Uncovering mechanisms of interorganelle lipid transport by enzymatic mass tagging.

Lipid trafficking is critical for the biogenesis and expansion of organelle membranes. Lipid transport proteins (LTPs) have been proposed to facilitate lipid transport at contact sites between organelles. Despite the fundamental importance of LTPs in cell physiology, our knowledge on the mechanisms of interorganelle lipid distribution remains poor due to the scarcity of assays to monitor lipid flux in vivo. In this review, we highlight the recent development of a versatile method named METALIC (Mass tagging-Enabled Tracking of Lipids in Cells), which uses a combination of enzymatic mass tagging and mass spectrometry to track lipid flux between organelles inside living cells. We discuss the methodology, its distinct advantages, limitations as well as its potential to unearth the pipelines of lipid transport and LTP function in vivo.

METALIC reveals interorganelle lipid flux in live cells by enzymatic mass tagging.

The distinct activities of organelles depend on the proper function of their membranes. Coordinated membrane biogenesis of different organelles necessitates lipid transport from their site of synthesis to their destination. Several factors have been proposed to participate in lipid distribution, but despite its basic importance, in vivo evidence linking the absence of putative transport pathways to specific transport defects remains scarce. A reason for this scarcity is the near absence of in vivo lipid trafficking assays. Here we introduce a versatile method named METALIC (Mass tagging-Enabled TrAcking of Lipids In Cells) to track interorganelle lipid flux inside cells. In this strategy, two enzymes, one directed to a 'donor' and the other to an 'acceptor' organelle, add two distinct mass tags to lipids. Mass-spectrometry-based detection of lipids bearing the two mass tags is then used to quantify exchange between the two organelles. By applying this approach, we show that the ERMES and Vps13-Mcp1 complexes have transport activity in vivo, and unravel their relative contributions to endoplasmic reticulum-mitochondria lipid exchange.

Is a staged reloading protocol effective to time the removal of circular frames? : a retrospective analysis.

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.

Rewiring phospholipid biosynthesis reveals resilience to membrane perturbations and uncovers regulators of lipid homeostasis.

The organelles of eukaryotic cells differ in their membrane lipid composition. This heterogeneity is achieved by the localization of lipid synthesizing and modifying enzymes to specific compartments, as well as by intracellular lipid transport that utilizes vesicular and non-vesicular routes to ferry lipids from their place of synthesis to their destination. For instance, the major and essential phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), can be produced by multiple pathways and, in the case of PE, also at multiple locations. However, the molecular components that underlie lipid homeostasis as well as the routes allowing their distribution remain unclear. Here, we present an approach in which we simplify and rewire yeast phospholipid synthesis by redirecting PE and PC synthesis reactions to distinct subcellular locations using chimeric enzymes fused to specific organelle targeting motifs. In rewired conditions, viability is expected to depend on homeostatic adaptation to the ensuing lipostatic perturbations and on efficient interorganelle lipid transport. We therefore performed genetic screens to identify factors involved in both of these processes. Among the candidates identified, we find genes linked to transcriptional regulation of lipid homeostasis, lipid metabolism, and transport. In particular, we identify a requirement for Csf1-an uncharacterized protein harboring a Chorein-N lipid transport motif-for survival under certain rewired conditions as well as lipidomic adaptation to cold, implicating Csf1 in interorganelle lipid transport and homeostatic adaptation.

Csf1: A Putative Lipid Transport Protein Required for Homeoviscous Adaptation of the Lipidome.

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.

Leri: A web-server for identifying protein functional networks from evolutionary couplings.

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.

Intraconal Metastasis Leading to Diagnosis of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the commontumor of the liver and the third most common cause of cancer-related mortality worldwide. Patients with HCC may have metastasis to different sites. Intrahepatic and extrahepatic metastases are found in (~50-75%). Lung and regional lymph nodes are the most commonly involved sites. Metastasis to bone, skin, and adrenal glands are rare. Orbit metastasis and intracranial invasion are extremely rare. We are presenting a case of HCC that metastasized to the orbital cavity. The patient presented with progressive proptosis of the eyeball with retrobulbar and intracranial invasion and involvement of the sub-scalp region. Based on the imaging findings, it was initially misdiagnosed as meningioma; however, histopathological examination of the biopsy specimen resulted in a definitive diagnosis of HCC metastasis. The present case reveals that the alternative diagnosis of metastasis must be considered when diagnosing retrobulbar lesions in patients with HCC.

