Spatial Chemoproteomics for Mapping the Active Proteome.

Functional regulation of cell signaling through dynamic changes in protein activity state as well as spatial organization represent two dynamic, complex, and conserved phenomena in biology. Seemingly separate areas of -omics method development have focused on building tools that can detect and quantify protein activity states, as well as map sub-cellular and intercellular protein organization. Integration of these efforts, through the development of chemical tools and platforms that enable detection and quantification of protein functional states with spatial resolution provide opportunities to better understand heterogeneity in the proteome within cell organelles, multi-cellular tissues, and whole organisms. This review provides an overview of and considerations for major classes of chemical proteomic probes and technologies that enable protein activity mapping from sub-cellular compartments to live animals.

PARK7 Catalyzes Stereospecific Detoxification of Methylglyoxal Consistent with Glyoxalase and Not Deglycase Function.

The protein PARK7 (also known as DJ-1) has been implicated in several diseases, with the most notable being Parkinson's disease. While several molecular and cellular roles have been ascribed to DJ-1, there is no real consensus on what its true cellular functions are and how the loss of DJ-1 function may contribute to the pathogenesis of Parkinson's disease. Recent reports have implicated DJ-1 in the detoxification of several reactive metabolites that are produced during glycolytic metabolism, with the most notable being the α-oxoaldehyde species methylglyoxal. While it is generally agreed that DJ-1 is able to metabolize methylglyoxal to lactate, the mechanism by which it does so is hotly debated with potential implications for cellular function. In this work, we provide definitive evidence that recombinant DJ-1 produced in human cells prevents the stable glycation of other proteins through the conversion of methylglyoxal or a related alkynyl dicarbonyl probe to their corresponding α-hydroxy carboxylic acid products. This protective action of DJ-1 does not require a physical interaction with a target protein, providing direct evidence for a glutathione-free glyoxalase and not a deglycase mechanism of methylglyoxal detoxification. Stereospecific liquid chromatography-mass spectrometry (LC-MS) measurements further uncovered the existence of nonenzymatic production of racemic lactate from MGO under physiological buffer conditions, whereas incubation with DJ-1 predominantly produces l-lactate. Collectively, these studies provide direct support for the stereospecific conversion of MGO to l-lactate by DJ-1 in solution with negligible or no contribution of direct protein deglycation.

Outcome of Operated Colorectal Cancers in Relation to the Type of Initial Referral.

Aim Since the introduction of the target referral system, there has been controversy about its value and whether it affected the short- and long-term outcomes of colorectal cancer surgeries. With contradicting results, this study highlights differences in personal and tumour characteristics, management differences, and outcomes in each referral pathway, including target pathway referrals for suspected cancers, emergency presentations, routine referrals, and incidentally discovered cancers during screening. Methods A retrospective study of colorectal cancer (CRC) patients operated on between January 1, 2010, and December 31, 2014, with records dating to the end of the five-year follow-up, was extracted anonymously from the database of CRC outcomes at the North Middlesex University Hospital NHS Trust, London. The total number of patients operated on through the four pathways was 176, with full records and competent follow-ups. Patients were classified according to the mode of referral: two-week wait (2WW or target), routine, emergency, and incidental discovery referrals. Comparisons were made between these groups with regard to personal and tumour characteristics, management, and outcome. Results It has been demonstrated by this study that target referrals present mainly with stage I cancers as compared to emergency referrals that present with more stage II (IIa+ IIb+ IIc). The highest percentage of cancer locations within the large bowel was rectal, followed by sigmoid in both target and emergency groups; 8.8% of target patients needed neoadjuvant chemoradiotherapy in the form of FOLFOX (folinic acid, 5-fluorouracil, and oxaliplatin) chemotherapy protocol with the addition of radiotherapy in patients with advanced rectal cancers, compared to 13.3% of emergency patients. Conclusion The colorectal 2WW system was the main pathway supplying colorectal cancer operations; it mostly showed earlier cancers than the other referral groups; its cancers were mostly rectosigmoid with less need for adjuvant chemotherapy; fewer recurrences; and it also showed a lower five-year mortality rate than the emergency group.

Experience of a District General Hospital With a Diverse Community in Operated Colorectal Cancers According to Ethnic Background.

