A New Orthonairovirus Associated with Human Febrile Illness.

BACKGROUND: In June 2019, a patient presented with persistent fever and multiple organ dysfunction after a tick bite at a wetland park in Inner Mongolia. Next-generation sequencing in this patient revealed an infection with a previously unknown orthonairovirus, which we designated Wetland virus (WELV). METHODS: We conducted active hospital-based surveillance to determine the prevalence of WELV infection among febrile patients with a history of tick bites. Epidemiologic investigation was performed. The virus was isolated, and its infectivity and pathogenicity were investigated in animal models. RESULTS: WELV is a member of the orthonairovirus genus in the Nairoviridae family and is most closely related to the tickborne Hazara orthonairovirus genogroup. Acute WELV infection was identified in 17 patients from Inner Mongolia, Heilongjiang, Jilin, and Liaoning, China, by means of reverse-transcriptase-polymerase-chain-reaction assay. These patients presented with nonspecific symptoms, including fever, dizziness, headache, malaise, myalgia, arthritis, and back pain and less frequently with petechiae and localized lymphadenopathy. One patient had neurologic symptoms. Common laboratory findings were leukopenia, thrombocytopenia, and elevated d-dimer and lactate dehydrogenase levels. Serologic assessment of convalescent-stage samples obtained from 8 patients showed WELV-specific antibody titers that were 4 times as high as those in acute-phase samples. WELV RNA was detected in five tick species and in sheep, horses, pigs, and Transbaikal zokors (Myospalax psilurus) sampled in northeastern China. The virus that was isolated from the index patient and ticks showed cytopathic effects in human umbilical-vein endothelial cells. Intraperitoneal injection of the virus resulted in lethal infections in BALB/c, C57BL/6, and Kunming mice. The Haemaphysalis concinna tick is a possible vector that can transovarially transmit WELV. CONCLUSIONS: A newly discovered orthonairovirus was identified and shown to be associated with human febrile illnesses in northeastern China. (Funded by the National Natural Science Foundation of China and the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences.).

Supervised and unsupervised machine learning approaches for monitoring subvisible particles within an aluminum-salt adjuvanted vaccine formulation.

Suspensions of protein antigens adsorbed to aluminum-salt adjuvants are used in many vaccines and require mixing during vial filling operations to prevent sedimentation. However, the mixing of vaccine formulations may generate undesirable particles that are difficult to detect against the background of suspended adjuvant particles. We simulated the mixing of a suspension containing a protein antigen adsorbed to an aluminum-salt adjuvant using a recirculating peristaltic pump and used flow imaging microscopy to record images of particles within the pumped suspensions. Supervised convolutional neural networks (CNNs) were used to analyze the images and create "fingerprints" of particle morphology distributions, allowing detection of new particles generated during pumping. These results were compared to those obtained from an unsupervised machine learning algorithm relying on variational autoencoders (VAEs) that were also used to detect new particles generated during pumping. Analyses of images conducted by applying both supervised CNNs and VAEs found that rates of generation of new particles were higher in aluminum-salt adjuvant suspensions containing protein antigen than placebo suspensions containing only adjuvant. Finally, front-face fluorescence measurements of the vaccine suspensions indicated changes in solvent exposure of tryptophan residues in the protein that occurred concomitantly with new particle generation during pumping.

Predicting Human Subcutaneous Bioavailability of Therapeutic Monoclonal Antibodies from Systemic Clearance and Volume of Distribution.

Subcutaneous delivery of monoclonal antibody therapeutics is often preferred to intravenous delivery due to better patient compliance and overall lower cost to the healthcare system. However, the systemic absorption of biologics dosed subcutaneously is often incomplete. The aim of this work was to describe a human bioavailability prediction method for monoclonal antibodies delivered subcutaneously that utilizes intravenous pharmacokinetic parameters as input. A two-compartment pharmacokinetic model featuring a parallel-competitive absorption pathway and a presystemic metabolism pathway was employed. A training data set comprised 19 monoclonal antibodies (geometric mean bioavailability of 68%), with previously reported human pharmacokinetic parameters, while a validation set included data compiled from 5 commercial drug products (geometric mean bioavailability of 69%). A single fitted absorption rate constant, paired with compound-specific estimates of presystemic metabolism rate proportional to compound-specific systemic clearance parameters, resulted in calculations of human subcutaneous bioavailability closely mimicking clinical data in the training data set with a root-mean-square error of 5.5%. Application of the same approach to the validation data set resulted in predictions characterized by 12.6% root-mean-square error. Factors that may have impacted the prediction accuracy include a limited number of validation data set compounds and an uncertainty in the absorption rate, which were subsequently discussed. The predictive method described herein provides an initial estimate of the subcutaneous bioavailability based exclusively on pharmacokinetic parameters available from intravenous dosing.

