Application of MPBT Assay for Multiplex Determination of Infectious Titers and for Selection of the Optimal Formulation for the Trivalent Novel Oral Poliovirus Vaccine.

Recently, a multiplex PCR-based titration (MPBT) assay was developed for simultaneous determination of infectious titers of all three Sabin strains of the oral poliovirus vaccine (OPV) to replace the conventional CCID50 assay, which is both time-consuming and laborious. The MPBT assay was shown to be reproducible, robust and sensitive. The conventional and MPBT assays showed similar results and sensitivity. The MPBT assay can be completed in two to three days, instead of ten days for the conventional assay. To prevent attenuated vaccine strains of poliovirus from reversion to virulence, a novel, genetically stable OPV (nOPV) was developed by modifying the genomes of conventional Sabin strains used in OPV. In this work, we evaluated the MPBT assay as a rapid screening tool to support trivalent nOPV (tnOPV) formulation development by simultaneous titration of the three nOPV strains to confirm stability as needed, for the selection of the lead tnOPV formulation candidate. We first assessed the ability of the MPBT assay to discriminate a 0.5 log10 titer difference by titrating the two tnOPV samples (undiluted and threefold-diluted) on the same plate. Once the assay was shown to be discriminating, we then tested different formulations of tnOPV drug products (DPs) that were subjected to different exposure times at 37 °C (untreated group and treated groups: 2 and 7 days at 37 °C), and to three freeze and thaw (FT) cycles. Final confirmation of the down selected formulation candidates was achieved by performing the conventional CCID50 assay, comparing the stability of untreated and treated groups and FT stability testing on the top three candidates. The results showed that the MPBT assay generates similar titers as the conventional assay. By testing two trivalent samples in the same plate, the assay can differentiate a 0.5 log10 difference between the titers of the tested nOPV samples. Also, the assay was able to detect the gradual degradation of nOPV viruses with different formulation compositions and under different time/temperature conditions and freeze/thaw cycles. We found that there were three tnOPV formulations which met the stability criteria of less than 0.5 log10 loss after 2 days' exposure to 37 ℃ and after three FT cycles, maintaining the potency of all three serotypes in these formulations. The ability of the MPBT assay to titrate two tnOPV lots (six viruses) in the same plate makes it cheaper and gives it a higher throughput for rapid screening. The assay detected the gradual degradation of the tnOPV and was successful in the selection of optimal formulations for the tnOPV. The results demonstrated that the MPBT method can be used as a stability indicating assay to assess the thermal stability of the nOPV. It can be used for rapid virus titer determination during the vaccine manufacturing process, and in clinical trials. The MPBT assay can be automated and applied for other viruses, including those with no cytopathic effect.

Development of a quantitative ELISA for SARS-CoV-2 vaccine candidate, NDV-HXP-S, with CpG 1018® adjuvant.

NDV-HXP-S is a Newcastle disease virus (NDV) vectored vaccine candidate which expresses the S-antigen of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This vaccine candidate is under evaluation in human clinical studies with and without cytosine phosphate guanine (CpG) 1018® adjuvant. Existing potency methods for NDV-HXP-S do not allow for quantification of the S-antigen when the adjuvant is present. To support evaluation of NDV-HXP-S with CpG 1018® adjuvant, an inhibition enzyme-linked immunosorbent assay (ELISA) was developed to allow for quantification and stability assessments of the vaccine. A pilot 6-month stability study was conducted on NDV-HXP-S vaccine with and without CpG 1018® adjuvant under refrigerated conditions (2°C to 8°C) and accelerated stability testing conditions (40°C). The vaccine was mixed with and without CpG 1018® adjuvant in saline and maintained S-antigen content at 2°C to 8°C for the entire 6-month period. Additionally, a pilot controlled temperature chain (CTC) stability study was conducted at the completion of the 6-month study and demonstrated the possibility for this vaccine candidate to attain CTC stability labeling.

Initial development and testing of an exhaled microRNA detection strategy for lung cancer case-control discrimination.

