The long noncoding RNA glycoLINC assembles a lower glycolytic metabolon to promote glycolysis.

Non-covalent complexes of glycolytic enzymes, called metabolons, were postulated in the 1970s, but the concept has been controversial. Here we show that a c-Myc-responsive long noncoding RNA (lncRNA) that we call glycoLINC (gLINC) acts as a backbone for metabolon formation between all four glycolytic payoff phase enzymes (PGK1, PGAM1, ENO1, and PKM2) along with lactate dehydrogenase A (LDHA). The gLINC metabolon enhances glycolytic flux, increases ATP production, and enables cell survival under serine deprivation. Furthermore, gLINC overexpression in cancer cells promotes xenograft growth in mice fed a diet deprived of serine, suggesting that cancer cells employ gLINC during metabolic reprogramming. We propose that gLINC makes a functional contribution to cancer cell adaptation and provide the first example of a lncRNA-facilitated metabolon.

Microbiota-Derived Short-Chain Fatty Acids Promote the Memory Potential of Antigen-Activated CD8+ T Cells.

Interactions with the microbiota influence many aspects of immunity, including immune cell development, differentiation, and function. Here, we examined the impact of the microbiota on CD8+ T cell memory. Antigen-activated CD8+ T cells transferred into germ-free mice failed to transition into long-lived memory cells and had transcriptional impairments in core genes associated with oxidative metabolism. The microbiota-derived short-chain fatty acid (SCFA) butyrate promoted cellular metabolism, enhanced memory potential of activated CD8+ T cells, and SCFAs were required for optimal recall responses upon antigen re-encounter. Mechanistic experiments revealed that butyrate uncoupled the tricarboxylic acid cycle from glycolytic input in CD8+ T cells, which allowed preferential fueling of oxidative phosphorylation through sustained glutamine utilization and fatty acid catabolism. Our findings reveal a role for the microbiota in promoting CD8+ T cell long-term survival as memory cells and suggest that microbial metabolites guide the metabolic rewiring of activated CD8+ T cells to enable this transition.

Sengers Syndrome-Associated Mitochondrial Acylglycerol Kinase Is a Subunit of the Human TIM22 Protein Import Complex.

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that catalyzes the phosphorylation of monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid, respectively. Mutations in AGK cause Sengers syndrome, which is characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis. Here we identified AGK as a subunit of the mitochondrial TIM22 protein import complex. We show that AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex, where it facilitates the import and assembly of mitochondrial carrier proteins. Mitochondria isolated from Sengers syndrome patient cells and tissues show a destabilized TIM22 complex and defects in the biogenesis of carrier substrates. Consistent with this phenotype, we observe perturbations in the tricarboxylic acid (TCA) cycle in cells lacking AGK. Our identification of AGK as a bona fide subunit of TIM22 provides an exciting and unexpected link between mitochondrial protein import and Sengers syndrome.

Reprogrammed mRNA translation drives resistance to therapeutic targeting of ribosome biogenesis.

Elevated ribosome biogenesis in oncogene-driven cancers is commonly targeted by DNA-damaging cytotoxic drugs. Our previous first-in-human trial of CX-5461, a novel, less genotoxic agent that specifically inhibits ribosome biogenesis via suppression of RNA polymerase I (Pol I) transcription, revealed single-agent efficacy in refractory blood cancers. Despite this clinical response, patients were not cured. In parallel, we demonstrated a marked improvement in the in vivo efficacy of CX-5461 in combination with PI3K/AKT/mTORC1 pathway inhibitors. Here, we reveal the molecular basis for this improved efficacy observed in vivo, which is associated with specific suppression of translation of mRNAs encoding regulators of cellular metabolism. Importantly, acquired resistance to this cotreatment is driven by translational rewiring that results in dysregulated cellular metabolism and induction of a cAMP-dependent pathway critical for the survival of blood cancers including lymphoma and acute myeloid leukemia. Our studies thus identify key molecular mechanisms underpinning the response of blood cancers to selective inhibition of ribosome biogenesis and define metabolic vulnerabilities that will facilitate the rational design of more effective regimens for Pol I-directed therapies.