An effective disease diagnostic model related to pyroptosis in ischemic cardiomyopathy.

Pyroptosis is involved in ischemic cardiomyopathy (ICM). The study aimed to investigate the pyroptosis-related genes and clarify their diagnostic value in ICM. The bioinformatics method identified the differential pyroptosis genes between the normal control and ICM samples from online datasets. Then, protein-protein interaction (PPI) and function analysis were carried out to explore the function of these genes. Following, subtype analysis was performed using ConsensusClusterPlus, functions, immune score, stromal score, immune cell proportion and human leukocyte antigen (HLA) genes between subtypes were investigated. Moreover, optimal pyroptosis genes were selected using the least absolute shrinkage and selection operator (LASSO) analysis to construct a diagnostic model and evaluate its effectiveness using receiver operator characteristic (ROC) analysis. Twenty-one differential expressed pyroptosis genes were identified, and these genes were related to immune and pyroptosis. Subtype analysis identified two obvious subtypes: sub-1 and sub-2. And LASSO identified 13 optimal genes used to construct the diagnostic model. The diagnostic model in ICM diagnosis with the area under ROC (AUC) was 0.965. Our results suggested that pyroptosis was tightly associated with ICM.

In vivo flow cytometry reveals an anti-metastatic effect of Rujifang in triple-negative breast cancer.

Breast cancer is the most common cancer, and triple-negative breast cancer (TNBC) has the highest metastasis and mortality rate among all breast cancer subtypes. Rujifang is a traditional Chinese medicine formula with many years of clinical application in breast cancer treatment. Here, we aim to investigate the effects of Rujifang on circulating tumor cell (CTC) dynamics and the tumor microenvironment in a ZsGreen/luciferase double-labeled TNBC orthotopic model. We report that the number of CTCs monitored by in vivo flow cytometry (IVFC) strongly correlates with disease progression. Rujifang treatment decreased the number of CTCs and suppressed the distant metastasis of TNBC. Moreover, immunofluorescence analysis revealed that Rujifang treatment could affect the tumor microenvironment by downregulating Kindlin-1, which has been reported to promote metastasis of TNBC. Our study provides evidence of the anti-metastatic effect of Rujifang against TNBC in an animal model using fluorescent cell lines. The results suggest the potential therapeutic value of Rujifang as an anti-metastatic drug, however, further clinical trials are needed to validate these findings in humans.

Predicting Colloidal Stability of High-Concentration Monoclonal Antibody Formulations in Common Pharmaceutical Buffers Using Improved Polyethylene Glycol Induced Protein Precipitation Assay.

Colloidal stability is an important consideration when developing high concentration mAb formulations. PEG-induced protein precipitation is a commonly used assay to assess the colloidal stability of protein solutions. However, the practical usefulness and the current theoretical model for this assay have yet to be verified over a large formulation space across multiple mAbs and mAb-based modalities. In the present study, we used PEG-induced protein precipitation assays to evaluate colloidal stability of 3 mAbs in 24 common formulation buffers at 20 and 5 °C. These prediction assays were conducted at low protein concentration (1 mg/mL). We also directly characterized high concentration (100 mg/mL) formulations for cold-induced phase separation, turbidity, and concentratibility by ultrafiltration. This systematic study allowed analysis of the correlation between the results of low concentration assays and the high concentration attributes. The key findings of this study include the following: (1) verification of the usefulness of three different parameters (Cmid, µB, and Tcloud) from PEG-induced protein precipitation assays for ranking colloidal stability of high concentration mAb formulations; (2) a new method to implement PEG-induced protein precipitation assay suitable for high throughput screening with low sample consumption; (3) improvement in the theoretical model for calculating robust thermodynamic parameters of colloidal stability (µB and εB) that are independent of specific experimental settings; (4) systematic evaluation of the effects of pH and buffer salts on colloidal stability of mAbs in common formulation buffers. These findings provide improved theoretical and practical tools for assessing the colloidal stability of mAbs and mAb-based modalities during formulation development.

