Outcomes in Patients with Spinal Metastases Managed with Surgical Intervention.

BACKGROUND: Spinal metastases are a significant cause of morbidity in patients with advanced cancer, and management often requires surgical intervention. Although prior studies have identified factors that influence outcomes with surgery, the ability of these factors to predict outcomes remains unclear in the era of contemporary therapies, and there is a need to better identify patients who are likely to benefit from surgery. METHODS: We performed a single-center, retrospective analysis to evaluate risk factors for poor outcomes in patients with spinal metastases treated with surgery. The primary outcome was mortality at 180 days. RESULTS: A total of 128 patients were identified. Age ≥ 65 years at surgery (p = 0.0316), presence of extraspinal metastases (p = 0.0110), and ECOG performance scores >1 (p = 0.0397) were associated with mortality at 180 days on multivariate analysis. These factors and BMI ≤ 30 mg/kg2 (p = 0.0008) were also associated with worse overall survival. CONCLUSIONS: Age > 65, extraspinal metastases, and performance status scores >1 are factors associated with mortality at 180 days in patients with spinal metastases treated with surgery. Patients with these factors and BMI ≤ 30 mg/kg2 had worse overall survival. Our results support multidisciplinary discussions regarding the benefits and risks associated with surgery in patients with these risk factors.

The PNUTS-PP1 complex acts as an intrinsic barrier to herpesvirus KSHV gene expression and replication.

Control of RNA Polymerase II (pol II) elongation is a critical component of gene expression in mammalian cells. The PNUTS-PP1 complex controls elongation rates, slowing pol II after polyadenylation sites to promote termination. The Kaposi's sarcoma-associated herpesvirus (KSHV) co-opts pol II to express its genes, but little is known about its regulation of pol II elongation. We identified PNUTS as a suppressor of a KSHV reporter gene in a genome-wide CRISPR screen. PNUTS depletion enhances global KSHV gene expression and overall viral replication. Mechanistically, PNUTS requires PP1 interaction, binds viral RNAs downstream of polyadenylation sites, and restricts transcription readthrough of viral genes. Surprisingly, PNUTS also represses productive elongation at the 5´ ends of the KSHV reporter and the KSHV T1.4 RNA. From these data, we conclude that PNUTS' activity constitutes an intrinsic barrier to KSHV replication likely by suppressing pol II elongation at promoter-proximal regions.

Genome-Wide CRISPR Screening to Identify Mammalian Factors that Regulate Intron Retention.

Intron retention (IR) regulates gene expression to control fundamental biological processes like metabolism, differentiation, and cell cycle. Despite a wide variety of genes controlled by IR, few techniques are available to identify regulators of IR in an unbiased manner. Here, we describe a CRISPR knockout screening method that can be applied to uncover regulators of IR. This method uses GFP reporter constructs containing a retained intron from a gene of interest such that GFP signal is regulated by IR in the same fashion as the endogenous gene. The GFP levels are then used as a readout for genome-wide CRISPR screening. We have successfully used this approach to identify novel regulator of IR of the MAT2A transcript and propose that similar screens will be broadly applicable for the identification of novel factors that control IR of specific transcripts.

A randomized feasibility study evaluating temozolomide circadian medicine in patients with glioma.

Background: Gliomas are the most common primary brain tumor in adults. Current treatments involve surgery, radiation, and temozolomide (TMZ) chemotherapy; however, prognosis remains poor and new approaches are required. Circadian medicine aims to maximize treatment efficacy and/or minimize toxicity by timed delivery of medications in accordance with the daily rhythms of the patient. We published a retrospective study showing greater anti-tumor efficacy for the morning, relative to the evening, administration of TMZ in patients with glioblastoma. We conducted this prospective randomized trial to determine the feasibility, and potential clinical impact, of TMZ chronotherapy in patients with gliomas (NCT02781792). Methods: Adult patients with gliomas (WHO grade II-IV) were enrolled prior to initiation of monthly TMZ therapy and were randomized to receive TMZ either in the morning (AM) before 10 am or in the evening (PM) after 8 pm. Pill diaries were recorded to measure compliance and FACT-Br quality of life (QoL) surveys were completed throughout treatment. Study compliance, adverse events (AE), and overall survival were compared between the two arms. Results: A total of 35 evaluable patients, including 21 with GBM, were analyzed (18 AM patients and 17 PM patients). Compliance data demonstrated the feasibility of timed TMZ dosing. There were no significant differences in AEs, QoL, or survival between the arms. Conclusions: Chronotherapy with TMZ is feasible. A larger study is needed to validate the effect of chronotherapy on clinical efficacy.

