Structural basis of acquired resistance to selpercatinib and pralsetinib mediated by non-gatekeeper RET mutations.

BACKGROUND: Selpercatinib (LOXO-292) and pralsetinib (BLU-667) are highly potent RET-selective protein tyrosine kinase inhibitors (TKIs) for treating advanced RET-altered thyroid cancers and non-small-cell lung cancer (NSCLC). It is critical to analyze RET mutants resistant to these drugs and unravel the molecular basis to improve patient outcomes. PATIENTS AND METHODS: Cell-free DNAs (cfDNAs) were analyzed in a RET-mutant medullary thyroid cancer (MTC) patient and a CCDC6-RET fusion NSCLC patient who had dramatic response to selpercatinib and later developed resistance. Selpercatinib-resistant RET mutants were identified and cross-profiled with pralsetinib in cell cultures. Crystal structures of RET-selpercatinib and RET-pralsetinib complexes were determined based on high-resolution diffraction data collected with synchrotron radiation. RESULTS: RETG810C/S mutations at the solvent front and RETY806C/N mutation at the hinge region were found in cfDNAs of an MTC patient with RETM918T/V804M/L, who initially responded to selpercatinib and developed resistance. RETG810C mutant was detected in cfDNAs of a CCDC6-RET-fusion NSCLC patient who developed acquired resistance to selpercatinib. Five RET kinase domain mutations at three non-gatekeeper residues were identified from 39 selpercatinib-resistant cell lines. All five selpercatinib-resistant RET mutants were cross-resistant to pralsetinib. X-ray crystal structures of the RET-selpercatinib and RET-pralsetinib complexes reveal that, unlike other TKIs, these two RET TKIs anchor one end in the front cleft and wrap around the gate wall to access the back cleft. CONCLUSIONS: RET mutations at the solvent front and the hinge are resistant to both drugs. Selpercatinib and pralsetinib use an unconventional mode to bind RET that avoids the interference from gatekeeper mutations but is vulnerable to non-gatekeeper mutations.

Spatial and molecular mapping of Pfkelch13 gene polymorphism in Africa in the era of emerging Plasmodium falciparum resistance to artemisinin: a systematic review.

The spread of Plasmodium falciparum isolates carrying mutations in the kelch13 (Pfkelch13) gene associated with artemisinin resistance (PfART-R) in southeast Asia threatens malaria control and elimination efforts. Emergence of PfART-R in Africa would result in a major public health problem. In this systematic review, we investigate the frequency and spatial distribution of Pfkelch13 mutants in Africa, including mutants linked to PfART-R in southeast Asia. Seven databases were searched (PubMed, Embase, Scopus, African Journal Online, African Index Medicus, Bioline, and Web of Science) for relevant articles about polymorphisms of the Pfkelch13 gene in Africa before January, 2019. Following PRISMA guidelines, 53 studies that sequenced the Pfkelch13 gene of 23 100 sample isolates in 41 sub-Saharan African countries were included. The Pfkelch13 sequence was highly polymorphic (292 alleles, including 255 in the Pfkelch13-propeller domain) but with mutations occurring at very low relative frequencies. Non-synonymous mutations were found in only 626 isolates (2·7%) from west, central, and east Africa. According to WHO, nine different mutations linked to PfART-R in southeast Asia (Phe446Ile, Cys469Tyr, Met476Ile, Arg515Lys, Ser522Cys, Pro553Leu, Val568Gly, Pro574Leu, and Ala675Val) were detected, mainly in east Africa. Several other Pfkelch13 mutations, such as those structurally similar to southeast Asia PfART-R mutations, were also identified, but their relevance for drug resistance is still unknown. This systematic review shows that Africa, thought to not have established PfART-R, reported resistance-related mutants in the past 5 years. Surveillance using PfART-R molecular markers can provide valuable decision-making information to sustain the effectiveness of artemisinin in Africa.

Variation in antagonism of the interferon response to rotavirus NSP1 results in differential infectivity in mouse embryonic fibroblasts.