In Situ Observation of Cellular Structure Changes in and Chain Segregations of Anabaena sp. PCC 7120 on TiO2 Films under a Photocatalytic Device.

Cyanobacteria outbreaks are serious water pollution events, causing water crises around the world. Photocatalytic disinfection, as an effective approach, has been widely used to inhibit blue algae growth. In this study, a tiny reaction room containing a TiO2 film was designed to fulfill in situ optical observation of the destruction process of a one-dimensional multicellular microorganism, Anabaena sp. PCC 7120, which is also a typical bacterial strain causing water blooms. It was found that the fragment number increased exponentially with the activation time. The fracture mechanics of the algae chains were hypothesized to be the combining functions of increased local tensile stress originated from the cell contracting as well as the oxidative attacks coming from reactive oxygen species (ROSs). It was assumed that the oxidative species were the root cause of cellular structure changes in and chain fractures of Anabaena sp. PCC 7120 in the photocatalytic inactivation activity.

Facile Synthesis of Cu-Doped ZnO Nanoparticles for the Enhanced Photocatalytic Disinfection of Bacteria and Fungi.

In this study, Cu-doped ZnO was prepared via the facile one-pot solvothermal approach. The structure and composition of the synthesized samples were characterized by XRD (X-ray diffraction), TEM (transmission electron microscopy), and XPS (X-ray photoelectron spectroscopy) analyses, revealing that the synthesized samples consisted of Cu-doped ZnO nanoparticles. Ultraviolet-visible (UV-vis) spectroscopy analysis showed that Cu-doping significantly improves the visible light absorption properties of ZnO. The photocatalytic capacity of the synthesized samples was tested via the disinfection of Escherichia coli, with the Cu-ZnO presenting enhanced disinfection compared to pure ZnO. Of the synthesized materials, 7% Cu-ZnO exhibited the best photocatalytic performance, for which the size was ~9 nm. The photocurrent density of the 7% Cu-ZnO samples was also significantly higher than that of pure ZnO. The antifungal activity for 7% Cu-ZnO was also tested on the pathogenic fungi of Fusarium graminearum. The macroconidia of F. graminearum was treated with 7% Cu-ZnO photocatalyst for 5 h, resulting in a three order of magnitude reduction at a concentration of 105 CFU/mL. Fluorescence staining tests were used to verify the survival of macroconidia before and after photocatalytic treatment. ICP-MS was used to confirm that Cu-ZnO met national standards for cu ion precipitation, indicating that Cu-ZnO are environmentally friendly materials.

Cervical alignment and clinical outcome of open-door laminoplasty vs. laminectomy and instrumentation in kyphotic multilevel cervical degenerative myelopathy.