Background This study aimed to investigate disparities in colorectal cancer (CRC) patients' demographics according to the five major ethnicities of patients living in the catchment area of North Middlesex Hospital. Methodology This retrospective study included CRC patients operated on between January 1, 2010, and December 31, 2014. Records dating to the end of the five-year follow-up were extracted anonymously from a database of CRC outcomes at the North Middlesex University Hospital NHS Trust. Comparisons were made according to ethnicity, patient demographics, type of presentation, cancer location, stage at diagnosis, recurrence, and mortality. Results A total of 176 adult patients were operated on for CRC between January 1, 2010, and December 31, 2014. The majority of the patients were referred as two-week wait target referrals. Emergency presentation of CRC was the highest in White non-UK patients. The White British Irish patients had their tumors mostly in the cecum, followed by the sigmoid colon, while the rectum followed by the sigmoid colon were the most common sites in the Black population. All study populations mainly presented with stage I disease, and the next highest incidence of cancers according to stage and ethnicity was stage IIIb in the Black population. Conclusions Differences in the ethnic background are important factors, especially in a diverse community, which can impact the age and mode of presentation of the disease, as well as the stage it starts to present. The location of the primary tumor, metastases, and recurrence sites are all affected by the ethnic background, which, subsequently, affect the survival of the patient.

The Accuracy of Different Modalities Used for Preoperative Primary Tumour Localisation in Operated Colorectal Cancer Patients.

Aim Colonoscopy and computed tomography (CT) scans of the abdomen and pelvis are routine pre-operative assessment tools in colorectal cancer (CRC) patients. There have been some discrepancies regarding the location of cancer when seen by colonoscopy versus CT scan. The purpose of this study was to compare the accuracy of a colonoscopy with a computed tomography (CT) scan of the abdomen and pelvis with contrast, which is done routinely before surgery to localise the exact site of the tumour within the large bowel, whilst comparing both to the operative, gross and histopathology findings of the exact location. Methods A retrospective study was carried out on 165 colorectal cancer patients operated on between January 1, 2010, and December 31, 2014, using electronic hospital records that were reviewed anonymously, comparing the location of cancer within the large bowels as was found on colonoscopy and CT scan of the abdomen and pelvis with contrast, comparing both to post-operative histopathology specimen or intra-operative assessment in cases where no resection of the primary tumour was performed. Results CT and colonoscopy were both accurate in diagnosing 70.5% of cases that had done both investigations pre-operatively. The best results were obtained when the cancer was located in the caecum as confirmed post-operatively; the combined accuracy rate was 100%. CT was accurate, whilst colonoscopy was not in eight (6.2%) cases (all are rectal or sigmoid cancers), and colonoscopy was accurate and CT was not in 12 cases, 10 of them were rectal and two were ascending colonic. Colonoscopy was not performed in 36 (21%) cases for a variety of reasons, including large bowel obstruction or perforation on presentation. In 32 of these cases, CT scan managed to accurately predict the location of cancer (mostly rectal and caecal), and CT scan was inaccurate in 20.6% of cases (34 out of 165), whilst colonoscopy was inaccurate in 13.9% of cases (18 out of 129). Conclusion Colonoscopy is more accurate in localising colorectal cancers than CT scan of the abdomen and pelvis with contrast. CT scan diagnoses regional and distant spread of colorectal cancers such as nodal status, invasion of neighbouring organs and/or peritoneum and the presence of liver metastases, whilst colonoscopy is limited to intraluminal diagnosis but can be both a diagnostic and therapeutic tool, with higher accuracy, in general, in localising colorectal cancers. Both CT scan and colonoscopy were equal in appendicular, caecal, splenic flexure and descending colon cancer localisation accuracy.

Widespread, Reversible Cysteine Modification by Methylglyoxal Regulates Metabolic Enzyme Function.

Methylglyoxal (MGO), a reactive metabolite byproduct of glucose metabolism, is known to form a variety of posttranslational modifications (PTMs) on nucleophilic amino acids. For example, cysteine, the most nucleophilic proteinogenic amino acid, forms reversible hemithioacetal and stable mercaptomethylimidazole adducts with MGO. The high reactivity of cysteine toward MGO and the rate of formation of such modifications provide the opportunity for mechanisms by which proteins and pathways might rapidly sense and respond to alterations in levels of MGO. This indirect measure of alterations in glycolytic flux would thereby allow disparate cellular processes to dynamically respond to changes in nutrient availability and utilization. Here we report the use of quantitative LC-MS/MS-based chemoproteomic profiling approaches with a cysteine-reactive probe to map the proteome-wide landscape of MGO modification of cysteine residues. This approach led to the identification of many sites of potential functional regulation by MGO. We further characterized the role that such modifications have in a catalytic cysteine residue in a key metabolic enzyme and the resulting effects on cellular metabolism.

