Results of a randomised Phase II trial of olaparib, chemotherapy or olaparib and cediranib in patients with platinum-resistant ovarian cancer.

BACKGROUND: OCTOVA compared the efficacy of olaparib (O) versus weekly paclitaxel (wP) or olaparib + cediranib (O + C) in recurrent ovarian cancer (OC). AIMS: The main aim of the OCTOVA trial was to determine the progression-free survival (PFS) of olaparib (O) versus the oral combination of olaparib plus cediranib (O + C) and weekly paclitaxel (wP) in recurrent ovarian cancer (OC). METHODS: In total, 139 participants who had relapsed within 12 months of platinum therapy were randomised to O (300 mg twice daily), wP (80 mg/m2 d1,8,15, q28) or O + C (300 mg twice daily/20 mg daily, respectively). The primary endpoint was progression-free survival (PFS) of olaparib (O) versus olaparib plus cediranib (O + C) or weekly paclitaxel (wP). The sample size was calculated to observe a PFS hazard ratio (HR) 0.64 in favour of O + C compared to O (20% one-sided type I error, 80% power). RESULTS: The majority had platinum-resistant disease (90%), 22% prior PARPi, 34% prior anti-angiogenic therapy, 30% germline BRCA1/2 mutations. The PFS was increased for O + C vs O (O + C 5.4 mo (2.3, 9.6): O 3.7 mo (1.8, 7.6) HR = 0.73; 60% CI: 0.59, 0.89; P = 0.1) and no different between wP and O (wP 3.9 m (1.9, 9.1); O 3.7 mo (1.8, 7.6) HR = 0.89, 60% CI: 0.72, 1.09; P = 0.69). The main treatment-related adverse events included manageable diarrhoea (4% Grade 3) and hypertension (4% Grade 3) in the O + C arm. DISCUSSION: OCTOVA demonstrated the activity of O + C in women with recurrent disease, offering a potential non-chemotherapy option. TRIAL REGISTRATION: ISRCTN14784018, registered on 19th January 2018 http://www.isrctn.com/ISRCTN14784018 .

Structure and Function of Alkane Monooxygenase (AlkB).

ConspectusEvery year, perhaps as much as 800 million tons of hydrocarbons enters the environment; alkanes make up a large percentage of it. Most are transformed by organisms that utilize these molecules as sources of energy and carbon. Both aerobic and anaerobic alkane transformation chemistries exist, capitalizing on the presence of alkanes in both oxic and anoxic environments. Over the past 40 years, tremendous progress has been made in understanding the structure and mechanism of enzymes that catalyze the transformation of methane. By contrast, progress involving enzymes that transform liquid alkanes has been slower with the first structures of AlkB, the predominant aerobic alkane hydroxylase in the environment, appearing in 2023. Because of the fundamental importance of C-H bond activation chemistries, interest in understanding how biology activates and transforms alkanes is high.In this Account, we focus on steps we have taken to understand the mechanism and structure of alkane monooxygenase (AlkB), the metalloenzyme that dominates the transformation of liquid alkanes in the environment (not to be confused with another AlkB that is an α-ketogluturate-dependent enzyme involved in DNA repair). First, we briefly describe what is known about the prevalence of AlkB in the environment and its role in the carbon cycle. Then we review the key findings from our recent high-resolution cryoEM structure of AlkB and highlight important similarities and differences in the structures of members of class III diiron enzymes. Functional studies, which we summarize, from a number of single residue variants enable us to say a great deal about how the structure of AlkB facilitates its function. Next, we overview work from our laboratories using mechanistically diagnostic radical clock substrates to characterize the mechanism of AlkB and contextualize the results we have obtained on AlkB with results we have obtained on other alkane-oxidizing enzymes and explain these results in light of the enzyme's structure. Finally, we integrate recent work in our laboratories with information from prior studies of AlkB, and relevant model systems, to create a holistic picture of the enzyme. We end by pointing to critical questions that still need to be answered, questions about the electronic structure of the active site of the enzyme throughout the reaction cycle and about whether and to what extent the enzyme plays functional roles in biology beyond simply initiating the degradation of alkanes.

