A single local delivery of paclitaxel and nucleic acids via an immunoactive polymer eliminates tumors and induces antitumor immunity.

Despite recent advances in cancer therapy, hard-to-reach, unidentified tumors remain a significant clinical challenge. A promising approach is to treat locatable and accessible tumors locally and stimulate antitumor immunity in situ to exert systemic effects against distant tumors. We hypothesize that a carrier of immunotherapeutics can play a critical role in activating antitumor immunity as an immunoadjuvant and a local retainer of drug combinations. Here, we develop a polyethyleneimine-lithocholic acid conjugate (2E'), which forms a hydrophobic core and cationic surface to codeliver hydrophobic small molecules and anionic nucleic acids and activates antigen-presenting cells via the intrinsic activities of 2E' components. 2E' delivers paclitaxel and small-interfering RNA (siRNA) targeting PD-L1 (or cyclic dinucleotide, [CDN]) to induce the immunogenic death of tumor cells and maintain the immunoactive tumor microenvironment, and further activates dendritic cells and macrophages, leveraging the activities of loaded drugs. A single local administration of 2E' or its combination with paclitaxel and PD-L1­targeting siRNA or CDN induces strong antitumor immunity, resulting in immediate regression of large established tumors, tumor-free survival, an abscopal effect on distant tumors, and resistance to rechallenge and metastasis in multiple models of murine tumors, including CT26 colon carcinoma, B16F10 melanoma, and 4T1 breast cancer. This study supports the finding that local administration of immunotherapeutics, when accompanied by the rationally designed carrier, can effectively protect the host from distant and recurrent diseases.

VDR promotes pancreatic cancer progression in vivo by activating CCL20-mediated M2 polarization of tumor associated macrophage.

BACKGROUND: Activation of VDR pathway was a promising anti-tumor therapy strategy. However, numerous clinical studies have demonstrated the effect of activating VDR is limited, which indicates that VDR plays a complex role in vivos. METHODS: We analyzed the TCGA database to examine the association between VDR expression and immune cell infiltration in pancreatic adenocarcinoma (PAAD). Western blot, ELISA, ChIP, and dual-luciferase reporter assays were performed to determine the mechanism of VDR regulating CCL20. Migration assay and immunofluorescence were used to investigate the role of CCL20 in M2 macrophage polarization and recruitment. We employed multiplexed immunohistochemical staining and mouse models to validate the correlation of VDR on macrophages infiltration in PAAD. Flow cytometry analysis of M2/M1 ratio in subcutaneous graft tumors. RESULTS: VDR is extensively expressed in PAAD, and patients with elevated VDR levels exhibited a significantly reduced overall survival. VDR expression in PAAD tissues was associated with increased M2 macrophages infiltration. PAAD cells overexpressing VDR promote macrophages polarization towards M2 phenotype and recruitment in vitro and vivo. Mechanistically, VDR binds to the CCL20 promoter and up-regulates its transcription. The effects of polarization and recruitment on macrophages can be rescued by blocking CCL20. Finally, the relationship between VDR and M2 macrophages infiltration was evaluated using clinical cohort and subcutaneous graft tumors. A positive correlation was demonstrated between VDR/CCL20/CD163 in PAAD tissues and mouse models. CONCLUSION: High expression of VDR in PAAD promotes M2 macrophage polarization and recruitment through the secretion of CCL20, which activates tumor progression. This finding suggests that the combination of anti-macrophage therapy may improve the efficacy of VDR activation therapy in PAAD.

Interpenetrating Networks of Collagen and Hyaluronic Acid That Serve as In Vitro Tissue Models for Assessing Macromolecular Transport.