For detecting field carcinogenesis non-invasively, early technical development and case-control testing of exhaled breath condensate microRNAs was performed. In design, human lung tissue microRNA-seq discovery was reconciled with TCGA and published tumor-discriminant microRNAs, yielding a panel of 24 upregulated microRNAs. The airway origin of exhaled microRNAs was topographically "fingerprinted", using paired EBC, upper and lower airway donor sample sets. A clinic-based case-control study (166 NSCLC cases, 185 controls) was interrogated with the microRNA panel by qualitative RT-PCR. Data were analyzed by logistic regression (LR), and by random-forest (RF) models. Feasibility testing of exhaled microRNA detection, including optimized whole EBC extraction, and RT and qualitative PCR method evaluation, was performed. For sensitivity in this low template setting, intercalating dye-based URT-PCR was superior to fluorescent probe-based PCR (TaqMan). In application, adjusted logistic regression models identified exhaled miR-21, 33b, 212 as overall case-control discriminant. RF analysis of combined clinical + microRNA models showed modest added discrimination capacity (1.1-2.5%) beyond clinical models alone: all subjects 1.1% (p = 8.7e-04)); former smokers 2.5% (p = 3.6e-05); early stage 1.2% (p = 9.0e-03), yielding combined ROC AUC ranging from 0.74 to 0.83. We conclude that exhaled microRNAs are qualitatively measureable, reflect in part lower airway signatures; and when further refined/quantitated, can potentially help to improve lung cancer risk assessment.

Preclinical Safety and Pharmacokinetics of Heat Stable Oxytocin in Sublingual Fast-Dissolving Tablet Formulation.

The work reported here focuses on an evaluation of a novel heat stable formulation of a uterotonic peptide drug oxytocin involving stability testing under elevated temperatures and toxicokinetic response generated by sublingual (SL) administration in rabbits. The formulation was thermotolerant, maintaining the potency of oxytocin in the form of a fast-dissolving tablet at the end of 2-year storage at 30 °C/65% relative humidity with less than 5% loss in oxytocin content based on analytical high performance liquid chromatography (HPLC). The toxicokinetic results in rabbits showed that the fast-dissolving tablet was safe without any reactogenicity or toxicity associated with SL administration or the excipients present in the formulation. The SL route elicited rapid absorption of oxytocin in plasma within 5 min of administration although lower than intramuscular (IM) administration. IM resulted in area under the curve (AUC) values approximately 5 times higher than SL oxytocin. However, due to the limitations encountered during SL administration in an anesthetized rabbit model, the relevance of heat stable oxytocin formulation that has the flexibility to be adapted in different formats may warrant a human clinical study to determine whether therapeutically relevant plasma levels for treating postpartum hemorrhage can be generated via alternate non-injectable routes of administration.

Nutrient drip irrigation for refractory hydrocarbon removal and microbial community shift in a historically petroleum-contaminated soil.

An adequate amount of nutrients is required to enable biodegradation of refractory hydrocarbons in petroleum-contaminated soil. In this study, a microcosm experiment was conducted using a drip fertigation method for petroleum-contaminated soil remediation. Nitrogen and phosphorus were homogeneously and periodically sprayed into a historically contaminated soil using a modified horticultural drip irrigation device. Various petroleum hydrocarbon fraction contents were then determined by gravimetry and gas chromatography (GC), and changes in the soil microbial community were analyzed by high throughput sequencing. After 90 days of remediation, the removal efficiencies of total petroleum hydrocarbon (TPH), saturates, aromatics, C7-C30 n-alkanes, and 16 PAHs were respectively enhanced by 21.5%, 25.5%, 12.4%, 10.4%, and 19.6% compared with the use of a single nutrient amendment application. The high throughput sequencing result showed that obvious changes had occurred in the soil microbial community compositions during drip fertigation; however, fungi were more sensitive to drip fertigation than bacteria. The resulting predominant bacterial and fungal genera were Dietzia, Nocardioides, Mycobacterium, Sphaerobacter, Leifsonia, and Aspergillus, Scolecobasidium, and Fusarium, respectively. Remediating polluted soils by regular fertigation ensures the automatic addition of even amounts of nutrients, which achieves high refractory hydrocarbon removal efficiencies. It is expected that this method can be applied in the in-situ remediation of petroleum-contaminated soil on a large scale.

