Generation of a bloodstream form Trypanosoma brucei double glycosyltransferase null mutant competent in receptor-mediated endocytosis of transferrin.

The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis.

A proposed pathway from D-glucose to D-arabinose in eukaryotes.

In eukaryotes, the D-enantiomer of arabinose (D-Ara) is an intermediate in the biosynthesis of D-erythroascorbate in yeast and fungi and in the biosynthesis of the nucleotide sugar GDP-α-D-arabinopyranose (GDP-D-Arap) and complex α-D-Arap-containing surface glycoconjugates in certain trypanosomatid parasites. Whereas the biosynthesis of D-Ara in prokaryotes is well understood, the route from D-glucose (D-Glc) to D-Ara in eukaryotes is unknown. In this paper, we study the conversion of D-Glc to D-Ara in the trypanosomatid Crithidia fasciculata using positionally labeled [13C]-D-Glc and [13C]-D-ribose ([13C]-D-Rib) precursors and a novel derivatization and gas chromatography-mass spectrometry procedure applied to a terminal metabolite, lipoarabinogalactan. These data implicate the both arms of pentose phosphate pathway and a likely role for D-ribulose-5-phosphate (D-Ru-5P) isomerization to D-Ara-5P. We tested all C. fasciculata putative sugar and polyol phosphate isomerase genes for their ability to complement a D-Ara-5P isomerase-deficient mutant of Escherichia coli and found that one, the glutamine fructose-6-phosphate aminotransferase (GFAT) of glucosamine biosynthesis, was able to rescue the E. coli mutant. We also found that GFAT genes of other trypanosomatid parasites, and those of yeast and human origin, could complement the E. coli mutant. Finally, we demonstrated biochemically that recombinant human GFAT can isomerize D-Ru-5P to D-Ara5P. From these data, we postulate a general eukaryotic pathway from D-Glc to D-Ara and discuss its possible significance. With respect to C. fasciculata, we propose that D-Ara is used not only for the synthesis of GDP-D-Arap and complex surface glycoconjugates but also in the synthesis of D-erythroascorbate.

The major surface protein of malaria sporozoites is GPI-anchored to the plasma membrane.

Glycosylphosphatidylinositol (GPI) anchor protein modification in Plasmodium species is well known and represents the principal form of glycosylation in these organisms. The structure and biosynthesis of GPI anchors of Plasmodium spp. has been primarily studied in the asexual blood stage of Plasmodium falciparum and is known to contain the typical conserved GPI structure of EtN-P-Man3GlcN-PI. Here, we have investigated the circumsporozoite protein (CSP) for the presence of a GPI anchor. CSP is the major surface protein of Plasmodium sporozoites, the infective stage of the malaria parasite. While it is widely assumed that CSP is a GPI-anchored cell surface protein, compelling biochemical evidence for this supposition is absent. Here, we employed metabolic labeling and mass-spectrometry-based approaches to confirm the presence of a GPI anchor in CSP. Biosynthetic radiolabeling of CSP with [3H]-palmitic acid and [3H]-ethanolamine, with the former being base-labile and therefore ester-linked, provided strong evidence for the presence of a GPI anchor on CSP, but these data alone were not definitive. To provide further evidence, immunoprecipitated CSP was analyzed for the presence of myo-inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for highly sensitive and quantitative detection. The single ion monitoring (SIM) method for GC-MS analysis confirmed the presence of the myo-inositol component in CSP. Taken together, these data provide confidence that the long-assumed presence of a GPI anchor on this important parasite protein is correct.

Identification of the glycosylphosphatidylinositol-specific phospholipase A2 (GPI-PLA2) that mediates GPI fatty acid remodeling in Trypanosoma brucei.

