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Abstract Understanding protein function is pivotal in comprehending the intricate mechanisms that underlie many crucial biological activities, with far-reaching implications in the fields of medicine, biotechnology, and drug development. However, more than 200 million proteins remain uncharacterized, and computational efforts heavily rely on protein structural information to predict annotations of varying quality. Here, we present a method that utilizes statistics-informed graph networks to predict protein functions solely from its sequence. Our method inherently characterizes evolutionary signatures, allowing for a quantitative assessment of the significance of residues that carry out specific functions. PhiGnet not only demonstrates superior performance compared to alternative approaches but also narrows the sequence-function gap, even in the absence of structural information. Our findings indicate that applying deep learning to evolutionary data can highlight functional sites at the residue level, providing valuable support for interpreting both existing properties and new functionalities of proteins in research and biomedicine.

The objective of the work presented here was to assess the feasibility of using glucosamine HCl as a solid-dispersion (SD) carrier to enhance the biopharmaceutical properties of a BCS class III/IV drug, acyclovir (ACV). The solid-dispersions of acyclovir and glucosamine HCl were prepared by an ethanol-based solvent evaporation method. The prepared formulations characterized by photomicroscopy, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier transforms infrared spectrophotometry (FTIR), powder x-ray diffractometry (PXRD) and drug content analysis. The functional characterization of ACV-SD was performed by aqueous solubility evaluation, dissolution studies, fasted versus fed state dissolution comparison, ex vivo permeability, and stability studies. Photomicroscopy and SEM analysis showed different surface morphologies for pure ACV, glucosamine HCl and ACV-SD. The physical-chemical characterization studies supported the formation of ACV-SD. A 12-fold enhancement in the aqueous solubility of ACV was observed in the prepared solid dispersions, compared to pure ACV. Results from in vitro dissolution demonstrated a significant increase in the rate and extent of ACV dissolution from the prepared ACV-SD formulations, compared to pure ACV. The rate and extent of ACV permeability across everted rat intestinal membrane were also found to be significantly increased in the ACV-SD formulations. Under fed conditions, the rate and extent of the in vitro dissolution of ACV from the formulation was appreciably greater compared to fasted conditions. Overall, the results from the study suggest the feasibility of utilizing glucosamine HCl as a solid dispersion carrier/excipient for enhancement of biopharmaceutical properties of acyclovir, and similar drugs with low solubility/permeability characteristics.

Aims: The timing of when to remove a circular frame is crucial; early removal results in refracture or deformity, while late removal increases the patient morbidity and delay in return to work. This study was designed to assess the effectiveness of a staged reloading protocol. We report the incidence of mechanical failure following both single-stage and two stage reloading protocols and analyze the associated risk factors. Methods: We identified consecutive patients from our departmental database. Both trauma and elective cases were included, of all ages, frame types, and pathologies who underwent circular frame treatment. Our protocol is either a single-stage or two-stage process implemented by defunctioning the frame, in order to progressively increase the weightbearing load through the bone, and promote full loading prior to frame removal. Before progression, through the process we monitor patients for any increase in pain and assess radiographs for deformity or refracture. Results: There were 244 frames (230 patients) included in the analyses, of which 90 were Ilizarov type frames and 154 were hexapods. There were 149 frames which underwent single-stage reloading and 95 frames which underwent a two-stage reloading protocol. Mechanical failure occurred after frame removal in 13 frames (5%), which suffered refracture. There were no cases of change in alignment. There was no difference between refracture patients who underwent single-stage or two-stage reloading protocols (p = 0.772). In all, 14 patients had failure prevented through identification with the reloading protocol. Conclusion: Our reloading protocol is a simple and effective way to confirm the timing of frame removal and minimize the rate of mechanical failure. Similar failure rates occurred between patients undergoing single-stage and two-stage reloading protocols. If the surgeon is confident with clinical and radiological assessment, it may be possible to progress directly to stage two and decrease frame time and patient morbidity. Cite this article: Bone Jt Open 2022;3(5):359–366.