INTRODUCTION: The aim of this study was to determine whether the sagittal lordotic alignment, clinical outcomes and axial symptoms (AS) could be improved by kyphotic correction through the posterior approach for the treatment of multilevel cervical degenerative myelopathy (CDM) and to further analyze the changes of cervical spinal alignment parameters after correction of kyphosis. The hypothesis was that correction of kyphosis can improve the severity of AS and neurological recovery. MATERIALS AND METHODS: We retrospectively reviewed 109 patients who suffered from multilevel CDM combined with kyphosis. The patients had undergone open-door laminoplasty (Group LP, 53 patients) and laminectomy with instrumentation (Group LI, 56 patients) between January 2014 and December 2018. Cervical spinal alignment parameters, including curvature index (CI), T1 slope, C2-7 Cobb angle, C2-7 SVA, were measured on the pre- and postoperative lateral radiographs. The recovery rate was calculated based on the Japanese Orthopedic Association (JOA) score. AS severity was quantified using Neck Disability Index (NDI). A P value less than 0.05 was considered to be significant. RESULTS: Analyses of postoperative follow-up data showed significant differences (P < 0.001) in CI, correction of CI, C2-7 Cobb angle, T1 slope, C2-7 SVA and NDI between Group LP and LI, but no significant differences in JOA score (P = 0.23) and recovery rate (P = 0.13). There were significant differences (P < 0.001) in CI, T1 slope, C2-7 Cobb angle, C2-7 SVA, JOA score, and NDI between pre- and postoperative follow-up in both groups. Correction of CI showed negative correlation with AS severity (r = -0.51, P < 0.001), and no association with recovery rate (r = 0.14, P = 0.15). CONCLUSIONS: Satisfied neurological improvement was achieved by LP and LI for multilevel CDM combined with kyphosis. Cervical kyphotic correction produced significant improvement of AS and increase of T1 slope and C2-7 SVA. However, the kyphotic correction may not be associated with better neurological recovery in the short-term postoperative period.

CircBCAR3 accelerates esophageal cancer tumorigenesis and metastasis via sponging miR-27a-3p.

RATIONALE: Circular RNAs (circRNAs) have been demonstrated to contribute to esophageal cancer progression. CircBCAR3 (hsa_circ_0007624) is predicted to be differentially expressed in esophageal cancer by bioinformatics analysis. We investigated the oncogenic roles and biogenesis of circBCAR3 in esophageal carcinogenesis. METHODS: Functions of circBCAR3 on cancer cell proliferation, migration, invasion, and ferroptosis were explored using the loss-of-function assays. A xenograft mouse model was used to reveal effects of circBCAR3 on xenograft growth and lung metastasis. The upstream and downstream mechanisms of circBCAR3 were investigated by bioinformatics analysis and confirmed by RNA immunoprecipitation and luciferase reporter assays. The dysregulated genes in hypoxia-induced esophageal cancer cells were identified using RNA-seq. RESULTS: CircBCAR3 was highly expressed in esophageal cancer tissues and cells and its expression was increased by hypoxia in vitro. Silencing of circBCAR3 repressed the proliferation, migration, invasion, and ferroptosis of esophageal cancer cells in vitro, as well as inhibited the growth and metastasis of esophageal xenograft in mice in vivo. The hypoxia-induced promotive effects on esophageal cancer cell migration and ferroptosis were rescued by circBCAR3 knockdown. Mechanistically, circBCAR3 can interact with miR-27a-3p by the competitive endogenous RNA mechanism to upregulate transportin-1 (TNPO1). Furthermore, our investigation indicated that splicing factor quaking (QKI) is a positive regulator of circBCAR3 via targeting the introns flanking the hsa_circ_0007624-formed exons in BCAR3 pre-mRNA. Hypoxia upregulates E2F7 to transcriptionally activate QKI. CONCLUSION: Our research demonstrated that splicing factor QKI promotes circBCAR3 biogenesis, which accelerates esophageal cancer tumorigenesis via binding with miR-27a-3p to upregulate TNPO1. These data suggested circBCAR3 as a potential target in the treatment of esophageal cancer. Hypoxia induces the upregulation of E2F7, which transcriptionally activates QKI in esophageal cancer cells. QKI increases the formation of circBCAR3 by juxtaposing the circularized exons. CircBCAR3 binds with miR-27a-3p to promote TNPO1 expression. CircBCAR3 promoted the proliferation, migration, invasion, and ferroptosis of esophageal cancer cells by miR-27a-3p.

Machine learning approaches to root cause analysis, characterization, and monitoring of subvisible particles in monoclonal antibody formulations.