The biosynthesis of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) in the parasitic protozoan Trypanosoma brucei involves fatty acid remodeling of the GPI precursor molecules before they are transferred to protein in the endoplasmic reticulum. The genes encoding the requisite phospholipase A2 and A1 activities for this remodeling have thus far been elusive. Here, we identify a gene, Tb927.7.6110, that encodes a protein that is both necessary and sufficient for GPI-phospholipase A2 (GPI-PLA2) activity in the procyclic form of the parasite. The predicted protein product belongs to the alkaline ceramidase, PAQR receptor, Per1, SID-1, and TMEM8 (CREST) superfamily of transmembrane hydrolase proteins and shows sequence similarity to Post-GPI-Attachment to Protein 6 (PGAP6), a GPI-PLA2 that acts after transfer of GPI precursors to protein in mammalian cells. We show the trypanosome Tb927.7.6110 GPI-PLA2 gene resides in a locus with two closely related genes Tb927.7.6150 and Tb927.7.6170, one of which (Tb927.7.6150) most likely encodes a catalytically inactive protein. The absence of GPI-PLA2 in the null mutant procyclic cells not only affected fatty acid remodeling but also reduced GPI anchor sidechain size on mature GPI-anchored procyclin glycoproteins. This reduction in GPI anchor sidechain size was reversed upon the re-addition of Tb927.7.6110 and of Tb927.7.6170, despite the latter not encoding GPI precursor GPI-PLA2 activity. Taken together, we conclude that Tb927.7.6110 encodes the GPI-PLA2 of GPI precursor fatty acid remodeling and that more work is required to assess the roles and essentiality of Tb927.7.6170 and the presumably enzymatically inactive Tb927.7.6150.

Cyclin-dependent kinase 12 is a drug target for visceral leishmaniasis.

Visceral leishmaniasis causes considerable mortality and morbidity in many parts of the world. There is an urgent need for the development of new, effective treatments for this disease. Here we describe the development of an anti-leishmanial drug-like chemical series based on a pyrazolopyrimidine scaffold. The leading compound from this series (7, DDD853651/GSK3186899) is efficacious in a mouse model of visceral leishmaniasis, has suitable physicochemical, pharmacokinetic and toxicological properties for further development, and has been declared a preclinical candidate. Detailed mode-of-action studies indicate that compounds from this series act principally by inhibiting the parasite cdc-2-related kinase 12 (CRK12), thus defining a druggable target for visceral leishmaniasis.

A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and activity are essential in parasitic Leishmania.

Glycoconjugates play major roles in the infectious cycle of the trypanosomatid parasite Leishmania While GDP-Fucose synthesis is essential, fucosylated glycoconjugates have not been reported in Leishmania major [H. Guo et al., J. Biol. Chem. 292, 10696-10708 (2017)]. Four predicted fucosyltransferases appear conventionally targeted to the secretory pathway; SCA1/2 play a role in side-chain modifications of lipophosphoglycan, while gene deletion studies here showed that FUT2 and SCAL were not essential. Unlike most eukaryotic glycosyltransferases, the predicted α 1-2 fucosyltransferase encoded by FUT1 localized to the mitochondrion. A quantitative "plasmid segregation" assay, expressing FUT1 from the multicopy episomal pXNG vector in a chromosomal null ∆fut1- background, established that FUT1 is essential. Similarly, "plasmid shuffling" confirmed that both enzymatic activity and mitochondrial localization were required for viability, comparing import-blocked or catalytically inactive enzymes, respectively. Enzymatic assays of tagged proteins expressed in vivo or of purified recombinant FUT1 showed it had a broad fucosyltransferase activity including glycan and peptide substrates. Unexpectedly, a single rare ∆fut1- segregant (∆fut1s ) was obtained in rich media, which showed severe growth defects accompanied by mitochondrial dysfunction and loss, all of which were restored upon FUT1 reexpression. Thus, FUT1 along with the similar Trypanosoma brucei enzyme TbFUT1 [G. Bandini et al., bioRxiv, https://www.biorxiv.org/content/10.1101/726117v2 (2021)] joins the eukaryotic O-GlcNAc transferase isoform as one of the few glycosyltransferases acting within the mitochondrion. Trypanosomatid mitochondrial FUT1s may offer a facile system for probing mitochondrial glycosylation in a simple setting, and their essentiality for normal growth and mitochondrial function renders it an attractive target for chemotherapy of these serious human pathogens.

Common and unique features of glycosylation and glycosyltransferases in African trypanosomes.

