Potential impurities in drug substances: Compound-specific toxicology limits for 20 synthetic reagents and by-products, and a class-specific toxicology limit for alkyl bromides.

This paper provides compound-specific toxicology limits for 20 widely used synthetic reagents and common by-products that are potential impurities in drug substances. In addition, a 15 µg/day class-specific limit was developed for monofunctional alkyl bromides, aligning this with the class-specific limit previously defined for monofunctional alkyl chlorides. Both the compound- and class-specific toxicology limits assume a lifetime chronic exposure for the general population (including sensitive subpopulations) by all routes of exposure for pharmaceuticals. Inhalation-specific toxicology limits were also derived for acrolein, formaldehyde, and methyl bromide because of their localized toxicity via that route. Mode of action was an important consideration for a compound-specific toxicology limit. Acceptable intake (AI) calculations for certain mutagenic carcinogens assumed a linear dose-response for tumor induction, and permissible daily exposure (PDE) determination assumed a non-linear dose-response. Several compounds evaluated have been previously incorrectly assumed to be mutagenic, or to be mutagenic carcinogens, but the evidence reported here for such compounds indicates a lack of mutagenicity, and a non-mutagenic mode of action for tumor induction. For non-mutagens with insufficient data to develop a toxicology limit, the ICH Q3A qualification thresholds are recommended. The compound- and class-specific toxicology limits described here may be adjusted for an individual drug substance based on treatment duration, dosing schedule, severity of the disease and therapeutic indication.

Interpretation of scanning tunneling quasiparticle interference and impurity states in cuprates.

We apply a recently developed method combining first principles based Wannier functions with solutions to the Bogoliubov-de Gennes equations to the problem of interpreting STM data in cuprate superconductors. We show that the observed images of Zn on the surface of Bi_{2}Sr_{2}CaCu_{2}O_{8} can only be understood by accounting for the tails of the Cu Wannier functions, which include significant weight on apical O sites in neighboring unit cells. This calculation thus puts earlier crude "filter" theories on a microscopic foundation and solves a long-standing puzzle. We then study quasiparticle interference phenomena induced by out-of-plane weak potential scatterers, and show how patterns long observed in cuprates can be understood in terms of the interference of Wannier functions above the surface. Our results show excellent agreement with experiment and enable a better understanding of novel phenomena in the cuprates via STM imaging.

A Nanoscale-Localized Ion Damage Josephson Junction Using Focused Ion Beam and Ion Implanter.

High-T(c) Josephson junctions were fabricated by nanolithography using focused ion beam (FIB) milling and ion implantation. The junctions were formed in a YBa2Cu3O7-x, thin film in regions defined using a gold-film mask with 50-nm-wide (top) slits, engraved by FIB. The focused ion beam system parameters for dwell time and passes were set to remove gold up to a precise depth. 150 keV oxygen ions were implanted at a nominal dose of up to 5 x 10(13) ions/cm2 into YBa2Cu3O7-x microbridges through the nanoscale slits. The current-voltage curves of the ion implantation junctions exhibit resistive-shunted-junction-like behavior at 77 K. The junction had an approximately linear temperature dependence of critical current. Shapiro steps were observed under microwave irradiation. A 50-nm-wide slit and 0-20-nm-thick buffer layers were chosen in order to make Josephson junctions due to the V-shape of the FIB-milled trench.

Relevance of the Heisenberg-Kitaev model for the honeycomb lattice iridates A2IrO3.

Combining thermodynamic measurements with theoretical calculations we demonstrate that the iridates A2IrO3 (A=Na, Li) are magnetically ordered Mott insulators where the magnetism of the effective spin-orbital S=1/2 moments can be captured by a Heisenberg-Kitaev (HK) model with interactions beyond nearest-neighbor exchange. Experimentally, we observe an increase of the Curie-Weiss temperature from θ≈-125 K for Na2IrO3 to θ≈-33 K for Li2IrO3, while the ordering temperature remains roughly the same T(N)≈15 K. Using functional renormalization group calculations we show that this evolution of θ and T(N) as well as the low temperature zigzag magnetic order can be captured within this extended HK model. We estimate that Na2IrO3 is deep in a magnetically ordered regime, while Li2IrO3 appears to be close to a spin-liquid regime.