Processing stresses on therapeutic proteins may cause formation of subvisible particles. Different stress mechanisms generate particle populations with characteristic morphological "fingerprints," and machine learning techniques like convolutional neural networks (CNNs) allow classification of microscopy images of these particles according to known stresses at their root cause. Using CNNs to classify novel particle types not included during network training may lead to inaccurate classification, however, using CNNs to monitor the presence of particulate matter not explicitly used in training could serve as a useful process analytical technology. We used CNNs to classify and identify the root cause of particles generated by subjecting three monoclonal antibodies (mAbs) to various common manufacturing stresses. We probed the generality of particles generated by stressing different mAbs in different formulations and showed that CNN analyses were sensitive not only to the applied stress, but also the buffer conditions and the particular mAb that generated particle populations. Thus, models trained on images of particles created with one mAb and buffer system may not provide accurate root cause analysis when applied to particles generated by other mAb and buffer systems. A lever-rule analysis of CNN-derived fingerprints was used to characterize the composition of mixtures of particle types. Finally, we monitored the temporal evolution of CNN-derived fingerprints when novel populations of particles, which were not included during training, were generated by pumping mAb solutions through a peristaltic pump.

Occluding morphologically complicated left atrial appendage using the small-umbrella LAmbre device.

BACKGROUND: Percutaneous left atrial appendage (LAA) occlusion is effective for stroke prevention in patients with atrial fibrillation. LAA can have a complex anatomy, such as multiple lobes or a large orifice, which may render it unsuitable for occlusion using regular devices. We aimed to investigate the feasibility, safety, and short-term efficacy of the small-umbrella LAmbre device for morphologically complicated LAA. METHODS: We retrospectively enrolled 129 consecutive patients who underwent LAA occlusion using the LAmbre device; the small-umbrella LAmbre device was used in 30 of these patients. We analyzed patients' characteristics, procedural details, and outcomes. RESULTS: Twenty-two patients (73.3%) had multilobed (≥ 2) LAA. The umbrella of the occluder was anchored in the branch in 9 patients and in the common trunks of branches in 13 patients. The landing zone and orifice diameters were 19.0 ± 4.39 mm and 27.4 ± 3.95 mm, respectively. The sizes of the umbrella and occluder cover were 22.0 ± 3.42 mm and 34.3 ± 2.75 mm, respectively. At 3-month follow-up transesophageal echocardiography in 24 patients, no peri-device residual flow was reported. Device thrombosis was detected in one patient at 3 months and disappeared after 3 months of anticoagulation. Ischemic stroke occurred in one patient; no other adverse events were reported. CONCLUSIONS: Occlusion of morphologically complicated LAA using the small-umbrella LAmbre device was feasible, safe, and effective in patients with atrial fibrillation in this study. This occluder provides an alternative for patients who cannot be treated with regular-sized LAA occlusion devices.

Single Cell Metabolite Detection Using Inertial Microfluidics-Assisted Ion Mobility Mass Spectrometry.

Single-cell metabolite measurement remains highly challenging due to difficulties related to single cell isolation, metabolite detection, and identification of low levels of metabolites. Here, as a first step of the technological development, we propose a novel strategy integrating spiral inertial microfluidics and ion mobility mass spectrometry (IM-MS) for single-cell metabolite detection and identification. Cells in methanol suspension are inertially focused into a single stream in the spiral microchannel. This stream of separated cells is delivered to the nanoelectrospray needle to be lysed and ionized and subsequently analyzed in real time by IM-MS. This analytical system enables six to eight single-cell metabolic fingerprints to be collected per minute, including gas-phase collisional cross section (CCS) measurements as an additional molecular descriptor, giving increased confidence in metabolite identification. As a proof of concept, the metabolic profiles of three types of cancer cells (U2OS, HepG2, and HepG2.215) were successfully screened, and 19 distinct lipids species were identified with CCS value filtering. Furthermore, principal component analysis (PCA) showed differentiation of the three cancer cell lines, mainly due to cellular surface phospholipids. Taken together, our technology platform offers a simple and efficient method for single-cell lipid profiling, with additional ion mobility separation of lipids significantly improving the confidence toward identification of metabolites.

Study on spin angular momentum balance in harmonics generated from counter-rotating two-color laser fields.

High-order harmonics generated from Xe driven by counter-rotating two-color driving fields are studied in the frame of a quantum-field scattering theory, and the spin angular momentum transfer is discussed. The driving field is composed by a circularly polarized (CP) mode and an elliptically polarized (EP) mode. We treat the EP mode as a compostition of counter-rotating CP fields of unequal intensity. We use a pair of phased generalized Bessel functions to describe the harmonic generation amplitude, and the conservation of the spin angular momentum during harmonic generation in the two-color field is derived in a solid base and in a straightforward way. The experimentally observed V-type and Λ-type distributions of the harmonic spectra with ellipticity are recovered theoretically. Balance pattern of the spin angular momentum is disclosed substantially.