High-fidelity preclinical in vitro tissue models can reduce the failure rate of drugs entering clinical trials. Collagen and hyaluronic acid (HA) are major components of the extracellular matrix of many native tissues and affect therapeutic macromolecule diffusion and recovery through tissues. Although collagen and HA are commonly used in tissue engineering, the physical and mechanical properties of these materials are variable and depend highly on processing conditions. In this study, HA was chemically modified and crosslinked via hydrazone bonds to form interpenetrating networks of crosslinked HA (HAX) with collagen (Col). These networks enabled a wide range of mechanical properties, including stiffness and swellability, and microstructures, such as pore morphology and size, that can better recapitulate diverse tissues. We utilized these interpenetrating ColHAX hydrogels as in vitro tissue models to examine macromolecular transport and recovery for early-stage drug screening. Hydrogel formulations with varying collagen and HAX concentrations imparted different gel properties based on the ratio of collagen to HAX. These gels were stable and swelled up to 170% of their original mass, and the storage moduli of the ColHAX gels increased over an order of magnitude by increasing collagen and HA concentration. Interestingly, when HAX concentration was constant and collagen concentration increased, both the pore size and spatial colocalization of collagen and HA increased. HA in the system dominated the ζ-potentials of the gels. The hydrogel and macromolecule properties impacted the mass transport and recovery of lysozyme, ß-lactoglobulin, and bovine serum albumin (BSA) from the ColHAX gels─large molecules were largely impacted by mesh size, whereas small molecules were influenced primarily by electrostatic forces. Overall, the tunable properties demonstrated by the ColHAX hydrogels can be used to mimic different tissues for early-stage assays to understand drug transport and its relationship to matrix properties.

Chemical preparation, degradation analysis, computational docking and biological activities of novel sulfonylureas with 2,5-disubstituted groups.

Based on the previous finding that a substitution at 5-position of the benzene ring is favorable to enhance the degradation rates of sulfonylurea herbicides, a total of 16 novel 2,5-disubsituted sulfonylurea compounds were chemically synthesized and fully characterized by means of 1H NMR, 13C NMR, HRMS and X-ray diffraction. By using HPLC analysis, the degradation behavior of M03, a compound belonging to this family, was studied and confirmed that chlorsulfuron itself is not a degraded product of the 2,5-disubstituted sulfonylureas. Inhibition constants against plant acetohydroxyacid synthase (AHAS) were determined for selected compounds, among which SU3 showed seven times stronger activity against the mutant W574L enzyme than chlorsulfuron. Molecular docking suggested that the substituted group at 5-position of benzene ring is likely to interact with the surrounding residues Met200 and Asp376 of AtAHAS. From the greenhouse herbicidal assay and crop safety test, SU5 and SU6 are considered as herbicide candidates to control dicotyledon weeds in corn, while SU3 is likely to be a promising candidate to control dicotyledon weed species and barnyard grass in wheat. The present research has therefore provided some new insights to understand the structure-activity relationships of herbicidal sulfonylureas with di-substitutions at benzene ring.

Degradation of 5-Dialkylamino-Substituted Chlorsulfuron Derivatives in Alkaline Soil.

Sulfonylurea herbicides are widely used as acetolactate synthase (ALS) inhibitors due to their super-efficient activity. However, some sulfonylurea herbicides show toxicity under crop rotation due to their long degradation time, for example, chlorsulfuron. Our research goal is to obtain chlorsulfuron-derived herbicides with controllable degradation time, good crop safety and high herbicidal activities. Based on our previously reported results in acidic soil, we studied the degradation behaviors of 5-dialkylamino-substituted chlorsulfuron derivatives (NL101-NL108) in alkaline soil (pH 8.39). The experimental data indicate that addition of the 5-dialkylamino groups on the benzene ring of chlorsulfuron greatly accelerated degradation in alkaline soil. These chlorsulfuron derivatives degrade 10.8 to 51.8 times faster than chlorsulfuron and exhibit excellent crop safety on wheat and corn (through pre-emergence treatment). With a comprehensive consideration of structures, bioassay activities, soil degradation and crop safety, it could be concluded that 5-dialkylamino-substituted chlorsulfuron derivatives are potential green sulfonylurea herbicides for pre-emergence treatment on both wheat and corn. The study also provides valuable information for the discovery of new sulfonylurea herbicides for crop rotation.

Selective magnetic responses of silicon nanoparticles modulated by waveguide structures.

