RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer.

Background: BRCA1 and BRCA2 (BRCA1/2)-deficient tumors display impaired homologous recombination repair (HRR) and enhanced sensitivity to DNA damaging agents or to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi). Their efficacy in germline BRCA1/2 (gBRCA1/2)-mutated metastatic breast cancers has been recently confirmed in clinical trials. Numerous mechanisms of PARPi resistance have been described, whose clinical relevance in gBRCA-mutated breast cancer is unknown. This highlights the need to identify functional biomarkers to better predict PARPi sensitivity. Patients and methods: We investigated the in vivo mechanisms of PARPi resistance in gBRCA1 patient-derived tumor xenografts (PDXs) exhibiting differential response to PARPi. Analysis included exome sequencing and immunostaining of DNA damage response proteins to functionally evaluate HRR. Findings were validated in a retrospective sample set from gBRCA1/2-cancer patients treated with PARPi. Results: RAD51 nuclear foci, a surrogate marker of HRR functionality, were the only common feature in PDX and patient samples with primary or acquired PARPi resistance. Consistently, low RAD51 was associated with objective response to PARPi. Evaluation of the RAD51 biomarker in untreated tumors was feasible due to endogenous DNA damage. In PARPi-resistant gBRCA1 PDXs, genetic analysis found no in-frame secondary mutations, but BRCA1 hypomorphic proteins in 60% of the models, TP53BP1-loss in 20% and RAD51-amplification in one sample, none mutually exclusive. Conversely, one of three PARPi-resistant gBRCA2 tumors displayed BRCA2 restoration by exome sequencing. In PDXs, PARPi resistance could be reverted upon combination of a PARPi with an ataxia-telangiectasia mutated (ATM) inhibitor. Conclusion: Detection of RAD51 foci in gBRCA tumors correlates with PARPi resistance regardless of the underlying mechanism restoring HRR function. This is a promising biomarker to be used in the clinic to better select patients for PARPi therapy. Our study also supports the clinical development of PARPi combinations such as those with ATM inhibitors.

Daily acute intermittent hypoxia improves breathing function with acute and chronic spinal injury via distinct mechanisms.

Daily acute intermittent hypoxia (dAIH) elicits respiratory plasticity, enhancing respiratory motor output and restoring breathing capacity after incomplete cervical spinal injuries (cSCI). We hypothesized that dAIH-induced functional recovery of breathing capacity would occur after both acute (2 weeks) and chronic (8 weeks) cSCI, but through distinct cellular mechanisms. Specifically, we hypothesized that dAIH-induced breathing recovery would occur through serotonin-independent mechanisms 2wks post C2 cervical hemisection (C2Hs), versus serotonin-dependent mechanisms 8wks post C2Hs. In two independent studies, dAIH or sham (normoxia) was initiated 1 week (Study 1) or 7 weeks (Study 2) post-C2Hs to test our hypothesis. Rats were pre-treated with intra-peritoneal vehicle or methysergide, a broad-spectrum serotonin receptor antagonist, to determine the role of serotonin signaling in dAIH-induced functional recovery. Our data support the hypothesis that dAIH-induced recovery of breathing capacity transitions from a serotonin-independent mechanism with acute C2Hs to a serotonin-dependent mechanism with chronic C2Hs. An understanding of shifting mechanisms giving rise to dAIH-induced respiratory motor plasticity is vital for clinical translation of dAIH as a therapeutic modality.

Enhanced recovery of breathing capacity from combined adenosine 2A receptor inhibition and daily acute intermittent hypoxia after chronic cervical spinal injury.