Selective recognition of DNA parallel G-quadruplexes by 3,8a-disubstituted indolizinones.

Owing to its potential biological relevance, DNA G-quadruplex has been considered as a prospective anti-cancer target. Some known G-quadruplex-interactive N-containing compounds with low cytotoxicity have become prospective anticancer drugs. Here we reported a new type of N-containing alkaloids 3,8a-disubstituted indolizinones, and investigated their substituent effects at 3- and 8a-positions in targeting to DNA c-myc G-quadruplex. And then we used 3-naphtyl-8a-(pyridin-2-yl) substrate I8 as an example, and investigated its ability in targeting to DNA parallel G-quadruplexes in vitro.

Comparative Blood and Urine Metabolomics Analysis of Healthy Elderly and Young Male Singaporeans.

Comparative metabolomics analysis of biofluids could provide information about the metabolic alterations in aging. To investigate the signature of multiple metabolic profiles associated with aging in an Asian population, we performed a pilot study in healthy Singaporeans, including 33 elderly and 33 young males. Fasting whole bloods were analyzed by routine hematology; the serum and urine metabolome profiles were obtained using NMR-based nontargeted metabolomics analysis and targeted lipoprotein analysis. Among the 90 identified compounds in serum and urine samples, 32 were significantly different between the two groups. The most obvious age-related metabolic signatures include decreased serum levels of albumin lysyl and essential amino acids and derivatives but increased levels of N-acetyl glycoproteins and several lipids and elevated urine levels of trimethylamine N-oxide, scyllo-inositol, citrate, and ascorbic acid but decreased levels of several amino acids, acetate, etc. Among 112 lipoprotein subfractions, 65 were elevated, and 2 were lower in the elderly group. These significantly age-varying metabolites, especially in the amino acid and fatty acid metabolism pathways, suggest that the regulation of these pathways contributes to the aging process in Chinese Singaporeans. Further multiomics studies including the gut microbiome and intervention studies in a larger cohort are needed to elucidate the possible mechanisms in the aging process.

Long non-coding RNA LINC01194 promotes the proliferation, migration and invasion of lung adenocarcinoma cells by targeting miR-641/SETD7 axis.

BACKGROUND: It is increasingly evidenced that long non-coding RNAs (lncRNAs) play an important role in various diseases. LncRNA LINC01194 acts as an oncogene in several cancer types. Nevertheless, the role of LINC01194 in lung adenocarcinoma (LUAD) has not yet been revealed. METHODS: qRT-PCR was used to detect the expression of LINC01194, miR-641 and SETD7 mRNA, while western blot was exploited to examine SETD7 protein level. Cell proliferation was detected by colony formation and EdU assays. Transwell assays detected cell migration and invasion. TUNEL assay and flow cytometry analysis were used to detect cell apoptosis. RIP, RNA pull down and luciferase reporter assays detected the binding among LINC01194, miR-641 and SETD7. RESULTS: LINC01194 was significantly upregulated in LUAD tissues and cell lines. Knockdown of LINC01194 resulted in decreased cell proliferation, migration and invasion, and increased apoptosis. Mechanistic experiments unveiled that LINC01194 augmented SETD7 expression in LUAD cells by competitively interacting with miR-641. Rescue experiments showed that miR-641 inhibition and SETD7 overexpression rescued the repressing impacts on LUAD cell proliferation, migration and invasion caused by LINC01194 knockdown. CONCLUSION: LINC01194 promotes the progression of LUAD by enhancing miR-641-targeted SETD7. The LINC01194/miR-641/SETD7 axis might provide new molecular targets for treating LUAD.

Islatravir Case Study for Enhanced Screening of Thermodynamically Stable Crystalline Anhydrate Phases in Pharmaceutical Process Development by Hot Melt Extrusion.