Rotavirus NSP1 has been shown to function as an E3 ubiquitin ligase that mediates proteasome-dependent degradation of interferon (IFN) regulatory factors (IRF), including IRF3, -5, and -7, and suppresses the cellular type I IFN response. However, the effect of rotavirus NSP1 on viral replication is not well defined. Prior studies used genetic analysis of selected reassortants to link NSP1 with host range restriction in the mouse, suggesting that homologous and heterologous rotaviruses might use their different abilities to antagonize the IFN response as the basis of their host tropisms. Using a mouse embryonic fibroblast (MEF) model, we demonstrate that heterologous bovine (UK and NCDV) and porcine (OSU) rotaviruses fail to effectively degrade cellular IRF3, resulting in IRF3 activation and beta IFN (IFN-beta) secretion. As a consequence of this failure, replication of these viruses is severely restricted in IFN-competent wild-type, but not in IFN-deficient (IFN-alpha/beta/gamma receptor- or STAT1-deficient) MEFs. On the other hand, homologous murine rotaviruses (ETD or EHP) or the heterologous simian rotavirus (rhesus rotavirus [RRV]) efficiently degrade cellular IRF3, diminish IRF3 activation and IFN-beta secretion and are not replication restricted in wild-type MEFs. Genetic reassortant analysis between UK and RRV maps the distinctive phenotypes of IFN antagonism and growth restriction in wild-type MEFs to NSP1. Therefore, there is a direct relationship between the replication efficiencies of different rotavirus strains in MEFs and strain-related variations in NSP1-mediated antagonism of the type I IFN response.

Role of interferon in homologous and heterologous rotavirus infection in the intestines and extraintestinal organs of suckling mice.

Recent studies demonstrated that viremia and extraintestinal rotavirus infection are common in acutely infected humans and animals, while systemic diseases appear to be rare. Intraperitoneal infection of newborn mice with rhesus rotavirus (RRV) results in biliary atresia (BA), and this condition is influenced by the host interferon response. We studied orally inoculated 5-day-old suckling mice that were deficient in interferon (IFN) signaling to evaluate the role of interferon on the outcome of local and systemic infection after enteric inoculation. We found that systemic replication of RRV, but not murine rotavirus strain EC, was greatly enhanced in IFN-alpha/beta and IFN-gamma receptor double-knockout (KO) or STAT1 KO mice but not in mice deficient in B- or T-cell immunity. The enhanced replication of RRV was associated with a lethal hepatitis, pancreatitis, and BA, while no systemic disease was observed in strain EC-infected interferon-deficient mice. In IFN-alpha/beta receptor KO mice the extraintestinal infection and systemic disease were only moderately increased, while RRV infection was not augmented and systemic disease was not present in IFN-gamma receptor KO mice. The increase of systemic infection in IFN-deficient mice was also observed during simian strain SA11 infection but not following bovine NCDV, porcine OSU, or murine strain EW infection. Our data indicate that the requirements for the interferon system to inhibit intestinal and extraintestinal viral replication in suckling mice vary among different heterologous and homologous rotavirus strains, and this variation is associated with lethal systemic disease.

Protection against lipopolysaccharide-induced endothelial dysfunction in resistance and conduit vasculature of iNOS knockout mice.

Endothelial dysfunction is a characteristic of, and may be pathogenic in, inflammatory cardiovascular diseases, including sepsis. The mechanism underlying inflammation-induced endothelial dysfunction may be related to the expression and activity of inducible nitric oxide synthase (iNOS). This possibility was investigated in isolated resistance (mesenteric) and conduit (aorta) arteries taken from lipopolysaccharide (LPS)-treated (12.5 mg/kg i.v.) or saline-treated iNOS knockout (KO) and wild-type (WT) mice. LPS pretreatment (for 15 h, but not 4 h) profoundly suppressed responses to acetylcholine (ACh) and significantly reduced sensitivity to the NO donor spermine-NONOate (SPER-NO) in aorta and mesenteric arteries of WT mice. This effect was temporally associated with iNOS protein expression in both conduit and resistance arteries and with a 10-fold increase in plasma NOx levels. In contrast, no elevation of plasma NOx was observed in LPS-treated iNOS KO animals, and arteries dissected from these animals did not express iNOS or display hyporeactivity to ACh or SPER-NO. The mechanism underlying this phenomenon may be suppression of eNOS expression, as observed in arteries of WT animals, that was absent in arteries of iNOS KO animals. These results clearly demonstrate that iNOS induction plays an integral role in mediation of the endothelial dysfunction associated with sepsis in both resistance and conduit arteries.

Conditioning with rabbit versus horse ATG dramatically alters clinical outcomes in identical twins with severe aplastic anemia transplanted with the same allogeneic donor.