Experimental observation of the crystallization of a paired holon state.

An excitation at 201 meV is observed in the doped-hole ladder cuprate Sr14Cu24O41, using ultraviolet resonance Raman scattering with incident light at 3.7 eV polarized along the rungs. The excitation is of charge nature, with a temperature independent excitation energy, and can be understood via an intraladder pair-breaking process. The intensity tracks closely the order parameter of the charge density wave in the ladder CDW(L), but persists above its transition temperature T(CDW(L)), indicating a strong local pairing above the T(CDW(L)). The 201 meV excitation vanishes in La6Ca8Cu24O(41+δ), and La5Ca9Cu24O41 which are samples with no holes in the ladders. Our results suggest that the doped holes in the ladder are composite bosons consisting of paired holons that order below T(CDW).

Room-temperature ferromagnetism of Cu-doped ZnO films probed by soft X-ray magnetic circular dichroism.

We report direct evidence of room-temperature ferromagnetic ordering in O-deficient ZnO:Cu films by using soft x-ray magnetic circular dichroism and x-ray absorption. Our measurements have revealed unambiguously two distinct features of Cu atoms associated with (i) magnetically ordered Cu ions present only in the oxygen-deficient samples and (ii) magnetically disordered regular Cu2+ ions present in all the samples. We find that a sufficient amount of both oxygen vacancies (V(O)) and Cu impurities is essential to the observed ferromagnetism, and a non-negligible portion of Cu impurities is uninvolved in the magnetic order. Based on first-principles calculations, we propose a microscopic "indirect double-exchange" model, in which alignments of localized large moments of Cu in the vicinity of the V(O) are mediated by the large-sized vacancy orbitals.

Atherosclerotic cardiovascular disease screening and management protocols among adult HIV clinics in Asia.

OBJECTIVES: Integration of HIV and non-communicable disease services improves the quality and efficiency of care in low- and middle-income countries (LMICs). We aimed to describe current practices for the screening and management of atherosclerotic cardiovascular disease (ASCVD) among adult HIV clinics in Asia. METHODS: Sixteen LMIC sites included in the International Epidemiology Databases to Evaluate AIDS - Asia-Pacific network were surveyed. RESULTS: Sites were mostly (81%) based in urban public referral hospitals. Half had protocols to assess tobacco and alcohol use. Protocols for assessing physical inactivity and obesity were in place at 31% and 38% of sites, respectively. Most sites provided educational material on ASCVD risk factors (between 56% and 75% depending on risk factors). A total of 94% reported performing routine screening for hypertension, 100% for hyperlipidaemia and 88% for diabetes. Routine ASCVD risk assessment was reported by 94% of sites. Protocols for the management of hypertension, hyperlipidaemia, diabetes, high ASCVD risk and chronic ischaemic stroke were in place at 50%, 69%, 56%, 19% and 38% of sites, respectively. Blood pressure monitoring was free for patients at 69% of sites; however, most required patients to pay some or all the costs for other ASCVD-related procedures. Medications available in the clinic or within the same facility included angiotensin-converting enzyme inhibitors (81%), statins (94%) and sulphonylureas (94%). CONCLUSION: The consistent availability of clinical screening, diagnostic testing and procedures and the availability of ASCVD medications in the Asian LMIC clinics surveyed are strengths that should be leveraged to improve the implementation of cardiovascular care protocols.

High-Resolution X-ray Observations of the Orion Nebula.

Observations of the Trapezium region in the Orion Nebula obtained with the high-resolution x-ray imaging instrument on board the Einstein Observatory reveal at least 58 sources of x-ray emission. All but two of the sources can be identified with visible stars. The strongest x-ray source is the star Theta(1)C, which excites the emission nebula. Its x-ray luminosity is 6 x 10(32) ergs per second. The rest of the x-ray sources may be identified with stars of all spectral types. Strong x-ray emission is not observed from members of the infrared cluster embedded within the Orion molecular cloud.

Genotoxicity profiles of common alkyl halides and esters with alkylating activity.

