Improved Structural Characterization of Glycerophospholipids and Sphingomyelins with Real-Time Library Searching.

In mass spectrometry-based lipidomics, complex lipid mixtures undergo chromatographic separation, are ionized, and are detected using tandem MS (MSn) to simultaneously quantify and structurally characterize eluting species. The reported structural granularity of these identified lipids is strongly reliant on the analytical techniques leveraged in a study. For example, lipid identifications from traditional collisionally activated data-dependent acquisition experiments are often reported at either species level or molecular species level. Structural resolution of reported lipid identifications is routinely enhanced by integrating both positive and negative mode analyses, requiring two separate runs or polarity switching during a single analysis. MS3+ can further elucidate lipid structure, but the lengthened MS duty cycle can negatively impact analysis depth. Recently, functionality has been introduced on several Orbitrap Tribrid mass spectrometry platforms to identify eluting molecular species on-the-fly. These real-time identifications can be leveraged to trigger downstream MSn to improve structural characterization with lessened impacts on analysis depth. Here, we describe a novel lipidomics real-time library search (RTLS) approach, which utilizes the lipid class of real-time identifications to trigger class-targeted MSn and to improve the structural characterization of phosphotidylcholines, phosphotidylethanolamines, phosphotidylinositols, phosphotidylglycerols, phosphotidylserine, and sphingomyelins in the positive ion mode. Our class-based RTLS method demonstrates improved selectivity compared to the current methodology of triggering MSn in the presence of characteristic ions or neutral losses.

Hypo-fractionated accelerated radiotherapy with concurrent and maintenance temozolomide in newly diagnosed glioblastoma: updated results from phase II HART-GBM trial.

BACKGROUND: Glioblastoma (GBM) patients have poor survival outcomes despite treatment advances and most recurrences occur within the radiation field. Survival outcomes after dose escalation through hypofractionated accelerated RT(HART) were evaluated in this study. We previously reported the study's initial results showing similar survival outcomes with acceptable toxicities. Updated results after 5 years are being analysed to determine long-term survival trends. PATIENTS AND METHODS: 89 patients of newly diagnosed GBM after surgery were randomized to conventional radiotherapy (CRT) or HART. CRT arm received adjuvant RT 60 Gy in 30 fractions over 6 weeks and the HART arm received 60 Gy in 20 fractions over 4 weeks, both with concurrent and adjuvant temozolomide. RESULTS: 83 patients were eligible for analysis. After a median follow-up of 18.9 months, the median OS was 26.5 months and 22.4 months in the HART and CRT arms respectively. 5 year OS was 18.4% in the HART arm versus 3.8% in the CRT arm. This numerical difference in overall survival between the two arms was not statistically significant. The median PFS was not significantly different. CONCLUSION: The long-term results of the trial support HART as a promising treatment option with comparable survival outcomes to the current standard of care. Phase III trials are required for further validation of this regimen which has the potential to become the new standard of care in GBM.

Does testosterone influence the association between sleep and frailty in men: results from the European Male Aging Study.

BACKGROUND: Previous studies have suggested an association between sleep disturbance and frailty. The mechanism is unknown, although it has been suggested that hormonal factors may play a role. METHODS: The aim was to determine the association between sleep duration, sleep quality and frailty, and to determine whether testosterone influenced this association. Males aged 40-79 years were recruited from eight European centres to the European Male Aging Study (EMAS). Subjects completed an interviewer-assisted questionnaire including questions regarding sleep quality and duration. Sleep quality was scored 0-20 and categorised as 0-4, 5-9, 10-14, and 15-20, with higher scores indicating poorer quality. A 39-component frailty index (FI) was constructed. Total testosterone levels were measured. The association between sleep duration, sleep quality and the FI was assessed using negative binomial regression, with adjustment for putative confounders including testosterone level. RESULTS: Two thousand three hundred ninety-three participants contributed data to the analysis. The mean age was 63.3 years and mean sleep duration was 7.01 h. The mean frailty index was 0.15. Mean testosterone levels declined with decreasing sleep quality. After adjustment, compared to those with a sleep score of 0-4, the FI was 57% (95% CI 38%, 78%) higher among those with a sleep score of 15-20. After adjustment compared to those with normal sleep duration (6-9 h), those with a short (< 6 h) and long (≥ 9 h) sleep duration had a 16% (95% CI 6%, 28%) and 11% (95% CI 0%, 23%) higher FI, respectively. Adjustment for testosterone did not influence the strength of either association. CONCLUSION: Frailty is associated with impaired sleep quality and sleep duration. The association cannot, however, be explained by variation in testosterone levels.