Daily acute intermittent hypoxia (dAIH) improves breathing capacity after C2 spinal hemisection (C2HS) in rats. Since C2HS disrupts spinal serotonergic innervation below the injury, adenosine-dependent mechanisms underlie dAIH-induced functional recovery 2weeks post-injury. We hypothesized that dAIH-induced functional recovery converts from an adenosine-dependent to a serotonin-dependent, adenosine-constrained mechanism with chronic injury. Eight weeks post-C2HS, rats began dAIH (10, 5-min episodes, 10.5% O2; 5-min intervals; 7days) followed by AIH 3× per week (3×wAIH) for 8 additional weeks with/without systemic A2A receptor inhibition (KW6002) on each AIH exposure day. Tidal volume (VT) and bilateral diaphragm (Dia) and T2 external intercostal motor activity were assessed in unanesthetized rats breathing air and during maximum chemoreflex stimulation (MCS: 7% CO2, 10.5% O2). Nine weeks post-C2HS, dAIH increased VT versus time controls (p<0.05), an effect enhanced by KW6002 (p<0.05). dAIH increased bilateral Dia activity (p<0.05), and KW6002 enhanced this effect in contralateral (p<0.05) and ipsilateral Dia activity (p<0.001), but not T2 inspiratory activity. Functional benefits of combined AIH plus systemic A2A receptor inhibition were maintained for 4weeks. Thus, in rats with chronic injuries: 1) dAIH improves VT and bilateral diaphragm activity; 2) VT recovery is enhanced by A2A receptor inhibition; and 3) functional recovery with A2A receptor inhibition and AIH "reminders" last 4weeks. Combined dAIH and A2A receptor inhibition may be a simple, safe, and effective strategy to accelerate/enhance functional recovery of breathing capacity in patients with respiratory impairment from chronic spinal injury.

Respiratory outcomes after mid-cervical transplantation of embryonic medullary cells in rats with cervical spinal cord injury.

Respiratory motor output after cervical spinal cord injury (cSCI) is profoundly influenced by spinal serotonin. We hypothesized that intraspinal transplantation of embryonic midline brainstem (MB) cells rich in serotonergic raphé neurons would improve respiratory outcomes after cSCI. One week after hemisection of the 2nd cervical segment (C2Hx) a suspension of either embryonic (E14) MB cells, fetal spinal cord cells (FSC), or media only (sham) was delivered to the dorsal C3 spinal cord of adult male rats. Six weeks later, ventilation was evaluated using plethysmography; phrenic nerve activity was evaluated in a subset of rats. Seven of 12 rats receiving MB-derived grafts had clear histological evidence of serotonin-positive neurons in the C3-4 dorsal white matter. The transplantations had no impact on baseline breathing patterns, but during a brief respiratory challenge (7% inspired CO2) rats with successful MB grafts had increased ventilation compared to rats with failed MB grafts, FSC or sham grafts. Recordings from the phrenic nerve ipsilateral to C2Hx also indicated increased output during respiratory challenge in rats with successful MB grafts. We conclude that intraspinal allografting of E14 MB cells can have a positive impact on respiratory motor recovery following high cSCI.

Impact of KRAS codon subtypes from a randomised phase II trial of selumetinib plus docetaxel in KRAS mutant advanced non-small-cell lung cancer.

BACKGROUND: Selumetinib (AZD6244, ARRY-142886)+docetaxel increases median overall survival (OS) and significantly improves progression-free survival (PFS) and objective response rate (ORR) compared with docetaxel alone in patients with KRAS mutant, stage IIIB/IV non-small-cell lung cancer (NSCLC; NCT00890825). METHODS: Retrospective analysis of OS, PFS, ORR and change in tumour size at week 6 for different sub-populations of KRAS codon mutations. RESULTS: In patients receiving selumetinib+docetaxel and harbouring KRAS G12C or G12V mutations there were trends towards greater improvement in OS, PFS and ORR compared with other KRAS mutations. CONCLUSION: Different KRAS mutations in NSCLC may influence selumetinib/docetaxel sensitivity.

Daily acute intermittent hypoxia elicits functional recovery of diaphragm and inspiratory intercostal muscle activity after acute cervical spinal injury.