The non-vesicular transport of lipids between organelles mediated by lipid transport proteins (LTPs) is a key determinant of organelle biogenesis and function. Despite performing a vital function in organelle homeostasis, none of the LTP-encoding genes identified so far are truly essential, even in the simple genome of yeast, suggesting widespread redundancy. In line with this fact, it has been found that a number of LTPs have overlapping functions, making it challenging to assign unique roles for an individual LTP in lipid distribution. In our genetic screens under stringent conditions in which the distinct function of an LTP might become essential, we stumbled upon Csf1, a highly conserved protein with a Chorein-N motif found in other lipid transporters and unraveled a new function for Csf1 in lipid remodeling and homeoviscous adaptation of the lipidome. Here, we further speculate on the potential mechanisms of how the putative function of Csf1 in lipid transport could be intimately connected to its role in lipid remodeling across organelles.

Information on the co-evolution of amino acid pairs in a protein can be used for endeavors such as protein engineering, mutation design, and structure prediction. Here we report a method that captures significant determinants of proteins using estimated co-evolution information to identify networks of residues, termed ”residue communities”, relevant to protein function. On the benchmark dataset (67 proteins with both catalytic and allosteric residues), the Pearson’s correlation between the identified residues in the communities at functional sites is 0.53, and it is higher than 0.8 by taking account of conserved residues derived from the method. On the endoplasmic reticulum-mitochondria encounter structure complex, the results indicate three distinguishable residue communities that are relevant to functional roles in the protein family, suggesting that the residue communities could be general evolutionary signatures in proteins. Based on the method, we provide a webserver for the scientific community to explore the signatures in protein families, which establishes a powerful tool to analyze residue-level profiling for the discovery of functional sites and biological pathway identification. This web-server is freely available for non-commercial users at https://kornmann.bioch.ox.ac.uk/leri/services/ecs.html, neither login nor e-mail required.

The objective of the work presented here was to assess the feasibility of using glucosamine HCl as a solid-dispersion (SD) carrier to enhance the biopharmaceutical properties of a BCS class III/IV drug, acyclovir (ACV). The solid-dispersions of acyclovir and glucosamine HCl were prepared by an ethanol-based solvent evaporation method. The prepared formulations characterized by photomicroscopy, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier transform infrared spectrophotometry (FTIR), powder x-ray diffractometry (PXRD) and drug content analysis. The functional characterization of ACV-SD was performed by aqueous solubility evaluation, dissolution studies, fasted versus fed state dissolution comparison, ex vivo permeability, and stability studies. Photomicroscopy and SEM analysis showed different surface morphologies for pure ACV, glucosamine HCl and ACV-SD. The physical-chemical characterization studies supported the formation of ACV-SD. A 12-fold enhancement in the aqueous solubility of ACV was observed in the prepared solid dispersions, compared to pure ACV. Results from in vitro dissolution demonstrated a significant increase in the rate and extent of ACV dissolution from the prepared ACV-SD formulations, compared to pure ACV. The rate and extent of ACV permeability across everted rat intestinal membrane were also found to be significantly increased in the ACV-SD formulations. Under fed conditions, the rate and extent of the in vitro dissolution of ACV from the formulation was appreciably greater compared to fasted conditions. Overall, the results from the study suggest the feasibility of utilizing glucosamine HCl as a solid dispersion carrier/excipient for enhancement of biopharmaceutical properties of acyclovir, and similar drugs with low solubility/permeability characteristics.

Objectives: To report on unique cases of spinal compression fractures due to pregnancy associated osteoporosis (PAO) and to suggest a satisfactory treatment modality. Materials and Methods: A single center retrospective study. We reviewed the data of 535 patients with osteoporotic spinal compression fractures over a period of 5 year. Two patients who developed spinal compression fractures due to PAO were identified and treated. Results: The clinical presentation and blood investigations ruled out other causes of osteoporosis. Dual energy X ray absorptiometry was used to confirm the diagnosis. All patients improved with medical management. Conclusion: Vertebral fractures due to PAO should be considered as a differential diagnosis in patients with back pain who are in the third trimester of pregnancy or in postpartum. Early recognition and appropriate conservative management would be necessary to prevent complications such as new vertebral fractures and chronic back pain.