Development of Aplastic Anemia during Treatment of Anaplastic Astrocytoma with Temozolomide.

Temozolomide (TMZ) is an oral alkylating agent that is considered the standard therapy in primary intracranial malignancies. The medication is well tolerated with a most common side effect of bone marrow suppression that is encountered in a small proportion of patients, often reversible with medication discontinuation and supportive treatment. Rarely, aplastic anemia can develop during treatment with TMZ. Here, we present a case of a patient who developed aplastic anemia following treatment with TMZ. We offer a review of the existing literature to have a better understanding of the causative effect and to examine the characteristics and outcomes when aplastic anemia develops during treatment with TMZ.

Development of Assay Systems for Amber Codon Decoding at the Steps of Initiation and Elongation in Mycobacteria.

Genetic analysis of the mechanism of protein synthesis in Gram-positive bacteria has remained largely unexplored because of the unavailability of appropriate in vivo assay systems. We developed chloramphenicol acetyltransferase (CAT)-based in vivo reporter systems to study translation initiation and elongation in Mycobacterium smegmatis The CAT reporters utilize specific decoding of amber codons by mutant initiator tRNA (i-tRNA, metU) molecules containing a CUA anticodon (metUCUA). The assay systems allow structure-function analyses of tRNAs without interfering with the cellular protein synthesis and function with or without the expression of heterologous GlnRS from Escherichia coli We show that despite their naturally occurring slow-growth phenotypes, the step of i-tRNA formylation is vital in translation initiation in mycobacteria and that formylation-deficient i-tRNA mutants (metUCUA/A1, metUCUA/G72, and metUCUA/G72G73) with a Watson-Crick base pair at the 1·72 position participate in elongation. In the absence of heterologous GlnRS expression, the mutant tRNAs are predominantly aminoacylated (glutamylated) by nondiscriminating GluRS. Acid urea gels show complete transamidation of the glutamylated metUCUA/G72G73 tRNA to its glutaminylated form (by GatCAB) in M. smegmatis In contrast, the glutamylated metUCUA/G72 tRNA did not show a detectable level of transamidation. Interestingly, the metUCUA/A1 mutant showed an intermediate activity of transamidation and accumulated in both glutamylated and glutaminylated forms. These observations suggest important roles for the discriminator base position and/or a weak Watson-Crick base pair at 1·72 for in vivo recognition of the glutamylated tRNAs by M. smegmatis GatCAB.IMPORTANCE Genetic analysis of the translational apparatus in Gram-positive bacteria has remained largely unexplored because of the unavailability of appropriate in vivo assay systems. We developed chloramphenicol acetyltransferase (CAT)-based reporters which utilize specific decoding of amber codons by mutant tRNAs at the steps of initiation and/or elongation to allow structure-function analysis of the translational machinery. We show that formylation of the initiator tRNA (i-tRNA) is crucial even for slow-growing bacteria and that i-tRNA mutants with a CUA anticodon are aminoacylated by nondiscriminating GluRS. The discriminator base position, and/or a weak Watson-Crick base pair at the top of the acceptor stem, provides important determinants for transamidation of the i-tRNA-attached Glu to Gln by the mycobacterial GatCAB.

Sustenance of Escherichia coli on a single tRNAMet.