Early-life house dust mite aeroallergen exposure augments cigarette smoke-induced myeloid inflammation and emphysema in mice.

BACKGROUND: Longitudinal studies have identified childhood asthma as a risk factor for obstructive pulmonary disease (COPD) and asthma-COPD overlap (ACO) where persistent airflow limitation can develop more aggressively. However, a causal link between childhood asthma and COPD/ACO remains to be established. Our study aimed to model the natural history of childhood asthma and COPD and to investigate the cellular/molecular mechanisms that drive disease progression. METHODS: Allergic airways disease was established in three-week-old young C57BL/6 mice using house dust mite (HDM) extract. Mice were subsequently exposed to cigarette smoke (CS) and HDM for 8 weeks. Airspace enlargement (emphysema) was measured by the mean linear intercept method. Flow cytometry was utilised to phenotype lung immune cells. Bulk RNA-sequencing was performed on lung tissue. Volatile organic compounds (VOCs) in bronchoalveolar lavage-fluid were analysed to screen for disease-specific biomarkers. RESULTS: Chronic CS exposure induced emphysema that was significantly augmented by HDM challenge. Increased emphysematous changes were associated with more abundant immune cell lung infiltration consisting of neutrophils, interstitial macrophages, eosinophils and lymphocytes. Transcriptomic analyses identified a gene signature where disease-specific changes induced by HDM or CS alone were conserved in the HDM-CS group, and further revealed an enrichment of Mmp12, Il33 and Il13, and gene expression consistent with greater expansion of alternatively activated macrophages. VOC analysis also identified four compounds increased by CS exposure that were paradoxically reduced in the HDM-CS group. CONCLUSIONS: Early-life allergic airways disease worsened emphysematous lung pathology in CS-exposed mice and markedly alters the lung transcriptome.

Remodeling of Carbon Metabolism during Sulfoglycolysis in Escherichia coli.

Sulfoquinovose (SQ) is a major metabolite in the global sulfur cycle produced by nearly all photosynthetic organisms. One of the major pathways involved in the catabolism of SQ in bacteria such as Escherichia coli is a variant of the glycolytic Embden-Meyerhof-Parnas (EMP) pathway termed the sulfoglycolytic EMP (sulfo-EMP) pathway, which leads to the consumption of three of the six carbons of SQ and the excretion of 2,3-dihydroxypropanesulfonate (DHPS). Comparative metabolite profiling of aerobically glucose (Glc)-grown and SQ-grown E. coli cells was undertaken to identify the metabolic consequences of the switch from glycolysis to sulfoglycolysis. Sulfoglycolysis was associated with the diversion of triose phosphates (triose-P) to synthesize sugar phosphates (gluconeogenesis) and an unexpected accumulation of trehalose and glycogen storage carbohydrates. Sulfoglycolysis was also associated with global changes in central carbon metabolism, as indicated by the changes in the levels of intermediates in the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway (PPP), polyamine metabolism, pyrimidine metabolism, and many amino acid metabolic pathways. Upon entry into stationary phase and the depletion of SQ, E. coli cells utilize their glycogen, indicating a reversal of metabolic fluxes to allow glycolytic metabolism. IMPORTANCE The sulfosugar sulfoquinovose is estimated to be produced on a scale of 10 billion metric tons per annum, making it a major organosulfur species in the biosulfur cycle. The microbial degradation of sulfoquinovose through sulfoglycolysis allows the utilization of its carbon content and contributes to the biomineralization of its sulfur. However, the metabolic consequences of microbial growth on sulfoquinovose are unclear. We use metabolomics to identify the metabolic adaptations that Escherichia coli undergoes when grown on sulfoquinovose versus glucose. This revealed the increased flux into storage carbohydrates through gluconeogenesis and the reduced flux of carbon into the TCA cycle and downstream metabolism. These changes are relieved upon entry into stationary phase and reversion to glycolytic metabolism. This work provides new insights into the metabolic consequences of microbial growth on an abundant sulfosugar.

