insights into the mechanism of action of glycoconjugates.

Multiple functions of glomalin released by arbuscular mycorrhizal fungi are well-recognized, whereas the role of exogenous glomalins including easily extractable glomalin-related soil protein (EE-GRSP) and difficultly extractable glomalin-related soil protein (DE-GRSP) is unexplored for plant responses. Our study was carried out to assess the effects of exogenous EE-GRSP and DE-GRSP at varying strengths on plant growth and chlorophyll concentration of trifoliate orange (Poncirus trifoliata) seedlings, along with changes in root nutrient acquisition, auxin content, auxin-related enzyme and transporter protein gene expression, and element contents of purified GRSP. Sixteen weeks later, exogenous GRSP displayed differential effects on plant growth (height, stem diameter, leaf number, and biomass production): the increase by EE-GRSP and the decrease by DE-GRSP. The best positive effect on plant growth occurred at exogenous EE-GRSP at ½ strength. Similarly, the GRSP application also differently affected total chlorophyll content, root morphology (total length, surface area, and volume), and root N, P, and K content: positive effect by EE-GRSP and negative effect by DE-GRSP. Exogenous EE-GRSP accumulated more indoleacetic acid (IAA) in roots, which was associated with the upregulated expression of root auxin synthetic enzyme genes (PtTAA1, PtYUC3, and PtYUC4) and auxin influx transporter protein genes (PtLAX1, PtLAX2, and PtLAX3). On the other hand, exogenous DE-GRSP inhibited root IAA and indolebutyric acid (IBA) content, associated with the downregulated expression of root PtTAA1, PtLAX1, and PtLAX3. Root IAA positively correlated with root PtTAA1, PtYUC3, PtYUC4, PtLAX1, and PtLAX3 expression. Purified EE-GRSP and DE-GRSP showed similar element composition but varied in part element (C, O, P, Ca, Cu, Mn, Zn, Fe, and Mo) concentration. It concluded that exogenous GRSP triggered differential effects on growth response, and the effect was associated with the element content of pure GRSP and the change in auxins and root morphology. EE-GRSP displays a promise as a plant growth biostimulant in citriculture.

Despite the recent successes in siRNA therapeutics, targeted delivery beyond the liver remains the major hurdle for the widespread application of siRNA in vivo. Current cationic liposome or polymer-based delivery agents are restricted to the liver and suffer from off-target effect, poor clearance, low serum stability, and high toxicity. In this study, we have genetically engineered a non-cationic tumor-targeted universal siRNA nanocarrier. This protein nanocarrier consists of three function domains: dsRNA binding domain (dsRBD) (from human protein kinase R) for any siRNA binding, 18-histidines for endosome escape, and two RGD peptides at N-and C-termini for targeting tumor and tumor neovasculature. We showed that cloned dual-RGD-dsRBD-18his (dual-RGD) protein protects siRNA against RNases, induces effective siRNA endosomal escape, specific targets on integrin αvβ3 expressing cells in vitro, and homes siRNA to tumor in vivo. The delivered siRNA leads target gene knockdown in the cell lines and tumor xenografts with low toxicity. This multifunctional, biomimetic, charge-neutral siRNA carrier is biodegradable, low toxic, suitable for mass production by fermentation, and serum stable, holding great potential to provide a widely applicable siRNA carrier for tumor-targeted siRNA delivery.

The functional and structural evaluation of AryC, the cytochrome P450 performing biaryl coupling in biosynthetic arylomycin assembly, and its application in the chemo-enzymatic synthesis of arylomycin A2 is described. AryC efficiently converts free substrates without the requirement of any protein interaction partner, likely enabled by a strongly hydrophobic cavity at the surface of AryC pointing to the substrate tunnel. The resulting reactivity of AryC is unprecedented in cytochrome P450-mediated biaryl construction in non-ribosomal peptides, in which PCP-tethering so far was crucial both in vivo and in vitro. Our work thus provides a basis for the development of general biocatalytic platforms for the efficient biocatalytic synthesis of biaryl peptide antibiotics.