A Review of the Diagnostic Use of Fine-needle Aspiration Cytology for Tuberculosis Epididymo-orchitis: To Do or Not to Do.

Isolated tuberculous epididymo-orchitis is a rare manifestation of the vast extrapulmonary tuberculosis (EPTB) disease spectrum, especially in developed nations, making it prone to delayed diagnosis or misdiagnosis and inadvertent orchiectomy. Several observational studies and case reports have been reported with the successful use of fine-needle aspiration cytology (FNAC) in diagnosing tuberculosis orchitis, thus avoiding inadvertent orchiectomy. Because tuberculous epididymo-orchitis can mimic testicular neoplasm, the use of FNAC is not prevalent in developed countries for fear of the seeding of tumor cells and there is a lack of consensus on the use of FNAC for diagnostic purposes in such patients. We report a case of a 27-year-old man with an atypical presentation of genitourinary tuberculosis (TB) and its management. The case report also reviews the literature to discuss the available evidence and tries to answer the long-standing question on the role of FNAC in the diagnosis of tuberculous epididymo-orchitis. The currently available literature has demonstrated the safety and efficacy of FNAC in diagnosing TB epididymo-orchitis and, based on our review, the benefits of differentiating TB epididymo-orchitis from testicular malignancy using FNAC exceeds its minimal risk and must be considered to minimize clinical diagnosis error and unnecessary orchiectomy in low-risk patients.

Facile synthesized novel hybrid graphene oxide/cobalt ferrite magnetic nanoparticles based surface coating material inhibit bacterial secretion pathway for antibacterial effect.

Nanomaterial based paints are in current demand in the area of surface protective coatings due to the significant advances made to improve their antibacterial and anticorrosion characteristics. In this work, we have developed magnetic graphene oxide (MGO) paint with the incorporation of cobalt ferrite (CF) and graphene oxide (GO) along with paint materials by using high energy ball milling (HEBM). Morphological, elemental and functional analysis of the MGO paint is studied with ESEM, AFM, Raman, FTIR spectroscopy. EDS and PIXE methods are used for elemental analysis. Thermal analysis shows that the MGO film was stable up to 100 °C. The saturation magnetization of CF MNP is observed as 76 emu/g and it is reduced to 12 emu/g for MGP paint. The detailed antibacterial study of the prepared MGO paint has performed with S. typhimurium and E. coli. The dead-live assessment shows the dead population for S. typhimurium is superior up to 82% whereas it is 20% for E. coli. The morphological damage of bacterial cells is studied using SEM technique. Flow cytometry analysis of reactive oxygen species (ROS) generation experiments and computational analysis supported the proposed mechanism of induced ROS for the damage of bacterial membrane via interaction of GO and CF with bacterial proteins leading to alteration in their functionality. The observed results indicate that the prepared MGO paint could be a better candidate in the area of nano paint for surface protective coatings.

Morphological Structure, Optical and Microbial Findings of Ferrites.

In this article, we report the interesting results on the effect of high energy ball milling (under 350 rpm for 0, 1, 3, 5, and 10 h) on the salt (NaCl:KCl) flux synthesized SrFe12O19 powder. The different processed powder was analyzed by powder X-ray diffraction (XRD), thermogravimetric analysis (TG/DTA), field emission scanning electron microscope (FESEM), Fourier-transform infrared (FT-IR) and near infra-red (NIR) reflectance spectroscopic techniques. From the present research findings, we note that the SrFe12O19 phase is susceptible to the high-energy ball milling process. The unmilled SrFe12O19 powder crystallized in hexagonal structure with the platelet-shaped SrFe12O19 grains exhibits high near-infrared (NIR) reflectance. The average crystalline size (XRD) of the unmilled (84.9 nm) SrFe12O19 powder is reduced to 22.2 nm after 1 h of ball milling. Ball milling of good quality SrFe12O19 powder for >1 h, mixed nanocrystalline phases of SrFe12O19 and α-Fe2O3 materials are formed. By increasing the ball milling time, the thermal features also vary accordingly. The platelet-shaped SrFe12O19 grains are strongly affected by ball milling of the salt flux synthesized SrFe12O19 powder for 1, 3, 5 and 10 h. Ball milling of SrFe12O19 powder has a strong effect on the NIR reflectance and color of the resultant ball-milled powders. The 1 h as-milled powder shows relatively strong inhibition zone as compared to other powder samples (0, 3, 5, 10 h) against Pseudomonas aeruginosa bacterium.