The use of Lutetium-177 PSMA radioligand therapy with high dose rate brachytherapy for locally recurrent prostate cancer after previous definitive radiation therapy: a randomized, single-institution, phase I/II study (ROADSTER).

BACKGROUND: Isolated local failure (ILF) can occur in patients who initially receive definitive radiation therapy for prostate cancer. Salvage therapy for ILF includes high dose rate (HDR) brachytherapy. Prostate Specific Membrane Antigen (PSMA) Positron Emission Tomography (PET) can accurately detect ILF and can exclude extraprostatic disease. Lutetium-177 PSMA Radioligand Therapy (RLT) is a novel treatment for prostate cancer that can target prostate cancer accurately, while sparing radiation dose to normal tissues. METHODS: ROADSTER is a phase I/II randomized, single-institution study. Patients with an ILF of prostate cancer after definitive initial radiation therapy are eligible. The ILF will be confirmed with biopsy, magnetic resonance imaging (MRI) and PSMA PET. Patients will be randomized between HDR brachytherapy in two fractions (a standard of care salvage treatment at our institution) (cohort 1) or one treatment of intravenous Lutetium-177 PSMA RLT, followed by one fraction of HDR brachytherapy (cohort 2). The primary endpoints for the phase I portion of the study (n = 12) will be feasibility, defined as 10 or more patients completing the study protocol within 24 months of study activation; and safety, defined as zero or one patients in cohort 2 experiencing grade 3 or higher toxicity in the first 6 months post-treatment. If feasibility and safety are achieved, the study will expand to a phase II study (n = 30 total) where preliminary efficacy data will be evaluated. Secondary endpoints include changes in prostate specific antigen levels, acute toxicity, changes in quality of life, and changes in translational biomarkers. Translational endpoints will include interrogation of blood, urine, and tissue for markers of DNA damage and immune activation with each treatment. DISCUSSION: ROADSTER explores a novel salvage therapy for ILF after primary radiotherapy with combined Lutetium-177 PSMA RLT and HDR brachytherapy. The randomized phase I/II design will provide a contemporaneous patient population treated with HDR alone to facilitate assessment of feasibility, tolerability, and biologic effects of this novel therapy. TRIAL REGISTRATION: NCT05230251 (ClinicalTrials.gov).

Quality-adjusted survival in women with gynecologic malignancies receiving IMRT after surgery: A Ppatient Rreported Ooutcome study of NRG oncology's RTOG 1203.

INTRODUCTION: NRG/RTOG 1203 compared 3-D conformal radiotherapy (3D CRT) to intensity-modulated radiotherapy (IMRT) in patients with endometrial or cervical cancer requiring post-operative radiotherapy after hysterectomy. The purpose of this study was to report the first quality-adjusted survival analysis comparing the two treatments. METHODS: NRG/RTOG 1203 randomized patients having undergone hysterectomy to either 3DCRT or IMRT. Stratification factors included RT dose, chemotherapy, and disease site. The EQ-5D, both index and visual analog scale (VAS), were obtained at baseline, 5 weeks after the start of RT, 4-6 weeks post RT and 1 and 3-years post RT. EQ-5D index and VAS scores along with quality-adjusted survival (QAS) were compared between treatment arms using the t-test at a two-sided significance level of 0.05. RESULTS: NRG/RTOG 1203 enrolled 289 patients of which 236 consented to participate in the patient reported outcome (PRO) assessments. QAS was higher in women treated with IMRT, 1374 vs 1333 days (p = 0.5) compared to patients treated with 3DCRT, but this difference was not statistically different. Patients treated with IMRT had less of a decline in VAS score 5 weeks post RT, -5.04, compared to patients treated with 3DCRT, -7.48, although not statistically significant (p = 0.38). CONCLUSION: This is the first report of the use of the EQ-5D comparing two radiotherapy techniques in the treatment of gynecologic malignancies after surgery. While there were no significant differences in QAS and VAS scores between patients who received IMRT vs. 3DCRT, RTOG 1203 was not powered to show statistical differences in these secondary endpoints.