Laparoscopic Management of Disseminated Peritoneal Leiomyomatosis.

STUDY OBJECTIVE: To show laparoscopic management of disseminated peritoneal leiomyomatosis (DPL). DESIGN: Stepwise demonstration of the technique with narrated video footage. SETTING: DPL is characterized by dissemination and proliferation of peritoneal and subperitoneal lesions primarily originating from smooth muscle cells [1]. Generally considered benign, cases of malignant transformation to leiomyosarcoma have been reported [2,3]. Iatrogenic DPL occurs because of unconfined morcellation resulting in small fragments of myoma that may implant on any organ and start deriving blood supply from it or may be pulled into port site while withdrawing laparoscopic cannulas [4]. It is estimated that the overall incidence of DPL after laparoscopic uncontained morcellation was 0.12% to 0.95% [5]. Mainstay of treatment is surgical resection of myomas and regular follow-up with imaging. A 28-year-old unmarried girl presented with complain of lump abdomen increasing in size for 1 year. She also complained of a 15 kg weight loss in the last 1 year; 4 years ago, patient had undergone laparoscopic myomectomy with unconfined morcellation for a 10 × 8 cm cervical myoma. Presently her menses were regular with a 28-day cycle and 3 to 4 days' average flow. Magnetic resonance imaging showed multiple nodular lesions of varying sizes in relation to small bowel, colon, uterus, and anterior abdominal wall  suggestive of DPL. Bilateral ovaries were normal. Tumor markers were as follows: CA 125 23.2 (<35) U/mL Carcinoembryonic antigen 1.67 (<8) ng/mL CA 19-9 47 (<37) U/mL Lactate dehydrogenase 809 (180-360) IU/L Alpha-fetoprotein 2.03 (<10) ng/mL Beta human chorionic gonadotropin 1.2(<2) mIU/mL Tru-cut biopsy was done elsewhere to rule out peritoneal carcinomatosis in view of raised CA 19-9 and lactate dehydrogenase, history of weight loss, and imaging showing multiple abdominal masses. Histopathological examination showed leiomyomatosis and immunohistochemistry for smooth muscle actin, desmin, and vimentin were positive. INTERVENTIONS: On laparoscopy the abdominal cavity was found studded with multiple leiomyomas of varying sizes deriving blood supply from ilium, transverse, descending and sigmoid colon, rectum, left tube, left ovary, pouch of Douglas, bilateral uterosacrals, uterovesical fold, and anterior abdominal wall. Large blood vessels were seen traversing between the descending and sigmoid colon and the myomas. Principles of surgery were as follows: 1. Complete removal of myomas 2. Cauterization of blood vessels feeding the parasitic myomas to minimize blood loss 3. Disscetion abutting the myoma to prevent injury to adjacent viscera. A total of 26 myomas were removed. All the myomas were retrieved by morcellation in a bag. Histopathology confirmed the diagnosis of diffuse peritoneal leiomyomatosis. Follow-up ultrasound at 6 months showed no recurrence of leiomyomatosis. CONCLUSION: Proper mapping of lesions and surgery for complete removal of all masses is the mainstay of treatment. Contained morcellation in bag should be the norm to prevent iatrogenic DPL. Regular follow-up with imaging is required to rule out recurrence.

Spinal atypical teratoid rhabdoid tumor-narrative review and report of a rare case managed with multimodality approach.