A review on applications of chitosan-based Schiff bases.

Biopolymers have become very attractive as they are degradable, biocompatible, non-toxic and renewable. Due to the intrinsic reactive amino groups, chitosan is vibrant in the midst of other biopolymers. Using the versatility of these amino groups, various structural modifications have been accomplished on chitosan through certain chemical reactions. Chemical modification of chitosan via imine functionalization (RR'CNR″; R: alkyl/aryl, R': H/alkyl/aryl and R″: chitosan ring) is significant as it recommends the resultant chitosan-based Schiff bases (CSBs) for the important applications in the fields like biology, catalysis, sensors, water treatment, etc. CSBs are usually synthesized by the Schiff condensation reaction between chitosan's amino groups and carbonyl compounds with the removal of water molecules. In this review, we first introduce the available synthetic approaches for the preparation of CSBs. Then, we discuss the biological applications of CSBs including antimicrobial activity, anticancer activity, drug carrier ability, antioxidant activity and tissue engineering capacity. Successively, the applications of CSBs in other fields such as catalysis, adsorption and sensors are demonstrated.

Enhanced transdermal permeation and anti-inflammatory potential of phospholipids complex-loaded matrix film of umbelliferone: Formulation development, physico-chemical and functional characterization.

In the present study, umbelliferone - phospholipids complex - loaded matrix film (UPLC - MF) was developed with a goal of improving transdermal permeation and anti-inflammatory potential of umbelliferone (UMB). Umbelliferone - phospholipids complex (UPLC) was prepared using solvent evaporation method. UPLC-MF was prepared by simple and reproducible solvent casting method. Prepared UPLC and UPLC-MF were both physico-chemically characterized by Fourier transforms infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), powder x-ray diffraction (PXRD), proton nuclear magnetic resonance spectroscopy (1H NMR), weight variation, thickness, tensile strength, folding endurance, % elongation, moisture content and uptake Functional characterization of UPLC and UPLC-MF was carried out by solubility analysis, in vitro dissolution, diffusion, and ex vivo permeation via dialysis and biological membrane. UPLC - MF was also evaluated for in vivo anti-inflammatory activity using carrageenan-induced Albino rat paw model. Design-based optimal values for formulation and process variables of UPLC were observed to be 1:1.78, 50 °C and 2 h, respectively. Physico-chemical characterization confirmed the formation of the complex and the film. UPLC demonstrated a higher aqueous solubility (~11-fold), compared to pure UMB. Rate and extent of dissolution of UMB from UPLC was enhanced significantly to that of pure UMB. Compared to UMB-MF, the diffusion and permeation rate of UMB from UPLC-MF enhanced significantly. The UPLC - MF improved the anti-inflammatory potential of UMB by significant enhancement of edema inhibition (%), compared to UMB-MF. The obtained results showed that the present combined formulation system could be employed as a promising strategy for improving transdermal permeation of UMB.

Positron Annihilation Studies on Chemically Synthesized FeCo Alloy.

Equiatomic flower-like FeCo magnetic nanoparticles are synthesized through a modified one-pot polyol technique. The as-prepared samples are annealed at 700 and 800 °C under reducing atmosphere. The saturation magnetization and coercivity of the flower-like FeCo are found to be 198 (1) emu/g and 243 (10) Oe respectively. The magnetic properties of FeCo approach the bulk behavior with annealing. Positron lifetime studies on the chemically synthesized equiatomic FeCo magnetic nanoparticles with flower-like morphology are reported and compared with Fe, Co and FeCo annealed at various temperatures. The FeCo is characterized by different lifetime components corresponding to positron annihilation events in vacancies and various open volume defects due to their unique morphology. The studies suggest defects arising out of cluster vacancies and interpetal gap that reduce on annealing. The average pore size obtained from positron annihilation studies closely matches with the interpetal distance obtained from the electron microscopic analysis for the flower-like FeCo.