Parents' Perception of Barriers to the Comprehensive Management of Children With Cleft Lip and Palate in Bogota, Colombia.

OBJECT: To identify the perception of barriers to the comprehensive management of cleft lip and palate (CLP) by parents/caregivers of Colombian children with this condition. SETTING AND SAMPLE POPULATION: Fifty parents/caregivers of children with CLP under 12 years attending a center specialized in the management of craniofacial congenital conditions in Bogota, Colombia. MATERIALS AND METHODS: This study consisted of 2 phases: a quantitative phase (descriptive cross-sectional) and a qualitative phase (focus group [FG]). Chi-square and Fisher exact tests were used to analyze the association variables. The barriers and alternative ways to overcome barriers were analyzed in the FG. RESULTS: Comprehensive management was mostly defined as access to multiple treatments (54%), and this concept was expanded in the FG toward understanding CLP at all levels. Monoparental families spend their income on treatments (29%) than nuclear families (0%) (P = .001). All parents with high education levels were familiar with healthcare centers specialized in CLP as opposed to 66.7% of parents with basic primary education (P < .05). Regarding the timeliness of appointments, 12.2% of parents earning between 1 and 2 minimum wages reported some kind of difficulty, whereas those earning less than one minimum wage reported difficulties in 66.7% of cases (P = .046). CONCLUSIONS: All participants reported barriers, which increased or decreased depending on their socioeconomic status. The FG allowed the discussion of alternatives to overcome barriers, such as structural, solidarity, and self-management actions.

Posts Supporting Anti-Environmental Policy in Brazil are Shared More on Social Media.

Weakening environmental laws supported by disinformation are currently of concern in Brazil. An example of disinformation is the case of the "firefighter cattle". Supporters of this idea believe that by consuming organic mass, cattle decrease the risk of fire in natural ecosystems. This statement was cited by a member of the Bolsonaro government in response to the unprecedented 2020 fires in the Pantanal, as well as in support of a new law that enables extensive livestock in protected areas of this biome. By suggesting that grazing benefits the ecosystem, the "firefighter cattle" argument supports the interests of agribusiness. However, it ignores the real costs of livestock production on biodiversity. We analysed the social repercussion of the "firefighter cattle" by analysing public reactions to YouTube, Facebook, and Google News posts. These videos and articles and the responses to them either agreed or disagreed with the "firefighter cattle". Supportive posts were shared more on social media and triggered more interactions than critical posts. Even though many netizens disagreed with the idea of "firefighter cattle", it has gone viral, and was used as a tool to strengthen anti-environmental policies. We advocate that government institutions should use resources and guidelines provided by the scientific community to raise awareness. These materials include international reports produced by the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services (IPBES) and the Intergovernmental Panel on Climate Change (IPCC). We need to curb pseudoscience and misinformation in political discourse, avoiding misconceptions that threaten natural resources and confuse global society.

Proteome analysis of human adipocytes identifies depot-specific heterogeneity at metabolic control points.