Eukaryotic protein glycosylation is mediated by glycosyl- and oligosaccharyl-transferases. Here, we describe how African trypanosomes exhibit both evolutionary conservation and significant divergence compared with other eukaryotes in how they synthesise their glycoproteins. The kinetoplastid parasites have conserved components of the dolichol-cycle and oligosaccharyltransferases (OSTs) of protein N-glycosylation, and of glycosylphosphatidylinositol (GPI) anchor biosynthesis and transfer to protein. However, some components are missing, and they process and decorate their N-glycans and GPI anchors in unique ways. To do so, they appear to have evolved a distinct and functionally flexible glycosyltransferases (GT) family, the GT67 family, from an ancestral eukaryotic ß3GT gene. The expansion and/or loss of GT67 genes appears to be dependent on parasite biology. Some appear to correlate with the obligate passage of parasites through an insect vector, suggesting they were acquired through GT67 gene expansion to assist insect vector (tsetse fly) colonisation. Others appear to have been lost in species that subsequently adopted contaminative transmission. We also highlight the recent discovery of a novel and essential GT11 family of kinetoplastid parasite fucosyltransferases that are uniquely localised to the mitochondria of Trypanosoma brucei and Leishmania major. The origins of these kinetoplastid FUT1 genes, and additional putative mitochondrial GT genes, are discussed.

A Trypanosoma brucei ß3 glycosyltransferase superfamily gene encodes a ß1-6 GlcNAc-transferase mediating N-glycan and GPI anchor modification.

The parasite Trypanosoma brucei exists in both a bloodstream form (BSF) and a procyclic form (PCF), which exhibit large carbohydrate extensions on the N-linked glycans and glycosylphosphatidylinositol (GPI) anchors, respectively. The parasite's glycoconjugate repertoire suggests at least 38 glycosyltransferase (GT) activities, 16 of which are currently uncharacterized. Here, we probe the function(s) of the uncharacterized GT67 glycosyltransferase family and a ß3 glycosyltransferase (ß3GT) superfamily gene, TbGT10. A BSF-null mutant, created by applying the diCre/loxP method in T. brucei for the first time, showed a fitness cost but was viable in vitro and in vivo and could differentiate into the PCF, demonstrating nonessentiality of TbGT10. The absence of TbGT10 impaired the elaboration of N-glycans and GPI anchor side chains in BSF and PCF parasites, respectively. Glycosylation defects included reduced BSF glycoprotein binding to the lectin ricin and monoclonal antibodies mAb139 and mAbCB1. The latter bind a carbohydrate epitope present on lysosomal glycoprotein p67 that we show here consists of (-6Galß1-4GlcNAcß1-)≥4 poly-N-acetyllactosamine repeats. Methylation linkage analysis of Pronase-digested glycopeptides isolated from BSF wild-type and TbGT10 null parasites showed a reduction in 6-O-substituted- and 3,6-di-O-substituted-Gal residues. These data define TbGT10 as a UDP-GlcNAc:ßGal ß1-6 GlcNAc-transferase. The dual role of TbGT10 in BSF N-glycan and PCF GPI-glycan elaboration is notable, and the ß1-6 specificity of a ß3GT superfamily gene product is unprecedented. The similar activities of trypanosome TbGT10 and higher-eukaryote I-branching enzyme (EC 2.4.1.150), which belong to glycosyltransferase families GT67 and GT14, respectively, in elaborating N-linked glycans, are a novel example of convergent evolution.

Elimination of GPI2 suppresses glycosylphosphatidylinositol GlcNAc transferase activity and alters GPI glycan modification in Trypanosoma brucei.

Many eukaryotic cell-surface proteins are post-translationally modified by a glycosylphosphatidylinositol (GPI) moiety that anchors them to the cell membrane. The biosynthesis of GPI anchors is initiated in the endoplasmic reticulum by transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol. This reaction is catalyzed by GPI GlcNAc transferase, a multisubunit complex comprising the catalytic subunit Gpi3/PIG-A as well as at least five other subunits, including the hydrophobic protein Gpi2, which is essential for the activity of the complex in yeast and mammals, but the function of which is not known. To investigate the role of Gpi2, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote and important model organism that initially provided the first insights into GPI structure and biosynthesis. We generated insect-stage (procyclic) trypanosomes that lack TbGPI2 and found that in TbGPI2-null parasites, (i) GPI GlcNAc transferase activity is reduced, but not lost, in contrast with yeast and human cells, (ii) the GPI GlcNAc transferase complex persists, but its architecture is affected, with loss of at least the TbGPI1 subunit, and (iii) the GPI anchors of procyclins, the major surface proteins, are underglycosylated when compared with their WT counterparts, indicating the importance of TbGPI2 for reactions that occur in the Golgi apparatus. Immunofluorescence microscopy localized TbGPI2 not only to the endoplasmic reticulum but also to the Golgi apparatus, suggesting that in addition to its expected function as a subunit of the GPI GlcNAc transferase complex, TbGPI2 may have an enigmatic noncanonical role in Golgi-localized GPI anchor modification in trypanosomes.