Discovery of arylamide-5-anilinoquinazoline-8-nitro derivatives as VEGFR-2 kinase inhibitors: Synthesis, in vitro biological evaluation and molecular docking.

Herein, we embarked on a structural optimization campaign aiming at the discovery of novel anticancer agents with our previously reported XL-6f as a lead compound. A library of 23 compounds has been synthesized based on the highly conserved active site of VEGFR-2. Several title compounds exhibited selective inhibitory activities against VEGFR-2, which also displayed selective anti-proliferation potency against HepG2 cell. All synthesized compounds were evaluated for anti-angiogenesis capability. Compound 7o showed the most potent anti-angiogenesis ability, the efficient cytotoxic activities (in vitro against HUVEC and HepG2 cell lines with IC50 values of 0.58 and 0.23 µM, respectively). The molecular docking analysis revealed 7o is a Type-II inhibitor of VEGFR-2 kinase. In general, these results indicated these arylamide-5-anilinoquinazoline-8-nitro derivatives are promising inhibitors of VEGFR-2 for the potential treatment of anti-angiogenesis.

Design, synthesis and in vitro evaluation of 6-amide-2-aryl benzoxazole/benzimidazole derivatives against tumor cells by inhibiting VEGFR-2 kinase.

Herein, we have carried out a structural optimization campaign to discover the novel anti-tumor agents with our previously screened YQY-26 as the hit compound. A library of thirty-seven 6-amide-2-aryl benzoxazole/benzimidazole derivatives has been designed and synthesized based on the highly conserved active site of VEGFR-2. Several title compounds exhibited selective inhibitory activities against VEGFR-2 than EGFR kinases, which also displayed selective anti-proliferation potency against the HUVEC and HepG2 than the A549 and MDA-MB-231 cancer cell lines. The newly synthesized compounds were evaluated for anti-angiogenesis capability by chick chorioallantoic membrane (CAM) assay. Among them, compounds 9d showed the most potent anti-angiogenesis ability (79% inhibition at 10 nM/eggs), the efficient cytotoxic activities (in vitro against the HUVEC and HepG2 cell lines with IC50 values of 1.47 and 2.57 µM, respectively), and excellent VEGFR-2 kinase inhibition (IC50 = 0.051 µM). The molecular docking analysis revealed that compound 9d is a Type II inhibitor of VEGFR-2 kinase. These results indicated that the 6-amide-2-arylbenzoxazole and 6-amide-2-aryl benzimidazole derivatives are promising inhibitors of VEGFR-2 kinase for the potential treatment of anti-angiogenesis.

Discovery of 6-Arylurea-2-arylbenzoxazole and 6-Arylurea-2-arylbenzimidazole Derivatives as Angiogenesis Inhibitors: Design, Synthesis and in†vitro Biological Evaluation.

We embarked on a structural optimization campaign aimed at the discovery of novel anti-angiogenesis agents with previously reported imidazole kinase inhibitors as a lead compound. A library of 29 compounds was synthesized. Several title compounds exhibited selective inhibitory activities against vascular endothelial growth factor receptor 2 (VEGFR-2) over epidermal growth factor receptor (EGFR) kinase; these compounds also displayed selective and potent antiproliferative activity against three cancer cell lines. The newly synthesized compounds were evaluated for anti-angiogenesis activity by chick chorioallantoic membrane (CAM) assay. Among them, 1-(2-(2-chlorophenyl)benzo[d]oxazol-5-yl)-3-(4-(trifluoromethoxy)phenyl)urea (compound 5 n) showed the most potent anti-angiogenesis capacity, efficient cytotoxic activities (in vitro against human umbilical vein endothelial cells (HUVEC), H1975, A549, and HeLa cell lines, with respective IC50 values of 8.46, 1.40, 7.61, and 0.28 µm), and an acceptable level of VEGFR-2 kinase inhibition (IC50 =0.25 µm). Molecular docking analysis revealed 5 n to be a type II inhibitor of VEGFR-2 kinase. In general, these results indicate that these 6-arylurea-2-arylbenzoxazole/benzimidazole derivatives are promising inhibitors of VEGFR-2 kinase for potential development into anti-angiogenesis drugs.