Adipose tissue is a primary regulator of energy balance and metabolism. The distribution of adipose tissue depots is of clinical interest because the accumulation of upper-body subcutaneous (ASAT) and visceral adipose tissue (VAT) is associated with cardiometabolic diseases, whereas lower-body glutealfemoral adipose tissue (GFAT) appears to be protective. There is heterogeneity in morphology and metabolism of adipocytes obtained from different regions of the body, but detailed knowledge of the constituent proteins in each depot is lacking. Here, we determined the human adipocyte proteome from ASAT, VAT, and GFAT using high-resolution Sequential Window Acquisition of all Theoretical (SWATH) mass spectrometry proteomics. We quantified 4,220 proteins in adipocytes, and 2,329 proteins were expressed in all three adipose depots. Comparative analysis revealed significant differences between adipocytes from different regions (6% and 8% when comparing VAT vs. ASAT and GFAT, 3% when comparing the subcutaneous adipose tissue depots, ASAT and GFAT), with marked differences in proteins that regulate metabolic functions. The VAT adipocyte proteome was overrepresented with proteins of glycolysis, lipogenesis, oxidative stress, and mitochondrial dysfunction. The GFAT adipocyte proteome predicted the activation of peroxisome proliferator-activated receptor α (PPARα), fatty acid, and branched-chain amino acid (BCAA) oxidation, enhanced tricarboxylic acid (TCA) cycle flux, and oxidative phosphorylation, which was supported by metabolomic data obtained from adipocytes. Together, this proteomic analysis provides an important resource and novel insights that enhance the understanding of metabolic heterogeneity in the regional adipocytes of humans.NEW & NOTEWORTHY Adipocyte metabolism varies depending on anatomical location and the adipocyte protein composition may orchestrate this heterogeneity. We used SWATH proteomics in patient-matched human upper- (visceral and subcutaneous) and lower-body (glutealfemoral) adipocytes and detected 4,220 proteins and distinguishable regional proteomes. Upper-body adipocyte proteins were associated with glycolysis, de novo lipogenesis, mitochondrial dysfunction, and oxidative stress, whereas lower-body adipocyte proteins were associated with enhanced PPARα activation, fatty acid, and BCAA oxidation, TCA cycle flux, and oxidative phosphorylation.

A Mycoplasma gallisepticum Glycerol ABC Transporter Involved in Pathogenicity.

MalF has been shown to be required for virulence in the important avian pathogen Mycoplasma gallisepticum To characterize the function of MalF, predicted to be part of a putative ABC transporter, we compared metabolite profiles of a mutant with a transposon inserted in malF (MalF-deficient ST mutant 04-1; ΔmalF) with those of wild-type bacteria using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Of the substrates likely to be transported by an ABC transport system, glycerol was detected at significantly lower abundance in the ΔmalF mutant, compared to the wild type. Stable isotope labeling using [U-13C]glycerol and reverse transcription-quantitative PCR analysis indicated that MalF was responsible for the import of glycerol into M. gallisepticum and that, in the absence of MalF, the transcription of gtsA, which encodes a second transporter, GtsA, was upregulated, potentially to increase the import of glycerol-3-phosphate into the cell to compensate for the loss of MalF. The loss of MalF appeared to have a global effect on glycerol metabolism, suggesting that it may also play a regulatory role, and cellular morphology was also affected, indicating that the change to glycerol metabolism may have a broader effect on cellular organization. Overall, this study suggests that the reduced virulence of the ΔmalF mutant is due to perturbed glycerol uptake and metabolism and that the operon including malF should be reannotated as golABC to reflect its function in glycerol transport.IMPORTANCE Many mycoplasmas are pathogenic and cause disease in humans and animals. M. gallisepticum causes chronic respiratory disease in chickens and infectious sinusitis in turkeys, resulting in economic losses in poultry industries throughout the world. Expanding our knowledge about the pathogenesis of mycoplasma infections requires better understanding of the specific gene functions of these bacteria. In this study, we have characterized the metabolic function of a protein involved in the pathogenicity of M. gallisepticum, as well as its effect on expression of selected genes, cell phenotype, and H2O2 production. This study is a key step forward in elucidating why this protein plays a key role in virulence in chickens. This study also emphasizes the importance of functional characterization of mycoplasma proteins, using tools such as metabolomics, since prediction of function based on homology to other bacterial proteins is not always accurate.

SdrA, an NADP(H)-regenerating enzyme, is crucial for Coxiella burnetii to resist oxidative stress and replicate intracellularly.