Long-term flight depends heavily on intensive energy metabolism in animals; however, the neuroendocrine mechanisms underlying efficient substrate utilization remain elusive. Here, we report that the adipokinetic hormone/corazonin-related peptide (ACP) can facilitate muscle lipid utilization in a famous long-term migratory flighting species, Locusta migratoria. By peptidomic analysis and RNAi screening, we identified brain-derived ACP as a key flight-related neuropeptide. ACP gene expression increased notably upon sustained flight. CRISPR/Cas9-mediated knockout of ACP gene and ACP receptor gene (ACPR) significantly abated prolonged flight of locusts. Transcriptomic and metabolomic analyses further revealed that genes and metabolites involved in fatty acid transport and oxidation were notably downregulated in the flight muscle of ACP mutants. Finally, we demonstrated that a fatty-acid-binding protein (FABP) mediated the effects of ACP in regulating muscle lipid metabolism during long-term flight in locusts. Our results elucidated a previously undescribed neuroendocrine mechanism underlying efficient energy utilization associated with long-term flight., eLife digest Flight allows insects to find food or seek a better environment. Some insects have developed the ability of ‘long-term flight’, which allows them to make continuous journeys over large distances. For example, one locust species regularly crosses the Red Sea which is up to 300 km wide – a spectacular feat for insects only a few inches long. However, flight is an energy-intensive activity, and insects’ muscles need the right sort of chemical fuel to work properly. Previous work has shown that this ‘fuel consumption’ is highly dynamic and happens in two stages. First, immediately after take-off, the muscles rapidly consume carbohydrates (sugars); then, during the prolonged phase of the flight, muscles switch to exclusively consume lipids (fats). How the flight muscles ‘know’ when to start using fats for energy remains largely unclear. It has been suggested that this switch may involve hormone-like chemicals made in the brain called neuroendocrine peptides. Hou et al. therefore set out to test this hypothesis, using the locust species Locusta migratoria as a representative migratory insect. Initial experiments used an abundance detection technique to determine which of the neuroendocrine peptides were active in adult locusts. Further analysis, looking specifically at locusts that had just been flying, revealed that the gene for a peptide called ACP became much more active after one hour of continuous flight. Further evidence that the ACP hormone could indeed be helping to power long-term flight came from locusts with a mutated, ‘switched-off’ version of the gene. These insects could only fly for half the time, and half the distance, compared to locusts that did not have mutations in the gene for ACP. Biochemical studies of the ACP mutant locusts confirmed that their flight muscle cells could not transport and break down fatty acids normally. These experiments also showed that ACP was acting through a type of carrier protein called FABP, which is present in many different insects and normally ‘ferries’ lipids to the places they are needed. These findings shed new light on the biological mechanisms that control long-term flight in migratory insects. The ability to move over long distances is key to the outbreak of locust plagues, which in turn cause widespread crop damage around the world. Hou et al. therefore hope that this knowledge will one day help develop effective strategies for locust pest control.

Structure-based drug discovery efforts require knowledge of where drug-binding sites are located on target proteins. To address the challenge of finding druggable sites, we developed a machine-learning algorithm called TACTICS (Trajectory-based Analysis of Conformations To Identify Cryptic Sites), which uses an ensemble of molecular structures (such as molecular dynamics simulation data) as input. First, TACTICS uses k-means clustering to select a small number of conformations that represent the overall conformational heterogeneity of the data. Then, TACTICS uses a random forest model to identify potentially bindable residues in each selected conformation, based on protein motion and geometry. Lastly, residues in possible binding pockets are scored using fragment docking. As proof-of-principle, TACTICS was applied to the analysis of simulations of the SARS-CoV-2 main protease and methyltransferase and the Yersinia pestis aryl carrier protein. Our approach recapitulates known small-molecule binding sites and predicts the locations of sites not previously observed in experimentally determined structures. The TACTICS code is available at https://github.com/Albert-Lau-Lab/tactics_protein_analysis.