A major cause of mortality after spinal cord injury is respiratory failure. In normal rats, acute intermittent hypoxia (AIH) induces respiratory motor plasticity, expressed as diaphragm (Dia) and second external intercostal (T2 EIC) long-term facilitation (LTF). Dia (not T2 EIC) LTF is enhanced by systemic adenosine 2A (A2A) receptor inhibition in normal rats. We investigated the respective contributions of Dia and T2 EIC to daily AIH-induced functional recovery of breathing capacity with/without A2A receptor antagonist (KW6002, i.p.) following C2 hemisection (C2HS). Rats received daily AIH (dAIH: 10, 5-min episodes, 10.5% O2; 5-min normoxic intervals; 7 successive days beginning 7days post-C2HS) or daily normoxia (dNx) with/without KW6002, followed by weekly (reminder) presentations for 8weeks. Ventilation and EMGs from bilateral diaphragm and T2 EIC muscles were measured with room air breathing (21% O2) and maximum chemoreceptor stimulation ( MCS: 7% CO2, 10.5% O2). dAIH increased tidal volume (VT) in C2HS rats breathing room air (dAIH+vehicle: 0.47±0.02, dNx+vehicle: 0.40±0.01ml/100g; p<0.05) and MCS (dAIH+vehicle: 0.83±0.01, dNx+vehicle: 0.73±0.01ml/100g; p<0.001); KW6002 had no significant effect. dAIH enhanced contralateral (uninjured) diaphragm EMG activity, an effect attenuated by KW6002, during room air breathing and MCS (p<0.05). Although dAIH enhanced contralateral T2 EIC EMG activity during room air breathing, KW6002 had no effect. dAIH had no statistically significant effects on diaphragm or T2 EIC EMG activity ipsilateral to injury. Thus, two weeks post-C2HS: 1) dAIH enhances breathing capacity by effects on contralateral diaphragm and T2 EIC activity; and 2) dAIH-induced recovery is A2A dependent in diaphragm, but not T2 EIC. Daily AIH may be a useful in promoting functional recovery of breathing capacity after cervical spinal injury, but A2A receptor antagonists (e.g. caffeine) may undermine its effectiveness shortly after injury.

Rapid diaphragm atrophy following cervical spinal cord hemisection.

A cervical (C2) hemilesion (C2Hx), which disrupts ipsilateral bulbospinal inputs to the phrenic nucleus, was used to study diaphragm plasticity after acute spinal cord injury. We hypothesized that C2Hx would result in rapid atrophy of the ipsilateral hemidiaphragm and increases in mRNA expression of proteolytic biomarkers. Diaphragm tissue was harvested from male Sprague-Dawley rats at 1 or 7 days following C2Hx. Histological analysis demonstrated reduction in cross-sectional area (CSA) of type I and IIa fibers in the ipsilateral hemidiaphragm at 1 but not 7 days. Type IIb/x fibers, however, had reduced CSA at 1 and 7 days. A targeted gene array was used to screen mRNA changes for genes associated with skeletal muscle myopathy and myogenesis; this was followed by qRT-PCR validation. Changes in diaphragm gene expression suggested that profound myoplasticity is initiated immediately following C2Hx including activation of both proteolytic and myogenic pathways. We conclude that an immediate myoplastic response occurs in the diaphragm after C2Hx with atrophy occurring in ipsilateral myofibers within 1 day.

Recovery of inspiratory intercostal muscle activity following high cervical hemisection.