High-refractive-index nanoparticles (NPs), such as silicon NPs, were considered as effective carriers in their response to a magnetic field at optical frequencies. Such NPs play an important role in many state-of-the-art technologies in nano-optics. Although the resonance properties of these NPs when varying their structural parameters have been studied intensely in the past few years, their interaction with the underlying substrate has seldom been discussed, in particular, when the substrate is a waveguide structure that significantly modulates the optical responses of the NPs. We proposed and studied a selective magnetic coupling system comprising a Si-NP on a metal-dielectric waveguide (MDW). The MDW structure supports either a transverse electric (TE) or a transverse magnetic (TM) mode that induces a large polarization dependence in the magnetic resonance. A new manifestation of the optical spin Hall effect was demonstrated in which a vertical rotating magnetic dipole excites a TE-type waveguide mode with a specific unidirectional emission. Making use of this polarization response, we developed a scanning imaging system that can selectively map the transverse or longitudinal magnetic field component of a focused beam depending on the type of MDW used in the system. This selective magnetic resonance coupling system is expected to be valuable for studying the fundamental interactions between the magnetic field and matter and for developing related nano-applications.

Camouflaging Nanoparticles for Ratiometric Delivery of Therapeutic Combinations.

Combination therapy is a common clinical practice in the management of malignancies. Synergistic therapeutic outcomes are achieved only when tumor cells are exposed to drugs in an optimal ratio and sequence; therefore, carriers coencapsulating multiple drugs are widely pursued for their coordinated delivery. However, it is challenging to coload drugs with different physicochemical properties in a single carrier with specific ratios. It is not even beneficial to load them in one carrier if they need to be released at different times. We propose to load drugs into chemically compatible carriers separately, equalize different carriers by a simple, rapid, and versatile camouflage technique based on natural polyphenol tannic acid (TA), and administer them in desirable ratios and sequences. To demonstrate this potential, different nanoparticles (NPs) with different charges and material basis, such as polymeric (carboxyl-terminated or amine-terminated cationic polystyrene NPs or poly(lactic- co-glycolic acid (PLGA) NPs), inorganic (mesoporous silica NPs (MSNs)), and liposomal NPs, are camouflaged with TA layers and further modified with folate-conjugated polyethylene glycol to aid in the delivery to tumors. The camouflaged NPs show similar physicochemical properties and interactions with KB cells despite the difference in core platforms, and their mixtures interact with common cell targets in a ratiometric manner. In KB-tumor-bearing mice, the camouflaged PLGA NPs and MSNs show near-perfect colocalization in tumors. These results support that TA helps equalize different NPs with high versatility and enables their ratiometric delivery to common targets. This approach can relieve technical challenges in ratiometric codelivery or sequential delivery of therapeutic agents with distinct physicochemical properties.

Low-loss metal-dielectric waveguide mode enabled structured illumination microscopy with 0.18λ0 resolution.

Structured illumination microscopy (SIM) is a powerful super-resolved imaging technique which enables to perform fast and in vivo imaging of bio-samples. In order to achieve a better resolution of a SIM system, evanescent waves with larger in-plane wave-vector are preferred for SIM, among which the total internal reflection (TIRF-SIM) and the plasmonic SIM (pSIM) configurations are widely studied. Here, we demonstrated a metal-dielectric waveguide (MDW) based SIM system - termed as MDW-SIM, which can achieve a good compromise between TIRF-SIM and pSIM. The MDW can support a low-loss waveguide mode at an aqueous environment, with an evanescent tail existing above the water/dielectric interface for SIM. A proof-of-concept imaging experiment was performed on fluorescent beads, where a spatial resolution of 86nm was achieved at a 473nm illumination wavelength and a 1.45 numerical aperture objective lens. The proposed MDW-SIM has a great potential for the bio-imaging applications.

Mapping the weak plasmonic transverse field by a dielectric-nanoparticle-on-film structure with ultra-high precision.

Highly confined electromagnetic fields play a significant role in modern nano-optics, among which surface plasmon polaritons (SPPs) are outstanding because of their subwavelength and enhancement nature. While many state-of-the-art methods have been proposed to uncover the field distribution of SPPs, it still faces challenge to map the weak transverse field component (the field tangential to the interface) of SPPs with high contrast and precision. We propose a direct imaging technique, which employs a dielectric-nanoparticle-on-metal-film (DNP-MF) structure as a near-field probe, to overcome this difficulty. The angular distribution of the scattering radiation from the structure is strongly polarization dependent. By extracting the scattering signals that are mainly induced by the horizontal polarization, the imaging of the weak plasmonic transverse field with high precision can be achieved. The mappings of SPPs distributions excited by various vector beams were performed in experiment, which accord excellent with theory. This technique provides a new approach for near-field imaging with high contrast and reliability, which is expected to be valuable for studying the vectorial features of SPPs such as transverse spin, spin-orbit interactions, etc.