Drug synthesis and/or formulation can generate genotoxic impurities. For instance, strong acid/alcohol interactions during the process of drug salt formation produce alkylating agents such as alkyl halides and alkyl esters of alkyl sulfonic acids. The genotoxicity of a few classic alkylating agents such as methyl and ethyl methanesulfonate have been previously well characterized, whereas the majority of compounds from this class have only been tested in the Salmonella reversion assay. Therefore, the goal of this study was to investigate clastogenicity and DEL recombination profiles of 22 halogenated alkanes and alkylesters of sulfuric and alkane-, aryl-sulfonic acids using a battery of cellular and molecular assays. The in-vitro micronucleus assay in CHO cells was used to measure clastogenicity and the deletion recombination (DEL) assay in S. cerevisiae provided a measure of DNA deletions. We also examined the compounds' reactivity towards 4-(p-nitrobenzyl)pyridine (NBP), a surrogate molecule for biological ring nitrogens. Methylating agents were most potent in all three assays and the alkyl chlorides evaluated in our study were negative in all three assays. Also, a strong correlation was found between the MN, DEL and NBP assays. In summary, this study contributes to a better understanding of the genotoxic properties of common alkyl halides and alkyl esters with alkylating activity and might provide guidance for managing risk of genotoxic process-related impurities of drug substances and products.

Chemoproteomics-enabled covalent ligand screen reveals a cysteine hotspot in reticulon 4 that impairs ER morphology and cancer pathogenicity.

Chemical genetics has arisen as a powerful approach for identifying novel anti-cancer agents. However, a major bottleneck of this approach is identifying the targets of lead compounds that arise from screens. Here, we coupled the synthesis and screening of fragment-based cysteine-reactive covalent ligands with activity-based protein profiling (ABPP) chemoproteomic approaches to identify compounds that impair colorectal cancer pathogenicity and map the druggable hotspots targeted by these hits. Through this coupled approach, we discovered a cysteine-reactive acrylamide DKM 3-30 that significantly impaired colorectal cancer cell pathogenicity through targeting C1101 on reticulon 4 (RTN4). While little is known about the role of RTN4 in colorectal cancer, this protein has been established as a critical mediator of endoplasmic reticulum tubular network formation. We show here that covalent modification of C1101 on RTN4 by DKM 3-30 or genetic knockdown of RTN4 impairs endoplasmic reticulum and nuclear envelope morphology as well as colorectal cancer pathogenicity. We thus put forth RTN4 as a potential novel colorectal cancer therapeutic target and reveal a unique druggable hotspot within RTN4 that can be targeted by covalent ligands to impair colorectal cancer pathogenicity. Our results underscore the utility of coupling the screening of fragment-based covalent ligands with isoTOP-ABPP platforms for mining the proteome for novel druggable nodes that can be targeted for cancer therapy.

The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein.

BACKGROUND: Colicin E7 (ColE7) is one of the bacterial toxins classified as a DNase-type E-group colicin. The cytotoxic activity of a colicin in a colicin-producing cell can be counteracted by binding of the colicin to a highly specific immunity protein. This biological event is a good model system for the investigation of protein recognition. RESULTS: The crystal structure of a one-to-one complex between the DNase domain of colicin E7 and its cognate immunity protein Im7 has been determined at 2.3 A resolution. Im7 in the complex is a varied four-helix bundle that is identical to the structure previously determined for uncomplexed Im7. The structure of the DNase domain of ColE7 displays a novel alpha/beta fold and contains a Zn2+ ion bound to three histidine residues and one water molecule in a distorted tetrahedron geometry. Im7 has a V-shaped structure, extending two arms to clamp the DNase domain of ColE7. One arm (alpha1(*)-loop12-alpha2(*); where * represents helices in Im7) is located in the region that displays the greatest sequence variation among members of the immunity proteins in the same subfamily. This arm mainly uses acidic sidechains to interact with the basic sidechains in the DNase domain of ColE7. The other arm (loop 23-alpha3(*)-loop 34) is more conserved and it interacts not only with the sidechain but also with the mainchain atoms of the DNase domain of ColE7. CONCLUSIONS: The protein interfaces between the DNase domain of ColE7 and Im7 are charge-complementary and charge interactions contribute significantly to the tight and specific binding between the two proteins. The more variable arm in Im7 dominates the binding specificity of the immunity protein to its cognate colicin. Biological and structural data suggest that the DNase active site for ColE7 is probably near the metal-binding site.