BACKGROUND: Spinal atypical teratoid rhabdoid tumor (AT/RT) is an extremely rare tumor and represents less than 2% of all AT/RTs. METHODS: Available medical literature on spinal AT/RT in English was retrieved from PubMed and comprehensively reviewed. Clinical presentation, diagnosis, management, prognosis, and outcome in patients with spinal AT/RT have been elucidated by citing a case of extradural AT/RT of the cervicodorsal spine. RESULTS: The age at presentation is usually less than 3 years. The most common site is the cervicodorsal spine. The most frequent tumor location is intradural extramedullary. A contrast-enhanced magnetic resonance imaging (MRI) of the entire neuraxis is the imaging modality of choice. The incidence of leptomeningeal dissemination is high (15-30%). Histopathological examination shows an admixture of primitive neuroectodermal, mesenchymal, and epithelial elements along with rhabdoid cells. Loss of SMARCB1/INI1 is considered pathognomonic of AT/RT. Maximal safe resection of tumor is the initial management of choice. Thereafter focal radiotherapy for localized tumor or craniospinal irradiation for leptomeningeal dissemination should be considered. Post-operative intensive polychemotherapy including intrathecal and high-dose chemotherapy (with autologous stem cell rescue) is usually considered to optimize survival. Typically, the time to recurrence and overall survival are less than 6 and 12 months, respectively. However, with judicious multimodality management long-term survivors are increasingly being recognized. The illustrative patient was a 18-month-old girl diagnosed with extradural AT/RT of the cervicodorsal spine (C3-D1), who was managed with maximal safe resection of tumor, multiagent chemotherapy (ICE-ifosfamide, carboplatin, etoposide) and focal RT to the tumor bed-50.4 Gy/28 fractions/5.5 weeks. At the last follow-up visit, 30 months after surgery, she had complete clinicoradiological response. CONCLUSION: Multimodal treatment comprising maximal safe resection of tumor, multiagent chemotherapy (ICE), and focal RT can lead to successful outcome in patients with localized spinal AT/RT, under the age of 3 years.

Novel Real-Time Library Search Driven Data Acquisition Strategy for Identification and Characterization of Metabolites.

Structural characterization of novel metabolites in drug discovery or metabolomics is one of the most challenging tasks. Multilevel fragmentation (MSn) based approaches combined with various dissociation modes are frequently utilized for facilitating structure assignment of unknown compounds. As each of the MS precursors undergoes MSn, the instrument cycle time can limit the total number of precursors analyzed in a single LC run for complex samples. This necessitates splitting data acquisition into several analyses to target lower concentration analytes in successive experiments. Here we present a new LC/MS data acquisition strategy, termed Met-IQ, where the decision to perform an MSn acquisition is automatically made in real time based on the similarity between the experimental MS2 spectrum and a spectrum in a reference spectral library for the known compounds of interest. If similarity to a spectrum in the library is found, the instrument performs a decision-dependent event, such as an MS3 spectrum. Compared to an intensity-based, data-dependent MSn experiment, only a limited number of MS3 are triggered using Met-IQ, increasing the overall MS2 instrument sampling rate. We applied this strategy to an Amprenavir sample incubated with human liver microsomes. The number of MS2 spectra increased 2-fold compared to a data dependent experiment where MS3 was triggered for each precursor, resulting in identification of 14-34% more unique potential metabolites. Furthermore, the MS2 fragments were selected to focus likely sources of useful structural information, specifically higher mass fragments to maximize acquisition of MS3 data relevant for structure assignment. The described Met-IQ strategy is not limited to metabolism experiments and can be applied to analytical samples where the detection of unknown compounds structurally related to a known compound(s) is sought.

Effect of a quality improvement package on reducing newborn mortality from 26 to 14 in a Himalayan North Indian State.

Background: In India, newborn mortality remains high due to a number of factors, including poor quality of care at health facilities. The experience of executing complete neonatal care quality improvement (QI) package at selected hospitals in Himachal Pradesh and reduction in newborn mortality rate (NMR) is described in this study. Objective: The short-term objective was the participants' retention of knowledge and skills, and the achievement of uniform QI objectives following training and after a minimum of 6 months. Overall reduction in NMR was long-term objective. Methods: Newborn care QI package was implemented according to India Newborn Action Plan over a period of 48 months from 2013 to 2016, through infrastructure, trainings, and supportive supervision. Results: Total 13 health facilities were upgraded; 350 staff nurses and medical officers were trained. The mean posttraining knowledge score was 75% compared to 29% in the pretraining test, and 63% 1 year later. The competencies of health workers in the care of high-risk babies and 12 QI targets had improved, resulting in a 46% reduction in neonatal mortality in the state across all gestations and weights based on sample registration survey. Conclusion: Implementation of a bundle of evidence-based practices in low-resource setting for health system strengthening for intrapartum and neonatal care was linked to changed care behaviors among health-care providers, and reduction in NMR.