A tumor focused approach to resolving the etiology of DNA mismatch repair deficient tumors classified as suspected Lynch syndrome.

Routine screening of tumors for DNA mismatch repair (MMR) deficiency (dMMR) in colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumors leads to a significant proportion of unresolved cases classified as suspected Lynch syndrome (SLS). SLS cases (n = 135) were recruited from Family Cancer Clinics across Australia and New Zealand. Targeted panel sequencing was performed on tumor (n = 137; 80×CRCs, 33×ECs and 24xSSTs) and matched blood-derived DNA to assess for microsatellite instability status, tumor mutation burden, COSMIC tumor mutational signatures and to identify germline and somatic MMR gene variants. MMR immunohistochemistry (IHC) and MLH1 promoter methylation were repeated. In total, 86.9% of the 137 SLS tumors could be resolved into established subtypes. For 22.6% of these resolved SLS cases, primary MLH1 epimutations (2.2%) as well as previously undetected germline MMR pathogenic variants (1.5%), tumor MLH1 methylation (13.1%) or false positive dMMR IHC (5.8%) results were identified. Double somatic MMR gene mutations were the major cause of dMMR identified across each tumor type (73.9% of resolved cases, 64.2% overall, 70% of CRC, 45.5% of ECs and 70.8% of SSTs). The unresolved SLS tumors (13.1%) comprised tumors with only a single somatic (7.3%) or no somatic (5.8%) MMR gene mutations. A tumor-focused testing approach reclassified 86.9% of SLS into Lynch syndrome, sporadic dMMR or MMR-proficient cases. These findings support the incorporation of tumor sequencing and alternate MLH1 methylation assays into clinical diagnostics to reduce the number of SLS patients and provide more appropriate surveillance and screening recommendations.

Marine heatwave and reduced light scenarios cause species-specific metabolomic changes in seagrasses under ocean warming.

Climate change and extreme climatic events, such as marine heatwaves (MHWs), are threatening seagrass ecosystems. Metabolomics can be used to gain insight into early stress responses in seagrasses and help to develop targeted management and conservation measures. We used metabolomics to understand the temporal and mechanistic response of leaf metabolism in seagrasses to climate change. Two species, temperate Posidonia australis and tropical Halodule uninervis, were exposed to a combination of future warming, simulated MHW with subsequent recovery period, and light deprivation in a mesocosm experiment. The leaf metabolome of P. australis was altered under MHW exposure at ambient light while H. uninervis was unaffected. Light deprivation impacted both seagrasses, with combined effects of heat and low light causing greater alterations in leaf metabolism. There was no MHW recovery in P. australis. Conversely, the heat-resistant leaf metabolome of H. uninervis showed recovery of sugars and intermediates of the tricarboxylic acid cycle under combined heat and low light exposure, suggesting adaptive strategies to long-term light deprivation. Overall, this research highlights how metabolomics can be used to study the metabolic pathways of seagrasses, identifies early indicators of environmental stress and analyses the effects of environmental factors on plant metabolism and health.

Maximization of fitness by phenological and phenotypic plasticity in range expanding rabbitfishes (Siganidae).