[Microbial Community Structure Shift During Bioremediation of Petroleum Contaminated Soil Using High Throughput Sequencing].

The shift in microbial community structure during the bioremediation of oil-polluted soil was analyzed by high-throughput sequencing. The results demonstrated obvious changes in the soil microbial community structure and diversity during bioremediation. The species richness and evenness of the microbial community decreased substantially due to the bioaugmentation treatment. Proteobacteria became the predominant phylum, with a relative increase in abundance from 37.44% to 87.44%. Pseudomonas was the most dominant genus, which increased in abundance from 2.99% to 76.37%. In the biostimulation treated soil, the relative abundance of Proteobacteria decreased from 37.44% to 10.90%, while the phylum Firmicutes increased from 9.16% to 35.32%. At the genus level, the relative abundances of Exiguobacterium and Promicromonospora decreased from 8.49% and 18.96% to 2.19% and 14.97%, respectively. Nocardioides and Bacillus became the dominant genera and increased from 5.56% and 0.29% to 28.95% and 22.70%, respectively. The results indicated that bioaugmentation substantially influenced the soil microbial diversity and community structure. Additionally, the biostimulation treatment maintained the balance in the soil microbial community structure. The stabilization of bacteria community structure is beneficial to petroleum biodegradation in the soil.

[Isolation, Identification and Degradation Characteristics of a 17ß-estradiol Degrading Strain Fusarium sp. KY123915].

A 17ß-estradiol (E2) degrading strain (designated as Wu-SP1) was isolated from the activated sludge collected from a wastewater treatment plant (WWTP) in Xi'an. The strain was identified as Fusarium sp. according to 18S rDNA sequence and phylogenetic analysis. The optimal pH and temperature for E2 degradation were 6 and 30℃, respectively. Under these conditions, the E2 biodegradation rate of 2 mg·L-1 E2 amounted to 92.5% within 48 h by this strain. The kinetics of E2 degradation by the strain KY123915 were in good accord with the first-order equation, with the concentration ranged from 10 to 500 mg·L-1. UV spectrum analysis showed the strength of maximum absorption of metabolites became weak compared to E2, indicating that E2 may be degraded via estrone (E1) by Fusarium sp. KY123915.

Myelodysplastic Syndrome and Sweet's Syndrome Are Associated with a Mutation in Isocitrate Dehydrogenase 1.

BACKGROUND: Sweet's syndrome (SS) is a febrile neutrophilic dermatosis that has been clinically linked to hematological malignancies, particularly myelodysplastic syndrome (MDS), in a number of case series. Many epigenetic changes underlying MDS have been identified, such as a mutation in the isocitrate dehydrogenase 1 (IDH1) gene, which causes DNA hypermethylation and alteration of a number of genes that lead to leukemogenesis. However, the pathogenesis of malignancy-associated SS is unknown. CASE REPORT: We present two patients who were diagnosed with SS and concomitant IDH1-mutated MDS. Immunohistochemical staining of their skin lesions showed neutrophils diffusely positive for the IDH1 mutation. CONCLUSION: These cases demonstrate that IDH1 mutation may be implicated in the pathogenesis of malignancy-associated SS. Future investigation to elucidate this pathway is warranted. Establishing this molecular link can provide an earlier identification of patients with SS who are also at increased risk for developing MDS.

Heat-stable sublingual oxytocin tablets as a potential needle-free approach for preventing postpartum hemorrhage in low-resource settings.

Postpartum hemorrhage is a major cause of mortality and morbidity related to childbirth in developing countries. The recommended treatment includes administration of oxytocin; however, oxytocin is a heat-labile protein, and it must be given as an intramuscular injection by skilled health care providers. To address these challenges, we developed a freeze-dried oxytocin fast-dissolving tablet (FDT) for sublingual (SL) needle-free administration. Using methods developed previously, we produced a robust FDT that maintained oxytocin stability at 40 °C, 75% relative humidity for 12 months. This formulation contains 9% sucrose, 1.5% (hydroxypropyl)methyl cellulose, 9% mannitol, 4% dextran, 1% carbomer, 1% sodium taurocholate, and 100 IU oxytocin. An in vitro study showed a > 30% reduction in tissue transepithelial electrical resistance after treatment with the oxytocin FDT, implying an increase in the permeability of the mucosal tissue to oxytocin. Anesthetized Yucatan miniature swine were administered a SL FDT, and blood was periodically collected for a pharmacokinetic study. Higher plasma concentrations were seen when larger SL doses were given. The maximum concentrations for SL and intramuscular doses in anesthetized pigs were 207 and 612 pg/mL, respectively. Whether the levels attained will be sufficient to elicit beneficial results in humans is yet to be determined. This study demonstrates the feasibility of our approach for developing a heat-stable oxytocin tablet that can be administered successfully via the SL route.