OBJECTIVES: Background: South Asian Indians (SAI) have a higher prevalence of both chronic diseases and vitamin D deficiency compared to Caucasians. Previous research shows that vitamin D status is a determinant of metabolic health. SAIs have higher total fat, lower lean mass, and higher visceral or truncal fat compared to Caucasians. These factors are all associated with chronic health conditions at a younger age than their Caucasian counterparts. OBJECTIVE: The study aimed to determine the association between body composition parameters and the biomarkers of vitamin D status and whether these relationships differed between Caucasian and SAI cohorts. METHODS: Healthy SAIs and Caucasian men, who were 22 to 60 years of age, were enrolled. Anthropometric and body composition measurements, serum levels of 25 Hydroxy-vitamin D (25OHD), Parathyroid Hormone, and Vitamin D Binding-Protein were obtained. Independent t-test and Mann-Whitney u test were used to report the differences between groups. Spearman correlation was used to identify the possible association. A P value of less than 0.05 was considered significant. RESULTS: 30 SAIs and 30 Caucasians completed the study. Both groups did not significantly differ in their age and BMI. The 25OHD levels were statistically different between Caucasians (31.24 ± 9.14 ng/mL) and SAIs (22.94 ± 7.19 ng/mL), (P

International audience; Non-ribosomal peptide synthetases (NRPSs) are multienzymes that produce complex natural metabolites with many applications in medicine and agriculture. They are composed of numerous catalytic domains that elongate and chemically modify amino acid substrates or derivatives and of non-catalytic carrier protein domains that can tether and shuttle the growing products to the different catalytic domains. The intrinsic flexibility of NRPSs permits conformational rearrangements that are required to allow interactions between catalytic and carrier protein domains. Their large size coupled to this flexibility renders these multi-domain proteins very challenging for structural characterization. Here, we summarize recent studies that offer structural views of multi-domain NRPSs in various catalytically relevant conformations, thus providing an increased comprehension of their catalytic cycle. A better structural understanding of these multienzymes provides novel perspectives for their re-engineering to synthesize new bioactive metabolites.

Toxicity is one of the most important factors limiting the success of new drug development. In this paper, we built a fast and convenient new method (Carrier protein binding information-toxicity relationship, CPBITR) for predicting drug acute toxicity based on the perspective of binding information with carrier protein. First, we studied the binding information between carbamate pesticides and human serum albumin (HSA) through various spectroscopic methods and molecular docking. Then a total of 16 models were established to clarify the relationship between binding information with HSA and drug toxicity. The results showed that the binding information was related to toxicity. Finally we obtained the effective toxicity prediction model for carbamate pesticides. And the “Platform for Predicting Drug Toxicity Based on the Information of Binding with Carrier Protein” was established with the Back-propagation neural network model. We proposed and proved that it was feasible to predict drug toxicity from this new perspective: binding with carrier protein. According to this new perspective, toxicity prediction model of other drugs can also be established. This new method has the advantages of convenience and fast, and can be used to screen out low-toxic drugs quickly in the early stage. It is helpful for drug research and development.

Background and Aims Diabetes mellitus, either preexisting or developing after kidney transplantation remains a crucial clinical problem. The aim of this study is to compare the proteome of histologically normal glomeruli from normoglycemic (NG), T2DM, and PTDM patients one year after kidney transplantation to identify novel early biomarkers detectable prior to the development of histologically visible diabetic nephropathy. Method We comparatively analyzed the proteome of histologically normal glomeruli from normoglycemic (NG) recipients, and recipients with pre-existing type 2 diabetes mellitus (T2DM), or post-transplant diabetes mellitus (PTDM), in protocol biopsies obtained one year after kidney transplantation. Glomerular cross-sections were microdissected in core biopsies from 8 NG, 8 PTDM and 8 T2DM kidney transplant recipients. Proteome was determined by liquid chromatography-tandem mass spectrometry. Relative differences in protein abundance and significantly dysregulated pathways were analyzed. Results Proteins related to immune response and inflammation, transport regulation and cell organization and communication, including the nephrin family, were more abundant in NG, as compared to the combined groups of diabetic patients. Proteins involved in cell morphogenesis and adhesion were less abundant in PTDM, as compared to T2DM. In contrast, CCT3, CCT4 and CNDP2 diabetic nephropathy markers, LDHB and tacrolimus binding protein FKBP1A were significantly overrepresented in glomeruli from PTDM, as compared to T2DM patients. Conclusion These data suggest that glomerular proteome profile differentiates PTDM from NG and T2DM, and disruption of cell-cell interactions at molecular level represents an early event in PTDM development.