Anatomical and neurophysiological evidence indicates that thoracic interneurons can serve a commissural function and activate contralateral motoneurons. Accordingly, we hypothesized that respiratory-related intercostal (IC) muscle electromyogram (EMG) activity would be only modestly impaired by a unilateral cervical spinal cord injury. Inspiratory tidal volume (VT) was recorded using pneumotachography and EMG activity was recorded bilaterally from the 1st to 2nd intercostal space in anesthetized, spontaneously breathing rats. Studies were conducted at 1-3 days, 2 wks or 8 wks following C2 spinal cord hemisection (C2HS). Data were collected during baseline breathing and a brief respiratory challenge (7% CO(2)). A substantial reduction in inspiratory intercostal EMG bursting ipsilateral to the lesion was observed at 1-3 days post-C2HS. However, a time-dependent return of activity occurred such that by 2 wks post-injury inspiratory intercostal EMG bursts ipsilateral to the lesion were similar to age-matched, uninjured controls. The increases in ipsilateral intercostal EMG activity occurred in parallel with increases in VT following the injury (R=0.55; P<0.001). We conclude that plasticity occurring within a "crossed-intercostal" circuitry enables a robust, spontaneous recovery of ipsilateral intercostal activity following C2HS in rats.

The financial imperative of physicians to control demand of laboratory testing.

It is an integral component of doctor's duty of care to understand the significant impact laboratory testing has on the expense an ultimate quality of healthcare patients receive, yet the costs of these tests are poorly perceived. Utilising semi-structured interviews and questionnaires, we assessed surgeon's perceived costs of two commonly encountered clinical scenarios requiring out of hours laboratory testing. Of the 35 participants only 23.3% (n = 7) accurately estimated the overall cost. The most expensive test was "Type and Screen" at Euro 83, with 77.3% (n = 17) underestimating the cost. Non-consultant hospital doctors qualified for 3 years were more likely to underestimate on-call costs (p = 0.042). It is of utmost importance to improve the knowledge of all surgeons of the financial implications of investigations. Through education we can potentially reduce un-warranted costs and fulfill our duty of care in the most cost efficient manner.

Prenatal nicotine exposure alters respiratory long-term facilitation in neonatal rats.

Intermittent hypoxia can evoke persistent increases in ventilation (V (E)) in neonates (i.e. long-term facilitation, LTF) (Julien et al., 2008). Since prenatal nicotine (PN) exposure alters neonatal respiratory control (Fregosi and Pilarski, 2008), we hypothesized that PN would influence LTF of ventilation (V (E)) in neonatal rats. An osmotic minipump delivered nicotine 6 mg/kg per day or saline to pregnant dams. V (E) was assessed in unanesthetized pups via whole body plethysmography at post-natal (P) days 9-11 or 15-17 during baseline (BL, 21% O(2)), hypoxia (10 x 5 min, 5% O(2)) and 30 min post-hypoxia. PN pups had reduced BL V (E) (p<0.05) but greater increases in V (E) during hypoxia (p<0.05). Post-hypoxia V (E) (i.e. LTF) showed an agex treatment interaction (p<0.01) with similar values at P9-11 but enhanced LTF in saline (30+/-8%BL) vs. PN pups (6+/-5%BL; p=0.01) at P15-17. We conclude that the post-natal developmental time course of hypoxia-induced LTF is influenced by PN.

Respiratory recovery following high cervical hemisection.

In this paper we review respiratory recovery following C2 spinal cord hemisection (C2HS) and introduce evidence for ipsilateral (IL) and contralateral (CL) phrenic motor neuron (PhrMN) synchrony post-C2HS. Rats have rapid, shallow breathing after C2HS but ventilation ( logical or (E)) is maintained. logical or (E) deficits occur during hypercapnic challenge reflecting reduced tidal volume (VT), but modest recovery occurs by 12 wks post-injury. IL PhrMN activity recovers in a time-dependent manner after C2HS, and neuroanatomical evidence suggests that this may involve both mono- and polysynaptic pathways. Accordingly, we used cross-correlation to examine IL and CL PhrMN synchrony after C2HS. Uninjured rats showed correlogram peaks consistent with synchronous activity and common synaptic input. Correlogram peaks were absent at 2 wks post-C2HS, but by 12 wks 50% of rats showed peaks occurring with a 1.1+/-0.19ms lag from zero on the abscissa. These data are consistent with prolonged conduction time to IL (vs. CL) PhrMNs and the possibility of polysynaptic inputs to IL PhrMNs after chronic C2HS.