Epidemiological investigation of viral hepatitis E outbreak in two colocated training centers.

Background: A high number of jaundice cases were reported from two colocated training centers in North India. This outbreak was investigated to describe the epidemiology, identify risk factors, and recommend preventive and control measures. Methods: Initial line list was prepared, and case definition was defined as "the presence of icterus or passage of yellow-colored urine with fever/anorexia/vomiting/abdominal pain in a resident of Military Station A between 03/04/2016 to 06/06/2016". Case search was conducted through surveillance. An unmatched 1:1 case-control study was conducted to evaluate the associated risk factors. All cases were tested for hepatitis markers. Environmental investigation of food and water sources was conducted to identify the source of infection. Results: Of 172 cases, all were males from two co-located military training centers (attack rate, 4.7%). Clinical features included icterus (100%), yellowish discoloration of urine (98.9%), anorexia (97.22%), fever (80%), nausea/vomiting (56%), and abdominal pain (52.77%). Only one case (0.6%) had complication of fulminant hepatitis, and there were no deaths (CFR = 0%). Consumption of juice with ice from juice shops was significantly associated with illness (Odds Ratio-14.3 [95%CI 7.4-27.6]). Of 172 cases, 167 (97.1%) tested anti-HEV-IgM positive. Juice shops in training centers were using ice made from contaminated water with positive coliform test. All other water samples tested satisfactory. No cross-contamination of water pipelines with sewage was observed. Conclusion: Epidemiological evidence concludes that a large viral hepatitis E outbreak was likely caused by consumption of juice with contaminated ice. Early stoppage of contaminated ice usage led to timely control of the outbreak.

Manufacturing and characterization of whey and stevia-based popsicles enriched with concentrated beetroot juice.

The beet-root (Beta vulgaris) and whey powder together, can potentially use as a multifunctional ingredient in the manufacturing of the "Popsicles", due to their biochemical composition that can enhance the concentration of bioactive compounds. In the present study, beet-root juice concentrates were prepared at different time/temperature treatments viz 45 °C, 55 °C, and 65 °C for 120, 80 and 45 min. The effect of different time/temperature treatments on physicochemical composition, colour, antioxidant activity (%), bioactive compounds, spectral data and sensory acceptance were evaluated. The physicochemical parameters of popsicles (PTI, PT2, PT3) including protein, total phenols, betalain, radical scavenging activity %, colour and melting values were significantly affected (p ≤ 0.05) by the different time/temperature treatments. The concentration of betalain and protein in all the popsicles ranged from 1134 to 1299 mg/L and 1.92 to 1.54 g/100 g respectively. The reduction of bioactive components viz betacyanins, betaxanthins, betanin, oxalic and syringic acid was also observed in popsicle (PTI) as compared to control. Furthermore, popsicle (PT1) was prepared with beet-root juice concentrated at 45 °C showed maximum sensory acceptance. The physicochemical and organoleptic attributes of processed popsicles encourage the commercial usage of whey powder and concentrated beetroot juice.

Prevalence and predictors of adverse events with methotrexate mono- and combination-therapy for rheumatoid arthritis: a systematic review.

OBJECTIVES: This systematic review aims to summarize rates of adverse events (AEs) in patients with RA or inflammatory arthritis starting MTX as monotherapy or in combination with other csDMARDs, and to identify reported predictors of AEs. METHODS: Three databases were searched for studies reporting AEs in MTX-naïve patients with RA. Randomized controlled trials (RCTs) and observational cohort studies were included. Prevalence rates of AEs were pooled using random effects meta-analysis, stratified by study design. RESULTS: Forty-six articles (34 RCTs and 12 observational studies) were identified. The pooled prevalence of total AEs was 80.1% in RCTs (95% CI: 73.5, 85.9), compared with 23.1% in observational studies (95% CI: 12.3, 36.0). The pooled prevalence of serious AEs was 9.5% in RCTs (95% CI: 7.4, 11.7), and 2.1% in observational studies (95% CI: 1.0, 3.4). MTX discontinuation due to AEs was higher in observational studies (15.5%, 95% CI: 9.6, 22.3) compared with RCTs (6.7%, 95% CI: 4.7, 8.9). Gastrointestinal events were the most commonly reported AEs (pooled prevalence: 32.7%, 95% CI: 18.5, 48.7). Five studies examined predictors of AEs. RF status, BMI and HAQ score were associated with MTX discontinuation due to AEs; ACPA negativity, smoking and elevated creatinine were associated with increased risk of elevated liver enzymes. CONCLUSION: The review provides an up-to-date overview of the prevalence of AEs associated with MTX in patients with RA. The findings should be communicated to patients to help them make informed choices prior to commencing MTX.