Radiofrequency ablation for liver tumors abutting complex blood vessel structures: treatment protocol optimization using response surface method and computer modeling.

OBJECTIVE: To achieve a result of a large tumor ablation volume with minimal thermal damage to the surrounding blood vessels by designing a few clinically-adjustable operating parameters in radiofrequency ablation (RFA) for liver tumors abutting complex vascular structures. METHODS: Response surface method (RSM) was employed to correlate the ablated tumor volume (Ra) and thermal damage to blood vessels (Dt) based on RFA operating parameters: ablation time, electrode position, and insertion angle. A coupled electric-thermal-fluid RFA computer model was created as the testbed for RSM to simulate RFA process. Then, an optimal RFA protocol for the two conflicting goals, namely (1) large tumor ablation and (2) small thermal damage to the surrounding blood vessels, has been achieved under a specific ablation environment. RESULTS: Linear regression analysis confirmed that the RFA protocol significantly affected Ra and Dt (the adjusted coefficient of determination Radj2 = 93.61% and 95.03%, respectively). For a proposed liver tumor scenario (liver tumor with a dimension of 4×3×2.9 cm3 abutting a complex vascular structure), an optimized RFA protocol was found based on the regression results in RSM. Compared with a reference RFA protocol, in which the electrode was centered in the tumor with a 12-min ablation time, the optimized RFA protocol has increased Ra  from 98.1% to 99.6% and decreased Dt from 4.1% to 0.4%, achieving nearly the complete ablation of proposed liver tumor and ignorable thermal damages to vessels. CONCLUSION: This work showed that it is possible to design a few clinically-adjustable operating parameters of RFA for achieving a large tumor ablation volume while minimizing thermal damage to the surrounding blood vessels.

Lethal Electric Field Thresholds for Cerebral Cells With Irreversible Electroporation and H-FIRE Protocols: An In Vitro Three-Dimensional Cell Model Study.

The lethal electric field (LEF) thresholds for three typical cerebral cells, including a malignant glioblastoma (GBM) cell line and two cell lines from the healthy blood-brain barrier (BBB), treated by irreversible electroporation (IRE) or high-frequency irreversible electroporation (H-FIRE) protocols were investigated in an in vitro three-dimensional (3D) cell model. A conventional IRE protocol (90 pulses, 1 Hz, and 100-µs pulse duration) and three novel H-FIRE protocols (1-3-1, 0.5-1-0.5, and 1-1-1) were used to treat the cerebral cells in both 3D single-cell and two-cell models. The electrical conductivity of the 3D cell model under different electric field strengths were characterized with the method of electrochemical impedance spectroscopy (EIS). Based on EIS, a numerical electrothermal model of electroporation was built for the determination of the LEF threshold with different protocols and temperature monitoring. Cell viability was assessed by fluorescence staining 6 h after the treatment. The results showed no thermal lethal effect on cells when these protocols were used. The LEF threshold for GBM cells was significantly lower than that of the healthy BBB cells. These results suggest the possibility of selective ablation of human cerebral GBM by IRE and H-FIRE treatments with no injury or reversible injury to healthy cells, and the potential use of IRE or H-FIRE for transient disruption of the BBB to allow chemotherapy to reach the tumor.

Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition.

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.

An in vivo study of a custom-made high-frequency irreversible electroporation generator on different tissues for clinically relevant ablation zones.

PURPOSE: To examine the ablation zone, muscle contractions, and temperature increases in both rabbit liver and kidney models in vivo for a custom-made high-frequency irreversible electroporation (H-FIRE) generator. MATERIALS AND METHODS: A total of 18 New Zealand white rabbits were used to investigate five H-FIRE protocols (n = 3 for each protocol) and an IRE protocol (n = 3) for the performance of the designed H-FIRE device in both liver and kidney tissues. The ablation zone was determined by using histological analysis 72 h after treatment. The extent of muscle contractions and temperature change during the application of pulse energy were measured by a commercial accelerometer attached to animals and fiber optic temperature probe inserted into organs with IRE electrodes, respectively. RESULTS: All H-FIRE protocols were able to generate visible ablation zones without muscle contractions, for both liver and kidney tissues. The area of ablation zone generated in H-FIRE pulse protocols (e.g., 0.3-1 µs, 2000 V, and 90-195 bursts) appears similar to that of IRE protocol (100 µs, 1000 V, and 90 pulses) in both liver and kidney tissues. No significant temperature increase was noticed except for the protocol with the highest pulse energy (e.g., 1 µs, 2000 V, and 180 bursts). CONCLUSION: Our work serves to complement the current H-FIRE pulse waveforms, which can be optimized to significantly improve the quality of ablation zone in terms of precision for liver and kidney tumors in clinical setting.