Developing a Flexible Pediatric Dosage Form for Antiretroviral Therapy: A Fast-Dissolving Tablet.

Current presentations of the anti-HIV drugs lopinavir and ritonavir make appropriate dosing for children difficult. We conducted a feasibility study to develop a formulation for these drugs with child-safe excipients in a flexible dosage form for children across the pediatric age spectrum. The freeze-drying in blister approach was used to produce fast-dissolving tablets (FDTs), as these can be dispersed in fluids for easy administration, even to infants, and appropriate portions of the dispersion can be given for different ages/weights. We combined various ratios of polymers, surfactants, and bulking agents to incorporate the 2 highly hydrophobic drugs while maintaining drug stability, rapid disintegration, and good handling properties. The final FDT was robust and disintegrated in 0.5 mL of fluid in 10 s with up to 4 tablets dissolving in 2 mL to achieve varying doses accommodated in a common teaspoon. Drug recovery after dissolution in small volumes of liquid or fluid foods was 90%-105%. The final candidate FDT was stable at 40°C, 75% relative humidity for up to 3 months. FDTs are a promising flexible dosage form for antiretroviral treatment for pediatric patients, especially in low-resource settings.

Recombinant human G6PD for quality control and quality assurance of novel point-of-care diagnostics for G6PD deficiency.

BACKGROUND: A large gap for the support of point-of-care testing is the availability of reagents to support quality control (QC) of diagnostic assays along the supply chain from the manufacturer to the end user. While reagents and systems exist to support QC of laboratory screening tests for glucose-6-phosphate dehydrogenase (G6PD) deficiency, they are not configured appropriately to support point-of-care testing. The feasibility of using lyophilized recombinant human G6PD as a QC reagent in novel point-of-care tests for G6PD deficiency is demonstrated. METHODS: Human recombinant G6PD (r-G6PD) was expressed in Escherichia coli and purified. Aliquots were stored at -80°C. Prior to lyophilization, aliquots were thawed, and three concentrations of r-G6PD (representing normal, intermediate, and deficient clinical G6PD levels) were prepared and mixed with a protective formulation, which protects the enzyme activity against degradation from denaturation during the lyophilization process. Following lyophilization, individual single-use tubes of lyophilized r-G6PD were placed in individual packs with desiccants and stored at five temperatures for one year. An enzyme assay for G6PD activity was used to ascertain the stability of r-G6PD activity while stored at different temperatures. RESULTS: Lyophilized r-G6PD is stable and can be used as a control indicator. Results presented here show that G6PD activity is stable for at least 365 days when stored at -80°C, 4°C, 30°C, and 45°C. When stored at 55°C, enzyme activity was found to be stable only through day 28. CONCLUSIONS: Lyophilized r-G6PD enzyme is stable and can be used as a control for point-of-care tests for G6PD deficiency.

Preparing an Adjuvanted Thermoresponsive Gel Formulation for Sublingual Vaccination.

Thermoresponsive gels have unique physicochemical properties that may enable more effective mucosal delivery of active compounds. The thermoresponsive gel (TRG) formulation developed by our group for sublingual delivery maintains fluid-like liquid properties at 2 °C-8 °C and forms a gel at the physiological temperature (~37 °C) within a few seconds. Here, we describe the preparation of a thermoresponsive gel vaccine formulation. Our preclinical studies with various antigens suggest that the mucoadhesive, adjuvanted TRG formulation enabled increased contact of the vaccine antigen with the mucosa, resulting in increased mucosal response(s) with a potential for antigen dose reduction.