Severe aplastic anemia (SAA) is a rare disorder leading to bone marrow failure, which if left untreated, is invariably fatal. Conventional therapies with immunosuppressive therapy or allogeneic hematopoietic stem cell transplantation (HSCT) are highly effective. HSCT can offer a greater outcome in younger patients who have an available HLA match-related donor. Recent studies showing the addition of antithymocyte globulin (ATG) to the conditioning regimen improves engraftment and reduces the risk of graft-versus-host disease (GVHD).There are currently two ATG preparations in the USA, equine (or horse) and rabbit ATG. These agents are pharmacologically distinct, having significant differences in their pharmacokinetics and in vivo immunosuppressive effects [N Engl J Med 365(5):430-438, 2011]. Here, we report a case of two monozygotic twins with constitutional SAA that evolved to myelodysplastic syndrome (MDS) who both underwent allogeneic peripheral blood stem cell transplantation (PBSC) from the same single HLA antigen mismatched sibling donor with the only difference in the transplant regimen being the type of ATG used in the preparative regimen; one twin received horse ATG and the other received rabbit ATG during conditioning. This report emphasizes that dramatic differences in donor T cell chimerism and clinical outcomes including GVHD can occur as a consequence of the type of ATG that is utilized in the transplant conditioning regimen. These differences highlight that these agents should not be considered interchangeable drugs when used in this setting.

Food contains the bioactive peptide, cyclo(His-Pro).

Cyclo(His-Pro) (CHP) is a cyclic dipeptide with numerous biological activities. As small di- and tripeptides may be absorbed intact when ingested orally, we were interested in examining several common foods for the presence of cyclo(His-Pro)-like immunoreactivity (CHP-LI). In all foods tested, CHP-LI was found at levels 5-1500 times those previously found in human plasma. This CHP-LI was identical to authentic CHP by immunoidentity and chromatographic behavior. We conclude that 1) CHP-LI is readily detectable in several common food sources; 2) this CHP-LI is indistinguishable from authentic CHP; and 3) it is likely the CHP-LI in foods is absorbed in quantities sufficient to cause elevations of CHP-LI in plasma to biologically significant levels.

Attenuation of vasoconstriction by endogenous nitric oxide in rat caudal artery.

1. The effects of NG-nitro-L-arginine (L-NNA), NG-nitro-L-arginine methyl ester (L-NAME), haemoglobin and methylene blue have been examined on vascular reactivity in the rat isolated caudal artery. The effects of L-NNA and sodium nitroprusside were also investigated on the stimulation-induced (S-I) efflux of noradrenaline in the rat caudal artery. 2. L-NNA (10 microM) and L-NAME (10 microM) significantly attenuated the vasodilator responses to acetylcholine (1 nM-1 microM), but had no effect on vasodilator responses to papaverine (1-100 microM). 3. Vasoconstrictor responses to sympathetic nerve stimulation (3 Hz, 10 s), noradrenaline (0.01-1 microM), methoxamine (1-10 microM), 5-hydroxytryptamine (0.01-0.3 microM), phenylephrine (0.1-10 microM), endothelin-1 (10 nM) and KCl (40 mM) were significantly enhanced by 10 microM L-NNA. L-NAME (10 microM) caused a significant enhancement of vasoconstrictor responses to noradrenaline and sympathetic nerve stimulation in endothelium-intact, but not in endothelium-denuded tissues. 4. Haemoglobin and methylene blue (both 10 microM) enhanced the vasoconstrictor responses to sympathetic nerve stimulation and noradrenaline. The enhancements were absent in endothelium-denuded arterial segments. 5. In endothelium-denuded arterial segments precontracted with phenylephrine, the vasodilator responses to the nitric oxide donor, sodium nitroprusside (0.1-300 nM) were decreased by increasing the level of precontraction. 6. L-NNA (10 microM) had no effect on the S-I efflux of radioactivity from arteries in which transmitter stores had been labelled with [3H]-noradrenaline. 7. These results suggest that endothelial nitric oxide attenuates vasoconstrictor responses in the rat caudal artery through activation of soluble guanylate cyclase to decrease smooth muscle contractility. Therefore, the findings provide evidence that nitric oxide acts as a functional antagonist to oppose vasoconstriction.

Deficient nitric oxide responsible for reduced nerve blood flow in diabetic rats: effects of L-NAME, L-arginine, sodium nitroprusside and evening primrose oil.