Global warming is modifying the phenology, life-history traits and biogeography of species around the world. Evidence of these effects have increased over recent decades; however, we still have a poor understanding of the possible outcomes of their interplay across global climatic gradients, hindering our ability to accurately predict the consequences of climate change in populations and ecosystems. We examined the effect that changes in biogeography can have on the life-history traits of two of the most successful range-extending fish species in the world: the tropical rabbitfishes Siganus fuscescens and Siganus rivulatus. Both species have established abundant populations at higher latitudes in the northern and southern hemispheres and have been identified as important ecological engineers with the potential to alter the community structure of seaweed forests (Laminariales and Fucales) in temperate regions. Life-history trait information from across their global distribution was compiled from the published literature and meta-analyses were conducted to assess changes in (i) the onset and duration of reproductive periods, (ii) size at maturity, (iii) fecundity, (iv) growth rates, (v) maximum body sizes and (vi) longevity in populations at the leading edge of range expansion in relation to sea surface temperature and primary productivity (a common proxy for nutritional resource levels). Populations at highest latitudes had shortened their reproductive periods and reduced growth rates, taking longer to reach sexual maturity and maximum sizes, but compensated this with higher fecundity per length class and longer lifespans than populations in warmer environments. Low primary productivity and temperature in the Mediterranean Sea resulted in lower growth rates and body sizes for S. rivulatus, but also lower length at maturity, increasing life-time reproductive output. The results suggest that plasticity in the phenology and life-history traits of range-expanding species would be important to enhance their fitness in high latitude environments, facilitating their persistence and possible further poleward expansions. Quantifying the magnitude and direction of these responses can improve our understanding and ability to forecast species redistributions and its repercussions in the functioning of temperate ecosystems.

Impact of national guidelines on use of BRCA1/2 germline testing, risk management advice given to women with pathogenic BRCA1/2 variants and uptake of advice.

BACKGROUND: This nationwide study assessed the impact of nationally agreed cancer genetics guidelines on use of BRCA1/2 germline testing, risk management advice given by health professionals to women with pathogenic BRCA1/2 variants and uptake of such advice by patients. METHODS: Clinic files of 883 women who had initial proband screens for BRCA1/2 pathogenic variants at 12 familial cancer clinics between July 2008-July 2009 (i.e. before guideline release), July 2010-July 2011 and July 2012-July 2013 (both after guideline release) were audited to determine reason given for genetic testing. Separately, the clinic files of 599 female carriers without a personal history of breast/ovarian cancer who underwent BRCA1/2 predictive genetic testing and received their results pre- and post-guideline were audited to ascertain the risk management advice given by health professionals. Carriers included in this audit were invited to participate in a telephone interview to assess uptake of advice, and 329 agreed to participate. RESULTS: There were no significant changes in the percentages of tested patients meeting at least one published indication for genetic testing - 79, 77 and 78% of files met criteria before guideline, and two-, and four-years post-guideline, respectively (χ = 0.25, p = 0.88). Rates of documentation of post-test risk management advice as per guidelines increased significantly from pre- to post-guideline for 6/9 risk management strategies. The strategies with the highest compliance amongst carriers or awareness post-release of guidelines were annual magnetic resonance imaging plus mammography in women 30-50 years (97%) and annual mammography in women > 50 years (92%). Of women aged over 40 years, 41% had a risk-reducing bilateral mastectomy. Amongst women aged > 40 years, 75% had a risk-reducing salpingo-oophorectomy. Amongst women who had not had a risk-reducing bilateral mastectomy, only 6% took risk-reducing medication. Fear of side-effects was cited as the main reasons for not taking these medicines by 73% of women. CONCLUSIONS: Guidelines did not change the percentages of tested patients meeting genetic testing criteria but improved documentation of risk management advice by health professionals. Effective approaches to enhance compliance with guidelines are needed to improve risk management and quality of care.

Elevated zinc transporter ZnT3 in the dentate gyrus of mast cell-deficient mice.

Zinc is important in neurogenesis, but excessive levels can cause apoptosis and other pathologies leading to cognitive impairments. Mast cells are present in many brain regions including the hippocampus, an area rich in vesicular zinc. Mast cells contain zinc-rich granules and a well-developed mechanism for uptake of zinc ions; both features point to the potential for a role in zinc homeostasis. Prior work using the Timm stain supported this hypothesis, as increased labile zinc was detected in the hippocampus of mast cell-deficient mice compared to wild-type mice while no differences in total zinc were found between the two genotypes in the whole brain or other tissues. The current report further examines differences in zinc homeostasis between wild-type and mast cell-deficient mice by exploring the zinc transporter ZnT3, which transports labile zinc into synaptic vesicles. The first study used immunocytochemistry to localize ZnT3 within the mossy fibre layer of the hippocampus to determine whether there was differential expression of ZnT3 in wild-type versus mast cell-deficient mice. The second study used inductively coupled plasma mass spectrometry (ICP-MS) to determine total zinc content in the whole dentate gyrus of the two genotypes. The immunocytochemical results indicate that there are higher levels of ZnT3 localized to the mossy fibre layer of the dentate gyrus of mast cell-deficient mice than in wild-type mice. The ICP-MS data reveal no differences in total zinc in dentate gyrus as a whole. The results are consistent with the hypothesis that mast cells participate in zinc homeostasis at the level of synaptic vesicles.