Electroporation-Based Therapy for Brain Tumors: A Review.

Electroporation-based therapy (EBT), as a high-voltage-pulse technology has been prevalent with favorable clinical outcomes in the treatment of various solid tumors. This review paper aims to promote the clinical translation of EBT for brain tumors. First, we briefly introduced the mechanism of pore formation in a cell membrane activated by external electric fields using a single cell model. Then, we summarized and discussed the current in vitro and in vivo preclinical studies, in terms of (1) the safety and effectiveness of EBT for brain tumors in animal models, and (2) the blood-brain barrier (BBB) disruption induced by EBT. Two therapeutic effects could be achieved in EBT for brain tumors simultaneously, i.e., the tumor ablation induced by irreversible electroporation (IRE) and transient BBB disruption induced by reversible electroporation (RE). The BBB disruption could potentially improve the uptake of antitumor drugs thereby enhancing brain tumor treatment. The challenges that hinder the application of EBT in the treatment of human brain tumors are discussed in the review paper as well.

N-glycan microheterogeneity regulates interactions of plasma proteins.

Altered glycosylation patterns of plasma proteins are associated with autoimmune disorders and pathogenesis of various cancers. Elucidating glycoprotein microheterogeneity and relating subtle changes in the glycan structural repertoire to changes in protein-protein, or protein-small molecule interactions, remains a significant challenge in glycobiology. Here, we apply mass spectrometry-based approaches to elucidate the global and site-specific microheterogeneity of two plasma proteins: α1-acid glycoprotein (AGP) and haptoglobin (Hp). We then determine the dissociation constants of the anticoagulant warfarin to different AGP glycoforms and reveal how subtle N-glycan differences, namely, increased antennae branching and terminal fucosylation, reduce drug-binding affinity. Conversely, similar analysis of the haptoglobin-hemoglobin (Hp-Hb) complex reveals the contrary effects of fucosylation and N-glycan branching on Hp-Hb interactions. Taken together, our results not only elucidate how glycoprotein microheterogeneity regulates protein-drug/protein interactions but also inform the pharmacokinetics of plasma proteins, many of which are drug targets, and whose glycosylation status changes in various disease states.

Gluconeogenesis using glycerol as a substrate in bloodstream-form Trypanosoma brucei.

Bloodstream form African trypanosomes are thought to rely exclusively upon glycolysis, using glucose as a substrate, for ATP production. Indeed, the pathway has long been considered a potential therapeutic target to tackle the devastating and neglected tropical diseases caused by these parasites. However, plasma membrane glucose and glycerol transporters are both expressed by trypanosomes and these parasites can infiltrate tissues that contain glycerol. Here, we show that bloodstream form trypanosomes can use glycerol for gluconeogenesis and for ATP production, particularly when deprived of glucose following hexose transporter depletion. We demonstrate that Trypanosoma brucei hexose transporters 1 and 2 (THT1 and THT2) are localized to the plasma membrane and that knockdown of THT1 expression leads to a growth defect that is more severe when THT2 is also knocked down. These data are consistent with THT1 and THT2 being the primary routes of glucose supply for the production of ATP by glycolysis. However, supplementation of the growth medium with glycerol substantially rescued the growth defect caused by THT1 and THT2 knockdown. Metabolomic analyses with heavy-isotope labelled glycerol demonstrated that trypanosomes take up glycerol and use it to synthesize intermediates of gluconeogenesis, including fructose 1,6-bisphosphate and hexose 6-phosphates, which feed the pentose phosphate pathway and variant surface glycoprotein biosynthesis. We used Cas9-mediated gene knockout to demonstrate a gluconeogenesis-specific, but fructose-1,6-bisphosphatase (Tb927.9.8720)-independent activity, converting fructose 1,6-bisphosphate into fructose 6-phosphate. In addition, we observed increased flux through the tricarboxylic acid cycle and the succinate shunt. Thus, contrary to prior thinking, gluconeogenesis can operate in bloodstream form T. brucei. This pathway, using glycerol as a physiological substrate, may be required in mammalian host tissues.

