Reproductive strategy of Delta Smelt Hypomesus transpacificus and impacts of drought on reproductive performance.

Understanding reproductive biology and performance of fish is essential to formulate effective conservation and management programs. Here, we studied reproductive strategies of female Delta Smelt Hypomesus transpacificus, an endangered fish species in the State of California, the United States, focusing on (1) better understanding their distribution pattern during the winter and spring spawning season at very fine scale to predict their possible spawning grounds and (2) assessing impacts of a recent, severe drought on their reproductive performance. We formulated our hypotheses as follows; (1) female Delta Smelt migrate to particular locations for spawning so that mature females can be frequently found in those locations throughout the spawning season and (2) reproductive performance of individual female fish declined during the drought. To test the first hypotheses, we analyzed relationships between water quality parameters and maturity/distribution pattern of Delta Smelt. Salinity better explained the distribution pattern of Delta Smelt at subadult and adult stages compared with water temperature or turbidity. Although there are some freshwater locations where mature Delta Smelt can frequently be found during the spawning season, Delta Smelt at the final maturation stage (Stage 5: hydration) and post spawners appeared to be widespread in the area where salinity was below 1.0 during the spawning season. Therefore, Delta Smelt could theoretically spawn in any freshwater locations, with more specific spawning requirements in the wild (e.g., substrate type and depth) still unknown. Delta Smelt, which experienced dry and critically dry conditions (the 2013 and 2014 year-classes), showed smaller oocytes, and lower clutch size and gonadosomatic index compared with the fish caught in a wet year (2011 year-class) at the late vitellogenic stage (Stage 4 Late), suggesting reproductive performance was negatively affected by environmental conditions during the drought.

Gangrenous cholecystitis: innovative laparoscopic techniques to facilitate subtotal fenestrating cholecystectomy when a critical view of safety cannot be achieved.

BACKGROUND: Gangrenous cholecystitis is associated with a higher conversion rate of conversion from laparoscopic to open than acute non-gangrenous cholecystitis. New strategies and techniques are needed to decrease conversion rates and improve outcomes. METHODS: In this article, we provide a richly detailed, illustrated description of a modified fundus-first technique that we have developed over the last 15 years and now use routinely with rare conversions. We also compared outcomes of laparoscopic (LC) and open (OC) approaches for pathologically confirmed gangrenous cholecystitis in 146 patients during 1995-2005, the first 10 years during which these two approaches were performed contemporaneously at our institution on comparable patients. RESULTS: Among the 142 patients that met the inclusion criteria, laparoscopic procedures were started in 112 (79%) of these patients, with successful completion in 72 resulting in an overall conversion rate of 36%. During the last 5 years, however, in cases where the described laparoscopic technique was used, no patient has required conversion. The laparoscopic LC group had shorter average ICU stay (p < 0.05) and overall length of stay (2 vs 6 days, p < 0.001). Intraoperative cholangiography was completed in 37 of 72 LC patients (52%) versus 6 of 30 OC (20%). In five of the LC patients, a filling defect was seen on the cholangiogram and laparoscopic transcystic common bile duct stones, thereby avoiding a second anesthetic and endoscopic procedure. CONCLUSIONS: In the setting of severe inflammation, a number of procedural modifications can be incorporated to allow the surgeon to approach dissection of the gangrenous gallbladder using a flexible operative plan designed to optimize safe completion of this challenging procedure, with the expected improvement in surgical outcomes.

Characterizing and Overriding the Structural Mechanism of the Quizartinib-Resistant FLT3 "Gatekeeper" F691L Mutation with PLX3397.