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification.

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification.

The Enigmatic P450 Decarboxylase OleT Is Capable of, but Evolved To Frustrate, Oxygen Rebound Chemistry.

OleT is a cytochrome P450 enzyme that catalyzes the removal of carbon dioxide from variable chain length fatty acids to form 1-alkenes. In this work, we examine the binding and metabolic profile of OleT with shorter chain length (n ≤ 12) fatty acids that can form liquid transportation fuels. Transient kinetics and product analyses confirm that OleT capably activates hydrogen peroxide with shorter substrates to form the high-valent intermediate Compound I and largely performs C-C bond scission. However, the enzyme also produces fatty alcohol side products using the high-valent iron oxo chemistry commonly associated with insertion of oxygen into hydrocarbons. When presented with a short chain fatty acid that can initiate the formation of Compound I, OleT oxidizes the diagnostic probe molecules norcarane and methylcyclopropane in a manner that is reminiscent of reactions of many CYP hydroxylases with radical clock substrates. These data are consistent with a decarboxylation mechanism in which Compound I abstracts a substrate hydrogen atom in the initial step. Positioning of the incipient substrate radical is a crucial element in controlling the efficiency of activated OH rebound.

Function of Metallothionein-3 in Neuronal Cells: Do Metal Ions Alter Expression Levels of MT3?

A study of factors proposed to affect metallothionein-3 (MT3) function was carried out to elucidate the opaque role MT3 plays in human metalloneurochemistry. Gene expression of Mt2 and Mt3 was examined in tissues extracted from the dentate gyrus of mouse brains and in human neuronal cell cultures. The whole-genome gene expression analysis identified significant variations in the mRNA levels of genes associated with zinc homeostasis, including Mt2 and Mt3. Mt3 was found to be the most differentially expressed gene in the identified groups, pointing to the existence of a factor, not yet identified, that differentially controls Mt3 expression. To examine the expression of the human metallothioneins in neurons, mRNA levels of MT3 and MT2 were compared in BE(2)C and SH-SY5Y cell cultures treated with lead, zinc, cobalt, and lithium. MT2 was highly upregulated by Zn2+ in both cell cultures, while MT3 was not affected, and no other metal had an effect on either MT2 or MT3.

Comparative gene co-expression networks show enrichment of brassinosteroid and vitamin B processes in a seagrass under simulated ocean warming and extreme climatic events.

Introduction: Ocean warming combined with extreme climatic events, such as marine heatwaves and flash flooding events, threaten seagrasses globally. How seagrasses cope with these challenges is uncertain, particularly for range-edge populations of species such as Posidonia australis in Shark Bay, Western Australia. Analyzing gene expression while manipulating multiple stressors provides insight into the genetic response and resilience of seagrasses to climate change. We conducted a gene expression study on a polyploid clone of P. australis during an 18-week mesocosm experiment to assess the responses to single and combined future climate change-associated stressors. Methods: Plants were exposed to (1) future ocean warming temperature (baseline +1.5°C) followed by a simulated marine heat wave (baseline +5.5°C), (2) light deprivation simulating observed marine heatwave driven turbidity (95% shade) at baseline temperatures, or (3) both stressors simultaneously. Basal leaf meristems were sampled for gene expression analysis using RNA-seq at four time points during the experiment. Weighted gene co-expression network analysis, GO term enrichment, and KEGG pathway enrichment analyses were used to identify stress responses. Results: Shaded plants showed specific gene enrichment for shade avoidance (programmed cell death) after three weeks of stress, and before any heated tanks showed a specific heat response. Shaded plants were positively correlated with programmed cell death and stress-related processes at the end of the experiment. Once ocean warming temperatures (+1.5°C) were in effect, gene enrichment for heat stress (e.g., ROS scavenging and polyamine metabolism) was present. Vitamin B processes, RNA polymerase II processes. and light-related meristematic phase changes were expressed with the addition of simulated MHW. Heated plants showed meristematic growth signatures as well as trehalose and salicylic acid metabolism. Brassinosteroid-related processes were significantly enriched in all stressor treatments at all time points, except for the isolated heat-stressed plants three weeks after stressor initiation. Discussion: Gene expression responses to the interaction between heat waves and turbidity-induced light reduction support the observed geographical scale mortality in seagrasses observed for P. australis in Shark Bay, suggesting that even this giant polyploid clone will be negatively impacted by more extreme climate change projections.