The current work describes the formulation and evaluation of a phospholipid complex of kaempferol to enhance the latter’s aqueous solubility, in vitro dissolution rate, in vivo antioxidant and hepatoprotective activities, and oral bioavailability. The kaempferol-phospholipid complex was synthesized using a freeze-drying method with the formulation being optimized using a full factorial design (32) approach. Our results include the validation of the mathematical model in order to ascertain the role of specific formulation and process variables that contribute favorably to the formulation’s development. The final product was characterized and confirmed by Differential Scanning Calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR), Proton Nuclear Magnetic Resonance Spectroscopy (1H-NMR), and Powder X-ray Diffraction (PXRD) analysis. The aqueous solubility and the in vitro dissolution rate were enhanced compared to that of pure kaempferol. The in vivo antioxidant properties of the kaempferol-phospholipid complex were evaluated by measuring its impact on carbon tetrachloride (CCl4)-intoxicated rats. The optimized phospholipid complex improved the liver function test parameters to a significant level by restoration of all elevated liver marker enzymes in CCl4-intoxicated rats. The complex also enhanced the in vivo antioxidant potential by increasing levels of GSH (reduced glutathione), SOD (superoxide dismutase), catalase and decreasing lipid peroxidation, compared to that of pure kaempferol. The final optimized phospholipid complex also demonstrated a significant improvement in oral bioavailability demonstrated by improvements to key pharmacokinetic parameters, compared to that of pure kaempferol.

BACKGROUND: In the search for more potent and safer tuberculosis vaccines, CAF01 was identified as a remarkable formulation. Based on cationic liposomes and including a synthetic mycobacterial glycolipid as TLR-independent immunomodulator, it induces strong and protective T helper-1 and T helper-17 adult murine responses to Ag85B-ESAT-6, a major mycobacterial fusion protein. Here, we assessed whether these properties extend to early life and how CAF01 mediates its adjuvant properties in vivo. METHODS/FINDINGS: Following adult or neonatal murine immunization, Ag85B-ESAT-6/CAF01 similarly reduced the post-challenge bacterial growth of M. bovis BCG, whereas no protection was observed using Alum as control. This protection was mediated by the induction of similarly strong Th1 and Th17 responses in both age groups. Multifunctional Th1 cells were already elicited after a single vaccine dose and persisted at high levels for at least 6 months even after neonatal priming. Unexpectedly, this potent adjuvanticity was not mediated by a massive targeting/activation of dendritic cells: in contrast, very few DCs in the draining lymph nodes were bearing the labeled antigen/adjuvant. The increased expression of the CD40 and CD86 activation markers was restricted to the minute portion of adjuvant-bearing DCs. However, vaccine-associated activated DCs were recovered several days after immunization. CONCLUSION: The potent adult and neonatal adjuvanticity of CAF01 is associated in vivo with an exquisite but prolonged DC uptake and activation, fulfilling the preclinical requirements for novel tuberculosis vaccines to be used in early life.

BACKGROUND: With the exception of some live vaccines, e.g. BCG, subunit vaccines formulated with "classical" adjuvants do not induce similar responses in neonates as in adults. The usual neonatal profile is characterized by lower levels of TH1-associated biomarkers. This has hampered the development of new neonatal vaccines for diseases that require early protection. Tuberculosis is one of the major targets for neonatal immunization. In this study, we assessed the immunogenicity of a novel candidate vaccine comprising a mycobacterial fusion protein, Ag85B-ESAT-6, in a neonatal murine immunization model. METHODS/FINDINGS: The Ag85B-ESAT-6 fusion protein was formulated either with a classical alum based adjuvant or with the novel IC31 adjuvant. Following neonatal or adult immunization, 3 parameters were studied in vivo: (1) CD4(+) T cell responses, (2) vaccine targeting/activation of dendritic cells (DC) and (3) protection in a surrogate mycobacterial challenge model. Conversely to Alum, IC31 induced in both age groups strong Th1 and Th17 responses, characterized by multifunctional T cells expressing IL-2 and TNF-alpha with or without IFN-gamma. In the draining lymph nodes, a similarly small number of DC contained the adjuvant and/or the antigen following neonatal or adult immunization. Expression of CD40, CD80, CD86 and IL-12p40 production was focused on the minute adjuvant-bearing DC population. Again, DC targeting/activation was similar in adults and neonates. These DC/T cell responses resulted in an equivalent reduction of bacterial growth following infection with M. bovis BCG, whereas no protection was observed when Alum was used as adjuvant. CONCLUSION: Neonatal immunization with the IC31-adjuvanted Ag85B-ESAT-6 subunit vaccine elicited adult-like multifunctional protective anti-mycobacterial T cell responses through the induction of an adult pattern of in vivo DC activation.