Living organisms possess two types of tRNAs for methionine. Initiator tRNAs bind directly into the ribosomal P-site to initiate protein synthesis, and the elongators bind to the A-site during the elongation step. Eubacterial initiators (tRNAfMet) are unique in that the methionine attached to them is formylated to facilitate their binding to initiation factor 2 (IF2), and to preclude them from binding to elongation factor Tu (EFTu). However, in mammalian mitochondria, protein synthesis proceeds with a single dual function tRNAMet. Escherichia coli possesses four tRNAfMet (initiator) and two tRNAMet (elongator) genes. Free-living organisms possessing the mitochondrion like system of single tRNAMet are unknown. We characterized mutants of E. coli tRNAfMet that function both as initiators and elongators. We show that some of the tRNAfMet mutants sustain E. coli lacking all four tRNAfMet and both tRNAMet genes, providing a basis for natural occurrence of mitochondria like situation in free living organisms. The tRNA mutants show in vivo binding to both IF2 and EFTu, indicating how they carry out these otherwise mutually exclusive functions by precise regulation of their in vivo formylation. Our results provide insights into how distinct initiator and elongator methionine tRNAs might have evolved from a single 'dual function' tRNA.

Utilisation of 10-formyldihydrofolate as substrate by dihydrofolate reductase (DHFR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) tranformylase/IMP cyclohydrolase (PurH) in Escherichia coli.

Dihydrofolate reductase (DHFR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/IMP cyclohydrolase (PurH) play key roles in maintaining folate pools in cells, and are targets of antimicrobial and anticancer drugs. While the activities of bacterial DHFR and PurH on their classical substrates (DHF and 10-CHO-THF, respectively) are known, their activities and kinetic properties of utilisation of 10-CHO-DHF are unknown. We have determined the kinetic properties (kcat/Km) of conversion of 10-CHO-DHF to 10-CHO-THF by DHFR, and to DHF by PurH. We show that DHFR utilises 10-CHO-DHF about one third as efficiently as it utilises DHF. The 10-CHO-DHF is also utilised (as a formyl group donor) by PurH albeit slightly less efficiently than 10-CHO-THF. The utilisation of 10-CHO-DHF by DHFR is ~50 fold more efficient than its utilisation by PurH. A folate deficient Escherichia coli (∆pabA) grows well when supplemented with adenine, glycine, thymine and methionine, the metabolites that arise from the one-carbon metabolic pathway. Notably, when the ∆pabA strain harboured a folate transporter, it grew in the presence of 10-CHO-DHF alone, suggesting that it (10-CHO-DHF) can enter one-carbon metabolic pathway to provide the required metabolites. Thus, our studies reveal that both DHFR and PurH could utilise 10-CHO-DHF for folate homeostasis in E. coli.

Association between treatment-related lymphopenia and overall survival in elderly patients with newly diagnosed glioblastoma.

Management of patients with glioblastoma (GBM) often includes radiation (RT) and temozolomide (TMZ). The association between severe treatment-related lymphopenia (TRL) after the standard chemoradiation and reduced survival has been reported in GBM patients with the median age of 57. Similar findings were described in patients with head and neck, non-small cell lung, and pancreatic cancers. This retrospective study is designed to evaluate whether elderly GBM patients (age ≥65) develop similar TRL after RT/TMZ and whether such TRL is associated with decreased survival. Serial total lymphocyte counts (TLC) were retrospectively reviewed in patients (age ≥65) with newly diagnosed GBM undergoing RT/TMZ and associated with treatment outcomes. Seventy-two patients were eligible: median KPS 70, median age 71 years (range 65-86) with 56 % of patients >70 years, 53% female, 31% received RT ≤45 Gy. Baseline median TLC was 1100 cells/mm(3) which fell by 41% to 650 cells/mm(3) 2 months after initiating RT/TMZ (p < 0.0001). Patients with TLC <500 cells/mm(3) at 2 months had a shorter survival than those with higher TLCs with a median overall survival of 4.6 versus 11.6 months, respectively. Multivariate analysis revealed a significant association between TRL and survival (HR 2.76, 95% CI 1.30-5.86, p = 0.008). Treatment-related lymphopenia is frequent, severe, and an independent predictor for survival in elderly patients with GBM. These findings add to the body of evidence that immunosuppression induced by chemoradiation is associated with inferior clinical outcomes. Prospective studies are needed to confirm these findings suggesting that immune preservation is important in this cancer.