Metallothionein-3 attenuates the effect of Cu2+ ions on actin filaments.

Metallothionein 3 (MT-3) is a cysteine-rich metal-binding protein that is expressed in the mammalian central nervous system and kidney. Various reports have posited a role for MT-3 in regulating the actin cytoskeleton by promoting the assembly of actin filaments. We generated purified, recombinant mouse MT-3 of known metal compositions, either with zinc (Zn), lead (Pb), or copper/zinc (Cu/Zn) bound. None of these forms of MT-3 accelerated actin filament polymerization in vitro, either with or without the actin binding protein profilin. Furthermore, using a co-sedimentation assay, we did not observe Zn-bound MT-3 in complex with actin filaments. Cu2+ ions on their own induced rapid actin polymerization, an effect that we attribute to filament fragmentation. This effect of Cu2+ is reversed by adding either EGTA or Zn-bound MT-3, indicating that either molecule can chelate Cu2+ from actin. Altogether, our data indicate that purified recombinant MT-3 does not directly bind actin but it does attenuate the Cu-induced fragmentation of actin filaments.

Structure and mechanism of the alkane-oxidizing enzyme AlkB.

Alkanes are the most energy-rich form of carbon and are widely dispersed in the environment. Their transformation by microbes represents a key step in the global carbon cycle. Alkane monooxygenase (AlkB), a membrane-spanning metalloenzyme, converts straight chain alkanes to alcohols in the first step of the microbially-mediated degradation of alkanes, thereby playing a critical role in the global cycling of carbon and the bioremediation of oil. AlkB biodiversity is attributed to its ability to oxidize alkanes of various chain lengths, while individual AlkBs target a relatively narrow range. Mechanisms of substrate selectivity and catalytic activity remain elusive. Here we report the cryo-EM structure of AlkB, which provides a distinct architecture for membrane enzymes. Our structure and functional studies reveal an unexpected diiron center configuration and identify molecular determinants for substrate selectivity. These findings provide insight into the catalytic mechanism of AlkB and shed light on its function in alkane-degrading microorganisms.

Exploring the impact of the reclassification of a hereditary cancer syndrome gene variant: emerging themes from a qualitative study.

The complexity of genetic variant interpretation means that a proportion of individuals who undergo genetic testing for a hereditary cancer syndrome will have their test result reclassified over time. Such a reclassification may involve a clinically significant upgrade or downgrade in pathogenicity, which may have significant implications for medical management. To date, few studies have examined the psychosocial impact of a reclassification in a hereditary cancer syndrome context. To address this gap, semi-structured telephone interviews were performed with eighteen individuals who had a BRCA1, BRCA2 or Lynch syndrome-related (MLH1, MSH2, MSH6 or PMS2) gene variant reclassified. The interviews were analysed utilising an inductive, qualitative approach and emergent themes were identified by thematic analysis. Variable levels of recall amongst participants were found. Common motivations for initial testing included a significant personal and/or family history of cancer and a desire to "find an answer". No individual whose uncertain result was upgraded reported negative psychosocial outcomes; most reported adapting to their reclassified result and appraised their genetic testing experience positively. However, individuals whose likely pathogenic/pathogenic results were downgraded reported feelings of anger, shock and sadness post reclassification, highlighting that additional psychosocial support may be required for some. Genetic counselling issues and recommendations for clinical practice are outlined.