Gut Microbiota Dysfunction as Reliable Non-invasive Early Diagnostic Biomarkers in the Pathophysiology of Parkinson's Disease: A Critical Review.

Recent investigations suggest that gut microbiota affects the brain activity through the microbiota-gut-brain axis under both physiological and pathological disease conditions like Parkinson's disease. Further dopamine synthesis in the brain is induced by dopamine producing enzymes that are controlled by gut microbiota via the microbiota-gut-brain axis. Also alpha synuclein deposition and the associated neurodegeneration in the enteric nervous system that increase intestinal permeability, oxidative stress, and local inflammation, accounts for constipation in Parkinson's disease patients. The trigger that causes blood brain barrier leakage, immune cell activation and inflammation, and ultimately neuroinflammation in the central nervous system is believed to be due to the chronic low-grade inflammation in the gut. The non-motor symptoms that appear years before motor symptoms could be reliable early biomarkers, if they could be correlated with the established and reliable neuroimaging techniques or behavioral indices. The future directions should therefore, focus on the exploration of newer investigational techniques to identify these reliable early biomarkers and define the specific gut microbes that contribute to the development of Parkinson's disease. This ultimately should pave the way to safer and novel therapeutic approaches that avoid the complications of the drugs delivered today to the brain of Parkinson's disease patients.

Development of real-time PCR assay for the detection of Mycoplasma bovis.

Mycoplasma bovis is one of the important bovine mycoplasma involved in economically important clinical conditions like respiratory diseases, otitis media, and mastitis. The present study was undertaken with the objective of developing a SYBR Green dye-based real-time PCR assay targeting uvrC gene for the diagnosis of M. bovis. The analytical sensitivity and specificity of the assay were evaluated. The test showed 103-fold more sensitivity than conventional PCR and detected down to 100 fg level of DNA. It was found to be specific, as no cross reactivity was shown with other related bacteria and Mycoplasma species. The developed assay was able to detect down to 40 copies of uvrC gene from spiked bovine milk samples. At present, this developed assay may be used as a valuable diagnostic tool for the detection of Mycoplasma bovis.

Design and evaluation of dental films of PEGylated rosin derivatives containing sparfloxacin for periodontitis.

OBJECTIVE: In this study, PEGylated rosin derivatives (PRDs) namely D1 and D2 were synthesized and evaluated for their application to produce sustained-release antibacterial films containing sparfloxacin for periodontitis. SIGNIFICANCE: PRDs are biodegradable and biocompatible, and therefore sustained-release dental implant of PRD-sparfloxacin can provide an effectual treatment for periodontitis. METHODS: Films were produced by solvent casting technique and characterized for morphology, swelling-index, in vitro degradation and drug release kinetics. The impact of type of PRD, concentration of PRDs, and addition of plasticizer (dibutyl phthalate) on various film properties was evaluated. The films were also subjected to stability study at 30 °C and 40 °C for 90 days. RESULTS: Both D1 and D2 produced smooth and non-porous films with sparfloxacin. The D1 films, due to lower amount of polyethylene glycol 400 in D1, exhibited lower swelling-index, slower degradation, and slower drug release compared to D2 films. An increase in PRDs concentration decreased swelling-index, prolonged degradation time, and decreased drug release rate of films; addition of plasticizer showed the similar effect. At pH 7.6, D1 and D2 films showed complete degradation at the end of 58 and 51 days, respectively. At the end of 21 days, D1 and D2 films released 41.85% and 61.53% sparfloxacin, respectively. The drug release from D1 films followed Higuchi square-root kinetics, while D2 films released drug by the zero order kinetics. The stability conditions did not significantly alter PRDs-film properties. CONCLUSION: Results revealed that PRDs can be used successfully to produce sustained-release antibacterial films containing sparfloxacin for the treatment of periodontitis.