Coxiella burnetii, the causative agent of the zoonotic disease Q fever, is a Gram-negative bacterium that replicates inside macrophages within a highly oxidative vacuole. Screening of a transposon mutant library suggested that sdrA, which encodes a putative short-chain dehydrogenase, is required for intracellular replication. Short-chain dehydrogenases are NADP(H)-dependent oxidoreductases, and SdrA contains a predicted NADP+ binding site, suggesting it may facilitate NADP(H) regeneration by C. burnetii, a key process for surviving oxidative stress. Purified recombinant 6×His-SdrA was able to convert NADP+ to NADP(H) in vitro. Mutation to alanine of a conserved glycine residue at position 12 within the predicted NADP binding site abolished significant enzymatic activity. Complementation of the sdrA mutant (sdrA::Tn) with plasmid-expressed SdrA restored intracellular replication to wild-type levels, but expressing enzymatically inactive G12A_SdrA did not. The sdrA::Tn mutant was more susceptible in vitro to oxidative stress, and treating infected host cells with L-ascorbate, an anti-oxidant, partially rescued the intracellular growth defect of sdrA::Tn. Finally, stable isotope labelling studies demonstrated a shift in flux through metabolic pathways in sdrA::Tn consistent with the presence of increased oxidative stress, and host cells infected with sdrA::Tn had elevated levels of reactive oxygen species compared with C. burnetii NMII.

3D image-guided interstitial brachytherapy for primary vaginal cancer: A multi-institutional experience.

PURPOSE/OBJECTIVES: High dose rate (HDR) interstitial brachytherapy (ISBT) boost is integral for definitive radiation treatment of primary vaginal cancer. Technological advances with CT or MRI guidance provide improved precision and ability to treat more extensively invasive tumors over historical techniques, but reported experience is limited. We sought to provide updated outcome and toxicity data for women with primary vaginal cancer undergoing treatment with a modern ISBT technique. MATERIAL/METHODS: Databases of primary vaginal carcinoma patients treated at two Canadian academic cancer institutions were combined including patient, tumor and treatment characteristics, and survival outcomes and toxicity data. Descriptive statistics, survival estimates based on the Kaplan-Meier method, and univariable/multivariable Cox proportional hazards regression analyses are reported. RESULTS: Between 2002 and 2017, 67 women with primary vaginal cancer were treated with 3D HDR ISBT. FIGO stage distribution was I (22.4%), II (50.8%), III (17.9%), IVa (9.0%). All patients received external beam radiotherapy and HDR ISBT of 500-750 cGy per fraction over 2-4 fractions. Median follow-up was 2.68 years (95% confidence interval: 2.04-6.04). Cumulative rate of grade 3-4 genitourinary/gastrointestinal toxicity was 10.4%. Four patients developed vaginal fistula. Progression-free survival at 2 and 3 years was 73.5% and 66.4% for all patients, 78.3% and 75.0% for stage I-II and 61.6% and 46.2% for stage III-IVa, respectively (log-rank p = 0.252). CONCLUSIONS: Use of 3D image-guided HDR ISBT boost was safe and resulted in improved survival outcomes compared to historical rates in this series of primary vaginal cancer patients. Prospective study is warranted to better define clinical and dosimetric predictors of local control.

Patient and family financial burden associated with cancer treatment in Canada: a national study.

GOAL: To determine patient-reported financial and family burden associated with treatment of cancer in the previous 28 days across Canada. METHODS: A self-administered questionnaire (P-SAFE v7.2.4) was completed by 901 patients with cancer from twenty cancer centres nationally (344 breast, 183 colorectal, 158 lung, 216 prostate) measuring direct and indirect costs related to cancer treatment and foregone care. Monthly self-reported out-of-pocket-costs (OOPCs) included drugs, homecare, homemaking, complementary/ alternative medicines, vitamins/supplements, family care, accommodations, devices, and "other" costs. Travel and parking costs were captured separately. Patients indicated if OOPC, travel, parking, and lost income were a financial burden. RESULTS: Mean 28-day OOPCs were CA$518 (US Purchase Price Parity [PPP] $416), plus CA$179 (US PPP $144) for travel and CA$84 (US PPP $67) for parking. Patients self-reporting high financial burden had total OOPCs (33%), of CA$961 (US PPP $772), while low-burden participants (66%) had OOPCs of CA$300 (US PPP $241). "Worst burden" respondents spent a mean of 50.7% of their monthly income on OOPCs (median 20.8%). Among the 29.4% who took time off work, patients averaged 18.0 days off. Among the 26.0% of patients whose caregivers took time off work, caregivers averaged 11.5 days off. Lastly, 41% of all patients had to reduce spending. Fifty-two per cent of those who reduced spending were families earning < CA$50,000/year. CONCLUSIONS: In our Canadian sample, high levels of financial burden exist for 33% of patients, and the severity of burden is higher for those with lower household incomes.