Mycobacterium tuberculosis MutT1 (Rv2985) and ADPRase (Rv1700) proteins constitute a two-stage mechanism of 8-oxo-dGTP and 8-oxo-GTP detoxification and adenosine to cytidine mutation avoidance.

Approximately one third of the world population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. A better understanding of the pathogen biology is crucial to develop new tools/strategies to tackle its spread and treatment. In the host macrophages, the pathogen is exposed to reactive oxygen species, known to damage dGTP and GTP to 8-oxo-dGTP and 8-oxo-GTP, respectively. Incorporation of the damaged nucleotides in nucleic acids is detrimental to organisms. MutT proteins, belonging to a class of Nudix hydrolases, hydrolyze 8-oxo-G nucleoside triphosphates/diphosphates to the corresponding nucleoside monophosphates and sanitize the nucleotide pool. Mycobacteria possess several MutT proteins. However, a functional homolog of Escherichia coli MutT has not been identified. Here, we characterized MtuMutT1 and Rv1700 proteins of M. tuberculosis. Unlike other MutT proteins, MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP, and 8-oxo-GTP to 8-oxo-GDP. Rv1700 then converts them to the corresponding nucleoside monophosphates. This observation suggests the presence of a two-stage mechanism of 8-oxo-dGTP/8-oxo-GTP detoxification in mycobacteria. MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP with a Km of ∼50 µM and Vmax of ∼0.9 pmol/min per ng of protein, and Rv1700 converts 8-oxo-dGDP to 8-oxo-dGMP with a Km of ∼9.5 µM and Vmax of ∼0.04 pmol/min per ng of protein. Together, MtuMutT1 and Rv1700 offer maximal rescue to E. coli for its MutT deficiency by decreasing A to C mutations (a hallmark of MutT deficiency). We suggest that the concerted action of MtuMutT1 and Rv1700 plays a crucial role in survival of bacteria against oxidative stress.

Distinctive contributions of the ribosomal P-site elements m2G966, m5C967 and the C-terminal tail of the S9 protein in the fidelity of initiation of translation in Escherichia coli.

The accuracy of pairing of the anticodon of the initiator tRNA (tRNA(fMet)) and the initiation codon of an mRNA, in the ribosomal P-site, is crucial for determining the translational reading frame. However, a direct role of any ribosomal element(s) in scrutinizing this pairing is unknown. The P-site elements, m(2)G966 (methylated by RsmD), m(5)C967 (methylated by RsmB) and the C-terminal tail of the protein S9 lie in the vicinity of tRNA(fMet). We investigated the role of these elements in initiation from various codons, namely, AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG with tRNA(fMet(CAU) (tRNA(fMet) with CAU anticodon); CAC and CAU with tRNA(fMet(GUG); UAG with tRNA(fMet(CAU) ; UAC with tRNA(fMet(GUG) ; and AUC with tRNA(fMet(GUG) using in vivo and computational methods. Although RsmB deficiency did not impact initiation from most codons, RsmD deficiency increased initiation from AUA, CAC and CAU (2- to 3.6-fold). Deletion of the S9 C-terminal tail resulted in poorer initiation from UUG, GUG and CUG, but in increased initiation from CAC, CAU and UAC codons (up to 4-fold). Also, the S9 tail suppressed initiation with tRNA(fMet(CAU) lacking the 3GC base pairs in the anticodon stem. These observations suggest distinctive roles of 966/967 methylations and the S9 tail in initiation.