Syntheses and biological evaluation of 1,2,3-triazole and 1,3,4-oxadiazole derivatives of imatinib.

Three novel series of 1,2,3-triazole and 1,3,4-oxadiazole derivatives of imatinib were prepared and evaluated in vitro for their cytostatic effects against a human chronic myeloid leukemia (K562), acute myeloid leukemia (HL60), and human leukemia stem-like cell line (KG1a). The structure-activity relationship was analyzed by determining the inhibitory rate of each imatinib analog. Benzene and piperazine rings were necessary groups in these compounds for maintaining inhibitory activities against the K562 and HL60 cell lines. Introducing a trifluoromethyl group significantly enhanced the potency of the compounds against these two cell lines. Surprisingly, some compounds showed significant inhibitory activities against KG1a cells without inhibiting common leukemia cell lines (K562 and HL60). These findings suggest that these compounds are able to inhibit leukemia stem-like cells.

Delivery of STING agonists for cancer immunotherapy.

Agonists of the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-Stimulator of Interferon Genes (STING) pathway, a critical mediator of innate immune response to foreign invaders with DNA, have gained significant interest in cancer immunotherapy. STING agonists are envisioned as a way of complementing the antitumor activity of the patient's immune system and immune checkpoint blockade therapy. However, their clinical development has been challenging due to the poor pharmacokinetic and physicochemical properties. This review discusses drug delivery efforts to circumvent the challenges, their accomplishment, and unmet needs based on the last five years of literature.

Perceived parental warmth attenuates the link between perceived parental rejection and rumination in Chinese early adolescents: two conditional moderation models.

Background: Prior studies have explored the association between perceived parental rejection-warmth and adolescents' rumination, but it is unclear whether the interaction between perceived parental rejection and warmth can predict adolescents' rumination in a Chinese context and whether this interaction varies by children's gender during the post-COVID-19 era. Objective: This study aimed to address these issues in Chinese early adolescents from a family system perspective. Methods: A total of 910 adolescents (M age = 13.63, 48.50% female) from two middle schools in Chongqing and Changsha, China participated in the survey, answering measures for demographics, perceived parental rejection-warmth, and rumination. Results: The results show that adolescents' rumination was positively related to perceived paternal rejection (r = 0.326, p <.001) and maternal rejection (r = 0.343, p <.001), and negatively related to perceived paternal warmth (r = -.184, p <.001) and maternal warmth (r = -0.125, p <.001). Moreover, perceived maternal warmth significantly moderated the link between perceived paternal rejection and adolescents' rumination (boot effect = -0.066, 95CI% = [-0.124, -0.010]), but this moderating effect is only presented in boys not in girls (boot effect = -0.063, 95CI% = [-0.015, 0.140]). However, perceived paternal warmth moderated the link between perceived maternal rejection and rumination in adolescents (boot effect = -0.052, 95CI% = [-0.103, -0.001]), and this interaction varied by adolescents' gender (boot effect = 0.103, 95CI% = [0.029, 0.177]). Conclusions: Perceived Parental rejection and parental warmth co-exist in the Chinese family system, and they exert an interactive effect on adolescents' rumination depending on their gender. It implies that both parents should be more accepting, caring, considerate, and warm toward their daughters, and it is also in line with the tradition and status quo of parenting in Chinese families. These findings have implications for Chinese parental co-parenting practices in families with adolescents and adolescence mental health counseling work.

Systemic Delivery of Paclitaxel by Find-Me Nanoparticles Activates Antitumor Immunity and Eliminates Tumors.