UNLABELLED: Tyrosine kinase domain mutations are a common cause of acquired clinical resistance to tyrosine kinase inhibitors (TKI) used to treat cancer, including the FLT3 inhibitor quizartinib. Mutation of kinase "gatekeeper" residues, which control access to an allosteric pocket adjacent to the ATP-binding site, has been frequently implicated in TKI resistance. The molecular underpinnings of gatekeeper mutation-mediated resistance are incompletely understood. We report the first cocrystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved Asp-Phe-Gly motif in the activation loop. This reliance makes quizartinib critically vulnerable to gatekeeper and activation loop substitutions while minimizing the impact of mutations elsewhere. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less vitally on specific interactions with the gatekeeper position. SIGNIFICANCE: We report the first cocrystal structure of FLT3 with a kinase inhibitor, elucidating the structural mechanism of resistance due to the gatekeeper F691L mutation. PLX3397 is a novel FLT3 inhibitor with in vitro activity against this mutation but is vulnerable to kinase domain mutations in the FLT3 activation loop.

Crenolanib is a selective type I pan-FLT3 inhibitor.

Tyrosine kinase inhibitors (TKIs) represent transformative therapies for several malignancies. Two critical features necessary for maximizing TKI tolerability and response duration are kinase selectivity and invulnerability to resistance-conferring kinase domain (KD) mutations in the intended target. No prior TKI has demonstrated both of these properties. Aiming to maximize selectivity, medicinal chemists have largely sought to create TKIs that bind to an inactive (type II) kinase conformation. Here we demonstrate that the investigational type I TKI crenolanib is a potent inhibitor of Fms tyrosine kinase-3 (FLT3) internal tandem duplication, a validated therapeutic target in human acute myeloid leukemia (AML), as well as all secondary KD mutants previously shown to confer resistance to the first highly active FLT3 TKI quizartinib. Moreover, crenolanib is highly selective for FLT3 relative to the closely related protein tyrosine kinase KIT, demonstrating that simultaneous FLT3/KIT inhibition, a prominent feature of other clinically active FLT3 TKIs, is not required for AML cell cytotoxicity in vitro and may contribute to undesirable toxicity in patients. A saturation mutagenesis screen of FLT3-internal tandem duplication failed to recover any resistant colonies in the presence of a crenolanib concentration well below what has been safely achieved in humans, suggesting that crenolanib has the potential to suppress KD mutation-mediated clinical resistance. Crenolanib represents the first TKI to exhibit both kinase selectivity and invulnerability to resistance-conferring KD mutations, which is unexpected of a type I inhibitor. Crenolanib has significant promise for achieving deep and durable responses in FLT3-mutant AML, and may have a profound impact upon future medicinal chemistry efforts in oncology.

Activity of ponatinib against clinically-relevant AC220-resistant kinase domain mutants of FLT3-ITD.

Secondary point mutations in the Fms-like tyrosine kinase 3 (FLT3) tyrosine kinase domain (KD) are common causes of acquired clinical resistance to the FLT3 inhibitors AC220 (quizartinib) and sorafenib. Ponatinib (AP24534) is a multikinase inhibitor with in vitro and clinical activity in tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia, irrespective of BCR-ABL KD mutation. Ponatinib has demonstrated early clinical efficacy in chemotherapy-resistant acute myeloid leukemia (AML) patients with internal tandem duplication (ITD) mutations in FLT3. We assessed the in vitro activity of ponatinib against clinically relevant FLT3-ITD mutant isoforms that confer resistance to AC220 or sorafenib. Substitution of the FLT3 "gatekeeper" phenylalanine with leucine (F691L) conferred mild resistance to ponatinib, but substitutions at the FLT3 activation loop (AL) residue D835 conferred a high degree of resistance. Saturation mutagenesis of FLT3-ITD exclusively identified FLT3 AL mutations at positions D835, D839, and Y842. The switch control inhibitor DCC-2036 was similarly inactive against FLT3 AL mutations. On the basis of its in vitro activity against FLT3 TKI-resistant F691 substitutions, further clinical evaluation of ponatinib in TKI-naïve and select TKI-resistant FLT3-ITD+ AML patients is warranted. Alternative strategies will be required for patients with TKI-resistant FLT3-ITD D835 mutations.

Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia.