Stability of live attenuated rotavirus vaccine with selected preservatives and primary containers.

Rotavirus infection, which can be prevented by vaccination, is responsible for a high burden of acute gastroenteritis disease in children, especially in low-income countries. An appropriate formulation, packaging, and delivery device for oral rotavirus vaccine has the potential to reduce the manufacturing cost of the vaccine and the logistical impact associated with introduction of a new vaccine, simplify the vaccination procedure, and ensure that the vaccine is safely and accurately delivered to children. Single-dose prefilled presentations can be easy to use; however, they are typically more expensive, can be a bottleneck during production, and occupy a greater volume per dose vis-à-vis supply chain storage and medical waste disposal, which is a challenge in low-resource settings. Multi-dose presentations used thus far have other issues, including increased wastage of vaccine and the need for separate delivery devices. In this study, the goals were to evaluate both the technical feasibility of using preservatives to develop a liquid multi-dose formulation and the primary packaging alternatives for orally delivered, liquid rotavirus vaccines. The feasibility evaluation included evaluation of commonly used preservatives for compatibility with rotavirus vaccines and stability testing of rotavirus vaccine in various primary containers, including Lameplast's plastic tubes, BD's oral dispenser version of Uniject™ (Uniject DP), rommelag's blow-fill-seal containers, and MEDInstill's multi-dose vial and pouch. These presentations were compared to a standard glass vial. The results showed that none of the preservatives tested were compatible with a live attenuated rotavirus vaccine because they had a detrimental effect on the viability of the virus. In the presence of preservatives, vaccine virus titers declined to undetectable levels within 1 month. The vaccine formulation without preservatives maintained a stability profile over 12 months in all primary containers that was similar to its profile in standard glass vials. This study demonstrates that there are multiple options for the primary container for rotavirus vaccines intended for oral delivery. Selection of an optimal primary container should take into consideration additional factors, including stability as well as cold chain volume, usability, cost, and manufacturing feasibility.

Genome Wide Methylome Alterations in Lung Cancer.

Aberrant cytosine 5-methylation underlies many deregulated elements of cancer. Among paired non-small cell lung cancers (NSCLC), we sought to profile DNA 5-methyl-cytosine features which may underlie genome-wide deregulation. In one of the more dense interrogations of the methylome, we sampled 1.2 million CpG sites from twenty-four NSCLC tumor (T)-non-tumor (NT) pairs using a methylation-sensitive restriction enzyme- based HELP-microarray assay. We found 225,350 differentially methylated (DM) sites in adenocarcinomas versus adjacent non-tumor tissue that vary in frequency across genomic compartment, particularly notable in gene bodies (GB; p<2.2E-16). Further, when DM was coupled to differential transcriptome (DE) in the same samples, 37,056 differential loci in adenocarcinoma emerged. Approximately 90% of the DM-DE relationships were non-canonical; for example, promoter DM associated with DE in the same direction. Of the canonical changes noted, promoter (PR) DM loci with reciprocal changes in expression in adenocarcinomas included HBEGF, AGER, PTPRM, DPT, CST1, MELK; DM GB loci with concordant changes in expression included FOXM1, FERMT1, SLC7A5, and FAP genes. IPA analyses showed adenocarcinoma-specific promoter DMxDE overlay identified familiar lung cancer nodes [tP53, Akt] as well as less familiar nodes [HBEGF, NQO1, GRK5, VWF, HPGD, CDH5, CTNNAL1, PTPN13, DACH1, SMAD6, LAMA3, AR]. The unique findings from this study include the discovery of numerous candidate The unique findings from this study include the discovery of numerous candidate methylation sites in both PR and GB regions not previously identified in NSCLC, and many non-canonical relationships to gene expression. These DNA methylation features could potentially be developed as risk or diagnostic biomarkers, or as candidate targets for newer methylation locus-targeted preventive or therapeutic agents.

RICTOR Amplification Defines a Novel Subset of Patients with Lung Cancer Who May Benefit from Treatment with mTORC1/2 Inhibitors.