EirA Is a Novel Protein Essential for Intracellular Replication of Coxiella burnetii.

The zoonotic bacterial pathogen Coxiella burnetii is the causative agent of Q fever, a febrile illness which can cause a serious chronic infection. C. burnetii is a unique intracellular bacterium which replicates within host lysosome-derived vacuoles. The ability of C. burnetii to replicate within this normally hostile compartment is dependent on the activity of the Dot/Icm type 4B secretion system. In a previous study, a transposon mutagenesis screen suggested that the disruption of the gene encoding the novel protein CBU2072 rendered C. burnetii incapable of intracellular replication. This protein, subsequently named EirA (essential for intracellular replication A), is indispensable for intracellular replication and virulence, as demonstrated by infection of human cell lines and in vivo infection of Galleria mellonella The putative N-terminal signal peptide is essential for protein function but is not required for localization of EirA to the bacterial inner membrane compartment and axenic culture supernatant. In the absence of EirA, C. burnetii remains viable but nonreplicative within the host phagolysosome, as coinfection with C. burnetii expressing native EirA rescues the replicative defect in the mutant strain. In addition, while the bacterial ultrastructure appears to be intact, there is an altered metabolic profile shift in the absence of EirA, suggesting that EirA may impact overall metabolism. Most strikingly, in the absence of EirA, Dot/Icm effector translocation was inhibited even when EirA-deficient C. burnetii replicated in the wild type (WT)-supported Coxiella containing vacuoles. EirA may therefore have a novel role in the control of Dot/Icm activity and represent an important new therapeutic target.

Brucella Exposure Risk Events in 10 Clinical Laboratories, New York City, USA, 2015 to 2017.

From 2015 to 2017, 11 confirmed brucellosis cases were reported in New York City, leading to 10 Brucella exposure risk events (Brucella events) in 7 clinical laboratories (CLs). Most patients had traveled to countries where brucellosis is endemic and presented with histories and findings consistent with brucellosis. CLs were not notified that specimens might yield a hazardous organism, as the clinicians did not consider brucellosis until they were notified that bacteremia with Brucella was suspected. In 3 Brucella events, the CLs did not suspect that slow-growing, small Gram-negative bacteria might be harmful. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), which has a limited capacity to identify biological threat agents (BTAs), was used during 4 Brucella events, which accounted for 84% of exposures. In 3 of these incidents, initial staining of liquid media showed Gram-positive rods or cocci, including some cocci in chains, suggesting streptococci. Over 200 occupational exposures occurred when the unknown isolates were manipulated and/or tested on open benches, including by procedures that could generate infectious aerosols. During 3 Brucella events, the CLs examined and/or manipulated isolates in a biological safety cabinet (BSC); in each CL, the CL had previously isolated Brucella Centers for Disease Control and Prevention recommendations to prevent laboratory-acquired brucellosis (LAB) were followed; no seroconversions or LAB cases occurred. Laboratory assessments were conducted after the Brucella events to identify facility-specific risks and mitigations. With increasing MALDI-TOF MS use, CLs are well-advised to adhere strictly to safe work practices, such as handling and manipulating all slow-growing organisms in BSCs and not using MALDI-TOF MS for identification until BTAs have been ruled out.

Is hypofractionated whole pelvis radiotherapy (WPRT) as well tolerated as conventionally fractionated WPRT in prostate cancer patients? The HOPE trial.