HLJ1 is an endogenous Src inhibitor suppressing cancer progression through dual mechanisms.

HLJ1 (DNAJB4), a DNAJ/Hsp40 chaperone, has emerged as a novel prognostic marker in lung cancers; however, the molecular contribution and functionality in neoplastic diseases remain to be established. This study demonstrated that HLJ1 inhibits epithelial-mesenchymal transition in vitro and reduces lung cancer metastasis in vivo. Using shRNA silencing and ectopic expression of HLJ1, we found that HLJ1 not only suppresses catalytic activity of Src but also downregulates the formation of oncogenic complexes associated with the EGFR, FAK and STAT3 signaling pathways. A screen of specimens from HLJ1-knockout mice and lung cancer patients validated that HLJ1 expression is inversely correlated with Src activity. Mechanistically, HLJ1 protein directly bound to catalytic and protein-binding domains of Src through its amino acid Y172 and the P301/P304 motif. Following Src-induced HLJ1 phosphorylation at Y172, HLJ1-Src interaction was elevated, resulting in Src inhibition and malignancy suppression. Interestingly, both Src-binding regions also occurred in other DNAJB family members and contributed to anti-invasive activities of DNAJB proteins. We conclude that HLJ1 is an endogenous Src inhibitor that can suppress cancer metastasis through complex interacting mechanisms. This HLJ1-Src complex might provide a promising molecular model for developing new anticancer strategies.

Changes in lectin binding by differentiating cutaneous keratinocytes from the newborn rat.

Surface glycoconjugates of cells from the basal layer of the skin of the newborn rat bind the isolectin I-B4 from Griffonia simplicifolia (GS I-B4) (alpha-D-galactosyl specificity). Surface glycoconjugates of the differentiated cells from the spinous and lower granular layers bind Ulex europeus agglutinin I (UEA) (alpha-L-fucosyl specificity). The change from GS I-B4 binding to UEA binding was studied in rat keratinocytes that were cultured as a monolayer in low-calcium medium until confluence, and then induced to stratify and terminally differentiate by raising the calcium concentration of the medium. The cells in the monolayer had basal cell morphology and exhibited surface binding of GS I-B4. However, at confluence, 30-40% of these cells also showed surface binding of UEA. There was an increase with time in the number of cells which bound both GS I-B4 and UEA. Raising the calcium concentration of the medium resulted in an increase in UEA binding. Cells of the upper layers of the stratifying cultures showed intense UEA binding but did not show any GS I-B4 binding. Double staining of frozen sections of newborn rat skin with fluorescein-conjugated GS I-B4 and rhodamine-conjugated UEA revealed that the surfaces of cells from the lower spinous layer bound both lectins. Thirty percent of the major glycoprotein fraction, that was isolated from the membranes of the epidermal cells of the newborn rat and was bound to an affinity column of UEA-Sepharose 4B, was also bound to an affinity column of GS I-B4-Sepharose 4B. These results indicate that surface glycoconjugates of rat keratinocytes differentiating in culture exhibit a change from GS I-B4 binding to UEA binding; the change in the cell surface glycoconjugates that results in the appearance of UEA binding, a feature of differentiated cells, occurs independently of stratification; and the change from GS I-B4 binding to UEA binding probably involves an "intermediate" glycoconjugate that binds both GS I-B4 and UEA and is found on the surface of cells from the lower spinous layer of the epidermis of the newborn rat.