Successful Multimodality Management of Atypical Teratoid/Rhabdoid Tumour of the Lower Dorsal Spine with Spinal Drop Metastasis: Illustrated Review.

INTRODUCTION: Spinal atypical teratoid/rhabdoid tumour (AT/RT) is exquisitely rare and constitutes 2% of all AT/RTs. CASE PRESENTATION: A 6-year-old boy presented with low backache for the last 5 months. MRI of the spine showed a 1.5 × 1.5 × 4.7 cm intradural extramedullary mass extending from D10 to D12, causing compression of the conus medullaris. With a preoperative diagnosis of ependymoma, a gross total resection (GTR) of tumour was performed. Post-operative histopathology showed AT/RT. The tumour cells were immunopositive for cytokeratin, epithelial membrane antigen, smooth muscle actin, and p53 and immunonegative for MIC2, desmin, glial fibrillary acidic protein, and INI1. Post-operative neuraxis MRI revealed post-operative changes (D10-D12) with a 9 mm enhancing lesion at L5-S1 junction suggesting drop metastasis. There was no lesion in brain. Cerebrospinal fluid cytology did not show any malignant cell. The metastatic work-up was normal. He received 3 cycles of chemotherapy with ICE regimen (ifosfamide, carboplatin, and etoposide). Subsequently, he received craniospinal irradiation (CSI)-36 Gy/20 fractions/4 weeks followed by focal boost to primary tumour bed and spinal drop metastasis-14.4 Gy/8 fractions/1.5 weeks. Thereafter, he received 3 more cycles of ICE regimen. End-of-treatment MRI spine showed post-op changes (D10-D12) and 38.9% reduction of the L5-S1 lesion suggesting partial response. Six monthly spinal MRI showed serial reduction of the metastatic lesion leading to complete response (CR) 1 year after completion of treatment. On last follow-up (30 months from the initial diagnosis), he was neurologically intact and in CR. CONCLUSION: Multimodality management comprising GTR of tumour, CSI followed by focal boost, and multiagent chemotherapy (ICE) can lead to successful outcome in patients with this rare and aggressive spinal tumour.

Ultraviolet Photodissociation for Non-Target Screening-Based Identification of Organic Micro-Pollutants in Water Samples.

Non-target screening (NTS) based on the combination of liquid chromatography coupled to high-resolution mass spectrometry has become the key method to identify organic micro-pollutants (OMPs) in water samples. However, a large number of compounds remains unidentified with current NTS approaches due to poor quality fragmentation spectra generated by suboptimal fragmentation methods. Here, the potential of the alternative fragmentation technique ultraviolet photodissociation (UVPD) to improve identification of OMPs in water samples was investigated. A diverse set of water-relevant OMPs was selected based on k-means clustering and unsupervised artificial neural networks. The selected OMPs were analyzed using an Orbitrap Fusion Lumos equipped with UVPD. Therewith, information-rich MS2 fragmentation spectra of compounds that fragment poorly with higher-energy collisional dissociation (HCD) could be attained. Development of an R-based data analysis workflow and user interface facilitated the characterization and comparison of HCD and UVPD fragmentation patterns. UVPD and HCD generated both unique and common fragments, demonstrating that some fragmentation pathways are specific to the respective fragmentation method, while others seem more generic. Application of UVPD fragmentation to the analysis of surface water enabled OMP identification using existing HCD spectral libraries. However, high-throughput applications still require optimization of informatics workflows and spectral libraries tailored to UVPD.

Neprosin, a Selective Prolyl Endoprotease for Bottom-up Proteomics and Histone Mapping.