DNA Mismatch Repair Gene Variant Classification: Evaluating the Utility of Somatic Mutations and Mismatch Repair Deficient Colonic Crypts and Endometrial Glands.

Germline pathogenic variants in the DNA mismatch repair (MMR) genes (Lynch syndrome) predispose to colorectal (CRC) and endometrial (EC) cancer. Lynch syndrome specific tumor features were evaluated for their ability to support the ACMG/InSiGHT framework in classifying variants of uncertain clinical significance (VUS) in the MMR genes. Twenty-eight CRC or EC tumors from 25 VUS carriers (6xMLH1, 9xMSH2, 6xMSH6, 4xPMS2), underwent targeted tumor sequencing for the presence of microsatellite instability/MMR-deficiency (MSI-H/dMMR) status and identification of a somatic MMR mutation (second hit). Immunohistochemical testing for the presence of dMMR crypts/glands in normal tissue was also performed. The ACMG/InSiGHT framework reclassified 7/25 (28%) VUS to likely pathogenic (LP), three (12%) to benign/likely benign, and 15 (60%) VUS remained unchanged. For the seven re-classified LP variants comprising nine tumors, tumor sequencing confirmed MSI-H/dMMR (8/9, 88.9%) and a second hit (7/9, 77.8%). Of these LP reclassified variants where normal tissue was available, the presence of a dMMR crypt/gland was found in 2/4 (50%). Furthermore, a dMMR endometrial gland in a carrier of an MSH2 exon 1-6 duplication provides further support for an upgrade of this VUS to LP. Our study confirmed that identifying these Lynch syndrome features can improve MMR variant classification, enabling optimal clinical care.

A tumor focused approach to resolving the etiology of DNA mismatch repair deficient tumors classified as suspected Lynch syndrome.

Routine screening of tumors for DNA mismatch repair (MMR) deficiency (dMMR) in colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumors leads to a significant proportion of unresolved cases classified as suspected Lynch syndrome (SLS). SLS cases (n=135) were recruited from Family Cancer Clinics across Australia and New Zealand. Targeted panel sequencing was performed on tumor (n=137; 80xCRCs, 33xECs and 24xSSTs) and matched blood-derived DNA to assess for microsatellite instability status, tumor mutation burden, COSMIC tumor mutational signatures and to identify germline and somatic MMR gene variants. MMR immunohistochemistry (IHC) and MLH1 promoter methylation were repeated. In total, 86.9% of the 137 SLS tumors could be resolved into established subtypes. For 22.6% of these resolved SLS cases, primary MLH1 epimutations (2.2%) as well as previously undetected germline MMR pathogenic variants (1.5%), tumor MLH1 methylation (13.1%) or false positive dMMR IHC (5.8%) results were identified. Double somatic MMR gene mutations were the major cause of dMMR identified across each tumor type (73.9% of resolved cases, 64.2% overall, 70% of CRC, 45.5% of ECs and 70.8% of SSTs). The unresolved SLS tumors (13.1%) comprised tumors with only a single somatic (7.3%) or no somatic (5.8%) MMR gene mutations. A tumor-focused testing approach reclassified 86.9% of SLS into Lynch syndrome, sporadic dMMR or MMR-proficient cases. These findings support the incorporation of tumor sequencing and alternate MLH1 methylation assays into clinical diagnostics to reduce the number of SLS patients and provide more appropriate surveillance and screening recommendations.

The clinical utility and costs of whole-genome sequencing to detect cancer susceptibility variants-a multi-site prospective cohort study.