Haplotype analysis of the myotonic dystrophy type 1 (DM1) locus in Taiwan: implications for low prevalence and founder mutations of Taiwanese myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disorder caused by a CTG trinucleotide expansion at the DM1 locus. In this study, we investigated the frequency distribution of various CTG repeats in normal alleles and haplotyped the normal and expanded DM1 locus in a group of Taiwanese people. In the 496 normal chromosomes examined, up to 18 alleles with different CTG lengths from 5 to 30 repeats were found and the frequency of (CTG)(>18) alleles was only 1.4% (7/496), predicting a low prevalence of DM1. In addition, there is no absolute association between (CTG)(5-19) alleles and Alu insertion/deletion polymorphism observed on normal chromosomes. All DM1 alleles examined, however, were found to be associated with the Alu insertion. Further detailed genetic analysis demonstrated that at least eight haplotypes, including a new haplotype (L), were present in the Taiwanese population and that all DM1 alleles were with the same haplotype (haplotype A) as that identified in Canadian and Japanese DM1 populations. These findings support the notion that the out-of-Africa DM1 alleles were originated by stepwise expansion from a pool of large-sized normal chromosomes with haplotype A.

Lanosterol 14alpha-demethylase (CYP51), NADPH-cytochrome P450 reductase and squalene synthase in spermatogenesis: late spermatids of the rat express proteins needed to synthesize follicular fluid meiosis activating sterol.

Lanosterol 14alpha-demethylase (CYP51) is a cytochrome P450 enzyme involved primarily in cholesterol biosynthesis. CYP51 in the presence of NADPH-cytochrome P450 reductase converts lanosterol to follicular fluid meiosis activating sterol (FF-MAS), an intermediate of cholesterol biosynthesis which accumulates in gonads and has an additional function as oocyte meiosis-activating substance. This work shows for the first time that cholesterogenic enzymes are highly expressed only in distinct stages of spermatogenesis. CYP51, NADPH-P450 reductase (the electron transferring enzyme needed for CYP51 activity) and squalene synthase (an enzyme preceding CYP51 in the pathway) proteins have been studied. CYP51 was detected in step 3-19 spermatids, with large amounts in the cytoplasm/residual bodies of step 19 spermatids, where P450 reductase was also observed. Squalene synthase was immunodetected in step 2-15 spermatids of the rat, indicating that squalene synthase and CYP51 proteins are not equally expressed in same stages of spermatogenesis. Discordant expression of cholesterogenic genes may be a more general mechanism leading to transient accumulation of pathway intermediates in spermatogenesis. This study provides the first evidence that step 19 spermatids and residual bodies of the rat testis have the capacity to produce MAS sterols in situ.

The reproductive toxicity of boric acid.

Previous studies on the reproductive toxicity of boric acid have indicated that male rodents suffer testicular atrophy after treatment. There were, however, no studies of the potential effects on female fertility or on the neonate. In addition, no study described the development of the testicular lesion, thought to be related to the mechanism of toxicity. A Reproductive Assessment by Continuous Breeding (RACB) study using mice exposed to boric acid at 1000, 4500, and 9000 ppm in the diet indicated that there are probably multiple sites of action, although male fertility appears very sensitive. Possible effects on female fertility cannot be separated from potential developmental toxicity and need additional investigation. Decrements in sperm motility were observed at all exposure levels, and testicular atrophy was confirmed in high- and middle-dose-group males. This was investigated further by timed serial-sacrifice studies using 9000 ppm in the diet of rats, which found that the first lesion seen in the testis was an inhibition of spermiation (release of mature spermatids). With continued dosing, this was followed by a disorganization of the normal ordered layering of the seminiferous epithelium, germ cell sloughing and death, and finally, atrophy. Subsequent studies using additional doses (2000, 3000, 4500, 6000, and 9000 ppm) found that it was possible to observe inhibited spermiation that did not progress to atrophy (4500 ppm and below) within the 9-week exposure period.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of the testicular toxicity of boric acid in rats: in vivo and in vitro studies.