Trypsin dominates bottom-up proteomics, but there are reasons to consider alternative enzymes. Improving sequence coverage, exposing proteomic "dark matter," and clustering post-translational modifications in different ways and with higher-order drive the pursuit of reagents complementary to trypsin. Additionally, enzymes that are easy to use and generate larger peptides that capitalize upon newer fragmentation technologies should have a place in proteomics. We expressed and characterized recombinant neprosin, a novel prolyl endoprotease of the DUF239 family, which preferentially cleaves C-terminal to proline residues under highly acidic conditions. Cleavage also occurs C-terminal to alanine with some frequency, but with an intriguingly high "skipping rate." Digestion proceeds to a stable end point, resulting in an average peptide mass of 2521 units and a higher dependence upon electron-transfer dissociation for peptide-spectrum matches. In contrast to most proline-cleaving enzymes, neprosin effectively degrades proteins of any size. For 1251 HeLa cell proteins identified in common using trypsin, Lys-C, and neprosin, almost 50% of the neprosin sequence contribution is unique. The high average peptide mass coupled with cleavage at residues not usually modified provide new opportunities for profiling clusters of post-translational modifications. We show that neprosin is a useful reagent for reading epigenetic marks on histones. It generates peptide 1-38 of histone H3 and peptide 1-32 of histone H4 in a single digest, permitting the analysis of co-occurring post-translational modifications in these important N-terminal tails.

SEXUAL AND REPRODUCTIVE HEALTH CONCERNS OF PERSONS WITH DISABILITY IN INDIA: AN ISSUE OF DEEP-ROOTED SILENCE.

Global estimates suggest that over a billion people live with a disability that is significant enough to affect their daily lives. According to the 2011 Indian Census, India alone has about 26.8 million people with disabilities. Research suggests that persons with disabilities (PwDs) in India are among the most neglected, stigmatized, poor and least educated of the world's population, and women with disabilities in India are the most marginalized, both socially and economically. They bear the triple burden of being discriminated against through being 'women' (socially marginal beings), 'disabled' (incapacitated, inefficient and undesirable) and 'women with disabilities' (the weakest of the weak), often becoming socially invisible. Although there has been a general recognition over the years that the educational and employment opportunities of PwDs in India need to be improved, their sexual needs and aspirations, sexuality concerns and sexual and reproductive health and rights have been largely ignored. The objective of this paper is to highlight the paucity of research on the sexual and reproductive health concerns of PwDs, particularly women, in the Indian context using existing literature on India, and to identify the possible reasons of this neglect. The study describes the obstacles faced by PwDs, particularly women, to acquiring good sexual and reproductive information and services, based on the results of empirical studies. Given the lack of research on this in India, the evidence largely comes from studies conducted elsewhere in the world. Lack of information and education about sexual health concerns, physical and/or infrastructural inaccessibility, judgemental provider attitudes, limited provider knowledge about disability issues and individual factors, including inhibitions about seeking health care and financial barriers, are identified as factors inhibiting the sexual and reproductive rights of people with disabilities in India.

Biosynthesized composites of Au-Ag nanoparticles using Trapa peel extract induced ROS-mediated p53 independent apoptosis in cancer cells.

The current study highlights rapid, sustainable, and cost-effective biosynthesis of silver (Ag), gold (Au) nanoparticles (NPs), and bimetallic Au-AgNPs composites using bio-waste extract of Trapa natans. Growth of the NPs was monitored spectrophotometrically and peak was observed at ∼525 nm, ∼450 nm, and ∼495 nm corresponding to Plasmon absorbance of AuNPs, AgNPs, and Au-AgNPs, respectively. Transmission electron microscopy (TEM) revealed the size of AgNPs (∼15 nm), AuNPs (∼25 nm), and Au-AgNPs (∼26-90 nm). Synthesized NPs follow the Gaussian bell curve and its crystalline nature was identified by X-ray diffraction (XRD). Furthermore, Au-AgNPs induced cytotoxicity in various cancer cells (HCT116, MDA-MB-231, and HeLa) effectively at 200 µg/mL. Au-AgNPs-exposed cancer cells exhibited apoptotic features such as nuclear condensation, mitochondrial membrane potential loss, and cleavage of casp-3 and poly (ADP-ribose) polymerase-1 (PARP). Au-AgNPs exposure enhanced reactive oxygen species (ROS) and upon inhibition of ROS, apoptosis was reduced effectively. NPs treatment killed HCT116 WT and p53 knockout cells without any significant difference. Mechanistically, Au-AgNPs derived with Trapa peel extract significantly enhance ROS which trigger p53-independent apoptosis in various cancer cells effectively. Our study explores the use of bio-waste for the green synthesis of NPs, which can be attractive candidates for cancer therapy.

Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques.

Targeted top-down (TD) and middle-down (MD) mass spectrometry (MS) offer reduced sample manipulation during protein analysis, limiting the risk of introducing artifactual modifications to better capture sequence information on the proteoforms present. This provides some advantages when characterizing biotherapeutic molecules such as monoclonal antibodies, particularly for the class of biosimilars. Here, we describe the results obtained analyzing a monoclonal IgG1, either in its ∼150 kDa intact form or after highly specific digestions yielding ∼25 and ∼50 kDa subunits, using an Orbitrap mass spectrometer on a liquid chromatography (LC) time scale with fragmentation from ion-photon, ion-ion, and ion-neutral interactions. Ultraviolet photodissociation (UVPD) used a new 213 nm solid-state laser. Alternatively, we applied high-capacity electron-transfer dissociation (ETD HD), alone or in combination with higher energy collisional dissociation (EThcD). Notably, we verify the degree of complementarity of these ion activation methods, with the combination of 213 nm UVPD and ETD HD producing a new record sequence coverage of ∼40% for TD MS experiments. The addition of EThcD for the >25 kDa products from MD strategies generated up to 90% of complete sequence information in six LC runs. Importantly, we determined an optimal signal-to-noise threshold for fragment ion deconvolution to suppress false positives yet maximize sequence coverage and implemented a systematic validation of this process using the new software TDValidator. This rigorous data analysis should elevate confidence for assignment of dense MS2 spectra and represents a purposeful step toward the application of TD and MD MS for deep sequencing of monoclonal antibodies.

Hypofractionated accelerated radiotherapy (HART) with concurrent and adjuvant temozolomide in newly diagnosed glioblastoma: a phase II randomized trial (HART-GBM trial).

INTRODUCTION: Maximal safe surgical resection followed by adjuvant chemoradiation has been standard for newly diagnosed glioblastoma multiforme (GBM). Hypofractionated accelerated radiotherapy (HART) has the potential to improve outcome as it reduces the overall treatment time and increases the biological effective dose. METHODS: Between October 2011 and July 2017, a total of 89 newly diagnosed GBM patients were randomized to conventional fractionated radiotherapy (CRT) or HART. Radiotherapy was delivered in all patients with a three-dimensional conformal radiotherapy technique in CRT arm (60 Gy in 30 fractions over 6 weeks @ 2 Gy/per fraction) or simultaneous integrated boost intensity modulated radiotherapy in HART arm (60 Gy in 20 fractions over 4 weeks @ 3 Gy/per fraction to high-risk planning target volume (PTV) and 50 Gy in 20 fractions over 4 weeks @ 2.5 Gy/per fraction to low-risk PTV). The primary endpoint of the trial was overall survival (OS). RESULTS: After a median follow-up of 11.4 months (Range: 2.9-42.5 months), 26 patients died and 39 patients had progression of the disease. Median OS for the entire cohort was 23.4 months. Median OS in the CRT and HART arms were 18.07 months (95% CI 14.52-NR) and 25.18 months (95% CI 12.89-NR) respectively, p = 0.3. Median progression free survival (PFS) for the entire cohort was 13.5 months (Range: 11.7-15.7 months). In multivariate analysis patients younger than 40 years of age, patients with a gross total resection of tumor and a mutated IDH-1 had significantly better OS. PFS was significantly better for patients with a gross total resection of tumor and a mutated IDH-1. All patients included in the trial completed the planned course of radiation. Only two patients required hospital admission for features of raised intracranial tension. One patient in the HART arm required treatment interruption. CONCLUSION: HART is comparable to CRT in terms of survival outcome. HART arm had no excess treatment interruption and minimal toxicity. Dose escalation, reduction in overall treatment time, is the advantages with use of HART.