Modest spontaneous recovery of ventilation following chronic high cervical hemisection in rats.

Following C2 spinal hemisection (C2HS) in adult rats, ipsilateral phrenic motoneuron (PhMN) recovery occurs through a time-dependent activation of latent, crossed-spinal collaterals (i.e., spontaneous crossed phrenic phenomenon; sCPP) from contralateral bulbospinal axons. Ventilation is maintained during quiet breathing after C2HS, but the ability to increase ventilation during a respiratory stimulation (e.g. hypercapnia) is impaired. We hypothesized that long-term expression of the sCPP would correspond to a progressive normalization in ventilatory patterns during respiratory challenge. Breathing was assessed via plethsymography in unanesthetized animals and phrenic motor output was measured in urethane-anesthetized, paralyzed and vagotomized rats. At 2-week post-C2HS, minute ventilation (VE) was maintained during baseline (room air) conditions as expected but was substantially blunted during hypercapnic challenge (68+/-3% of VE in uninjured, weight-matched rats). However, by 12 weeks the spinal-lesioned rats achieved a hypercapnic VE response that was 85+/-7% of control (p=0.017 vs. 2 wks). These rats also exhibited augmented breaths (AB's) or "sighs" more frequently (p<0.05) than controls; however, total AB volume was significantly less than control at 2- and 12-week post-injury (69+/-4% and 80+/-5%, p<0.05, respectively). We also noted that phrenic neurograms demonstrated a consistent delay in onset of the ipsilateral vs. contralateral inspiratory phrenic burst at 2-12-week post-injury. Finally, the ipsilateral phrenic response to respiratory challenge (hypoxia) was greater, though not normalized, at 4-12- vs. 2-week post-injury. We conclude that recovery of ventilation deficits occurs over 2-12-week post-C2HS; however, intrinsic neuroplasticity remains insufficient to concurrently restore a normal level of ipsilateral phrenic output.

Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis.

The complete genome sequence of Enterococcus faecalis V583, a vancomycin-resistant clinical isolate, revealed that more than a quarter of the genome consists of probable mobile or foreign DNA. One of the predicted mobile elements is a previously unknown vanB vancomycin-resistance conjugative transposon. Three plasmids were identified, including two pheromone-sensing conjugative plasmids, one encoding a previously undescribed pheromone inhibitor. The apparent propensity for the incorporation of mobile elements probably contributed to the rapid acquisition and dissemination of drug resistance in the enterococci.

Actinobacillus actinomycetemcomitans harbours type IV secretion system genes on a plasmid and in the chromosome.

Nine contiguous genes encoding a potential type IV secretion system have been identified in the chromosome of Actinobacillus actinomycetemcomitans strain VT747 and on a plasmid (pVT745) in strain VT745. Seven of these genes encode predicted proteins that share significant homology with type IV secretion proteins in Bordetella pertussis (ptl operon), Brucella melitensis biovar suis and Agrobacterium tumefaciens (virB operons), where they are involved in protein secretion, pathogen intracellular survival and multiplication, and DNA transport, respectively. Results of previous studies have demonstrated that pVT745 is a conjugative plasmid and that a secondary plasmid, pMMB67, can be mobilized from strain VT745. Given these results, it was hypothesized that (1) the type IV secretion genes on pVT745 are responsible for these two functions and (2) the type IV VT747 chromosomal genes also play a role in the transport of DNA. Wild-type and mutant strains of VT745 were evaluated for their conjugative abilities. Wild-type mating efficiency was 10(-6) transconjugants per donor, while the mutant strain yielded no transconjugants. Wild-type VT745 harbouring a co-resident plasmid, pMMB67, mobilized pMMB67 at a frequency of 10(-6), while VT747 was unable to mobilize this plasmid. These results support the hypothesis that the plasmid-encoded type IV secretion system on pVT745 is involved in DNA transport. However, the chromosomally encoded secretion system may not play a role in DNA transport in strain VT747. While the precise function of these chromosomal genes in strain VT747 has not been determined, Northern blot analyses demonstrated that these genes are expressed in both ACT: actinomycetemcomitans strains VT745 and VT747.