Protection of the transplant kidney during cold perfusion with doxycycline: proteomic analysis in a rat model.

BACKGROUND: It has been previously shown that doxycycline (Doxy) protects the kidney from preservation injury by inhibition of matrix metalloproteinase. However, the precise molecular mechanism involved in this protection from injury is not known. We used a pharmaco-proteomics approach to identify potential molecular targets associated with kidney preservation injury. METHODS: Rat kidneys were cold perfused with or without doxycycline (Doxy) for 22 h. Kidneys perfusates were analyzed for the presence of injury markers such as lactate dehydrogenase (LDH), and neutrophil-gelatinase associated lipocalin (NGAL). Proteins extracted from kidney tissue were analyzed by 2-dimensional gel electrophoresis. Proteins of interest were identified by mass spectrometry. RESULTS: Triosephosphate isomerase, PGM, dihydropteridine reductase-2, pyridine nucleotide-disulfide oxidoreductase, phosphotriesterase-related protein, and aminoacylase-1A were not affected by cold perfusion. Perfusion with Doxy increased their levels. N(G),N(G)-dimethylarginine dimethylaminohydrolase and phosphoglycerate kinase 1 were decreased after cold perfusion. Perfusion with Doxy led to an increase in their levels. CONCLUSIONS: This study revealed specific metabolic enzymes involved in preservation injury and in the mechanism whereby Doxy protects the kidney against injury during cold perfusion.

Proteomic Analysis of the Cell Cycle of Procylic Form Trypanosoma brucei.

We describe a single-step centrifugal elutriation method to produce synchronous Gap1 (G1)-phase procyclic trypanosomes at a scale amenable for proteomic analysis of the cell cycle. Using ten-plex tandem mass tag (TMT) labeling and mass spectrometry (MS)-based proteomics technology, the expression levels of 5325 proteins were quantified across the cell cycle in this parasite. Of these, 384 proteins were classified as cell-cycle regulated and subdivided into nine clusters with distinct temporal regulation. These groups included many known cell cycle regulators in trypanosomes, which validates the approach. In addition, we identify 40 novel cell cycle regulated proteins that are essential for trypanosome survival and thus represent potential future drug targets for the prevention of trypanosomiasis. Through cross-comparison to the TrypTag endogenous tagging microscopy database, we were able to validate the cell-cycle regulated patterns of expression for many of the proteins of unknown function detected in our proteomic analysis. A convenient interface to access and interrogate these data is also presented, providing a useful resource for the scientific community. Data are available via ProteomeXchange with identifier PXD008741 (https://www.ebi.ac.uk/pride/archive/).

What Information Are Patients Receiving from the Internet about the Operative and Nonoperative Management of Acute Appendicitis?

INTRODUCTION: Recent studies suggest that nonoperative management of appendicitis (NOMA) may be a reasonable option for managing uncomplicated acute appendicitis. We examined the Internet to see if patients are likely to find the information they need to make an informed decision between the 2 options. METHODS: A list of 29 search terms was established by a focus group and then entered into Google, resulting in 49 unique webpages, each reviewed by 3 reviewers. Consensus was obtained for bias (surgery, NOMA, or balanced), webpage type, JAMA score, reading grade, and DISCERN score, a measure of quality of written information for patients. RESULTS: Thirty of the 49 websites (61%) favored surgery, while 13 (27%) favored NOMA, and 6 sites (12%) provided balanced information. Twelve of 49 sites (24%) did not list NOMA as an option. The majority of patient-directed (11/12 = 92%) and physician-directed (7/9 = 78%) webpages favored surgery, whereas academic webpages presented a more balanced distribution. Academic and physician-directed webpages ranked higher than commercial and news webpages (median ranks 3 and 4 vs. 7.5 and 8). Only 8/49 sites (16%) mentioned that the presence of a fecalith predicts the failure of NOMA. Reading grades were almost all well above the recommended grade 8 level. CONCLUSION: Most of the webpages available on the Internet do not provide enough information, nor are they sufficiently understandable to allow most patients to make an informed decision about the current options for the management of acute appendicitis.