High-dose boric acid (BA) exposure produces testicular lesions in adult rats characterized by inhibited spermiation (IS) that may progress to atrophy. In vivo and in vitro studies addressed possible mechanisms. In vivo, boron tissue disposition was examined, since no detailed data existed, and relevant boron concentrations for in vitro studies needed to be set. Since BA induces riboflavinuria and also affects calcium/phosphorus homeostasis, and testis zinc appears essential for normal testis function, we examined BA effects on flavin status and testis levels of phosphorus (P), calcium (Ca) and zinc (Zn). Data showed that the testicular toxicity and central nervous system (CNS) hormonal effect were not due to selective boron accumulation in testis or brain/hypothalamus, with testis boron concentrations at approximately 1 to 2 mM; that riboflavin deficiency is not involved, due to both the absence of overt signs of deficiency and effects on tissue flavin content during BA exposure; and that changes in testis P, Ca and Zn levels did not precede atrophy, and are therefore unlikely to be mechanistically relevant. In vitro studies addressed the hallmarks of the BA testicular toxicity: the mild hormone effect, the initial IS, and atrophy. No effect of BA on the steroidogenic function of isolated Leydig cells was observed, supporting the contention of a CNS-mediated rather than a direct hormone effect. Since increased testicular cyclic adenosine monophosphate (cAMP) produces IS, and a role for the serine proteases plasminogen activators (PAs) in spermiation has been proposed, we examined in vitro BA effects on both Sertoli cell cAMP accumulation and PA activity, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Prospective evaluation of extraocular motility following double-plate molteno implantation.

OBJECTIVE: To determine the incidence and type of extraocular motility disturbance after double-plate Molteno implantation. METHODS: In a prospective clinical series, we evaluated preoperative and postoperative ocular motility at 3 and 6 months in 24 eyes of 24 patients undergoing double-plate Molteno implantation. Visual acuity, motility testing, and subjective and objective diplopia were evaluated at each examination. RESULTS: Within the first 6 months postoperatively, new or worse strabismus developed in 11 (46%) of the 24 study patients. Three of the 11 patients had a generalized restriction of the superior rectus and the superior oblique muscles, all of which persisted 6 months after surgery. Four patients had clinical features consistent with an acquired Brown syndrome, and 6 months after surgery, 3 of the 4 patients had a residual deviation, although the deviation in 1 patient resolved. A superior oblique palsy developed in 3 patients, and a lateral rectus palsy developed in 1 patient. All 4 of the muscle palsies resolved or were resolving during the follow-up period, which ranged from 6 to 12 months. CONCLUSIONS: Extraocular motility disturbances are not rare after double-plate Molteno surgery. Muscle palsies, acquired Brown syndromes, and generalized restrictions occurred in similar proportions. CLINICAL RELEVANCE: Patients should be counseled before Molteno surgery concerning the risk of strabismus and diplopia.

Castration induces a marked reduction in the incidence of thyroid cancers.

Although thyroid disease in humans predominates in females, the prognosis of thyroid cancer in males is much less favorable. To further explore the role of male sex hormones, we utilized the experimental model of radiation-induced follicular cancer in the rat, studying the effects of castration on tumor development. Microscopic evidence of tumor formation was correlated with the biochemical parameters serum thyroxine (T4), thyrotropin (TSH), and thyrocalcitonin (TC) at 24 months. The incidence of follicular carcinomas in intact males ws 58%, compared to 28% in comparable females. Castration of males caused a reduction in the incidence to 22%. Mean circulating levels of TSH were markedly elevated in all three irradiated groups. Mean T4 levels were reduced significantly only in irradiated castrated animals. TC levels were reduced in all irradiated groups. We conclude that although elevated levels of TSH probably play a significant role in the development of radioactive iodine-triggered follicular thyroid neoplasms in the rat, male sex hormone may also play an important role beyond their known effect on basal and thyrotropin-releasing hormone-stimulated TSH.

Administration of dehydroepiandrosterone to burned mice preserves normal immunologic competence.

Burned individuals display a reduced ability to elicit cellular and humoral immune responses and a depression in the vitro production of certain T-cell lymphokines. Treatment of burned mice with 100 micrograms of dehydroepiandrosterone within 1 hour after injury resulted in preserving a completely normal capacity to produce T-cell-derived lymphokines and to generate cellular immune responses. In addition, dehydroepiandrosterone-treated thermally injured mice demonstrated an above-normal ability to resist an induced infection with the intracellular pathogen, Listeria monocytogenes. Dehydroepiandrosterone-treated animals also did not exhibit the sustained plasma levels of interleukin 6 that normally accompany thermal injury and infection. Because of its antiglucocorticoid effects and positive immunoregulatory influences, we believe dehydroepiandrosterone to be a beneficial form of therapy for thermally injured individuals.