Vps13-Mcp1 interact at vacuole-mitochondria interfaces and bypass ER-mitochondria contact sites.

Membrane contact sites between endoplasmic reticulum (ER) and mitochondria, mediated by the ER-mitochondria encounter structure (ERMES) complex, are critical for mitochondrial homeostasis and cell growth. Defects in ERMES can, however, be bypassed by point mutations in the endosomal protein Vps13 or by overexpression of the mitochondrial protein Mcp1. How this bypass operates remains unclear. Here we show that the mitochondrial outer membrane protein Mcp1 functions in the same pathway as Vps13 by recruiting it to mitochondria and promoting its association to vacuole-mitochondria contacts. Our findings support a model in which Mcp1 and Vps13 work as functional effectors of vacuole-mitochondria contact sites, while tethering is mediated by other factors, including Vps39. Tethered and functionally active vacuole-mitochondria interfaces then compensate for the loss of ERMES-mediated ER-mitochondria contact sites.

Formulation and characterization of an apigenin-phospholipid phytosome (APLC) for improved solubility, in vivo bioavailability, and antioxidant potential.

The apigenin-phospholipid phytosome (APLC) was developed to improve the aqueous solubility, dissolution, in vivo bioavailability, and antioxidant activity of apigenin. The APLC synthesis was guided by a full factorial design strategy, incorporating specific formulation and process variables to deliver an optimized product. The design-optimized formulation was assayed for aqueous solubility, in vitro dissolution, pharmacokinetics, and antioxidant activity. The pharmacological evaluation was carried out by assessing its effects on carbon tetrachloride-induced elevation of liver function marker enzymes in a rat model. The antioxidant activity was assessed by studying its effects on the liver antioxidant marker enzymes. The developed model was validated using the design-optimized levels of formulation and process variables. The physical-chemical characterization confirmed the formation of phytosomes. The optimized formulation demonstrated over 36-fold higher aqueous solubility of apigenin, compared to that of pure apigenin. The formulation also exhibited a significantly higher rate and extent of apigenin release in dissolution studies. The pharmacokinetic analysis revealed a significant enhancement in the oral bioavailability of apigenin from the prepared formulation, compared to pure apigenin. The liver function tests indicated that the prepared phytosome showed a significantly improved restoration of all carbon tetrachloride-elevated rat liver function marker enzymes. The prepared formulation also exhibited antioxidant potential by significantly increasing the levels of glutathione, superoxide dismutase, catalase, and decreasing the levels of lipid peroxidase. The study shows that phospholipid-based phytosome is a promising and viable strategy for improving the delivery of apigenin and similar phytoconstituents with low aqueous solubility.

Critical Role of the Sorting Polymer in Carbon Nanotube-Based Minority Carrier Devices.

A prerequisite for carbon nanotube-based optoelectronic devices is the ability to sort them into a pure semiconductor phase. One of the most common sorting routes is enabled through using specific wrapping polymers. Here we show that subtle changes in the polymer structure can have a dramatic influence on the figures of merit of a carbon nanotube-based photovoltaic device. By comparing two commonly used polyfluorenes (PFO and PFO-BPy) for wrapping (7,5) and (6,5) chirality SWCNTs, we demonstrate that they have contrasting effects on the device efficiency. We attribute this to the differences in their ability to efficiently transfer charge. Although PFO may act as an efficient interfacial layer at the anode, PFO-BPy, having the additional pyridine side groups, forms a high resistance layer degrading the device efficiency. By comparing PFO|C60 and C60-only devices, we found that presence of a PFO layer at low optical densities resulted in the increase of all three solar cell parameters, giving nearly an order of magnitude higher efficiency over that of C60-only devices. In addition, with a relatively higher contribution to photocurrent from the PFO-C60 interface, an open circuit voltage of 0.55 V was obtained for PFO-(7,5)-C60 devices. On the other hand, PFO-BPy does not affect the open circuit voltage but drastically reduces the short circuit current density. These results indicate that the charge transport properties and energy levels of the sorting polymers have to be taken into account to fully understand their effect on carbon nanotube-based solar cells.

Spinal compression fractures due to pregnancy-associated osteoporosis.

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.