Effective targeted cancer therapeutic development depends upon distinguishing disease-associated 'driver' mutations, which have causative roles in malignancy pathogenesis, from 'passenger' mutations, which are dispensable for cancer initiation and maintenance. Translational studies of clinically active targeted therapeutics can definitively discriminate driver from passenger lesions and provide valuable insights into human cancer biology. Activating internal tandem duplication (ITD) mutations in FLT3 (FLT3-ITD) are detected in approximately 20% of acute myeloid leukaemia (AML) patients and are associated with a poor prognosis. Abundant scientific and clinical evidence, including the lack of convincing clinical activity of early FLT3 inhibitors, suggests that FLT3-ITD probably represents a passenger lesion. Here we report point mutations at three residues within the kinase domain of FLT3-ITD that confer substantial in vitro resistance to AC220 (quizartinib), an active investigational inhibitor of FLT3, KIT, PDGFRA, PDGFRB and RET; evolution of AC220-resistant substitutions at two of these amino acid positions was observed in eight of eight FLT3-ITD-positive AML patients with acquired resistance to AC220. Our findings demonstrate that FLT3-ITD can represent a driver lesion and valid therapeutic target in human AML. AC220-resistant FLT3 kinase domain mutants represent high-value targets for future FLT3 inhibitor development efforts.

Increased CD62e(+) endothelial microparticle levels predict poor outcome in pulmonary hypertension patients.

BACKGROUND: Endothelial and leukocytes-derived microparticles (EMPs and LMPs, respectively) are increased in patients with pulmonary hypertension (PH). We hypothesized that the levels of circulating EMPs and LMPs could predict outcome in these patients. METHODS: Patients undergoing right heart catheterization for untreated pre-capillary PH were eligible for the study. Baseline hemodynamics and biologic and clinical parameters were measured at the time of enrollment. Measurements of CD62e(+), CD144(+) and CD31(+)/CD41(-) EMPs and CD45(+) LMPs were performed using flow cytometry in venous platelet-free plasma samples. After inclusion, patients were treated at the discretion of the physician and prospectively followed for 12 months. The primary end-point was the combined occurrence of death and re-admission for right heart failure (RHF) or worsening of RHF symptoms. RESULTS: Seven of 21 patients (mean age 54.1 +/- 3.5 years, 62% female) experienced the primary end-point during the study period. These patients had higher baseline levels of CD62e(+) EMPs, LMPs and hsCRP (high sensitivity C-reactive protein) compared to patients without events (p < 0.05), whereas no difference was observed for other microparticles and functional and hemodynamics parameters. Receiver operating curve analysis showed that baseline CD62e(+) EMPs levels of >353 events/microl predicted clinical complications. Kaplan-Meier analysis revealed that patients with baseline CD62e(+) EMPs above this cut-off value had a significantly worse prognosis compared with those subjects who had levels below this cut-off (p = 0.02, log-rank statistics). CONCLUSIONS: Elevated levels of circulating CD62e(+) EMPs but not LMPs in PH patients prior to treatment are associated with adverse clinical events. Assessment of CD62e(+) EMPs levels may represent a new tool for stratification of PH patients.

Mobilization of hematopoietic stem cells into the peripheral blood.

Hematopoietic stem cells can be mobilized out of the bone marrow into the blood for the reconstitution of hematopoiesis following high-dose therapy. Methods to improve mobilization efficiency and yields are rapidly emerging. Traditional methods include chemotherapy with or without myeloid growth factors. Plerixafor, a novel agent that disrupts the CXCR4-CXCL12 bond, the primary hematopoietic stem cell anchor in the bone marrow, has recently been US FDA-approved for mobilizing hematopoietic stem cells in patients with non-Hodgkin lymphoma and multiple myeloma. Plerixafor and myeloid growth factors as single agents appear safe to use in family or volunteer hematopoietic stem cells donors. Plerixafor mobilizes leukemic stem cells and is not approved for use in patients with acute leukemia. Patients failing to mobilize adequate hematopoietic stem cells with myeloid growth factors can often be successfully mobilized with chemotherapy plus myeloid growth factors or with plerixafor and granulocyte colony-stimulating factor.