The emergence of new active pharmaceutical ingredient (API) polymorphs in pharmaceutical development presents significant risks. Even with thorough polymorph screening, new pathways toward alternate crystal phases can present themselves over the course of formulation development; thus, further improvements in phase screening methods are needed. Herein, a case study is presented of a thermodynamically stable crystalline phase of the HIV drug Islatravir (MK-8591, EFdA) that was not isolated from initial pharmaceutical polymorph screening. In total, five Islatravir phases are identified: one monohydrate and four anhydrate phases. The new phase, anhydrate form IV, was unexpectedly discovered during hot melt extrusion (HME) process development of polymeric implant drug product formulations while probing extreme manufacturing process conditions (elevated shear forces). X-ray diffraction (XRD), differential scanning calorimetry (DSC), and solid-state nuclear magnetic resonance (ssNMR) were utilized as principal tools to identify the new polymorph. The result suggests that HME introduces conditions that may allow a thermodynamically stable crystalline phase to form and these conditions are not necessarily captured by routine pharmaceutical polymorph screening. Subsequent investigations identified procedures to generate the new anhydrate phase without HME equipment by the use of special thermal procedures. It is found that for a crystalline hydrate phase the rate of water loss as well as water entrapment in a heating vessel play a crucial role in phase conversions into different anhydrate polymorphs. Further, the polymer involved in the HME manufacturing process also plays a critical role in the phase conversion, likely by coating the API microparticles and thereby altering the phase conversion kinetics. Strategies presented herein to mimic phase changes during formulation manufacture hold promise for the identification of thermodynamically stable anhydrate forms in earlier stages of pharmaceutical development.

Metabolic Changes of Fusarium graminearum Induced by TPS Gene Deletion.

Fusarium head blight (FHB) mainly resulting from Fusarium graminearum (Fg) Schwabe is a notorious wheat disease causing huge losses in wheat production globally. Fg also produces mycotoxins, which are harmful to human and domestic animals. In our previous study, we obtained two Fg mutants, TPS1- and TPS2-, respectively, with a single deletion of trehalose 6-phosphate synthase (TPS1) and trehalose 6-phosphate phosphatase (TPS2) compared with the wild type (WT). Both mutants were unable to synthesize trehalose and produced fewer mycotoxins. To understand the other biochemical changes induced by TPS gene deletion in Fg, we comprehensively analyzed the metabolomic differences between TPS- mutants and the WT using NMR together with gas chromatography-flame ionization detection/mass spectrometry. The expression of some relevant genes was also quantified. The results showed that TPS1- and TPS2- mutants shared some common metabolic feature such as decreased levels for trehalose, Val, Thr, Lys, Asp, His, Trp, malonate, citrate, uridine, guanosine, inosine, AMP, C10:0, and C16:1 compared with the WT. Both mutants also shared some common expressional patterns for most of the relevant genes. This suggests that apart from the reduced trehalose biosynthesis, both TPS1 and TPS2 have roles in inhibiting glycolysis and the tricarboxylic acid cycle but promoting the phosphopentose pathway and nucleotide synthesis; the depletion of either TPS gene reduces the acetyl-CoA-mediated mycotoxin biosynthesis. TPS2- mutants produced more fatty acids than TPS1- mutants, suggesting different roles for TPS1 and TPS2, with TPS2- mutants having impaired trehalose biosynthesis and trehalose 6-phosphate accumulation. This may offer opportunities for developing new fungicides targeting trehalose biosynthesis in Fg for FHB control and mycotoxin reduction in the FHB-affected cereals.

Enhanced double ionization rate from O2 molecules driven by counter-rotating circularly polarized two-color laser fields.

We report that the nonsequential double ionization (NSDI) probability of an O 2 target can be enhanced greatly in a counter-rotating circularly polarized two-color driving field. The field is composed of a fundamental frequency and its third harmonic, and the combined electric field traces out a four-leaf-clover pattern. The electron ionized by such a field has more chances to collide with the valence electrons in the O 2 molecule, which significantly enhances the NSDI probability. This effect is more evident in low-intensity fields. We also find that the enhancement appears in a broad range of the field ratio of two colors and that both the NSDI yield and the underlying electronic behavior varies notably with the field ratio.