1. This study examined the potential role of impaired nitric oxide production and response in the development of endoneurial ischaemia in experimental diabetes. Rats were anaesthetized (Na pentobarbitone 45 mg kg-1, diazepam 2 mg kg-1) for measurement of sciatic nerve laser Doppler flux and systemic arterial pressure. Drugs were administered into the sciatic endoneurium via a microinjector attached to a glass micropipette. 2. In two separate studies comparing diabetic rats (streptozotocin-induced; 8-10 wk duration) with controls, nerve Doppler flux in diabetic rats (Study 1, 116.6 +/- 40.4 and Study 2, 90.1 +/- 34.7 (s.d.) in arbitrary units) was about half that measured in controls (219.6 +/- 52.4 and 212.8 +/- 95.5 respectively; P < 0.005 for both). There were no significant differences between the two in systemic arterial pressure. 3. Inhibition of nitric oxide production by microinjection of 1 nmol L-NAME into the endoneurium halved flux in controls (to 126.3 +/- 41.3 in Study 1 and 102.1 +/- 38.9 in Study 2; both P < 0.001), with no significant effect in diabetic rats, indicating markedly diminished tonic nitric oxide production in the latter. D-NAME was without effect on nerve Doppler flux. 4. L-Arginine (100 nmol), injected after L-NAME, markedly increased flux in controls (by 65.8% (P < 0.03) and 97.8% (P < 0.01) in the two studies) and by proportionally similar amounts in diabetic rats [75.8% (P < 0.001) and 60.2% (P < 0.02)]. The nitro-donor, sodium nitroprusside (SNP; 10 nmol) had similar effects to L-arginine in both groups (increases of 66.0% in controls and 77.5% in diabetics; both P < 0.002). 5. A second diabetic group, treated with evening primrose oil performed exactly like control rats in respect of responses to L-NAME, L-arginine and SNP. 6. These findings implicate deficient nitric oxide in nerve ischaemia of diabetes and suggest correction thereof as a mechanism of action of evening primrose oil.

Could dietary proteins serve as cyclo(His-Pro) precursors?

Cyclic dipeptides or diketopiperazines are readily generated during in vitro hydrolysis of proteins and polypeptides. This led us to examine whether cyclo(His-Pro) (CHP), a diketopiperazine containing histidine and proline, could be formed in vivo from dietary proteins. The data presented here show that at least in rat, neither urinary nor plasma concentration of CHP is elevated by consumption of a diet rich in proteins. Several dietary supplements derived from casein and/or soy protein hydrolysates, however, contain high levels of CHP-LI. Oral intake of one such supplement led to a sharp increase in the plasma level of CHP-LI.

Effects of adrenergic stimulation on sciatic nerve blood flow in diabetic and control rats.

Sciatic endoneurial blood flow is reduced in experimental diabetes. This study examined the possible involvement of noradrenergic mechanisms in this impairment. In anaesthetised rats (pentobarbitone sodium 50 mg/kg, diazepam 2 mg/kg), sciatic nerve laser Doppler flux and vascular resistance in diabetic rats (5-6 weeks) were lower (approximately 50%) and higher (approximately 42%), respectively, than that in age-matched control rats, indicating nerve ischaemia in the diabetic tissues. Tyramine (1 nmol), noradrenaline (0.001-1 nmol) and phenylephrine (0.01-10 nmol) produced significant increases of nerve vascular resistance in control rats. The responses to tyramine (1 nmol) were completely blocked by desipramine (10 nmol) and those to phenylephrine (10 nmol) were reversed by phentolamine (1 nmol). In streptozotocin-diabetic rats, responses to phenylephrine or noradrenaline were enhanced compared to control rats, but the enhancement failed to reach statistical significance. The findings demonstrate that adrenergic stimulation affects sciatic nerve endoneurial blood flow.

Endothelial nitric oxide attenuates vasoconstrictor responses to nerve stimulation and noradrenaline in the rat tail artery.

The effects of the nitric oxide synthesis inhibitor, NG-nitro-L-arginine (NOLA), have been examined in perfused segments of rat tail artery. NOLA (1 and 10 microM) significantly enhanced the vasoconstrictor responses to perivascular nerve stimulation (5 Hz, 10 s) and noradrenaline (10 ng). The enhancing effects of NOLA were prevented by L-arginine, but not by D-arginine, and were absent in endothelium-denuded artery segments. The results suggest that nitric oxide derived from endothelial cells attenuates vasoconstrictor responses to both nerve stimulation and noradrenaline.

Preautopsy magnetic resonance imaging: initial experience.

To our knowledge, there are no prospective data in the literature investigating the role of magnetic resonance imaging (MRI) in detecting abnormalities in cadavers to determine the feasibility of this concept. We prospectively studied six cadavers (three stillborn infants, one infant, and two adults) with a 0.15 T resistive magnet. The images obtained allowed detection of abnormalities in multiple organs. Although autopsy was superior to MRI in detecting very small abnormalities, MRI was equal to autopsy in detecting gross cranial, pulmonary, abdominal, and vascular pathology in this small series. In addition, MRI was superior to autopsy in detecting air and fluid in potential body spaces. Preautopsy MRI may be an alternate method in restricted or denied autopsies and may provide an additional MRI research and educational tool.