Local delivery of immune-activating agents has shown promise in overcoming an immunosuppressive tumor microenvironment (TME) and stimulating antitumor immune responses in tumors. However, systemic therapy is ultimately needed to treat tumors that are not readily locatable or accessible. To enable systemic delivery of immune-activating agents, we employ poly(lactic-co-glycolide) (PLGA) nanoparticles (NPs) with a track record in systemic application. The surface of PLGA NPs is decorated with adenosine triphosphate (ATP), a damage-associated molecular pattern to recruit antigen-presenting cells (APCs). The ATP-conjugated PLGA NPs (NPpD-ATP) are loaded with paclitaxel (PTX), a chemotherapeutic agent inducing immunogenic cell death to generate tumor antigens in situ. We show that the NPpD-ATP retains ATP activity in hostile TME and provides a stable "find-me" signal to recruit APCs. Therefore, the PTX-loaded NPpD-ATP helps populate antitumor immune cells in TME and attenuate the growth of CT26 and B16F10 tumors better than a mixture of PTX-loaded NPpD and ATP. Combined with anti-PD-1 antibody, PTX-loaded NPpD-ATP achieves complete regression of CT26 tumors followed by antitumor immune memory. This study demonstrates the feasibility of systemic immunotherapy using a PLGA NP formulation that delivers ICD-inducing chemotherapy and an immunostimulatory signal.

Hemophagocytic lymphohistiocytosis associated with brucellosis.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immune disorder categorized as familial HLH or secondary HLH. Our case report describes a 63-year-old woman with epilepsy whose clinical signs were unremitting fever and altered consciousness. Primary abnormalities consisted of fever, splenomegaly, cytopenia, hypertriglyceridemia, hyperferritinemia and hemophagocytosis in the bone marrow. Results of blood next generation sequencing and blood culture confirmed Brucella infection. This report illustrates a sHLH case caused by Brucella melitensis infection. Here, we review the classification, clinical features, diagnostic methods, treatment regimens, differential diagnosis, and prognosis of HLH and brucellosis.

A Five-gene Signature based on MicroRNA for Predicting Prognosis and Immunotherapy in Stomach Adenocarcinoma.

AIMS: We aimed to classify molecular subtypes and establish a prognostic gene signature based on miRNAs for the prognostic prediction and therapeutic response in Stomach adenocarcinoma (STAD). BACKGROUND: STAD is a common diagnosed gastrointestinal malignancy and its heterogeneity is a big challenge that influences prognosis and precision therapies. Present study was designed to classify molecular subtypes and construct a prognostic gene signature based on miRNAs for the prognostic prediction and therapeutic response in STAD. OBJECTIVE: The objective of this study is to investigate the molecular subtypes and prognostic model for STAD. METHODS: A STAD specific miRNA-messenger RNA (mRNA) competing endogenous RNA (ceRNA) network was generated using the RNA-Seq and miRNA expression profiles from The Cancer Genome Atlas (TCGA) database, in which miRNA-related mRNAs were screened. Molecular subtypes were then determined using miRNA-related genes. Through univariate Cox analysis and multivariate regression analysis, a prognostic model was established in GSE84437 Train dataset and validated in GSE84437 Test, TCGA, GSE84437 and GSE66229 datasets. Immunotherapy datasets were employed for assessing the performance of the risk model. Finally, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was applied to validate the expression of hub genes used for the risk score signature. RESULTS: We constructed a ceRNA network containing 84 miRNAs and 907 mRNAs and determined two molecular subtypes based on 26 genes from the intersection of TCGASTAD and GSE84437 datasets. Subtype S2 had poor prognosis, lower tumor mutational burden, higher immune score and lower response to immunotherapy. Subtype S1 was more sensitive to Sorafenib, Pyrimethamine, Salubrinal, Gemcitabine, Vinorelbine and AKT inhibitor VIII. Next, a five-gene signature was generated and its robustness was validated in Test and external datasets. This risk model also had a good prediction performance in immunotherapy datasets. CONCLUSION: This study promotes the underlying mechanisms of miRNA-based genes in STAD and offers directions for classification. A five-gene signature accurately predicts the prognosis and helps therapeutic options.

Terbium alginate encapsulated CsPbI3@Pb-MOF: a ratiometric fluorescent bead for detection and adsorption of Fe3.