Pathological deposition of collagen is a hallmark of renal fibrosis. We employed multimodal optical imaging to visualize and quantify collagen deposition in murine models of kidney fibrosis using the collagen-binding agent CNA35. For in vivo imaging, we used hybrid computed tomography-fluorescence molecular tomography (CT-FMT) and CNA35 labeled with the near-infrared fluorophore Cy7. Upon i.v. injection, CNA35-Cy7 accumulation was significantly higher in fibrotic kidneys compared to non-fibrotic kidneys. This difference was not detected for a non-specific scrambled version of CNA35-Cy7. Ex vivo, on kidney sections of animals and patients with renal fibrosis, CNA35-FITC co-localized with scar collagen type I and III, but not with the basement membrane collagen type IV. Upon i.v. injection, CNA35-FITC bound to both interstitial and perivascular fibrotic areas. In line with this perivascular accumulation, we observed significant perivascular fibrosis in animal models and patients using computer-based morphometry quantification. In conclusion, molecular imaging of collagen using CNA35 enables specific non-invasive quantification of renal fibrosis. Collagen imaging revealed significant perivascular fibrosis as a consistent component next to the more commonly assessed interstitial fibrosis. Our results lay the basis for further probe and protocol optimization towards the clinical translation of molecular imaging of kidney fibrosis.

Purpose. The prevalence of adrenal insufficiency (AI) in patients with decompensated liver cirrhosis is unknown. Because these patients have lower levels of cortisol-binding carrier proteins, their total serum cortisol (TSC) correlates poorly with free serum cortisol (FC). Salivary cortisol (SaC) correlates better with FC. We aimed to establish SaC thresholds for AI for the 250 g intravenous ACTH test and to estimate the prevalence of AI in non-critically ill cirrhotic patients. Methods. We included 39 patients with decompensated cirrhosis, 39 patients with known AI, and 45 healthy volunteers. After subjects fasted ≥8 hours, serum and saliva samples were collected for determinations of TSC and SaC at baseline 0’ (T0) and at 30-minute intervals after intravenous administration of 250

Enterococci have emerged as important nosocomial pathogens due to their resistance to the most commonly used antibiotics. Alternative treatments or prevention options are aimed at polysaccharides and surface-related proteins that play important roles in pathogenesis. Previously, we have shown that 2 Enterococcus faecium proteins, the secreted antigen A and the peptidyl-prolyl cis-trans isomerase, as well as the Enterococcus faecalis polysaccharide diheteroglycan, are able to induce opsonic and cross-protective antibodies. Here, we evaluate the use of glycoconjugates consisting of these proteins and an enterococcal polysaccharide to develop a vaccine with broader strain coverage. Diheteroglycan was conjugated to these 2 enterococcal proteins. Rabbit sera raised against these glycoconjugates showed Immunoglobulin G titers against the corresponding conjugate, as well as against the respective protein and carbohydrate antigens. Effective opsonophagocytic killing for the 2 sera was observed against different E. faecalis and E. faecium strains. Enzyme-linked immunosorbent assays against whole bacterial cells showed immune recognition of 22 enterococcal strains by the sera. Moreover, the sera conferred protection against E. faecalis and E. faecium strains in a mouse infection model. Our results suggest that these glycoconjugates are promising candidates for vaccine formulations with a broader coverage against these nosocomial pathogens and that the evaluated proteins are potential carrier proteins., Enterococcus faecium immunogenic proteins, the secreted antigen A and the peptidyl-prolyl cis-trans isomerase, conjugated to the immunogenic Enterococcus faecalis polysaccharide diheteroglycan are promising vaccine antigens with cross-species opsonic and protective potential to fight infections caused by these nosocomial pathogens.