Effects of acclimation temperature on enzymatic capacities and mitochondrial membranes from the body wall of the earthworm Lumbricus terrestris.

Many ectotherms respond to low temperature by adjusting capacities of enzymes from energy metabolism, restructuring membrane phospholipids and modulating membrane fluidity. Although much is known about the temperature biology of earthworms, it is not known to what extent earthworms employ compensatory changes in enzymatic capacities and membrane physical properties after exposure to low temperature. We examined activities of enzymes from glycolysis and central oxidative pathways as well as fluidity and phospholipid fatty acid composition of mitochondrial membranes prepared from the body wall of the temperate oligochaete Lumbricus terrestris after a one month acclimation to 5 degrees and 15 degrees C. No compensation occurs in central pathways of oxidative metabolism since activities of cytochrome-c oxidase and citrate synthase, when measured at a common temperature, are similar for 5 degrees C and 15 degrees C-acclimated animals. In contrast, activity of pyruvate kinase is elevated 1.3-fold after acclimation to 5 degrees C. Mitochondrial membranes display inverse compensation with respect to temperature (membranes from 5 degrees C animals are more ordered than membranes from 15 degrees C animals). Our results, in combination with earlier reports, indicate that routine metabolism in L. terrestris may be maintained at reduced temperatures with little or no change in enzymatic capacities and inverse compensation of mitochondrial membranes.

Finding drug targets in microbial genomes.

In this era of genomic science, knowledge about biological function is integrated increasingly with DNA sequence data. One area that has been significantly impacted by this accumulation of information is the discovery of drugs to treat microbial infections. Genome sequencing and bioinformatics is driving the discovery and development of novel classes of broad-spectrum antimicrobial compounds, and could enable medical science to keep pace with the increasing resistance of bacteria, fungi and parasites to current antimicrobials. This review discusses the use of genomic information in the rapid identification of target genes for antimicrobial drug discovery.

Complete genome sequence of a virulent isolate of Streptococcus pneumoniae.

The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.

Efflux in bacteria: what do we really know about it?

Efflux is the process in which bacteria transport compounds outside the cell which are potentially toxic, such as drugs or chemicals or compounds. Efflux pumps can be identified not only by biochemical, microbiological, or molecular means but with the availability of microbial genomic sequences, by the application of bioinformatics analysis of DNA sequences for key conserved structure motifs. Efflux has been identified as a relevant contributor to bacterial resistance in the clinic and is now recognised as one of the most important causes of intrinsic antibiotic resistance in bacteria, especially in Pseudomonas aeruginosa. With the recognition of efflux as a major factor in bacterial resistance, several companies have invested in the identification and development of bacterial efflux pump inhibitors. Among those, Microcide, Pfizer, Paratek and several academic laboratories are in the process of exploring efflux pump inhibitors from synthetic, natural products and peptidomimetics. Inhibiting bacterial efflux with a non-antibiotic inhibitor would restore activity of an antibiotic subject to efflux (similar to the use of beta-lactamase inhibitors to combat beta-lactamase production by bacteria). The feasibility of such an approach has been experimentally demonstrated in vitro and in vivo for efflux reversal of levofloxacin.

Exclusion limits on the WIMP-nucleon cross section from the cryogenic dark matter search.

The Cryogenic Dark Matter Search (CDMS) employs Ge and Si detectors to search for weakly interacting massive particles (WIMPs) via their elastic-scattering interactions with nuclei while discriminating against interactions of background particles. CDMS data, accounting for the neutron background, give limits on the spin-independent WIMP-nucleon elastic-scattering cross section that exclude unexplored parameter space above 10 GeV/c2 WIMP mass and, at >75% C.L., the entire 3sigma allowed region for the WIMP signal reported by the DAMA experiment.