Halide perovskite nanocrystals are innovative luminescent materials for fluorescent probes with high quantum yield and narrow emission bandwidth. However, the limited stability, single-signal response, and separation challenges obstruct their widespread use in water ion detection. Herein, a ratiometric fluorescence sensor based on terbium alginate gel beads (green fluorescent, namely Tb-AG) embedded with powdered CsPbI3@Pb-MOF (red fluorescent) was prepared for fluorescent determination and adsorption of Fe3+. Pb-MOF's protection notably enhances the water stability of CsPbI3, while the energy transfer between CsPbI3@Pb-MOF and Tb3+ elevates the optical performance of CsPbI3@Pb-MOF@Tb-AG. Significantly, Fe3+ markedly suppresses CsPbI3@Pb-MOF red fluorescence at 647 nm, while not noticeably affecting Tb-AG green emission at 528 nm. The sensor exhibited a strong linear response to Fe3+ concentrations ranging from 0 to 90 µM, with a detection limit of 0.44 µM and high selectivity. The CsPbI3@Pb-MOF@Tb-AG-based sensor has been effectively validated through its successful use in detecting Fe3+ in tap and river water samples. Furthermore, CsPbI3@Pb-MOF@Tb-AG demonstrates a notable adsorption capacity of 325.4 mg g-1 Fe3+. Finally, the mechanism of Fe3+ detection and adsorption was determined.

Using molecular characteristics to distinguish multiple primary lung cancers and intrapulmonary metastases.

Objectives: Multiple lung cancers may present as multiple primary lung cancers (MPLC) or intrapulmonary metastasis (IPM) with variations in clinical stage, treatment, and prognosis. However, the existing differentiation criteria based on histology do not fully meet the clinical needs. Next-generation sequencing (NGS) may play an important role in assisting the identification of different pathologies. Here, we extended the relevant data by combining histology and NGS to develop detailed identification criteria for MPLC and IPM. Materials and Methods: Patients with lung cancer (each patient had ≥2 tumors) were enrolled in the training (n = 22) and validation (n = 13) cohorts. Genomic profiles obtained from 450-gene-targeted NGS were analyzed, and the new criteria were developed based on our findings and pre-existing Martini & Melamed criteria and molecular benchmarks. Results: The analysis of the training cohort indicated that patients identified with MPLC had no (or <2) trunk or shared mutations. However, 98.02% of mutations were branch mutations, and 69.23% of MPLC had no common mutations. In contrast, a higher percentage of trunk (33.08%) or shared (9.02%) mutations were identified in IPM, suggesting significant differences among mutated components. Subsequently, eight MPLC and five IPM cases were identified in the validation cohort, aligning with the independent imaging and pathologic distinction. Overall, the percentage of trunk and shared mutations was higher in patients with IPM than in patients with MPLC. Based on these results and the establishment of new determination criteria for MPLC and IPM, we emphasize that the type and number of shared variants based on histologic consistency assist in identification. Conclusion: Determining genetic alterations may be an effective method for differentiating MPLC and IPM, and NGS can be used as a valuable assisting tool.

Trinuclear Cu-based covalent organic framework: π-conjugated framework regulating electron delocalization to promote photoreduction CO2.

Sunlight-driven CO2 reduction to value-added chemicals is an effective strategy to promote carbon recycling. The exploration of catalysts with efficient charge separation is crucially important for highly efficient CO2 photoreduction. In this work, the preparation of metal-cluster-based covalent organic framework (CuABD) integrated features from both metal organic frameworks (MOFs) and covalent organic frameworks (COFs) through the condensation of diamines and functionalized trinuclear copper clusters demonstrate a thoughtful design strategy. The reported yield of 1.3 mmol g-1 h-1 for formic acid (HCOOH) under simulated solar irradiation is impressive, surpassing the performance of many COF- and MOF-based catalysts previously reported. Compared to its isomorphic metal-free structure (named BDFTD) and bare trinuclear Cu cluster which present extremely poor catalytic activities, CuABD displays remarkably enhanced CO2 reduction activity. Experimental and theoretical investigations reveal that the efficient charge transfer between diamine monomer and cyclic trinuclear copper (I) units, and the electron delocalization of the π-conjugated framework are responsible for the appealing catalytic performance. In summary, the work presents a well-structured and scientifically sound exploration of a metal-cluster-based covalent organic framework for efficient CO2 reduction under sunlight.