Engineering microbial biosynthetic pathways represents a compelling route to gain access to expanded chemical diversity. Carrier proteins (CPs) play a central role in biosynthesis, but the fast motions of CPs make their conformational dynamics difficult to capture using traditional spectroscopic approaches. Here we present a low-resource method to directly reveal carrier protein-substrate interactions. Chemoenzymatic loading of commercially available, alkyne-containing substrates onto CPs enables rapid visualization of the molecular cargo’s local environment using Raman spectroscopy. This method could clarify the foundations of the chain sequestration mechanism, facilitate the rapid characterization of CPs, and enable visualization of the vectoral processing of natural products both in vitro and in vivo., Acyl carrier proteins (ACPs), a universal and highly conserved carrier of acyl intermediates during fatty acid and polyketide synthesis, are difficult to visualise. Here, the authors developed a facile, Raman spectroscopy-based method to detect ACP-substrate interactions.

The brain has specific properties that make it uniquely dependent upon transporters. This is the 3rd edition of a biennial special issue that originates from a scientific meeting devoted to studies of transporters and their relationship to brain function and to neurodevelopmental, neurologic, and psychiatric disorders. The field continues to rapidly evolve with advances in studies of structure that inform mechanism, with genetic analyses in humans revealing surprising aspects of biology, and with integrated cellular to whole animal analyses of the role of transporters in their control of physiology and pathophysiology. This special issue includes a sampling of review articles that address timely questions of the field followed by several primary research articles.

Among the age-dependent protein aggregation disorders, nine neurodegenerative diseases are caused by expansions of CAG repeats encoding polyglutamine (polyQ) tracts. We review the clinical, pathological, and biological features of these inherited disorders. We discuss insights into pathogenesis gleaned from studies of model systems and patients, highlighting work that informs efforts to develop effective therapies. An important conclusion from these analyses is that expanded CAG/polyQ domains are the primary drivers of neurodegeneration, with the biology of carrier proteins influencing disease-specific manifestations. Additionally, it has become apparent that CAG/polyQ repeat expansions produce neurodegeneration via multiple downstream mechanisms, involving both gain- and loss-of-function effects. This conclusion indicates that the likelihood of developing effective therapies targeting single nodes is reduced. The evaluation of treatments for premanifest disease will likely require new investigational approaches. We highlight the opportunities and challenges underlying ongoing work and provide recommendations related to the development of symptomatic and disease-modifying therapies and biomarkers that could inform future research.

Ultracentrifugation remains the gold standard for isolation of small extracellular vesicles (sEV), particularly for cancer applications. The objective of this study was to determine if a widely used ultracentrifugation protocol for isolation of serum sEV could be modified to reduce the number of ultracentrifugation cycles and increase efficiency, while maintaining equal or better sample purity and yield. Serum was obtained from two healthy subjects. sEVs were isolated from 1 mL aliquots using three different ultracentrifugation protocols. Co-isolation of RNA carrier protein was assessed by performing Western blots for ApoA-I, ApoB, and Ago2. Small RNA-sequencing was performed on the sEV isolates, and differential detection of small ncRNA was compared across isolation protocols. Reduction from three- to two-ultracentrifuge cycles with no sucrose cushion resulted in a much higher sEV yield but also had the highest levels of lipoprotein and Ago2 contamination. However, the two-ultracentrifugation cycle protocol that incorporated a 30% sucrose cushion into the first cycle resulted in slightly higher sEV yields with lower levels of protein contamination compared to the lengthier three-ultracentrifugation cycle approach, therefore presenting a more efficient alternative approach for isolation of serum sEVs. It was also notable that there were some differences in sEV ncRNA cargo according to protocol, although it was less than expected given the differences in co-isolated RNA carrier proteins. Our results suggest that use of the modified serum sEV isolation protocol with two ultracentrifugation cycles and incorporating a 30% sucrose cushion offers a more efficient approach in terms of efficiency and purity.