The breast-feeding dilemma and its impact on HIV-infected women and their children.

The rate of HIV transmission via breast-feeding ranges from 14% to 26%, depending on the timing of maternal infection. In settings where infant mortality rates from infectious diseases and malnutrition are low and relatively safe alternatives to breast-feeding are available, HIV-infected mothers should be advised not to breast-feed. Where breast-feeding by HIV-infected mothers and bottle-feeding both present serious risks of mortality, changing the conditions in which families live so that safe feeding alternatives become available must be a top priority. At the same time, these mothers need information about the relative risks and benefits of breast-feeding, early weaning, wet-nursing, and formula feeding. This article reviews the available research data and discusses critical gaps in current knowledge.

Lymphocyte cytotoxicity testing in patients and families with retinoblastoma.

This study tested lymphocyte cytotoxicity from patients with retinoblastoma. Lymphocytes were tested against two permanent cell lines, WERI and Y-79. Normal volunteers served as controls. The lymphocyte reactivity of both patients with retinoblastoma and their mothers showed significant elevation compared with the controls. This strongly suggests that transplacental sensitization had occurred in utero and that the tumor was present prior to birth. Two children suspected of having retinoblastoma was tested and found to have elevated cytotoxicity titers but were later shown to have nematode inflammatory reaction. Both of their mothers' lymphocytes were not reactive to the tumor cell line. These results indicate that retinoblastoma can be present in utero with sensitization of the mother to the tumor. This test, with further refinement, may be applicable as a screening test in the differential diagnosis of retinoblastoma.

The aging spine: clinical instability.

Clinical instability of the spine is an intensely controversial subject, and its diagnosis, especially in the aging, is difficult. Yet success in its management rests on accurate diagnosis. Because both clinical presentation and radiographic manifestations are nonspecific, the diagnosis of clinical instability lies in understanding the biomechanics involved, in recognizing the relevant radiographic manifestations, and, most importantly, in correlating those observations with the patient's clinical history and physical examination. Stabilization is the treatment of choice for clinical instability. Strengthening of the dynamic stabilizers, especially early in the course of the disease, may prevent or alleviate the incapacitating symptoms of instability, and further research into this area should be undertaken. Static stabilization by bracing has not proved effective, and spinal fusion carries a high risk of complication. Fusion should be reserved for patients whose diagnosis is clear and whose symptoms are recalcitrant to conservative management. Further understanding of clinical spinal instability in the aging will require more precise definition of terms and better standardization of criteria for its diagnosis, management, and research.

Lymphocyte toxicity in retinoblastoma.

Cytotoxicity can be used in the determination of patients with retinoblastoma. With further refinement, this type of test may become useful in aiding the physician in the detection of retinoblastoma.

The regeneration of peripheral noradrenergic nerves after chemical sympathectomy in diabetic rats: effects of nerve growth factor.

The study investigated the role of nerve growth factor (NGF) in the regeneration of noradrenergic nerves of the right atria from control and 8-week diabetic rats, after lesion caused by a single injection of 6-hydroxydopamine (6-OHDA, 100 mg/kg ip). This treatment caused a profound depletion of tissue noradrenaline (NA) of the right atria from both control and diabetic groups, followed by a progressive repletion that was not complete at 49 days. Immunoreactivity for the NGF receptors trkA and p75(NTR) was decreased and increased, respectively, between days 3 and 28 in right atria from diabetic rats and returned to pretreatment levels at day 49. Receptor levels were not significantly altered in controls. In contrast to tissue NA, at day 14 functional responses to electrical nerve stimulation of the right atria had completely returned to the pretreatment state in diabetic rats and were very close to normal in nondiabetic rats. NGF treatment (1 mg/kg, three times/week, for 2 weeks) increased tissue NA only in control rats; the pattern was similar after 6-OHDA. These findings are consistent with the hypothesis that NGF normally plays a role in the regulation of autonomic sympathetic nerves in the adult rat atrium and that mature and uninjured sympathetic neurons remain responsive to NGF. In injured noradrenergic neurons, NGF promotes regeneration in nondiabetic rats. The ability of NGF to promote regeneration of noradrenergic nerves is lost in diabetes and this may relate to the loss of trkA receptor on prejunctional nerve terminals after denervation.