BACKGROUND: Many families and individuals do not meet criteria for a known hereditary cancer syndrome but display unusual clusters of cancers. These families may carry pathogenic variants in cancer predisposition genes and be at higher risk for developing cancer. METHODS: This multi-centre prospective study recruited 195 cancer-affected participants suspected to have a hereditary cancer syndrome for whom previous clinical targeted genetic testing was either not informative or not available. To identify pathogenic disease-causing variants explaining participant presentation, germline whole-genome sequencing (WGS) and a comprehensive cancer virtual gene panel analysis were undertaken. RESULTS: Pathogenic variants consistent with the presenting cancer(s) were identified in 5.1% (10/195) of participants and pathogenic variants considered secondary findings with potential risk management implications were identified in another 9.7% (19/195) of participants. Health economic analysis estimated the marginal cost per case with an actionable variant was significantly lower for upfront WGS with virtual panel ($8744AUD) compared to standard testing followed by WGS ($24,894AUD). Financial analysis suggests that national adoption of diagnostic WGS testing would require a ninefold increase in government annual expenditure compared to conventional testing. CONCLUSIONS: These findings make a case for replacing conventional testing with WGS to deliver clinically important benefits for cancer patients and families. The uptake of such an approach will depend on the perspectives of different payers on affordability.

Parallel and competitive pathways for substrate desaturation, hydroxylation, and radical rearrangement by the non-heme diiron hydroxylase AlkB.

A purified and highly active form of the non-heme diiron hydroxylase AlkB was investigated using the diagnostic probe substrate norcarane. The reaction afforded C2 (26%) and C3 (43%) hydroxylation and desaturation products (31%). Initial C-H cleavage at C2 led to 7% C2 hydroxylation and 19% 3-hydroxymethylcyclohexene, a rearrangement product characteristic of a radical rearrangement pathway. A deuterated substrate analogue, 3,3,4,4-norcarane-d(4), afforded drastically reduced amounts of C3 alcohol (8%) and desaturation products (5%), while the radical rearranged alcohol was now the major product (65%). This change in product ratios indicates a large kinetic hydrogen isotope effect of ∼20 for both the C-H hydroxylation at C3 and the desaturation pathway, with all of the desaturation originating via hydrogen abstraction at C3 and not C2. The data indicate that AlkB reacts with norcarane via initial C-H hydrogen abstraction from C2 or C3 and that the three pathways, C3 hydroxylation, C3 desaturation, and C2 hydroxylation/radical rearrangement, are parallel and competitive. Thus, the incipient radical at C3 either reacts with the iron-oxo center to form an alcohol or proceeds along the desaturation pathway via a second H-abstraction to afford both 2-norcarene and 3-norcarene. Subsequent reactions of these norcarenes lead to detectable amounts of hydroxylation products and toluene. By contrast, the 2-norcaranyl radical intermediate leads to C2 hydroxylation and the diagnostic radical rearrangement, but this radical apparently does not afford desaturation products. The results indicate that C-H hydroxylation and desaturation follow analogous stepwise reaction channels via carbon radicals that diverge at the product-forming step.

An Overview of the Electron-Transfer Proteins That Activate Alkane Monooxygenase (AlkB).

Alkane-oxidizing enzymes play an important role in the global carbon cycle. Alkane monooxygenase (AlkB) oxidizes most of the medium-chain length alkanes in the environment. The first AlkB identified was from P. putida GPo1 (initially known as P. oleovorans) in the early 1970s, and it continues to be the family member about which the most is known. This AlkB is found as part of the OCT operon, in which all of the key proteins required for growth on alkanes are present. The AlkB catalytic cycle requires that the diiron active site be reduced. In P. putida GPo1, electrons originate from NADH and arrive at AlkB via the intermediacy of a flavin reductase and an iron-sulfur protein (a rubredoxin). In this Mini Review, we will review what is known about the canonical arrangement of electron-transfer proteins that activate AlkB and, more importantly, point to several other arrangements that are possible. These other arrangements include the presence of a simpler rubredoxin than what is found in the canonical arrangement, as well as two other classes of AlkBs with fused electron-transfer partners. In one class, a rubredoxin is fused to the hydroxylase and in another less well-explored class, a ferredoxin reductase and a ferredoxin are fused to the hydroxylase. We review what is known about the biochemistry of these electron-transfer proteins, speculate on the biological significance of this diversity, and point to key questions for future research.