International audience; Tetanus toxoid (TTd) plays an important role in the pharmaceutical world, especially in vaccines. The toxoid is obtained after formaldehyde treatment of the tetanus toxin. In parallel, current emphasis in the drug discovery field is put on producing well-defined and safer drugs, explaining the interest in finding new alternative proteins. The tetanus toxin fragment C (TTFC) has been extensively studied both as a neuroprotective agent for central nervous system disorders owing to its neuronal properties and as a carrier protein in vaccines. Indeed, it is derived from a part of the tetanus toxin and, as such, retains its immunogenic properties without being toxic. Moreover, this fragment has been well characterized, and its entire structure is known. Here, we propose a systematic review of TTFC by providing information about its structural features, its properties and its methods of production. We also describe the large uses of TTFC in the field of drug discovery. TTFC can therefore be considered as an attractive alternative to TTd and remarkably offers a wide range of uses, including as a carrier, delivery vector, conjugate, booster, inducer, and neuroprotector

The aim of this study was to examine the interaction of [Pd(2,2′-bipyridine) (morpholinedithiocarbamate)]NO3and [Pt (2,2′-bipyridine)(morpholinedithiocarbamate)]NO3with human serum albumin under physiological conditions by using fluorescence, absorption, and circular dichroism spectroscopic techniques. Spectroscopic analysis of the emission quenching at different temperatures demonstrated that the quenching mechanism was static quenching. From the circular dichroism results, thermal stability study, it was found that the interaction of the complexes with human serum albumin caused a conformational change of the protein reversibly. These 2 anticancer Pd and Pt complexes were activated against chronic myelogenous leukemia cell line K562, so that 50% cytotoxic concentration values of 16 and 26 μM for Pd and Pt complexes, respectively, were observed, which were much lower than that of cisplatin (154 μM). Biological activities of both Pd and Pt complexes were also assayed against selective microorganisms by the disc diffusion method. These results showed that the Pd(II) complex is antifungal agent but Pt(II) complex has antibacterial activity. Also, the interaction of both metal derivative complexes was studied by molecular docking. Complementary molecular docking results may be useful to determine the binding mechanism of human serum albumin in pharmaceutical and biophysical studies providing new insight in the novel pharmacology.

Many families of natural products are synthesized by large multidomain biological machines commonly referred to as megasynthases. While the advance of mechanism-based tools has opened new windows into the structural features within the protein • protein interfaces guiding carrier protein dependent enzymes, there is an immediate need for tools that can be engaged to link co-translated domains in a site-selective manner. Here, we demonstrate the use of silylcyanohydrins in a two-step, two-site selective crosslinking for the trapping of carrier protein interactions within megasynthases. This advance provides a new tool to trap intermediate states within multimodular systems, a key step toward understanding the specificities within fatty acid (FAS) and polyketide (PKS) synthases.

To compose the experiment, 160 male Topgen pigs - 80 surgically castrated (treatment 1) and 80 immunocastrated (treatment 2) - were randomly selected from a commercial swine farm at the moment of slaughter. Surgical castration was performed when the animals were seven days of age and immunocastration was performed by administering two doses (2 mL each) of immunocastration vaccine (analogue of GnRF linked to a carrier protein, development of anti-GnRF antibodies, 200 mg of a GnRF-protein conjugate/mL) when they were 104 and 132 days of age. Animals from both treatments were maintained in masonry stalls, where they received water and ad libitum diet (the same feed for both groups). The animals were slaughtered at 160 days of age, and the length and width of the testicles of immunocastrated animals were evaluated, along with the degree and number of carcass lesions, carcass traits and meat quality of both treatments. The means of these measurements were calculated and compared by Student´s t-test. For the immunocastrated treatment, Pearson´s correlation coefficients were also calculated for testicle length and width with backfat thickness. Approximately 80% of the immunocastrated animals had testicle widths of 11 cm or less. Immunocastrated animals showed higher degrees of lesions, pH (initial and 8 hours), hue, muscle depth and loin eye area and lower brightness, redness, chroma and backfat thickness than the surgically castrated animals. The correlation between testicle length and width with backfat thickness was inverse. Immunocastration can be an alternative to improve the proportion of lean meat (longissimus thoracis) instead of fat (backfat thickness) leading to better carcass and meat quality, since fat has become undesirable from a nutritional point of view in swine.