Ovarian PERK/NRF2/CX43/StAR/progesterone pathway activation mediates female reproductive dysfunction induced by cold exposure.

Ambient air temperature is a key factor affecting human health. Female reproductive disorders are representative health risk events under low temperature. However, the mechanism involving in cold-induced female reproductive disorders remains largely unknown. Female mice were intermittently exposed to cold conditions (4 °C) to address the health risk of low temperature on female reproductive system. Primary granulosa cells (GCs) were prepared and cultured under low temperature (35 °C) or exposed to ß3-adrenoreceptor agonist, isoproterenol, to mimic the condition of cold exposure. Western-blot, RT-PCR, co-IP, ELISA, pharmacological inhibition or siRNA-mediated knockdown of target gene were performed to investigate the possible role of hormones, gap conjunction proteins, and ER stress sensor protein in regulating female reproductive disorders under cold exposure. Cold exposure induced estrous cycle disorder and follicular dysplasia in female mice, accompanying with abnormal upregulation of progesterone and its synthetic rate-limiting enzyme, StAR, in the ovarian granulosa cells. Under the same conditions, an increase in connexin 43 (CX43) expressions in the GCs was also observed, which contributed to elevated progesterone levels in the ovary. Moreover, ER stress sensor protein, PERK, was activated in the ovarian GCs after cold exposure, leading to the upregulation of downstream NRF2-dependent CX43 transcription and aberrant increase in progesterone synthesis. Most importantly, blocking PERK expression in vivo significantly inhibited NRF2/CX43/StAR/progesterone pathway activation in the ovary and efficiently rescued the prolongation of estrous cycle and the increase in follicular atresia of the female mice induced by cold stress. We have elucidated the mechanism of ovarian PERK/NRF2/CX43/StAR/progesterone pathway activation in mediating female reproductive disorder under cold exposure. Targeting PERK might be helpful for maintaining female reproductive health under cold conditions.

Specific patterns of gyrA mutations determine the resistance difference to ciprofloxacin and levofloxacin in Klebsiella pneumoniae and Escherichia coli.

BACKGROUND: Wide use of ciprofloxacin and levofloxacin has often led to increased resistance. The resistance rate to these two agents varies in different clinical isolates of Enterobacteriaceae. Mutations of GyrA within the quinolone resistance-determining regions have been found to be the main mechanism for quinolone resistance in Enterobacteriaceae. It has been shown that only some of the mutations in the gyrA gene identified from clinical sources were involved in fluoroquinolone resistance. Whether different patterns of gyrA mutation are related to antimicrobial resistance against ciprofloxacin and levofloxacin is unclear. METHODS: The minimum inhibitory concentration (MIC) of ciprofloxacin and levofloxacin were determined by the agar dilution method followed by PCR amplification and sequencing of the quinolone resistance determining region of gyrA to identify all the mutation types. The correlation between fluoroquinolone resistance and the individual mutation type was analyzed. RESULTS: Resistance differences between ciprofloxacin and levofloxacin were found in 327 isolates of K. pneumoniae and E. coli in Harbin, China and in the isolates reported in PubMed publications. GyrA mutations were found in both susceptible and resistant isolates. For the isolates with QRDR mutations, the resistance rates to ciprofloxacin and levofloxacin were also statistically different. Among the 14 patterns of alterations, two single mutations (Ser83Tyr and Ser83Ile), and three double mutations (Ser83Leu+Asp87Asn, Ser83Leu+Asp87Tyr and Ser83Phe+Asp87Asn) were associated with both ciprofloxacin and levofloxacin resistance. Two single mutations (Ser83Phe and Ser83Leu) were related with ciprofloxacin resistance but not to levofloxacin. Resistance difference between ciprofloxacin and levofloxacin in isolates harboring mutation Ser83Leu+Asp87Asn were of statistical significance among all Enterobacteriaceae (P<0.001). CONCLUSIONS: Resistance rate to ciprofloxacin and levofloxacin were statistically different among clinical isolates of Enterobacteriaceae harboring GyrA mutations. Ser83Leu+Asp87Asn may account for the antimicrobial resistance difference between ciprofloxacin and levofloxacin.