The cubic phase of Magnesium oxide (MgO) nanoparticles is synthesized by the sol-gel method. The nanoparticles were subjected to annealing at 500∘C to enable stress and strain analysis. Characterization of the annealed nanoparticles was performed using powder X-ray diffraction (XRD). Through analysis of the XRD peak profile, key properties, such as crystallite size, lattice strain, deformation stress, and deformation energy density, were determined. These parameters were estimated using three models such as the Williamson–Hall-Isotropic Strain Model (W-H-ISM), the Williamson–Hall-Anisotropic Strain Model (W-H-ASM), and the Williamson–Hall-Energy Density Model (W-H-EDM). The estimations were based on the W-H plot derived from the powder XRD data. The findings of this study contribute to a better understanding of the structural and mechanical characteristics of annealed MgO nanoparticles, thus informing their potential applications. [ABSTRACT FROM AUTHOR]

Lipid transfer proteins (LTPs) are key players in cellular homeostasis and regulation, as they coordinate the exchange of lipids between different cellular organelles. Despite their importance, our mechanistic understanding of how LTPs function at the molecular level is still in its infancy, mostly due to the large number of existing LTPs and to the low degree of conservation at the sequence and structural level. In this work, we use molecular simulations to characterize a representative dataset of lipid transport domains (LTDs) of 12 LTPs that belong to 8 distinct families. We find that despite no sequence homology nor structural conservation, the conformational landscape of LTDs displays common features, characterized by the presence of at least 2 main conformations whose populations are modulated by the presence of the bound lipid. These conformational properties correlate with their mechanistic mode of action, allowing for the interpretation and design of experimental strategies to further dissect their mechanism. Our findings indicate the existence of a conserved, fold-independent mechanism of lipid transfer across LTPs of various families and offer a general framework for understanding their functional mechanism. Lipid Transfer Proteins (LTPs) coordinate the exchange of lipids between different cellular organelles to achieve cellular homeostasis, but the underlying mechanisms remain unclear. In this study, the authors characterize a representative set of structurally unrelated LTPs, identifying conserved, fold-independent mechanisms of lipid transfer that correlate with LTP conformational properties, thus providing a general framework of LTP mode of action. [ABSTRACT FROM AUTHOR]

Understanding protein function is pivotal in comprehending the intricate mechanisms that underlie many crucial biological activities, with far-reaching implications in the fields of medicine, biotechnology, and drug development. However, more than 200 million proteins remain uncharacterized, and computational efforts heavily rely on protein structural information to predict annotations of varying quality. Here, we present a method that utilizes statistics-informed graph networks to predict protein functions solely from its sequence. Our method inherently characterizes evolutionary signatures, allowing for a quantitative assessment of the significance of residues that carry out specific functions. PhiGnet not only demonstrates superior performance compared to alternative approaches but also narrows the sequence-function gap, even in the absence of structural information. Our findings indicate that applying deep learning to evolutionary data can highlight functional sites at the residue level, providing valuable support for interpreting both existing properties and new functionalities of proteins in research and biomedicine. Understanding protein function is vital for biomedicine. Here, authors develop a method using statistics-informed graph networks to predict functions from sequences. The method integrates evolutionary couplings and residue communities to improve the accuracy of function annotations for proteins. [ABSTRACT FROM AUTHOR]

The current work describes the formulation and evaluation of a phospholipid complex of kaempferol to enhance the latter’s aqueous solubility, in vitro dissolution rate, in vivo antioxidant and hepatoprotective activities, and oral bioavailability. The kaempferol-phospholipid complex was synthesized using a freeze-drying method with the formulation being optimized using a full factorial design (32) approach. Our results include the validation of the mathematical model in order to ascertain the role of specific formulation and process variables that contribute favorably to the formulation’s development. The final product was characterized and confirmed by Differential Scanning Calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR), Proton Nuclear Magnetic Resonance Spectroscopy (1H-NMR), and Powder X-ray Diffraction (PXRD) analysis. The aqueous solubility and the in vitro dissolution rate were enhanced compared to that of pure kaempferol. The in vivo antioxidant properties of the kaempferol-phospholipid complex were evaluated by measuring its impact on carbon tetrachloride (CCl4)-intoxicated rats. The optimized phospholipid complex improved the liver function test parameters to a significant level by restoration of all elevated liver marker enzymes in CCl4-intoxicated rats. The complex also enhanced the in vivo antioxidant potential by increasing levels of GSH (reduced glutathione), SOD (superoxide dismutase), catalase and decreasing lipid peroxidation, compared to that of pure kaempferol. The final optimized phospholipid complex also demonstrated a significant improvement in oral bioavailability demonstrated by improvements to key pharmacokinetic parameters, compared to that of pure kaempferol.