Biotin-decorated NIR-absorbing nanosheets for targeted photodynamic cancer therapy.

Targeted photodynamic therapy (PDT) is one of the promising approaches for the selective killing of cancerous cells without affecting the normal cells, and hence designing new strategies for targeted PDT is extremely important. Herein we report the design and synthesis of a new class of nanosheets derived from the self-assembly of the iodo-BODIPY-biotin conjugate as a photosensitizer for targeted PDT applications. The nanosheet exhibits a high extinction coefficient in the NIR region, high singlet oxygen efficiency, no toxicity in the dark and cell targeting ligands (biotin) on the surface, which are necessary features required for an ideal photosensitizer. Overexpression of sodium-dependent multivitamin transporters (SMVTs) in HeLa and A549 (biotin receptor positive cell lines) is explored for the selective uptake of the nanophotosensitizer through receptor mediated endocytosis (interaction between biotin and SMVT). Control experiments using a biotin receptor negative cell line (WI-38) are also carried out to confirm that the specific interaction between the SMVTs and biotin is mainly responsible for the selective uptake of the photosensitizer. Efficient killing of cancerous cells is demonstrated upon light irradiation through the generation of singlet oxygen and other reactive oxygen species around the cellular environment.

A DNA-based voltmeter for organelles.

The role of membrane potential in most intracellular organelles remains unexplored because of the lack of suitable tools. Here, we describe Voltair, a fluorescent DNA nanodevice that reports the absolute membrane potential and can be targeted to organelles in live cells. Voltair consists of a voltage-sensitive fluorophore and a reference fluorophore for ratiometry, and acts as an endocytic tracer. Using Voltair, we could measure the membrane potential of different organelles in situ in live cells. Voltair can potentially guide the rational design of biocompatible electronics and enhance our understanding of how membrane potential regulates organelle biology.

Post-operative outcomes of open reduction and internal fixation versus circular external fixation in treatment of tibial plafond fractures: A systematic review and meta-analysis.

INTRODUCTION: Tibial plafond fractures (TPF) are complex injuries often resulting in poor outcomes. Combination of articular impaction, metaphysealcomminution and soft-tissue injury results in a significant treatment challenge. The aim of this study was to conduct a systematic review and meta-analysis to compare post-operative complications and functional outcomes of open reduction and internal fixation (ORIF) versus circular external fixation (CEF) for treatment of TPF. METHODS: A comprehensive search of PubMed/MEDLINE, Embase, Scopus and Cochrane library was undertaken. All studies published in English language comparing ORIF with CEF for treatment of TPF were included. RESULTS: 5 comparative studies with 239 fractures met the inclusion criteria. Meta-analysis showed no significant difference in rates of non-union, malunion, superficial infection, deep infection, and secondary arthrodesis between the two treatment groups. Significantly higher rate of unplanned metalwork removal (RR 5.68, 95% CI 1.13 to 28.55, p = 0.04) and lower rate of post-traumatic arthritis (RR 0.48, 95% CI 0.30 to 0.78, p = 0.003) were found in patients that underwent ORIF. 1 study showed significantly lower functional outcomes scores with CEF (p< 0.05), whereas 3 studies found comparable functional outcomes between the two treatment groups. Overall, there was a preference in treating more severe injuries with CEF. CONCLUSION: CEF and ORIF are both acceptable treatment options for surgical management of TPF, with comparable post-operative complication rates and functional outcomes. This study highlights paucity of high-quality evidence regarding the optimal fixation method for TPF.

Heteronemin, a marine natural product, sensitizes acute myeloid leukemia cells towards cytarabine chemotherapy by regulating farnesylation of Ras.