Unabridged Analysis of Human Histone H3 by Differential Top-Down Mass Spectrometry Reveals Hypermethylated Proteoforms from MMSET/NSD2 Overexpression.

Histones, and their modifications, are critical components of cellular programming and epigenetic inheritance. Recently, cancer genome sequencing has uncovered driver mutations in chromatin modifying enzymes spurring high interest how such mutations change histone modification patterns. Here, we applied Top-Down mass spectrometry for the characterization of combinatorial modifications (i.e. methylation and acetylation) on full length histone H3 from human cell lines derived from multiple myeloma patients with overexpression of the histone methyltransferase MMSET as the result of a t(4;14) chromosomal translocation. Using the latest in Orbitrap-based technology for clean isolation of isobaric proteoforms containing up to 10 methylations and/or up to two acetylations, we provide extensive characterization of histone H3.1 and H3.3 proteoforms. Differential analysis of modifications by electron-based dissociation recapitulated antagonistic crosstalk between K27 and K36 methylation in H3.1, validating that full-length histone H3 (15 kDa) can be analyzed with site-specific assignments for multiple modifications. It also revealed K36 methylation in H3.3 was affected less by the overexpression of MMSET because of its higher methylation levels in control cells. The co-occurrence of acetylation with a minimum of three methyl groups in H3K9 and H3K27 suggested a hierarchy in the addition of certain modifications. Comparative analysis showed that high levels of MMSET in the myeloma-like cells drove the formation of hypermethyled proteoforms containing H3K36me2 co-existent with the repressive marks H3K9me2/3 and H3K27me2/3. Unique histone proteoforms with such "trivalent hypermethylation" (K9me2/3-K27me2/3-K36me2) were not discovered when H3.1 peptides were analyzed by Bottom-Up. Such disease-correlated proteoforms could link tightly to aberrant transcription programs driving cellular proliferation, and their precise description demonstrates that Top-Down mass spectrometry can now decode crosstalk involving up to three modified sites.

Clinico-hematological Profile of Hb E-ß Thalassemia-Prospective Analysis in a tertiary Care Centre.

INTRODUCTION: Hemoglobin E beta-thalassemia is one of the leading forms of severe thalassemia world wide. This disorder is more commonly found in South East Asia including north eastern states of India. Patients suffering from this disorder show marked clinical heterogeneity. MATERIALS AND METHODS: Referred cases of hemoglobin disorders from north India were evaluated prospectively. Details of clinical history and haematological findings including HPLC as well as mutation analysis were obtained. RESULTS: Twenty cases of E beta-thalassemia with widely variable clinical profile were included. The hematological parameters were also extremely variable with a wide range of hemoglobin (1.8-9.9 g/dl). Applying a severity scoring system all patients were classified the patients into mild (n=6), moderate (n=7) and severe (n=7) subclasses. We also correlated red cell Indies with HB E and HB F as well as age of onset of symptoms with HB E and HB F. IVS1-5(G-C) was found to be the most common thalassemia mutation associated with Hemoglobin E beta-thalassemia. CONCLUSION: Extremely variable clinical and haematological findings were observed in Hemoglobin E beta-thalassemia patients. These findings are comparable to other Indian studies. Appropriate knowledge of the clinical variability and unpredictable natural history can help better management of this group of patients.

Role of E-cadherin in Progression of Oral Squamous Cell Carcinoma: A Retrospective Immunohistochemical Study.

AIM: The aim of present study was to assess the role of E-cadherin in oral carcinogenesis by comparing their expressions in normal oral mucosa, oral epithelial dysplasia (OED), and oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: To elucidate the pattern of E-cadherin expression in oral carcinogenesis, 21 archival cases of formalin-fixed paraffin-embedded sections of OSCC, 21 OED, and 7 normal oral mucosa samples as control were used for the study. RESULTS: We observed reduction/loss of E-cadherin in membranous expression pattern and staining intensity with progression from dysplasia to oral cancer. CONCLUSION: A decrease in staining intensity and loss of E-cadherin membranous expression were noted from dysplasia to carcinoma, suggesting its role as a tumor suppressor gene. CLINICAL SIGNIFICANCE: E-cadherin can be used as a biomarker to assess and evaluate the progression and prognosis of oral dysplastic lesions and OSCC.