UNLABELLED: We identified amplification of RICTOR, a key component of the mTOR complex 2 (mTORC2), as the sole actionable genomic alteration in an 18-year-old never-smoker with lung adenocarcinoma. Amplification of RICTOR occurs in 13% of lung cancers (1,016 cases) in The Cancer Genome Atlas and at a similar frequency in an independent cohort of 1,070 patients identified by genomic profiling. In the latter series, 11% of cases harbored RICTOR amplification as the only relevant genomic alteration. Its oncogenic roles were suggested by decreased lung cancer cell growth both in vitro and in vivo with RICTOR ablation, and the transforming capacity of RICTOR in a Ba/F3-cell system. The mTORC1/2 inhibitors were significantly more active against RICTOR-amplified lung cancer cells as compared with other agents targeting the PI3K-AKT-mTOR pathway. Moreover, an association between RICTOR amplification and sensitivities to mTORC1/2 inhibitors was observed. The index patient has been treated with mTORC1/2 inhibitors that led to tumor stabilization for more than 18 months. SIGNIFICANCE: RICTOR amplification may define a novel and unique molecular subset of patients with lung cancer who may benefit from treatment with mTORC1/2 inhibitors.

Transforming growth factor ß1 and extracellular matrix protease expression in the uterosacral ligaments of patients with and without pelvic organ prolapse.

OBJECTIVES: This study was undertaken to evaluate the expression of transforming growth factor ß1 (TGF-ß1) and matrix metalloproteinase 9 (MMP-9), key regulators of the extracellular matrix composition, in the uterosacral ligaments (USLs) of women with pelvic organ prolapse (POP) compared with controls. METHODS: Under an institutional review board approval, USL samples were obtained from women undergoing vaginal hysterectomy for stage 2 or greater POP (cases, n = 21) and from women without POP undergoing vaginal hysterectomy for benign indications (controls, n = 19). Hematoxylin and eosin and trichrome staining were performed on the USL sections, and the distribution of smooth muscle and fibrous tissue were quantified. Immunohistochemical staining was performed using anti-TGF-ß1 and anti-MMP-9 antibodies. The expressions of TGF-ß1 and MMP-9 were evaluated by the pathologist, who was blinded to all clinical data. RESULTS: Transforming growth factor ß1 expression positively correlated with MMP-9 expression (R = 0.4, P = 0.01). The expressions of TGF-ß1 and MMP-9 were similar in subjects with POP versus controls. There was a significant increase in fibrous tissue (P = 0.008) and a corresponding decrease in smooth muscle (P = 0.03), associated with increasing age. The TGF-ß1 expression, but not MMP-9 expression, also significantly increased with age (P = 0.02). DISCUSSION: Although our study uncovered age-related alterations in USL composition and TGF-ß1 expression, there was no difference in the expression of TGF-ß1 or MMP-9 in the subjects with POP versus controls.

Serum and mucosal antibody responses to inactivated polio vaccine after sublingual immunization using a thermoresponsive gel delivery system.

Administering vaccines directly to mucosal surfaces can induce both serum and mucosal immune responses. Mucosal responses may prevent establishment of initial infection at the port of entry and subsequent dissemination to other sites. The sublingual route is attractive for mucosal vaccination, but both a safe, potent adjuvant and a novel formulation are needed to achieve an adequate immune response. We report the use of a thermoresponsive gel (TRG) combined with a double mutant of a bacterial heat-labile toxin (dmLT) for sublingual immunization with a trivalent inactivated poliovirus vaccine (IPV) in mice. This TRG delivery system, which changes from aqueous solution to viscous gel upon contact with the mucosa at body temperature, helps to retain the formulation at the site of delivery and has functional adjuvant activity from the inclusion of dmLT. IPV was administered to mice either sublingually in the TRG delivery system or intramuscularly in phosphate-buffered saline. We measured poliovirus type-specific serum neutralizing antibodies as well as polio-specific serum Ig and IgA antibodies in serum, saliva, and fecal samples using enzyme-linked immunosorbent assays. Mice receiving sublingual vaccination via the TRG delivery system produced both mucosal and serum antibodies, including IgA. Intramuscularly immunized animals produced only serum neutralizing and binding Ig but no detectable IgA. This study provides proof of concept for sublingual immunization using the TRG delivery system, comprising a thermoresponsive gel and dmLT adjuvant.