BACKGROUND: Patients with high-risk prostate cancer are at increased risk of lymph node metastasis and are thought to benefit from whole pelvis radiotherapy (WPRT). There has been recent interest in the use of hypofractionated radiotherapy in treating prostate cancer. However, toxicity and cancer outcomes associated with hypofractionated WPRT are unclear at this time. This phase II study aims to investigate the impact in quality of life associated with hypofractionated WPRT compared to conventionally fractionated WPRT. METHODS: Fifty-eight patients with unfavourable intermediate-, high- or very high-risk prostate cancer will be randomized in a 1:1 ratio between high-dose-rate brachytherapy (HDR-BT) + conventionally fractionated (45 Gy in 25 fractions) WPRT vs. HDR-BT + hypofractionated (25 Gy in 5 fractions) WPRT. Randomization will be performed with a permuted block design without stratification. The primary endpoint is late bowel toxicity and the secondary endpoints include acute and late urinary and sexual toxicity, acute bowel toxicity, biochemical failure-, androgen deprivation therapy-, metastasis- and prostate cancer-free survival of the hypofractionated arm compared to the conventionally fractionated arm. DISCUSSION: To our knowledge, this is the first study to compare hypofractionated WPRT to conventionally fractionated WPRT with HDR-BT boost. Hypofractionated WPRT is a more attractive and convenient treatment approach, and may become the new standard of care if demonstrated to be well-tolerated and effective. TRIAL REGISTRATION: This trial was prospectively registered in ClinicalTrials.gov as NCT04197141 on December 12, 2019.

Stereotactic Ablative Radiotherapy for Recurrent or Metastatic Gynecological Cancer: Extending Lives?

OPINION STATEMENT: Recent phase II clinical trials suggest that stereotactic ablative radiation therapy (SABR) can potentially improve survival for patients with oligometastatic cancer. However, these studies have mostly enrolled primaries other than gynecologic malignancies. While level I evidence is limited, recent publications exploring the use of SABR for oligometastatic gynecologic cancers have indicated a potential role for this treatment in para-aortic lymph node recurrences, and in visceral and brain metastases. The use of SABR for recurrences in the pelvis presents a number of challenges as these patients have often received previous radiation treatment. In these settings, care must be taken to avoid trespassing normal tissue tolerance with SABR leading to toxicity, especially as the potential benefit of SABR in this setting is not based on high-level evidence. Although SABR is feasible and in general safe for oligometastatic gynecologic malignancies, insufficient data are available to indicate whether it is associated with improved survival. Clinical judgment that incorporates patient and tumor factors is needed to determine if SABR is appropriate for selecting patients. Future directions include combining SABR with novel systemic therapies, determining optimal sequencing of treatments, and generating more robust randomized data pertaining to the use of SABR for oligometastatic gynecologic cancers.

Coxiella burnetii utilizes both glutamate and glucose during infection with glucose uptake mediated by multiple transporters.

Coxiella burnetii is a Gram-negative bacterium which causes Q fever, a complex and life-threatening infection with both acute and chronic presentations. C. burnetii invades a variety of host cell types and replicates within a unique vacuole derived from the host cell lysosome. In order to understand how C. burnetii survives within this intracellular niche, we have investigated the carbon metabolism of both intracellular and axenically cultivated bacteria. Both bacterial populations were shown to assimilate exogenous [13C]glucose or [13C]glutamate, with concomitant labeling of intermediates in glycolysis and gluconeogenesis, and in the TCA cycle. Significantly, the two populations displayed metabolic pathway profiles reflective of the nutrient availabilities within their propagated environments. Disruption of the C. burnetii glucose transporter, CBU0265, by transposon mutagenesis led to a significant decrease in [13C]glucose utilization but did not abolish glucose usage, suggesting that C. burnetii express additional hexose transporters which may be able to compensate for the loss of CBU0265. This was supported by intracellular infection of human cells and in vivo studies in the insect model showing loss of CBU0265 had no impact on intracellular replication or virulence. Using this mutagenesis and [13C]glucose labeling approach, we identified a second glucose transporter, CBU0347, the disruption of which also showed significant decreases in 13C-label incorporation but did not impact intracellular replication or virulence. Together, these analyses indicate that C. burnetii may use multiple carbon sources in vivo and exhibits greater metabolic flexibility than expected.