Cytarabine is a conventionally used chemotherapeutic agent for treating acute myeloid leukemia (AML). However, chemoresistance, toxic side-effects and poor patient survival rates retard the efficacy of its performance. The current study deals with the chemosensitization of AML cells using heteronemin, a marine natural product towards cytarabine chemotherapy. Heteronemin could effectively sensitize HL-60 cells towards sub-toxic concentration of cytarabine resulting in synergistic toxicity as demonstrated by MTT assay and [3H] thymidine incorporation studies, while being safe towards healthy blood cells. Flow cytometry for Annexin-V/PI and immunoblotting for caspase cleavage proved that the combination induces enhancement in apoptosis. Heteronemin being a farnesyl transferase inhibitor (FTI) suppressed cytarabine-induced, farnesyl transferase-mediated activation of Ras, as assessed by Ras pull-down assay. Upon pre-treating cells with a commercial FTI, L-744,832, the synergism was completely lost in the combination, confirming the farnesyl transferase inhibitory activity of heteronemin as assessed by thymidine incorporation assay. Heteronemin effectively down-regulated cytarabine-induced activation of MAPK, AP-1, NF-κB and c-myc, the down-stream targets of Ras signaling, which again validated the role of Ras in regulating the synergism. Hence we believe that the efficacy of cytarabine chemotherapy can be improved to a significant extent by combining sub-toxic concentrations of cytarabine and heteronemin.

Effects of sleep deprivation on cognitive and physical performance in university students.

Sleep deprivation is common among university students, and has been associated with poor academic performance and physical dysfunction. However, current literature has a narrow focus in regard to domains tested, this study aimed to investigate the effects of a night of sleep deprivation on cognitive and physical performance in students. A randomized controlled crossover study was carried out with 64 participants [58% male (n = 37); 22 ± 4 years old (mean ± SD)]. Participants were randomized into two conditions: normal sleep or one night sleep deprivation. Sleep deprivation was monitored using an online time-stamped questionnaire at 45 min intervals, completed in the participants' homes. The outcomes were cognitive: working memory (Simon game© derivative), executive function (Stroop test); and physical: reaction time (ruler drop testing), lung function (spirometry), rate of perceived exertion, heart rate, and blood pressure during submaximal cardiopulmonary exercise testing. Data were analysed using paired two-tailed T tests and MANOVA. Reaction time and systolic blood pressure post-exercise were significantly increased following sleep deprivation (mean ± SD change: reaction time: 0.15 ± 0.04 s, p = 0.003; systolic BP: 6 ± 17 mmHg, p = 0.012). No significant differences were found in other variables. Reaction time and vascular response to exercise were significantly affected by sleep deprivation in university students, whilst other cognitive and cardiopulmonary measures showed no significant changes. These findings indicate that acute sleep deprivation can have an impact on physical but not cognitive ability in young healthy university students. Further research is needed to identify mechanisms of change and the impact of longer term sleep deprivation in this population.

Folic acid conjugation improves the bioavailability and chemosensitizing efficacy of curcumin-encapsulated PLGA-PEG nanoparticles towards paclitaxel chemotherapy.

Nanoencapsulation has emerged as a novel strategy to enhance the pharmacokinetic and therapeutic potential of conventional drugs. Recent studies from our lab have established the efficacy of curcumin in sensitizing cervical cancer cells and breast cancer cells towards paclitaxel and 5-FU chemotherapy respectively. Factors that hinder the clinical use of curcumin as a sensitizer or therapeutic agent include its poor bioavailability and retention time. Earlier reports of improvement in bioavailability and retention of drugs upon nanoencapsulation have motivated us in developing various nanoformulations of curcumin, which were found to exhibit significant enhancement in bioavailability and retention time as assessed by our previous in vitro studies. Among the various formulations tested, curcumin-entrapped in PLGA-PEG nanoparticles conjugated to folic acid (PPF-curcumin) displayed maximum cell death. In the present study, we have demonstrated the efficacy of this formulation in augmenting the bioavailability and retention time of curcumin, in vivo, in Swiss albino mice. Further, the acute and chronic toxicity studies proved that the formulation is pharmacologically safe. We have also evaluated its potential in chemosensitizing cervical cancer cells to paclitaxel and have verified the results using cervical cancer xenograft model in NOD-SCID mice. Folic acid conjugation significantly enhanced the efficacy of curcumin in down-regulating various survival signals induced by paclitaxel in cervical cancer cells and have considerably improved its potential in inhibiting the tumor growth of cervical cancer xenografts. The non-toxic nature coupled with improved chemosensitization potential makes PPF-curcumin a promising candidate formulation for clinical trials.